WO2017020980A1 - Use of nanoparticles and cationically modified nucleic acid for the detection of a nucleic acid that is to be detected in a solution - Google Patents

Abstract

The invention relates to a use of at least two nanoparticles (2) and/or a cationically modified nucleic acid (6) for detecting a nucleic acid (10) that is to be detected in a solution. The invention also relates to a corresponding method and a kit for carrying out such a method.

Description

 Use of nanoparticles and cationically modified nucleic acid for detecting a nucleic acid to be detected in a solution

description

The invention is in the technical field of detecting nucleic acids of an at least partially known nucleotide sequence. Furthermore, the invention is in the technical field of detecting or determining a deviation of a nucleotide sequence of a nucleic acid from a predetermined nucleotide sequence and / or in the field of determining a concentration of a particular nucleic acid in a solution.

In particular, the invention is in one aspect of the art, the existence and / or concentration of nucleic acids such as e.g. Deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The detected nucleic acid may then, for example, provide information as to whether e.g. a disease germ was present in a sample to be tested and / or whether, e.g. a living being / organism carries a certain genetic predisposition or not. In the case of such nucleic acid detection, for example, the nucleic acid sequence to be detected may previously be wholly or at least partially known. Alternatively, for example, an objective may be to detect only the presence of a nucleic acid sequence to be detected in a sample and / or to determine any variations or deviations of the sequence from a predetermined sequence.

Furthermore, the invention may be in a technical field, in which a detection of a nucleic acids should take place directly, that is to carry out with sufficient amount of the nucleic acid to be detected, for example, without a previous example, enzymatic amplification. Without prior enzymatic amplification, for example, without a polymerase chain reaction (PCR) and / or isothermal amplification and / or prior transcription such as reverse transcription (RT), in which, for example by an enzymatic reaction, a type From nucleic acid such as RNA is rewritten in another type of nucleic acid such as DNA here, mean.

The invention may moreover be in a technical field in which the objective is an indirect detection of the nucleic acids, e.g. after and / or during e.g. enzymatic amplification reaction, e.g. PCR and / or isothermal amplification. This may be desirable if the nucleic acid to be detected is present, for example, only in small amounts in a sample to be examined and thus, for example, must first be duplicated, in order then to be available for a detection reaction in sufficient quantity. Hereby, it may e.g. be advantageous if the reaction to be detected takes place only above a certain minimum amount of nucleic acid to be detected or amplified and / or provides a detectable signal and thus, e.g. during a replication reaction, the time of occurrence of the detection reaction or the initial signal detection can be used as a measure of the amount of nucleic acid originally present (example real-time PCR or qPCR). Indirect detection of nucleic acids may also mean that before the actual detection, another step such as e.g. a prior transcription such as RT takes place, so a rewriting of RNA into DNA.

Various methods for the detection or detection of nucleic acids are known in the prior art. According to these conventional methods, the detection of nucleotides is usually carried out by means of dyes. Examples of such conventional methods are mentioned below.

For example, conventional methods employ intercalating dyes such as Sybr-Green, which have low baseline fluorescence when not in contact with double-stranded DNA but whose fluorescence increases greatly when incorporated into double-stranded DNA. Such a method is disclosed, for example, in US Pat. No. 5,436,134. Intercalating dyes such as Sybr-Green can also be used conventionally in a real-time PCR or qPCR to determine the amount of nucleic acid originally used by determining the time or the number of cycles passed through at that time, in which increases the fluorescence signal of the intercalating dye above a certain noise limit. Such a process is described in US Pat. No. 6,569,627 B2. However, such methods are often associated with the disadvantage that the detectable limit or the threshold of detectability can be achieved partially only at relatively high concentrations, for example of 10 nM of the amplificate in typical reaction volumes, whereby the duration of the amplification reaction to the earliest detection time very can be long.

For example, other conventional dye-based detection methods for nucleic acids utilize the fact that dyes exhibit different emission behaviors, depending on how close they are to another dye or quencher. For example, a quencher can strongly influence the emission behavior of a nearby dye by Förster resonance energy transfer (FRET). Based on this principle, there are also conventional real-time PCR or qPCR methods such as e.g. TaqMan (hydrolysis probes) or hybridization probes (also called "light cycler probes"), which keep depending on the concentration of the amplificate FRET partners at different distances from each other (TaqMan) or bring (hybridization probes) and thus produce different levels of fluorescence signals , Such a method is described, for example, in US Pat. No. 5,804,375.

Other conventional dye-based detection methods for nucleic acids have exploited the spatial localization of nucleic acids and make them visible there with dyes, for example in gel electrophoresis or array hybridization methods. Furthermore, methods are known in the art that detect nucleic acids without the use of dyes or other markers. For example, absorbance measurements in the ultraviolet (eg at a wavelength of 260 nm) allow determination of the concentration of nucleic acids, such as DNA and RNA.

In addition, other methods are known in the prior art, which use nanoparticles for the detection of nucleic acids. For example, US Pat. No. 6,812,334 B1 discloses such a method for detecting nucleic acids. According to this method, nanoparticles attached with oligonucleotides and one or more types of linking oligonucleotides are provided. Each compound oligonucleotide has two sections. The sequence of one segment is complementary to the sequence of one of the segments of nucleic acid and the sequence of the other segment is complementary to the sequence of oligonucleotides on the nanoparticles. The nanoparticle-oligonucleotide conjugates, the compound oligonucleotides, and the nucleic acid are brought together under hybridization conditions, resulting in a measurable change. The cited patent further discloses a kit for carrying out this method.

From Patent Application US 2010/0075335 A1, a colorimetric method for the detection of specific nucleic acid sequences, including mutations and single nucleotide polymorphisms (SNPs) in nucleotide sequences by aggregation of nanoparticles is known. In this procedure, identical nanopipes are used with oligonucleotides attached directly to gold nanoparticles, which are red in solution. The aggregation of these nanoprobes by an increase in the ionic strength leads to a color change from red to blue. The presence of the target DNA to be detected with a sequence that is completely complementary to the sequence of the nanoprobe prevents this aggregation, whereupon the solution remains red. For example, this method can be used to distinguish fully complementary sequences from sequences with an SNP. Furthermore, this patent application also discloses a kit for using said method. European Patent Application EP 1 870 478 A1 discloses a biosensor consisting of metal particles attached to a surface of a substrate. On these metal particles, a probe molecule is attached, which may be in particular a nucleic acid which forms a hairpin structure. The probe molecule has a fluorescent molecule. Before the reaction of the probe molecules with the target molecules, the distance between the metal particle and the fluorescent molecule is equal to or less than 5 nm, so that excitation energy is transferred from the fluorescent molecule to the metal particle. This quenches the fluorescence (quenched). The distance between fluorescent molecule and metal particles is greater after the reaction, so that the fluorescent molecule can fluoresce, with which then the reaction or the nucleic acid can be detected. In addition, the publication by R. Gill et al., "Fast, single-step, and surfactant-free oligonucleotide modification of gold nanoparticles using DNA with a positively charged tail", Chem. Commun., 2013, 49, 1400-1402 how nanoparticles can be functionalized and stabilized by ZNA with DNA. The thus functionalized nanoparticles can then be selectively linked to other nanoparticles by hybridization and thus produce a measurable optical change. In other words, the use of ZNA according to this conventional method promotes the "adhesion" of the DNA to a nanoparticle, whereby the connection of several nanoparticles is conventionally carried out by a hybridization of DNA which is applied to the various nanoparticles.

 Here, nucleic acids and nanoparticles are thus linked by ZNA, i. DNA hybridized by ZNA to nanoparticles.

The methods known from the prior art for detecting a nucleic acid adhere in particular to the disadvantages that they have a relatively low sensitivity and that the implementation of these methods often takes a great deal of time until the presence or absence or the concentration of a certain nucleic acid can be reliably assessed. In conventional methods, the detection of nucleotides is usually done by means of dyes. Dye-based methods, however, may have problems, for example with regard to fading and / or the stability of the dyes, quenching effects due to interaction with interfering substances in the sample, disturbing background fluorescence of other substances. Furthermore, conventional methods often have the need for often very complex detection methods. Avoiding dyes in methods for detecting nucleic acids is therefore desirable. It is the objective technical object of the present invention to provide a method and a kit which do not have the disadvantages of the conventional methods.

This object is achieved by an inventive use of nanoparticles according to claim 1, an inventive use of cationically modified nucleic acid according to claim 2, a method according to the invention with the steps of claim 3, and by a kit having the features of claim 15. Preferred embodiments are subject matters of the dependent claims.

In a first aspect, the invention relates to a use of at least two nanoparticles in combination with a cationically modified nucleic acid for detecting a nucleic acid to be detected in a solution. The use of the nanoparticles according to the invention has the advantage that the detection method of nucleic acids can be carried out particularly efficiently. In particular, the time duration which can be necessary to achieve a meaningful result can be significantly reduced by a use according to the invention. Furthermore, a use according to the invention has the advantage that preferably an implementation in conventional methods and / or the use of conventional detection devices and / or conventional measuring apparatuses is possible and thereby possibly no significant or no high cost for suitable devices to a use according to the invention are required. Furthermore, a use according to the invention has the technical advantage that, in particular during or within the scope of a detection method in a solution, the possibility of locally heating at least part of the solution can be combined with a particularly efficient detection method.

Nanoparticles are preferably those particles which have special optical properties due to their size. Preferably, their optical properties are mainly given by the size of the nanoparticles, but less by their material properties. The particular optical properties may be, for example, characteristic extinction spectra and / or scattering spectra, which are significantly influenced by the particle size and are not necessarily pronounced in the bulk material or macroscopic material. The nanoparticles are particularly preferably those nanoparticles whose optical properties are at least partially caused by the plasmonic properties of the nanoparticles. In this case, the nanoparticles preferably have a diameter of between 2 and 500 nm, more preferably between 3 and 300 nm, more preferably between 5 and 200 nm, even more preferably between 7 and 150 nm, most preferably between 10 and 100 nm spherical or spherical shape, but are not limited in their shape. For example, other forms of nanoparticles are conceivable, such as elongated or rod-shaped nanoparticles ("nanorods") or other geometric shapes.

In a preferred embodiment of the invention, at least a part of the nanoparticles comprises at least one metal and / or one semiconductor. The metal is preferably a noble metal, such as gold, silver, platinum or copper. The semiconductor may comprise, for example, silicon and / or germanium, as well as composite semiconductor materials, such as cadmium telluride or lead sulfide. Furthermore, at least a part of the nanoparticles can also be composed of different materials. For example, at least some of the nanoparticles can be used as shell-core nanoparticles ("core shell nanoparticle"). be formed, for example, the core comprises a metal and the shell comprises a semiconductor material, or the core comprises a semiconductor material and the shell comprises a metal. In a preferred embodiment, all nanoparticles used are of similar construction, in particular made of the same material. In a preferred embodiment, a nanoparticle may have on its surface pores which can be occupied by atoms or molecules having a size and charge suitable for the pores, for example if such atoms and / or molecules are present together with the nanoparticles in a solution. A nanoparticle should also comprise the atoms and / or molecules deposited on its surface.

In a particularly preferred embodiment, the nanoparticles are due to their special optical properties to absorb light at certain wavelengths particularly effective and / or scatter. In particular, the nanoparticles absorb or scatter light in a particularly efficient manner, whose wavelengths coincide with a plasmon resonance of the nanoparticles, wherein the wavelength of the plasmon resonance or the excitation energy of the plasmon resonance decisively by the size and / or shape and / or the material of the nanoparticles is determined. Excitation of the nanoparticles with light, whose photon energy approximately coincides with the plasmon resonance energy, is particularly suitable for transferring light energy to the nanoparticles or for allowing light energy to be absorbed by the nanoparticles. The terms nucleic acid and oligonucleotide include in the context of the present invention not only deoxyribonucleic acids or deoxyoligoribonucleotides, but also nucleic acids and oligonucleotides with one or more nucleotide analogues with modifications to their backbone (eg methylphosphonates, phosphorothioates or peptide nucleic acids (PNAs), in particular a sugar of the backbone). Further, they may also contain base analogues such as 7-deazapurines or universal bases such as nitroindole or modified natural bases such as N4-ethyl-cytosine. In another embodiment, the nucleic acids or oligonucleotides are conjugates or chimeras with non-nucleoside analogs, such as PNA. The nucleic acids or oligonucleotides may have at one or more positions non-nucleosidic units such as spacers, eg hexaethylene glycol or Cn spacers, where n is preferably a natural number between 3 and 6. Cn spacers are preferably spacers which comprise n carbon atoms. For example, Cn spacers may serve as spacers, especially between a binding member such as a thiol bond and the nucleic acid as such. For example, Cn spacers may be required for a synthesis of thiol oligomers or may be formed on thiol modifications. If the nucleic acids or oligonucleotides have modifications, these are preferably selected such that hybridization with natural DNA or RNA is also possible with the modification is. In further preferred embodiments, the nucleic acids and / or nucleotides may have modifications which influence the melting behavior, in particular the melting temperature. This may, for example, be advantageous in order to distinguish hybrids with different degrees of complementarity of their bases (mismatch discrimination). Further preferred modifications include LNA, 8-aza-7-deazapurines, 5-propynyl-uracil and cytosine and / or abasic disruptions or modifications in the nucleic acid or in the oligonucleotide. Further modifications within the meaning of the invention are, for. B. Modifications with biotin, thiol or thiols and fluorescence donor and fluorescence acceptor molecules. The cationically modified nucleic acid is preferably a nucleic acid which has a cationic block or a cationic section. This may be, for example, a nucleic acid which has a cationic extension on at least one end. The cationic extension preferably has an average charge density which is less anionic than the average charge density of a nucleic acid, in particular when both the nucleic acid and the cationic extension are present in a typical environment for the nucleic acid. In other words, the cationic extension is preferably more positively charged than a nucleic acid. The Nucleic acid with cationic modification or the cationically modified nucleic acid preferably further comprises a portion or a hybridization region, which comprises mainly a nucleic acid, and conjugated thereto a cationic extension.

In a preferred embodiment, the cationic extension of the cationically modified nucleic acid comprises cationic spermine. The cationic extension comprises between 1 and 20 spermine, preferably between 2 and 15 spermine, more preferably between 2 and 10 spermine, most preferably between 2 and 5 spermine. The mean electrical charge density of a cationically modified nucleic acid is less anionic than the mean electrical charge density of a non-cationically modified nucleic acid. In this way, for example, the electrostatic repulsion between a cationically modified nucleic acid and a non-cationically modified nucleic acid is less than the electrostatic repulsion between two non-cationically modified nucleic acids. Particularly preferably, a cationically modified nucleic acid and a non-cationically modified nucleic acid attract electrostatically. For example, a cationically modified nucleic acid comprises a nucleic acid of the type ZIP NUCLEIC ACID (ZNA) from the manufacturer METABION (METABION INTERNATIONAL AG, Planegg, Germany), which is described, for example, in the publications WO 2007 069 092 B1 or WO 2009 083 763 A1. Alternatively or additionally, a cationically modified nucleic acid may comprise, for example, at least one cationically charged dye and / or other cationic units. The addition of at least one cationic unit to a nucleic acid is preferably carried out at the 5 'end and / or at the 3' end of the nucleic acid. The detection of the nucleic acid to be detected can take place, for example, in that a detectable or measurable effect is detected, which occurs when the at least two nanoparticles are in the spatial proximity to at least one cationically modified nucleic acid, in particular if the at least two nanoparticles are at least partially bound to a cationically modified nucleic acid.

In a preferred embodiment of the invention, a nanoparticle is in particular in the. spatial proximity to a cationically modified nucleic acid when their electrostatic interaction with each other, i. their electrostatic repulsion and / or attraction forces, is influenced or if the electrostatic interaction between a nanoparticle and a cationically modified nucleic acid significantly affects the interaction of the nanoparticle to other, preferably adjacent, nanoparticles. The effective distance up to which electrostatic forces between the nanoparticle and the cationic modified nucleic acid have a significant influence on the interaction between the nanoparticle and the cationically modified nucleic acid may be influenced by their environment. For example, the salt concentration and / or the pH of the solution in which the nanoparticles and the cationically modified nucleic acids are located can have an influence on their electrostatic interaction. Preferably, the influence of the electrostatic interaction between a cationic modified nucleic acid and a nanoparticle for the preferred achievement of an intended effect according to the invention is preferably particularly pronounced if the distance between the cationic modified nucleic acid and the surface of the nanoparticle is less than 150 nm, more preferably less than 100 nm, more preferably less than 50 nm, more preferably less than 30 nm, most preferably less than 10 nm.

Preferably, the at least two nanoparticles have functionalized molecules on their surface to which a cationically modified nucleic acid can at least partially bind.

The solution in which the nanoparticles and / or the cationically modified nucleic acids are present preferably has suitable buffer conditions. The buffer conditions may be suitable, for example, if they have a Allow an amplification reaction to amplify a nucleic acid.

In a further aspect, the invention relates to a use of a cationically modified nucleic acid in combination with at least two nanoparticles for detecting a nucleic acid to be detected in a solution.

In a preferred embodiment, the use of a combination of the nanoparticles and the cationically modified nucleic acid can serve to detect a nucleic acid, in particular a further nucleic acid. In this case, for example, both the cationically modified nucleic acid and the nanoparticles can be placed in a solution in which the presence and / or concentration of a further nucleic acid is to be detected, wherein the nucleotide sequence of the nucleic acid to be detected is preferably at least partially known. In a preferred embodiment, conclusions can be drawn from the occurrence or non-occurrence of an interaction between the cationically modified nucleic acid and the nanoparticles or from the resulting measurable effect on the presence and / or absence and / or concentration of the nucleic acid to be detected in the solution to be pulled.

Furthermore, the invention relates to a method for detecting a nucleic acid to be detected in a solution, the method comprising the following steps:

 Providing at least two nanoparticles in the solution;

Providing a cationically modified nucleic acid in the solution;

Determining a change in at least one physical property of the solution wherein the physical property of the solution is at least partially changed by at least partially binding the at least two nanoparticles to the cationically modified nucleic acid and wherein the nucleic acid to be detected in the solution comprises at least partially binding the at least two nanoparticles at least partially promotes or at least partially inhibits the cationically modified nucleic acid. Preferably, the at least partial binding of the at least two nanoparticles to the cationically modified nucleic acid is more strongly inhibited or more promoted by a greater concentration of the nucleic acid to be detected in the solution than by a lower concentration of the nucleic acid to be detected in the solution. In other words, the concentration of the nucleic acid to be detected in the solution preferably increases or inhibits the at least partial binding of the at least two nanoparticles to the cationically modified nucleic acid. An interaction of the at least two nanoparticles with one another preferably changes at least partially when the at least two nanoparticles are at least partially bound to the cationically modified nucleic acid. Particularly preferably, the change in the physical property depends on the change in the interaction of the at least two nanoparticles with one another,

The change in the physical property of the solution is particularly preferably effected by a change in the interaction of the at least two nanoparticles, the change in the interaction of the at least two nanoparticles being influenced, in particular conditionally, by an at least partial binding of the at least two nanoparticles to the cationically modified nucleic acid.

Preferably, binding of the at least two nanoparticles to the cationically modified nucleic acid depends at least in part on a concentration of the nucleic acid to be detected in the solution.

In particular, the presence of the nucleic acid to be detected in the solution or the presence of a minimum concentration of the nucleic acid to be detected in the solution may lead to the two nanoparticles at least partially binding to at least partially to a cationically modified nucleic acid, while an absence of the detected Nucleic acid in the solution or the presence of a lower concentration than a minimum concentration of the nucleic acid to be detected in the solution does not cause the at least two nanoparticles at least partially bind to a cationically modified nucleic acid or at least partially bind to it.

Alternatively, the presence of the nucleic acid to be detected in the solution or the presence of a minimum concentration of the nucleic acid to be detected in the solution can result in the at least two nanoparticles not being able to bind at least partially to a cationically modified nucleic acid or at least partially unable to bind to it Absence of the nucleic acid to be detected in the solution or the presence of a lower concentration than the nucleic acid to be detected in the solution leads to the at least two nanoparticles at least partially binding to a cationically modified nucleic acid, or at least partially can not bind to it. In a preferred embodiment, the physical property of the solution is a property that can be measured. Preferably, the physical property of the solution is a macroscopic property of the solution. The physical property of the solution is particularly preferably a homogeneous property of the solution, ie the physical property of the solution can be measured in arbitrary partial volumes and / or at arbitrary points of the solution and the measurement then representative of the corresponding physical property of the solution entire solution is. The partial volumes are preferably at least so large that at least two nanoparticles and at least one cationically modified nucleic acid are contained in each partial volume. In practice, therefore, the partial volumes are preferably greater than 1 attoiter, more preferably greater than 1 femtoliter, more preferably greater than 1 picoliter, and most preferably greater than 1 nanoliter. For example, the physical property of the solution may be an optical property of the solution and / or a property of the composition of the solution, such as a property of the mixture and / or an occurrence of a failure product and / or sedimentation. In a preferred embodiment, at least one nucleic acid is bound to at least one of the nanoparticles.

In a preferred embodiment of the method, the at least one nucleic acid bound to the at least one nanoparticle is a nucleic acid which is to be characterized. In particular, the nucleic acid may be a nucleic acid whose nucleotide sequence is to be determined at least partially and / or by which the complementarity with another nucleic acid is to be determined. In a particularly preferred embodiment, the complementarity of the nucleotide sequence of the nucleic acid bound to the nanoparticle with at least part of the nucleotide sequence of the cationically modified nucleic acid is determined.

In further preferred embodiments, different nucleic acids having different nucleotide sequences may be attached to one nanoparticle each and / or different nucleic acids having different nucleotide sequences may be attached to different nanoparticles. In other words, a plurality of nanoparticles may be present, to each of which a nucleic acid of the same nucleotide sequence is bound and / or there may be a plurality of nanoparticles, to each of which nucleic acids having different nucleotide sequences are bound. Furthermore, the nanoparticles themselves may differ from one another, in particular in their size and / or in their shape and / or in their material. The nucleic acid bound to a nanoparticle can be attached to the nanoparticle in different ways. In a preferred embodiment, the nucleic acid is bound to the nanoparticle with a thiol bond and / or with a streptavidin-biotin bond and / or with a cationically modified nucleic acid. There may be further passive nucleotide sequences between an active recognition sequence or recognition nucleotide sequence of the nucleic acid and the surface of the nanoparticle. Such passive nucleotide sequences may be, for example, spacers and / or abasic modifications, such as Spacer9, dSpacer, polyethylene glycols and / or the like. In particular, passive nucleotide sequences can be formed from a same and / or a similar material, such as a recognition sequence or a hybridization region of the nucleic acid. For example, such spacers may be formed as DNA spacers and / or multi-adenine sequences. A spacer 9 is a modification which is based on a triethylene glycol chain with a length of 9 atoms, as is well known in the prior art.

In a further preferred embodiment, the nucleic acid bound to the at least one nanoparticle comprises at least one oligonucleotide.

In a particularly preferred embodiment, only one or a plurality of oligonucleotides are bound to the nanoparticle. If a plurality of oligonucleotides is bound to a nanoparticle, these may be identical and / or different oligonucleotides. For example, a plurality of different types of oligonucleotides can each be attached to a nanoparticle. The oligonucleotides may differ, for example, in their length and / or their binding and / or their nucleotide sequence. Preferably, between 1 and 5,000 oligonucleotides are attached to a nanoparticle. Between 2 and 4,000 oligonucleotides are particularly preferably attached to a nanoparticle. More preferably, between 10 and 3,000 oligonucleotides are attached to a nanoparticle. Even more preferably, between 20 and 2,000 oligonucleotides are attached to a nanoparticle. Particularly preferred are between 50 and 1000 oligonucleotides attached to a nanoparticle.

The larger the number of oligonucleotides attached to a nanoparticle and / or the greater the length or number of nucleotides per oligonucleotide attached to a nanoparticle, the greater the loading density of the nanoparticle. A larger loading density can have the beneficial effect of making the nanoparticles more stable in the solution. The nanoparticles are stable in a solution, in particular if they do not aggregate with one another, in particular at room temperature and without external influence. clump and / or fall out of solution. Such a foreign influence can be, for example, the compound with a cationically modified nucleic acid. In other words, a high loading density prevents or reduces the risk of aggregating nanoparticles. In a preferred embodiment, all stable nanoparticles present in a solution have at least one molecule bound to their surface. Preferably, the nanoparticles used have a loading density between 0.001 and 1, more preferably between 0.001 and 0.5, even more preferably between 0.001 and 0.2, more preferably between 0.001 and 0.1, most preferably between 0.01 and 0, 1 oligonucleotide per nm ² nanoparticle surface.

An oligonucleotide preferably comprises not more than 1,000 nucleotides, more preferably not more than 500 nucleotides, more preferably not more than 200 nucleotides, even more preferably not more than 100 nucleotides, most preferably not more than 80 nucleotides, most preferably not more as 50 nucleotides.

In a further preferred embodiment, the cationically modified nucleic acid is at least partially complementary to a nucleic acid to be detected in the solution and / or the at least one oligonucleotide is bound to the at least one nanoparticle, preferably at least partially complementary to the nucleic acid to be detected in the solution. When a first nucleic acid is partially complementary to a second nucleic acid, this means that at least a portion of the first nucleic acid is complementary to at least a portion of the second nucleic acid. In particular, this means that with a complementarity of two nucleic acids whose nucleotide sequences are designed such that these nucleic acids can hybridize with each other. Accordingly, if the cationically modified nucleic acid is at least partially complementary to the nucleic acid to be detected in the solution, a single strand of cationically modified single strand nucleic acid can be detected in the solution Hybridize nucleic acid. When the oligonucleotide attached to the nanoparticle is at least partially complementary to the nucleic acid to be detected in the solution, a single strand of the oligonucleotide attached to the nanoparticle may hybridize to a single strand of the nucleic acid to be detected in the solution. When the oligonucleotide attached to the nanoparticle hybridizes to the nucleic acid to be detected in the solution, the nucleic acid to be detected in the solution is linked to the nanoparticle. In a preferred embodiment, the at least one oligonucleotide is at least partially complementary to a first nucleotide sequence section of a nucleic acid to be detected in the solution, and the cationically modified nucleic acid is at least partially complementary to a second nucleotide sequence section of the nucleic acid to be detected in the solution.

For example, the first nucleotide sequence section and the second nucleotide sequence section can be formed on the same strand of the nucleic acid to be detected in the solution. In this case, when the oligonucleotide and the cationically modified nucleic acid combine with the first nucleotide sequence portion and the second nucleotide sequence portion, respectively, both the oligonucleotide and the cationically modified nucleic acid are hybridized on the same strand of the nucleic acid to be detected.

In a further preferred embodiment, the first nucleotide sequence section is formed on a first strand of the nucleic acid to be detected, and the second nucleotide sequence section is formed on a second strand of the nucleic acid to be detected which is different from the first strand. In a preferred embodiment, at least a portion of the at least one oligonucleotide and / or at least a portion of the cationically modified nucleic acid is a primer, such as a forward primer and / or a reverse primer. For example, the oligonucleotide attached to the nanoparticle may be a forward primer or reverse primer for the first strand of the nucleic acid to be detected. In addition, the cationically modified nucleic acid may be a forward primer or reverse primer for the second strand of the nucleic acid to be detected. According to this preferred embodiment, during an amplification reaction, preferably the oligonucleotide attached to the nanoparticle is elongated and / or the cationically modified nucleic acid is elongated during an amplification process. The resulting amplicons may then preferably hybridize with each other, wherein preferably the hybridization of an elongated oligonucleotide bound to a nanoparticle with an elongated nucleic acid having cationic modification may occur.

Preferably, during an annealing process of a PCR, the primers may hybridize to the first strand and / or the second strand of the nucleic acid to be detected. The oligonucleotide bound to the nanoparticle and / or the cationically modified nucleic acid may in this case comprise further parts or blocks or sections which essentially do not contribute to the function as a primer. For example, in the case of the oligonucleotide, this can be one or more sections with which the oligonucleotide is bound to the nanoparticle. Furthermore, the oligonucleotide may have one or more spacers which do not substantially contribute to the functionality as a primer. Furthermore, the cationically modified nucleic acid can also have spacers which do not contribute to the function as a primer. In a preferred embodiment, in particular the extension with the cationic modification does not contribute to the functionality as a primer as such. For example, a cationic extension can increase a melting point if the cationically modified nucleic acid hybridizes to a nucleic acid and / or influence, in particular increase, an affinity for a nucleic acid.

In a preferred embodiment, at least a portion of the at least one oligonucleotide and / or at least a portion of the cationically modified nucleic acid at the 3 'end carries one or more terminating modifications such as Dideoxycytidine (ddC) or phosphate or biotin. These modifications may prevent the 3 'extension of the at least one part of the at least one oligonucleotide and / or of the at least one part of the cationically modified nucleic acid by the polymerase, so that the at least one part of the at least one oligonucleotide and / or the at least one part the cationically modified nucleic acid can not serve as a primer. This can be advantageous in order to influence and / or disturb the amplification process as little as possible by the presence of the oligonucleotide and / or the cationically modified nucleic acid and / or to use the oligonucleotide and / or the cationically modified nucleic acid as hybridization probe. A hybridization probe here refers in particular to a nucleic acid and / or a part of a nucleic acid and / or a nucleotide sequence section of a nucleic acid whose hybridization with at least part of the nucleic acid to be detected can be used to check or detect the presence of the respective nucleic acid to be detected, the hybridization probe preferably hybridizing but can not be elongated.

In a further preferred embodiment, at least a portion of the nucleic acid linked to the nanoparticle is a primer, such as a forward primer and / or a reverse primer, wherein the cationically modified nucleic acid is not a primer or comprises a primer. According to this preferred embodiment, the cationically modified nucleic acid comprises a specific nucleotide sequence which serves as a specific hybridization probe. In other words, at least a portion of the cationically modified nucleic acid is substantially at least partially identical or complementary to the nucleotide sequence of the nucleic acid to be detected.

Alternatively, according to a preferred embodiment, at least a part of the cationically modified nucleic acid is a primer or comprises a primer, wherein the nucleic acid linked to the nanoparticle is not a primer or does not comprise a primer. According to this preferred embodiment, the nucleic acid linked to the nanoparticle comprises a specific nucleotide sequence which serves as a specific hybridization probe. In other words, at least a portion of the nucleic acid associated with the nanoparticle is substantially at least partially identical or complementary to the nucleotide sequence of the nucleic acid to be detected.

According to a particularly preferred embodiment, a nucleic acid to be detected in the solution can serve as a target in order to elongate the primer (s), for example in the course of an amplification reaction. elongate the nucleic acid or the cationically modified nucleic acid associated with the nanoparticle. The primer can be, for example, a forward primer. In addition, the solution may include another nucleic acid which also serves as a primer, for example as a reverse primer, and which is also elongated during the amplification reaction. The further nucleic acid may have a cationic modification or no cationic modification.

Preferably, according to this preferred embodiment, the amplification reaction alone, for example in the absence of a hybridization probe and / or in the absence of a nucleic acid to be detected, does not result in a sufficient measurable change in the physical property of the solution. If, for example, the corresponding complementary counterpart to the nanoparticle-bound nucleic acid or to the hybridization probe is formed during the amplification reaction in the elongated part of the forward primer with cationic modification, for example, the nanoparticle-bound hybridization probe can be bound to the part elongated during the amplification reaction Hybridize forward primers with cationic modification and result in a compound of nanoparticle-linked hybridization probe with the forward primer with cationic modification. From a sufficient amount of amplicon or elongated nucleic acid with cationic modification, there is a measurable change in the physikallischen Eigfenschaft of the solution. This method can be used, for example, either as an end point detection after completion of the amplification method or as a real-time method during the amplification reaction, which then preferably allows a quantification of the originally present nucleic acid concentration in the sample. Possibly. It may be advantageous to choose the amount of the reverse primer (which may be formed with or without cationic modification) lower than the amount of the cationic modification forward primer. This may be advantageous, for example, during the amplification reaction to generate an excess of the nucleic acid strand, which is produced by elongation of the cationic modification forward primer (single-stranded part of the amplicon) to which the nanoparticle-bound hybridization probe can hybridize without for example, to a competition with the opposite strand (single-stranded part of the amplicon by elongation of the reverse primer emerged) comes.

Preferably, the recognition sequence or hybridization probe sequence or hybridization probe nucleotide sequence and the primer sequences or nucleotide sequences which serve as primers do not overlap. Preferably, no contiguous portion of the recognition or hybridization probe sequence is greater than 7 nucleotide bases, more preferably greater than 5 nucleotide bases, even more preferably greater than 3, and most preferably greater than 2 Nucleotide bases complementary or identical to the primer sequences or the nucleotide sequences which serve as primers. Preferably, the nucleotide sequence of the hybridization probe, ie the hybridization probe sequence, is terminated at least at the 3 'end (eg by biotin, phosphate, ddC or other modifications which prevent elongation by the polymerase) or carries a protruding 3' Dangling end that is not complementary to the target binding sequence with at least one base. Thus, for example, it can be prevented that the hybridization probe sequence is involved in the exponential amplification, comparable, for example, to a TaqMan probe (see Example 6). For example, this preferred embodiment may have the advantage that the formation of primer dimers can be reduced and / or prevented. In particular, it can be prevented or at least partially avoided that the formation of primer dimers leads to a combination of nanoparticles with cationically modified nucleic acid. This may advantageously contribute to signal generation by mistake, although a nucleic acid to be detected is not present in the solution. In particular, in order to avoid a signal that occurs by mistake, the hybridization probe, ie the nucleic acid which is at least partially complementary to the nucleic acid to be detected or at least partially identical to the nucleic acid to be detected, ie the nucleic acid bound to the nanoparticle or the nucleic acid with cationic modification , is essentially not amplified according to this preferred embodiment. In a further preferred embodiment, at least a part of the cationically modified nucleic acid is not a primer or does not comprise a primer, and also the nucleic acid linked to the nanoparticle is not a primer or does not comprise a primer. According to this preferred embodiment, the nucleic acid linked to the nanoparticle comprises a specific nucleotide sequence which serves as a specific hybridization probe. In other words, at least a portion of the nucleic acid associated with the nanoparticle is substantially at least partially identical or complementary to the nucleotide sequence of the nucleic acid to be detected. In addition, according to this preferred embodiment, the cationically modified nucleic acid comprises a specific nucleotide sequence which serves as a specific hybridization probe. In other words, at least a portion of the cationically modified nucleic acid is substantially at least partially identical or complementary to the nucleotide sequence of the nucleic acid to be detected.

In a preferred embodiment, the method is used to determine the concentration of the nucleic acid to be detected in the solution, wherein the method preferably comprises a multiplication of the nucleic acid to be detected.

In particular, the detection of a nucleic acid to be detected in the solution can take place in that not or only the original or original or initially present nucleic acid itself is detected, but alternatively or additionally the amplicons or duplicates of the amplification reaction or amplification reaction generated original nucleic acid. This may, for example, make it possible to detect in the solution a nucleic acid whose initial concentration is too low to cause a measurable effect or a measurable change in the physical property of the solution without prior duplication. In particular, an amplification reaction may be advantageous if the change in the physical property of the solution resulting from the initially present nucleic acid to be detected is insufficient to reliably detect or measure this change.

In a preferred embodiment, the detection of the nucleic acid to be detected in the solution takes place during and / or after an amplification of the nucleic acid to be detected in the solution by means of a polymerase chain reaction (PCR).

In particular, the occurrence of a measurable change in the physical property of the solution in a real-time PCR can be used to determine from the serial number of the cycle at which this measurable change in the physical property of the solution occurs, the initial concentration of the nucleic acid to be detected.

Compared with conventional real-time PCR methods, the use according to the invention of nanoparticles and cationically modified nucleic acid can lead to earlier detectability, ie with a smaller number of cycles, of the optionally amplified nucleic acid to be detected and / or to a markedly pronounced, in particular steeper, signal increase. This can For example, allow a faster, improved and / or more reliable detection of the nucleic acid. A further advantage of the use according to the invention of nanoparticles and cationically modified nucleic acid may be that polymerase concentrations which are preferably not more than 20%, particularly preferably not more than 5%, of the polymerase typically recommended by the manufacturer of the polymerase can be sufficient correspond.

In a preferred embodiment, the amplification of the nucleic acid to be detected in the solution takes place by means of a PCR, wherein in PCR one cycle preferably comprises the steps of denaturation, annealing and elongation. Preferably, during a PCR, the cycle or the steps involved in the cycle are passed through several times. Most preferably, each cycle comprises the steps of denaturing, annealing and elongation once each. These steps may preferably take place at different temperatures of the solution. Furthermore, a cycle may consist solely of the steps denaturing, annealing and elongation. Alternatively, it is possible for one cycle to include denaturation at a first temperature and annealing and elongation together at a second temperature. A cycle may include further steps such as determining the physical property of the solution and / or detecting fluorescence and / or determining an amount of generated and / or existing amplicons. Preferably, the course of each cycle is identical to the other cycles. Alternatively, the cycles may also differ from each other, e.g. For example, the annealing temperature may be lower or higher in at least one cycle than in the previous cycle.

During denaturation, double-stranded nucleic acid present in the solution is dehybridized, or melted, or separated from one another. After denaturation, or melting, or dehybridization, the nucleic acid present in the solution is at least partially single-stranded. In particular, denaturation may be by global and / or local heating the solution done. Such local heating is described for example in the document DE 10 2013 215 166 B3.

A global heating of the solution can be carried out, for example, by heating the entire solution or at least part of the reaction vessel in which the solution is located.

During the annealing step, primers present in the solution attach to a single-stranded nucleic acid, as long as the nucleotide sequence of the particular primer is sufficiently complementary to at least a portion of the nucleic acid.

During the elongation, at least part of the strands of a nucleic acid in the solution, to which at least one primer in each case bears, is filled at least partially with free nucleotides by means of a polymerase.

Preferably, during real-time PCR, no more than 200 cycles, more preferably no more than 150 cycles, even more preferably no more than 100 cycles, more preferably no more than 60 cycles, even more preferably no more than 40 cycles, most preferably not go through more than 30 cycles, most preferably no more than 25 cycles.

In a preferred embodiment, an environment of the excited nanoparticles is locally heated by exciting at least a portion of the nanoparticles. Nanoparticles, which are excited to locally heat the solution, need not necessarily be the same or the same particles as the nanoparticles used for the detection of the physical property or a change in the physical property of the solution. For example, the nanoparticles may differ with regard to the oligonucleotides bound thereto and / or the material and / or the shape and / or the size.

The excitation of a nanoparticle preferably takes place by an optical excitation. The optical excitation of a nanoparticle, for example, by the absorption of photons by the nanoparticle happen. The absorption of a photon by a nanoparticle is particularly efficient, especially when the photon energy corresponds approximately to the excitation energy of a plasmone in the nanoparticle or the plasmon resonance of the nanoparticle. In particular, the photon energy substantially corresponds to the plasmon excitation energy if an at least partial overlap exists between the plasmon absorption band and the wavelength spectrum of the photon or of the incident light. The greater the spectral overlap of the incident light with the plasmon absorption spectrum of the nanoparticle, the greater the efficiency of the optical excitation of the nanoparticles by the incident light.

When heat is transferred to the environment of the nanoparticle by excitation of a nanoparticle, this means that energy is transferred to the nanoparticle and the nanoparticles heat their environment through the transfer of energy. Preferably, the excitation of the nanoparticle enhances the immediate environment of the nanoparticle heated as the wider environment of the nanoparticle. Typically, the nanoparticle is first heated by excitation, in particular by optical excitation, and then transfers the heat to its environment. Preferably, the environment of the nanoparticle is a spherical volume that is approximately one hundred times (100 times) the diameter of the nanoparticle that is at the center of this volume. More preferably, the volume is about ten times the diameter, more preferably about four times the diameter, most preferably less than twice the diameter of the nanoparticle.

The environment of the plurality of nanoparticles is preferably locally heated by the excitation of a plurality of nanoparticles. Particularly rapid temperature changes are possible in particular when the heated volume makes up only a small fraction of the total volume of the solution in which the nanoparticles are located. On the one hand, a high temperature difference can already be generated in this case with a small input of energy by the radiation or by the light. On the other hand, a very quick cooling of the heated volume possible if a sufficiently large, cold temperature reservoir in the irradiated volume or around the irradiated volume is present around to cool after irradiation, the nanoparticles and their environment again. This can be achieved, for example, by irradiating the nanoparticles with sufficient intensity (in order to achieve the desired temperature elevation) and sufficiently short (so that the heat remains localized). Advantageously, local heating makes it possible to expose the polymerases, which may also be present in the solution, to lower heat, so that PCR processes with a number of cycles of more than 80 can also be realized.

By way of example, such an optical excitation can take place by means of laser radiation, the photon energy preferably corresponding essentially to the excitation energy of a plasmon resonance of the at least one part of the nanoparticles. The excitation is preferably carried out by absorption of light or laser radiation through at least part of the nanoparticles. A local heating is present in particular if the duration of the optical excitation in the respectively irradiated volume (for example in the laser focus) t is selected to be shorter or equal to a critical excitation duration t1. Here, t1 is determined by a time that the heat takes to diffuse at an average nanoparticle distance from one nanoparticle to the next nanoparticle, multiplied by a scaling factor s1. At a mean nanoparticle distance | x | and a thermal diffusivity D of the medium between the nanoparticles is the critical excitation time t1 given by t1 = (s1 - | x |) ² / D, wherein the thermal diffusivity D typically in aqueous solution has a value of D = 10 ^"7 m ² / s The scaling factor s1 is preferably s1 100, particularly preferably s1> 10, particularly preferably s1> 1 and more preferably s1> 0, 1. For the definition of the local heating, the average nanoparticle distance in meters is calculated as \ x \ = (C _NP ^■ 1000 · ΝΑ ^~ 1/3, where CNP is the molar concentration of the nanoparticles and NA is the Avogadro constant.

In a preferred embodiment, the at least one physical property of the solution changes when at least a portion of the nanoparticles is associated with the at least one cationically modified nucleic acid, wherein the at least one property of the solution preferably comprises an optical property, and wherein the optical property is in particular an extinction and / or a scattering and / or an absorption. In a preferred embodiment, a nanoparticle is associated in particular with the cationically modified nucleic acid if the cationically modified nucleic acid is at least partially hybridized with an oligonucleotide attached to the nanoparticle. Alternatively, the cationically modified nucleic acid may be linked to a nanoparticle by the cationically modified nucleic acid being bound directly to the nanoparticle (eg via a thiol bond). In addition, in a preferred embodiment, a cationically modified nucleic acid can be associated with a nanoparticle in that an oligonucleotide attached to the nanoparticle is connected via a connecting nucleic acid with the cationically modified nucleic acid. This may be the case in particular when the oligonucleotide attached to the nanoparticle hybridizes to a first portion of the compound nucleic acid and the cationically modified nucleic acid hybridizes to a second portion of the compound nucleic acid. In a particularly preferred embodiment, a plurality of oligonucleotides may be bound to a nanoparticle. In particular, at least one cationically modified nucleic acid can bind directly and / or indirectly to at least some of the oligonucleotides bound to the nanoparticles. A direct binding may be present, for example, when the cationically modified nucleic acid hybridizes directly with the oligonucleotide attached to the nanoparticle. An indirect binding or compound may be present, in particular, when the cationically modified nucleic acid connects to the oligonucleotide attached to the nanoparticle via a connecting nucleic acid. In a further embodiment, different types of oligonucleotides may be attached to a nanoparticle, for example, to each of which one or more cationically modified nucleic acids can be attached. For example, the first oligonucleotides attached to the nanoparticle may have first cationically modified nucleic acids may be connected and connected to second nanoparticles attached to the second oligonucleotides second cationically modified nucleic acids, wherein the first oligonucleotides and the second oligonucleotides or the first modified nucleic acid and the second modified nucleic acid are different from each other. Furthermore, it is possible that the first cationically modified nucleic acid directly connects to one of the first oligonucleotides and / or the second cationically modified nucleic acid binds indirectly to one of the second oligonucleotides. The physical property of the solution may change, for example, in that the mean electrical charge density in the immediate vicinity of the nanoparticles and the associated nucleic acids changes if at least one cationically modified nucleic acid is associated with at least a portion of the nanoparticles. The immediate environment preferably comprises the spherical volume, which extends from the center of the nanoparticle to a distance from the nanoparticle surface of preferably 100 nm, more preferably 50 nm, more preferably 30 nm and most preferably 15 nm. When the term below refers to the average electric charge density of the nanoparticles, the charge density in the immediate vicinity of the nanoparticles is preferably always meant.

In a preferred embodiment, nanoparticles, which are each connected to at least one cationically modified nucleic acid, have a different electrostatic interaction with one another than nanoparticles have with one another, which are each not connected to a cationically modified nucleic acid. In a particularly preferred embodiment, a nanoparticle which is associated with at least one cationically modified nucleic acid, another interaction with a nanoparticle, which is not associated with a cationically modified nucleic acid, as the interaction between two nanoparticles, each not with a cationically modified nucleic acid are connected. In particular, the electrostatic repulsion of two nanoparticles, of which at least one nanoparticle is associated with at least one cationically modified nucleic acid is less than the electrostatic repulsion of two nanoparticles, each not associated with a cationically modified nucleic acid.

Preferably, the nanoparticles and / or the nucleic acids bound thereto are chosen such that they are stable in their normal environment or in the solution provided for this purpose, ie that they do not essentially aggregate with one another or do not clump together substantially. If a part of the nanoparticles is associated with at least one cationically modified nucleic acid, this particularly preferably results in at least the part of the nanoparticles which is associated with the at least one cationically modified nucleic acid aggregating with at least one other nanoparticle. Such aggregation preferably leads to a change in the physical property of the solution. If the physical property of the solution is at least one optical property, this change in the physical property of the solution can be, for example, a change in the amplitude and / or the spectral properties of the at least one optical property. For example, the strength of an extinction and / or a scattering and / or an absorption may change. Furthermore, a spectral property of the extinction and / or the scattering and / or the absorption may preferably change. For example, the spectral absorption band originating from the plasmon resonance of the nanoparticles can be subjected to a spectral broadening and / or a spectral redshift. In a particularly preferred embodiment, the concentration of the nanoparticles and / or the cationically modified nucleic acid is selected such that when a compound of at least a portion of the nanoparticles is combined with at least a portion of the cationically modified nucleic acid such a large change in the physical property can be caused that a Measurement or detection of the change in the physical property of the solution is possible. With particular preference, the concentration of the nanoparticles and / or the cationically modified nucleic acid is selected such that when at least a portion of the nanoparticles is combined with at least a portion of the cationically modified nucleic acid, such a large change in the physical property can be caused that the change is visible to the naked eye.

Particularly preferably, the measurement and / or determination of the change in the physical property of the solution takes place, for example, by measuring a change in the extinction at a longer wavelength than the extinction maximum of the plasmon resonance of the nanoparticles in the solution. Preferably, a wavelength is selected for this purpose, which has an increasing, in particular as high as possible, or reliably measurable extinction due to a redshift of the plasmon resonance due to a reduced average distance of at least two nanoparticles.

In a further preferred embodiment, the physical property of the solution is measured at different temperatures of the solution. For example, measuring the physical property of the solution at different temperatures can serve to determine the complementarity of two nucleic acids present in the solution. The greater the complementarity of two nucleic acids present in the solution, the higher is typically their melting temperature, when these are hybridized to a double strand. In this way, for example, by targeted, successive temperature increases can be determined at which temperature the melting point of two complementary or at least partially complementary nucleic acids in the solution and / or at which temperature the melting point is exceeded. Also, for example, measuring the physical property of the solution at different temperatures may serve to at least partially determine a sequence composition and / or length of two nucleic acids present in the solution. For DNA, it is typically believed that the greater the length and GC content, ie, the proportion of guanine cytosine base pairs, of two nucleic acids present in the solution, the higher their melting temperature typically is when hybridized to a double strand. Preferably, the melting curve is in the inventive use of nanoparticles and cationic modified nucleic acids sharper or more clearly recognizable or low in noise than a melting curve, which was recorded without the use of nanoparticles and cationically modified nucleic acids. In other words, in the case of the inventive use of nanoparticles and cationically modified nucleic acids according to a preferred embodiment, the half-width of the first derivative of the melting curve is preferably lower. Preferably, the half width of the first derivative of the melt curve is less than 7 ° C, more preferably less than 5 ° C, even more preferably less than 3 ° C, and most preferably less than 2 ° C. A steep melting curve, or in other words a small half-width of the first derivative of the melting curve, may be advantageous for better distinguishing melting points or melting temperatures of different nucleic acids, if they are close to each other or if the melting points or melting temperatures differ little. A further aspect of the invention relates to a kit for detecting a nucleic acid to be detected in a solution, comprising:

 at least one cationically modified nucleic acid; and

 at least two nanoparticles, wherein the at least two nanoparticles are designed to at least partially change the physical property of the solution by at least partially binding the at least two nanoparticles to the cationically modified nucleic acid and wherein the nucleic acid to be detected in the solution comprises at least partially binding the at least two nanoparticles Nanoparticles to the cationically modified nucleic acid at least partially promotes or at least partially inhibits.

The nanoparticles, on the one hand, and the modified nucleic acid, on the other hand, can be present separately from one another in the kit, or be packaged separately. In a preferred embodiment, the nanoparticles are present in a first solution and the cationically modified nucleic acid in a second solution different from the first solution. For example, the first and the second solution are merged by the user only when a method according to the invention is to be performed. Alternatively, the Nanoparticles and the cationically modified nucleic acid also exist together in a single solution.

In the following, individual preferred embodiments for solving the problem will be described by way of example with reference to the figures. Some of the embodiments described have features that are not necessarily required to carry out the claimed subject matter or method, but which provide desired properties in certain applications. Thus, embodiments are also to be regarded as falling under the described technical teaching, which does not have all the features of the embodiments described below. Further, in order to avoid unnecessary repetition, certain features are mentioned only with respect to each of the embodiments described below. It should be noted that the individual embodiments should therefore be considered not only in isolation but also in a synopsis. Based on this synopsis, those skilled in the art will recognize that individual embodiments may also be modified by incorporating one or more features of other embodiments. It should be understood that a systematic combination of the individual embodiments with single or multiple features described with respect to other embodiments may be desirable and useful, and therefore should be considered and also understood to be encompassed by the description.

It shows:

1A to 1G-2 are schematic representations of a first preferred embodiment and an effect of the method according to the first preferred embodiment;

2 shows a photographic representation of reaction vessels when carrying out a method according to the first preferred embodiment; 3A to 3D: a graphical representation of extinction spectra of solutions in carrying out a method according to a preferred embodiment; 4 shows a schematic representation of a second preferred embodiment;

5 shows a photographic representation of reaction vessels when carrying out a method according to the second preferred embodiment;

6 shows a schematic representation of a third preferred embodiment;

FIG. 7: a photographic representation of reaction vessels when carrying out a method according to the third preferred embodiment; FIG.

Fig. 8: a graphical. Representation of real-time PCR curves, which were recorded by means of a conventional, known from the prior art method; Figures 9A and 9B are a graphical representation of real-time PCR curves taken by a method according to a preferred embodiment of the invention;

10 is a graphical representation of real-time PCR curves taken by a method according to a preferred embodiment of the invention;

11 is a graphic representation of real-time PCR curves taken by a method according to a preferred embodiment of the invention for detecting genomic DNA; Fig. 12 is a graphic representation of real-time PCR curves taken by a method according to a preferred embodiment of the invention at a different MgC concentration of the solution; Fig. 13 is a graphical representation of the first derivative of melting curves taken by a method according to a preferred embodiment of the invention of false-positive and true-positive samples during a continuous temperature increase; FIGS. 14A and 14B are schematic diagrams of a measuring device suitable for performing a laser PCR; FIGS.

Fig. 15A is a graphical representation of real-time laser PCR curves measured by a method of the invention in a preferred embodiment;

Fig. 15B: first derivatives of the curves of Fig. 15A;

Fig. 16A is a graphic representation of real-time laser PCR curves measured by a method without a cationically modified nucleic acid;

Fig. 16B is a graph of other real-time laser PCR curves measured by a method without a cationically modified nucleic acid;

17 shows a graphic representation of real-time PCR curves which were measured by means of a method according to the invention in a further preferred embodiment.

In a first embodiment, nanoparticles are used, wherein at least a part of the nanoparticles carries at least one nucleic acid, ie at least one nanoparticle of the at least one part of the nanoparticles is at least one Bound nucleic acid. Preferably, at least one nucleic acid is bound to each nanoparticle. The nanoparticles are preferably brought into a solution. According to the first embodiment, cationically modified oligonucleotides whose nucleotide sequence is at least partially known are also used. The cationically modified oligonucleotides are brought into the solution in which the nanoparticles are located. With a high degree of complementarity between the nucleotide sequences of the nucleic acids attached to the nanoparticles and the oligonucleotides with cationic modification, preferably at least one of the oligonucleotides with cationic modification hybridizes to at least one of the nucleic acids attached to the nanoparticles. For example, there is a high degree of complementarity if the nucleotide sequence of the oligonucleotides has a sequence complementary to the nucleic acid over a whole hybridization region, which has less than 10% of the bases a non-complementary base pairing, preferably less than 5% of the bases more preferably, less than 3% of the bases have non-complementary base pairing, more preferably less than 1% of the bases have non-complementary base pairing, most preferably no non-complementary base pairing.

If a hybridization occurs, a change of at least one physical property of the solution occurs according to the first preferred embodiment. Preferably, the change in the physical property of the solution is measurable. If the measurable change in the physical property of the solution is measured, information about the complementarity between the nucleotide sequences of the cationically modified oligonucleotides and the nucleotide sequences of the nucleic acids attached to the nanoparticles can preferably be determined therefrom. For example, a method according to the first embodiment may be used to determine the degree of complementarity between the nucleic acids attached to the nanoparticles and the cationic modification oligonucleotides. Alternatively or additionally, a method according to the first embodiment can be used to determine the ability to hybridize between the nucleic acids attached to the nanoparticles with the cationic modification oligonucleotides. It is possible that the sequence of the nucleic acids attached to the nanoparticles is at least partially unknown.

1A to 1E show a schematic representation of the nanoparticles 2 with nucleic acids 4 bound thereto, the cationically modified oligonucleotides 6 and a schematic illustration of a basic principle of a method according to the invention according to the first preferred embodiment.

The schematic representation in FIG. 1A shows a nanoparticle 2 to which a plurality of nucleic acids 4 are attached or bonded or functionalized or conjugated. The nucleic acids 4 can be identical or different and can be attached to the nanoparticle 2 in the same or different orientation. FIG. 1A illustrates the nanoparticle 2 only in a two-dimensional projection or in a two-dimensional cross-section. The nucleic acids 4 are preferably distributed over the entire surface of the three-dimensional, preferably spherical, nanoparticle 2. Particularly preferably, the nucleic acids 4 attached to the nanoparticle 2 are single-stranded nucleic acids 4 and chosen such that they do not hybridize with other nucleic acids 4 bound to the nanoparticles 2 or to other nanoparticles 2. Particularly preferably, all of the nucleic acids 4 bound to the nanoparticles 2 are identical nucleic acids 4. FIG. 1B shows a schematic detailed representation of a cationically modified oligonucleotide 6. The size ratios in the figures or between different figures need not necessarily be such correspond to the actual proportions. The cationically modified oligonucleotide 6 in the embodiment shown comprises a hybridization region 6a and a cationic section 6b. The hybridization region 6a preferably comprises a nucleotide sequence. In a particularly preferred embodiment, the hybridization region 6a may comprise an oligonucleotide and / or in particular consist of an oligonucleotide. The cationic section 6b is preferably formed at one end, in particular at the 3 'end and / or at the 5' end, of the hybridization region 6a. The cationic section 6b comprises at least one cationic unit 6c. In the illustrated embodiment, the cationic portion 6b comprises three cationic units 6c. The cationic units 6c preferably comprise spermine or spermine units.

1C shows the nanoparticle 2 from FIG. 1A, wherein a plurality of cationically modified oligonucleotides 6 are hybridized to the nucleic acids 4 attached to the nanoparticle 2. At the shown nanoparticle 2 all conjugated nucleic acids are occupied with cationically modified oligonucleotides 6. However, this is not absolutely necessary, since only part of the nucleic acids 4 can be occupied by cationically modified oligonucleotides 6. In particular, it may be sufficient to achieve a measurable change in the physical property of the solution when only a portion of the oligonucleotide 4 attached to the nanoparticle 2 is occupied. The cationically modified oligonucleotides 6 preferably hybridize to the nucleic acids attached to the nanoparticle 2 in that the respective hybridization regions 6a of the cationically modified oligonucleotides 6 at least partially hybridize to the nucleic acids 4. Such a hybridization preferably takes place when the cationically modified oligonucleotides 6 and the nucleic acids 4 have a sufficient complementarity. Whether or when a complementarity is sufficient depends on various parameters, such as the temperature of the entire solution and / or the local environment. At a very high temperature, of at least 40 ° C, preferably at least 50 ° C, more preferably at least 60 ° C, even more preferably at least 70 ° C, most preferably at least 80 ° C, it may be necessary for the nucleotide sequences of Hybridization regions 6a of cationically modified oligonucleotides 6 are fully complementary to at least part of the nucleotide sequences of nucleic acids 4, ie that no non-complementary base pairings are present, while at lower temperatures hybridization can occur in the presence of single non-complementary base pairings.

FIG. 1 D shows a schematic representation of an interaction of two nanoparticles 2 in a solution, wherein a plurality of cationically modified oligonucleotides 6 which are at least partially complementary to the nucleic acids are hybridized on each of the two nanoparticles 2 or on the attached nucleic acids 4 4 are. When a cationically modified oligonucleotide ^is' 6 hybridized to a nanoparticle 2, this is intended to mean in the following that the cationically modified oligonucleotide is hybridized to a nucleic acid attached to the nanoparticles 2 4. 6

By a hybridization of at least one cationically modified oligonucleotide 6 to a nanoparticle 2, a mean electrical charge density of the complex of the nanoparticle 2 and the nucleic acids 4 or cationically modified oligonucleotides 6 attached to it changes, for example, those attached to a nanoparticle Nucleic acids 4, preferably electrically negatively charged, ie anionic. Accordingly, preferably, a plurality of nanoparticles 2 or a plurality of complexes of the nanoparticles 2 present in the solution repel one another due to a repulsive electrostatic interaction. In other words, the nanoparticles are preferably substantially stable in solution, i. they do not agglomerate and / or aggregate or aggregate only to such an extent that the physical property of the solution thereby preferably does not change or does not measurably change or the aggregation and de-aggregation are in equilibrium.

If at least part of the nanoparticles 2 hybridizes with at least one cationically modified oligonucleotide 6, this may preferably be a change in the mean electrical charge density of the Nanoparticles 2 or the complexes of nanoparticles 2 cause. In particular, the mean electrical charge density of the nanoparticles 2 or the complexes of the nanoparticles 2 with hybridized cationically modified oligonucleotides 6 may be less anionic or less electrically negative than the mean electrical charge density of the nanoparticles 2 or the complexes of the nanoparticles 2 without hybridized cationically modified Oligonucleotides 6. In other words, hybridization with cationically modified oligonucleotides 6 can lead to a more positive or cationic average electric charge density of the nanoparticles 2 or the complexes of the nanoparticles 2. This can preferably lead to the fact that the repulsive, electrostatic interaction of the hybridized with cationically modified oligonucleotides 6 nanoparticles 2 in the solution is lower. This can have the consequence that the mean distance between two nanoparticles 2 of the plurality of nanoparticles 2 in the solution decreases and / or the nanoparticles 2 at least partially aggregate and / or the nanoparticles 2 at least partially aggregate or clump together. Preferably, the mean distance between two preferably adjacent nanoparticles 2 of the plurality of nanoparticles 2 in the solution decreases to less than three times the average particle diameter of the nanoparticles 2, more preferably less than twice the average particle diameter, more preferably less than the length of the nanoparticles 2 average particle diameter, more preferably less than half the length of the average particle diameter, more preferably less than 20% of the average particle diameter. In this case, the distance between the respective points on the surfaces of the respective nanoparticles 2, which are closest to the other nanoparticles 2, is assumed as the distance between two nanoparticles.

In particular, a mean distance between two nanoparticles 2 of the plurality of nanoparticles 2 in the solution can be so small that the at least one optical property of the solution changes, in particular changes measurably. For example, such a property or change of the physical property may be a change of an optical or spectral property. For example, such a change may be a change in an optical Absorption and / or extinction and / or scattering. For example, such a change may be a redshift and / or spectral broadening of such an optical property. For example, such a change may also be a measurable reduction and / or a measurable increase in the measurable fluorescence of a dye, which may also be in the solution. A reduction or an increase in the fluorescence of a dye can be particularly pronounced, for example, if the fluorescence spectrum of the dye and the absorption spectrum of the plasmon resonance of nanoparticles 2 in the solution at least partially overlap.

For example, a change in an absorption band of the solution may result from a change in plasmon resonance of the nanoparticles 2 in the solution. For example, a smaller mean distance between two preferably adjacent nanoparticles 2 of the plurality of nanoparticles 2 in the solution can shift and / or widen an absorption band toward lower energies. In other words, a spectral redshift and / or spectral broadening of the plasmon resonance of the nanoparticles 2 can thereby occur. For example, this can be done by an interaction of plasmons or surface plasmons of the interacting nanoparticles 2. In particular, this can be done by a dipole / dipole interaction between the interacting nanoparticles 2. Such interactions of nanoparticles 2 are described, for example, in M. Quinten and U. Kreibig, Optical Properties of Aggregates of Small Metal Particles, Surface Science, Volume 172, Issue 3, 2 July 1986, pages 557-577.

Flg. 1E shows a schematic representation of a combination in which the nucleic acids 4 attached to the nanoparticles 2 are not and / or not sufficiently complementary with the cationically modified oligonucleotides 6. Due to the lack of complementarity of the nucleic acids 4 attached to the nanoparticles 2 with the cationically modified oligonucleotides 6, the cationically modified oligonucleotides 6 can not hybridize with the nucleic acids 4 attached to the nanoparticles 2. In this case, it is preferable not to decrease the average electric charge density of Nanoparticles 2 and / or not to a reduction in the average distance between two nanoparticles 2 of the plurality of nanoparticles 2 in the solution. Accordingly, preferably no or no measurable change in the physical property of the solution takes place.

In this way, a measurement of the physical property of the solution or the measurement of a change in the physical property of the solution can serve to provide information about a hybridization of the cationically modified oligonucleotides 6 with the nucleic acids 4 attached to the nanoparticles 2 and / or via a complementarity of the cationically modified oligonucleotides 6 with the nucleic acids 4 attached to the nanoparticles 2.

FIGS. 1F-1 and 1F-2 show a schematic representation of a method according to the invention in a preferred embodiment, in which an optical transmission of a solution containing nanoparticles 2 is measured. The incident light preferably has an intensity 11 and the optical spectrum of the incident light preferably at least partially overlaps with an absorption and / or extinction spectrum of at least part of the nanoparticles 2, such as the plasmon resonance of the nanoparticles 2 incident light around spectrally narrow-band light which is emitted, for example, by a light-emitting diode (LED) or a LASER. Particularly preferably, the central wavelength of the incident light is approximately equal to the wavelength of the plasmon resonance of the nanoparticles 2, if they do not essentially interact with one another and / or in particular do not aggregate with one another. Substantially non-interacting means in particular that the average distance between each two nanoparticles 2 is not so small that there is a measurable shift in a plasmon resonance of adjacent nanoparticles 2.

Fig. 1F-1 illustratively shows the case in which no such interaction between nanoparticles 2 takes place, since the distances between the nanoparticles are large enough not to efficiently such an interaction enable. The representation is not necessarily true to scale. If the incident light having the intensity 11 comprises a wavelength which can be efficiently absorbed by the nanoparticles due to the plasmon resonance, a measurable attenuation of the light intensity occurs within the reaction vessel R in the solution, so that the emergent, transmitted light has an intensity T1 , which is lower, in particular by a measurable factor, than the intensity 11 of the incident light. In this way, it can be demonstrated, for example, that the nanoparticles do not interact or at least do not completely or not interact with one another to a certain extent and / or aggregate and / or agglomerate and / or precipitated. The ratio of the intensity of the incident light 11 and the intensity of the transmitted light T1 is preferably a measure for a shift in the plasmon resonance of the nanoparticles 2 and / or for the mean distance between the nanoparticles 2 and / or for their aggregation.

Fig. 1 F-2 shows a schematic representation of the case in which in the solution of Fig. 1 F-1 additionally at least one nucleic acid with cationic modification 6 is present, which can connect to the nanoparticles 2. If the nucleic acid with cationic modification 6 hybridizes at least partially with the nanoparticles 2 or with a nucleic acid or an oligonucleotide 4 functionalized on the nanoparticles 2, then the mean distance between the nanoparticles can at least partially decrease. This can lead at least partially to an aggregation and / or precipitation and / or sedimentation of the nanoparticles 2. Particularly preferably, there is a shift and / or broadening of the plasmon resonance of the nanoparticles 2, which is why the absorption and / or the extinction of the solution or of the nanoparticles at least partially changes at the wavelength. This may result in that the attenuation of the incident light is less than in the case shown in Fig. 1F-1. Particularly preferably, the intensity of the transmitted light T2 can be greater than the intensity of the transmitted light T1 in the case shown in FIG. 1F-1, even if the intensity 11 of the incident light is the same in both cases. Preferably, the ratio of the intensity of the incident light 11 and the intensity of the transmitted light T2 is a measure for a shift in the plasmon resonance of the nanoparticles 2 and / or for the mean distance between the nanoparticles 2 and / or for their aggregation. Particularly preferred is a measure of a shift of the plasmon resonance of the nanoparticles 2 and / or for the average distance between the nanoparticles 2 and / or for their aggregation particularly efficient and particularly accurate from a comparison and / or a difference and / or a quotient of determine outgoing light intensities of the cases shown in Figs. 1 F-1 and 1 F-2. FIGS. 1G-1 and 1G-2 show a schematic representation of a method according to the invention in a further preferred embodiment, in which dyes 8 are additionally present in the solution and an intensity of an optical emission F1 or F2 of at least part of the dyes 8 is measured , As for the other properties, particularly with respect to the nanoparticles and the incident light, the cases shown in Figs. 1G-1 and 1G-2 correspond to the cases shown in Figs. 1F-1 and 1F-2, respectively. The absorption or excitation spectra and / or the emission spectra of the dyes 8 preferably overlap at least partially with the spectrum of the incident light and / or with the absorption spectrum of the nanoparticles 2, in particular with the spectrum of the plasmon resonance.

As shown in FIG. 1G-1, when the nanoparticles 2 are not aggregated, the nanoparticles efficiently absorb the incident light. In this way, the nanoparticles 2 preferably shield the dyes 8 from the incident light in a particularly efficient manner. Alternatively or additionally, the nanoparticles 2 preferably shield the emission of light caused by the dyes 8, which is caused, for example, by fluorescence of the dyes 8, in a particularly efficient manner. As a result, the emission emerging with intensity F1 from the solution can be at least partially attenuated or completely omitted. Preferably, the ratio of the intensity of the incident light 11 and the intensity of the light F1 emitted by the dyes 8 is a measure of a shift in the plasmon resonance of the nanoparticles 2 and / or for the mean distance between the nanoparticles 2 and / or for their aggregation. FIG. 1G-2 shows a schematic representation of the case in which in the solution from FIG. 1 G-1 additionally at least one nucleic acid with cationic modification 6 is present, which can combine with the nanoparticles 2. If the nucleic acid with cationic modification 6 is at least partially hybridized with the nanoparticles 2 or with nucleic acid or oligonucleotides 4 functionalized on the nanoparticles 2, then the average distance between the nanoparticles 2 can be at least partially reduced. This can lead at least partially to an aggregation and / or precipitation / and / or sedimentation of the nanoparticles 2. Particularly preferably, there is a shift and / or broadening of the plasmon resonance of the nanoparticles 2, which is why the absorption and / or extinction of the solution or the nanoparticles at the wavelength of the incident light and / or the absorption of the dyes 8 and / or the Emission of the dyes 8 at least partially changes. This preferably leads to a lesser shielding of the dyes 8 from the incident light and / or to a lower attenuation of the light emitted by the dyes 8. Particularly preferably, the intensity of the light F2 emitted by the dyes, which exits the reaction vessel R, may be greater than the intensity of the emitted light F1 in the case shown in FIG. 1G-1, even if the intensity 11 of the incident light in both cases the same size. The ratio of the intensity of the incident light 11 and the intensity of the emitted light F 2 is preferably a measure of a shift in the plasmon resonance of the nanoparticles 2 and / or for the mean distance between the nanoparticles 2 and / or for their aggregation. Particularly preferred is a measure of a shift of the plasmon resonance of the nanoparticles 2 and / or for the average distance between the nanoparticles 2 and / or for their aggregation particularly efficient and particularly accurate from a comparison and / or a difference and / or a quotient of determine emitted light intensities F1 and F2 of the cases shown in FIG. 1 G-1 and 1 G-2.

FIG. 2 shows an exemplary photographic representation of reaction vessels R1 to R8 in which a process according to the second preferred embodiment is carried out, the procedure being described in more detail below in Example 1 is described. Measurements of optical properties of the solution are shown in the extinction spectra shown in FIGS. 3A to 3C.

With reference to Fig. 4, a second preferred embodiment of the invention will be explained below. According to a second preferred embodiment of the invention, the nucleic acids 4 attached to at least one part of the nanoparticles each comprise an oligonucleotide. Preferably, the nucleic acids 4, which are attached to the nanoparticle 2, consist essentially of an oligonucleotide 4 and an adjoining bond, with which the oligonucleotide can bind to a surface of a nanoparticle 2. Preferably oligonucleotides 4 with known nucleotide sequence or with known nucleotide sequences are bound to the nanoparticles 2. In this case, all of the oligonucleotides 4 bound to the nanoparticles 2 may have the same or different nucleotide sequences.

4 shows a schematic representation of the second preferred embodiment. In the second preferred embodiment, the nanoparticles 2 are loaded with oligonucleotides 4, wherein the oligonucleotides 4 are at least partially complementary to the nucleic acid 10 to be detected. Furthermore, oligonucleotides 6 with cationic modification are preferably used in the second preferred embodiment, the nucleotide sequence or nucleotide sequences to the nucleotide sequence or the nucleotide sequences of attached to the nanoparticles 2 oligonucleotides 4 is at least partially complementary or are. In a particularly preferred embodiment, at least one type of complementary oligonucleotide 6 with cationic modification is provided for each type of oligonucleotide 4 attached to the nanoparticles 2.

If in the solution in which the nanoparticles 2 and the oligonucleotides 6 with cationic modification are present, no nucleic acids 10 to be detected or only a very low concentration of the nucleic acid 10 to be detected are present, the oligonucleotides 6 with cationic modification and the oligonucleotides 4 on the nanoparticles 2 hybridize with each other. As already With reference to Figures 1A-1E, hybridization of nanoparticles 2 or oligonucleotides 4 on nanoparticles 2 to cationic modification oligonucleotides 6 may cause a measurable change in the physical properties of the solution. If the nucleic acid 10 to be detected is present in an at least sufficiently high concentration in the solution, the hybridization of the oligonucleotides 4 on the nanoparticles 2 with the oligonucleotides 6 with cationic modification is at least partially prevented or prevented. As a result, furthermore, the measurable change in the physical property of the solution is at least partially prevented or at least partially prevented. This is due to the fact that the nucleic acid 10 to be detected according to the second preferred embodiment of the invention can hybridize with the oligonucleotides 6 with cationic modification and / or with the oligonucleotides 4 on the nanoparticles 2. The oligonucleotides 6 with cationic modification and / or the oligonucleotides 4 on the nanoparticles can thus be at least partially occupied by the nucleic acid 10 to be detected. Preferably, the nucleic acid 10 to be detected occupies such a large part of the oligonucleotides 4 on the nanoparticles 2 and / or the oligonucleotides 6 with cationic modification that the proportion of free or not occupied by the nucleic acid 10 to be detected Oiigonukleotide 4 on the nanoparticles or Oiigonucleotides 6 with cationic modification can not cause a measurable change in the physical property of the solution.

Preferably, the nucleic acid to be detected occurs in competition with the oligonucleotides 6 with cationic modification. In other words, an oligonucleotide 4 attached to the nanoparticle 2 can hybridize either with the nucleic acid 10 to be detected or with an oligonucleotide 6 with cationic modification, but not simultaneously with both. Although the oligonucleotide 6 with cationic modification is complementary to the oligonucleotide 4 on the nanoparticle 2, the oligonucleotide 6 with cationic modification may no longer bind to the oligonucleotide 4 on the nanoparticle 2, since this may already be occupied by a nucleic acid 10 to be detected is. Preferably also results in a sufficient concentration of Oligonucleotides 6 with cationic modification in the solution do not lead to a sufficient number of bonds between oligonucleotides 6 with cationic modification and oligonucleotides 4 on the nanoparticles 2 or with the nanoparticles 2, so that no measurable change in the physical property of the solution takes place.

The nucleic acid 10 to be detected or a solution with the nucleic acid 10 to be detected can be added to the solution with the nanoparticles either before or simultaneously with the oligonucleotides 6 with cationic modification. Particularly preferably, the nucleic acid 10 to be detected is added in time before the oligonucleotides 6 with cationic modification. It is also possible to add the nucleic acid 10 to be detected chronologically after the oligonucleotides 6 with cationic modification. Optionally, a heating step prior to a measurement of the change in the physical property of the solution can be inserted, ⁱⁿ order to solve any already existing bonds or hybridizations and to provide a new competitive situation during the cooling of the solution after the heating step. In particular, if the nucleic acids 10 to be detected are added to the solution only after the oligonucleotide 6 with cationic modification, such a heating step may be useful and / or necessary. Furthermore, such a heating step may be particularly useful if the nucleic acid 10 to be detected is initially present in very low concentration and is amplified during an amplification reaction, so that it can be reliably detected only during and / or after the amplification reaction. For example, in such a case, intermediate heating steps would make sense.

If the nucleic acid 10 to be detected is absent or not present in sufficient concentration in the solution and / or the nucleic acid to be detected is not sufficiently compatible with the oligonucleotides 4 on the nanoparticles 2 and / or the oligonucleotides 6 with cationic modification, a sufficient number of oligonucleotides may be preferred 6 with cationic modification to the nanoparticles 2 and the oligonucleotides 4 on the Nanoparticles 2 bind to cause a measurable change in the physical property of the solution.

A method according to the second preferred embodiment of the invention is preferably suitable for detecting a nucleic acid 10 to be detected in the solution or reaction solution. For this purpose, preferably the nanoparticles 2 with the oligonucleotides 4 attached thereto and the oligonucleotides 6 with cationic modification are brought into the solution in which the nucleic acid 10 to be detected is to be detected. The nanoparticles 2 and the oligonucleotides 6 with cationic modification need not necessarily be added simultaneously to the solution. Preferably, the nucleic acid 10 to be detected is initially free in the solution. According to the second preferred embodiment, at least part of the nucleotide sequence of the nucleic acid 10 to be detected is at least partially complementary to the nucleotide sequence of the oligonucleotides 4 on the nanoparticles 2 and / or to the nucleotide sequence of the oligonucleotides 6 with cationic modification.

FIG. 5 shows an exemplary photographic representation of reaction vessels R1 to R4 in which a process according to the second preferred embodiment is carried out, the procedure being described below in Example 2.

Hereinafter, a third preferred embodiment will be described with reference to FIG. In detail, FIG. 6 shows a two-dimensional cross-section or a two-dimensional projection of a nanoparticle 2, which is functionalized with oligonucleotides 4 having a known nucleotide sequence.

According to the third preferred embodiment, each oligonucleotide 4 bound to the nanoparticle has a known nucleotide sequence. Each oligonucleotide 6 with cationic modification also has a known nucleotide sequence. Preferably, the nucleotide sequence of the oligonucleotides 4 attached to the nanoparticle 2 is different from the known one Nucleotide sequence of oligonucleotides 6 with cationic modification. The known nucleotide sequences of the oligonucleotides 4, which are attached to the nanoparticles 2, and the nucleotide sequence of the oligonucleotides 6 with cationic modification are chosen such that the nucleotide sequence of the attached to the nanoparticle 2 oligonucleotide 4 at least partially complementary to a nucleotide sequence of a first portion of a in the Is nucleic acid to be detected 10 solution. The nucleotide sequence of the oligonucleotides 6 with cationic modification is preferably chosen such that it is complementary to a nucleotide sequence of a preferably adjacent second subregion of the nucleic acid 10 to be detected in the solution. Preferably, the first subregion and the second subregion of the nucleic acid 10 to be detected do not overlap.

The third preferred embodiment can serve, for example, to determine the complementarity of the nucleotide sequence of the nucleic acid 10 to be detected with the known nucleotide sequences of the oligonucleotides 4 attached to the nanoparticles 2 and the oligonucleotides 6 with cationic modification. With a high complementarity of the first subregion of the nucleic acid 10 to be detected with the oligonucleotides 4 attached to the nanoparticle 2 and a high complementarity of the second subregion of the nucleic acid 10 to be detected with the oligonucleotides 6 with cationic modification, preferably both an oligonucleotide 4 attached to a nanoparticle can be used , as well as an oligonucleotide 6 with cationic modification to one and the same strand of the nucleic acid to be detected 10 hybridize. In other words, with high complementarity, the nucleic acid 10 to be detected may serve as a compound nucleic acid to connect at least one oligonucleotide 4 attached to a nanoparticle and one oligonucleotide 6 having cationic modification. In other words, an oligonucleotide 4 attached to the nanoparticle is connected via the nucleic acid 10 to be detected with an oligonucleotide 6 with cationic modification. In other words, the nucleic acid 10 to be detected serves as a connecting nucleic acid to at least one oligonucleotide 4, which the nanoparticle is attached to connect with an oligonucleotide 6 with cationic modification.

Particularly preferably, there is a measurable change in the at least one physical property of the solution if at least one nanoparticle 2 or an oligonucleotide 4 attached thereto is connected via the nucleic acid 10 to be detected with at least one oligonucleotide 6 with cationic modification. In the absence or at too low a concentration of the nucleic acid 10 to be detected, not enough bonds between nanoparticles in 2 and oligonucleotides 6 with cationic modification can be generated despite a sufficient concentration of oligonucleotides 6 with cationic modification to cause a measurable change in the physical property of the solution can. The effect corresponds substantially to the effects that have already been described with reference to the first preferred embodiment and the second preferred embodiment.

Fig. 7 shows an exemplary photographic representation of reaction vessels R3 to R4, in which a method according to the second preferred embodiment is carried out, the implementation is described below in Example 3.

The present invention will be further illustrated by the following examples, but not limited to the following examples.

example 1

In Example 1, a method according to the first preferred embodiment of the invention was applied. Nucleic acids are attached to the nanoparticles, with the nanoparticles in solution. Furthermore, there are oligonucleotides with cationic modification in the solution, which have a known nucleotide sequence. For example, the method according to this example serves to measure the degree of complementarity or the ability to hybridize between the nucleic acid on the nanoparticles and the oligonucleotides with cationic modification. The sequence of the nucleic acid may be at least partially unknown. With high complementarity, the oligonucleotides with cationic modification hybridize to the nucleic acid on the nanoparticle and a measurable change is produced. Fig. 2 shows a photograph of 8 reaction vessels R1 to R8 in a multiwell plate, with a total volume or capacity of 20 μΙ per reaction vessel. Each vessel contains 40 pM (picomoles per liter) of 60 nm diameter gold nanoparticles (supplied by BBI). Each nanoparticle carries approximately 1,000 nucleic acids with Sequence 1 (see Appendix) functionalized via a thiol linkage to the nanoparticle surface (according to J. Hurst et al., Anal. Chem., 78 (24), 8313-8318, 2006 the content of which is incorporated herein by reference). After the functionalization, 6 washing steps are carried out. The nanoparticles are dissolved in a phosphate buffer (3.75 mM phosphate, pH 7) with 0.1% Tween 20, 5 mM MgC, 7.5 mM NaCl. Under these conditions, the nanoparticles are stable without addition of oligonucleotides with cationic modification and show a red color (see R1 and R2). In the upper row (R1, R3, R5 and R7), a ZNA (supplied by Metabion GmbH, Planegg, Germany) with sequence 2 (see Appendix) was added in different final concentrations as oligonucleotide with cationic modification. The concentrations are 100 nM in R7, 10 nM in R5, 1 nM in R3). Since sequences 1 and 2 have a high degree of complementarity with one another, the ZNA can hybridize well to the nanoparticle-bound nucleic acids. From a final concentration of the ZNA of 10 nM (R5 and R7), a clear decolorization of the nanoparticle solution is observable. In the lower row was added as oligonucleotide with cationic modification a ZNA (supplied by Metabion GmbH, Planegg, Germany) with the sequence 3 (see Appendix) in different final concentrations (100 nM in R8, 10 nM in R6, 1 nM in R4). , Since sequences 1 and 3 have very little complementarity with respect to each other, ZNA can not or only poorly hybridize to the nanoparticle-bound nucleic acids. None of these nanoparticle solutions in the bottom row (R2, R4, R6, R8) show significant discoloration. Both ZNA sequences used carry two cationic units in the ZNA modification. The image was taken 15 minutes after mixing together the reagents, ie the nanoparticles and the ZNA.

FIGS. 3A to 3D show extinction spectra of nanoparticle solutions. Figure 3A shows a solution with 60 nm diameter gold nanoparticles and 40 pM nanoparticle concentration (supplied by BBI). The extinction spectrum shown here are in arbitrary units, i. only qualitatively plotted against the wavelength. Each nanoparticle carries approximately 1,000 nucleic acids with sequence 4 (see Appendix), which have been functionalized via a thiol bond to the nanoparticle surface. The nanoparticles are dissolved in phosphate buffer (3.9 mM phosphate, pH 7) with 0.1% Tween 20, 5 mM MgC, 7.85 mM NaCl. The extinction spectrum (recorded with a Varian Cary 50 spectrometer in a quartz cuvette with 3 mm optical path) of a particle solution without addition of oligonucleotides with cationic modification (solid line) shows a plasmon resonance with maximum resonance at a wavelength of about 530 nm. The addition of an oligonucleotide with cationic modification (ZNA with sequence 5, see Appendix, final concentration 150 nM) 5 minutes after addition shows no significant change in the extinction spectrum (dotted line), since the sequences 4 and 5 to each other have a very low complementarity and the ZNA therefore not or only can hybridize poorly to the nanoparticle-bound nucleic acids.

In FIG. 3B, instead of the ZNA with sequence 5, a ZNA with sequence 6 (see Appendix) was added as an oligonucleotide with cationic modification (final concentration also 150 nM, same buffer system as in FIG. 3A). This shows already after 1 minute, a relatively large change in the extinction spectrum (dashed line) in comparison to the curve before adding the oligonucleotides with cationic modification (solid curve). After 5 minutes, the change is even more pronounced (dotted curve). Because the Sequences 4 and 6 have a high complementarity to each other, the ZNA can hybridize well or effectively to the nanoparticle-bound nucleic acids. The observable or measurable change in the extinction spectrum is typical for a shift of the plasmon resonance of the nanoparticles, which could be an indication of a smaller average distance of the nanoparticles from each other, which is caused by the binding of the oligonucleotides with cationic modification on the nanoparticles.

In Fig. 3C, the temperature of the sample of Fig. 3B was first raised to 60 ° C (absorbance spectrum shown as a dashed line). At the same time, the change in the optical properties initially remains (strongly broadened and red-shifted plasmon resonance in comparison to the extinction spectrum of a sample without oligonucleotides with cationic modification). In a further increase of the sample temperature to 70 ° C, however, the extinction spectrum changes substantially back to the original state with plasmon resonance maximum at a wavelength of about 530 nm (extinction spectrum shown as a solid line). This can be explained by the fact that the denaturation temperature of the DNA double strand from sequence 4 and 6 is reached and this double strand is separated. As a result, the oligonucleotides with cationic modification can be removed again from the nanoparticles. Accordingly, the local charge conditions on the nanoparticles and thus the cause for the bond between the nanoparticles can change again. As a result, the particles are released again, whereby the distance between the nanoparticles, for example, increases again by diffusion and Coulomb repulsion of the nanoparticles. This shows that the measurable change can also be reversible when the bond between cationic-modified oligonucleotides and nanoparticles is redissolved. In Figure 3D, oligonucleotides with cationic modification with Sequence 6 (which, in combination with nanoparticles functionalized with Sequence 4, produced a strong measurable change) were now functionalized to other nanoparticles functionalized with Sequence 11 (see Appendix) (final concentration of Oligonucleotide with cationic modification also 150 nM, nanoparticle concentration also 40 pM, same buffer system as in Fig. 3A and Fig. 3B), so no significant change is observed, since sequence 6 and 11 again have a very low complementarity to each other again.

Example 2

In Example 2, a method according to the second preferred embodiment was applied. In each case, the nanoparticles in the solution carry at least one oligonucleotide with a known nucleotide sequence. Furthermore, oligonucleotides with cationic modification having a known nucleotide sequence are added to the solution, the nucleotide sequence being at least partially complementary to the oligonucleotides attached to the nanoparticles. Furthermore, the solution may contain a nucleic acid to be detected which is initially free in solution and which is at least partially complementary to the oligonucleotides on the nanoparticles and / or to the oligonucleotides with cationic modification. Without nucleic acid or with only a low concentration of the nucleic acid, oligonucleotides with cationic modification and the oligonucleotides on the nanoparticles can hybridize and produce a measurable change. The presence of the nucleic acid or a sufficiently high concentration of the nucleic acid prevents this hybridization and thus the measurable change, since the nucleic acid can hybridize with the oligonucleotides with cationic modification and / or the oligonucleotides on the nanoparticles. The oligonucleotides with cationic modification and / or the oligonucleotides on the nanoparticles are thus at least partially occupied and can no longer (sufficiently) hybridize with each other and thus also cause no measurable change.

Fig. 5 shows a photograph of 4 reaction vessels R1, R2, R3 and R4 in a multiwell plate with a total volume or capacity of 20μΙ per reaction vessel. Each reaction tube contains 40 pM gold nanoparticles with 60 nm diameter (supplied by BBI), each nanoparticle carries about 1,000 oligonucleotides with sequence 4, which have been functionalized via a thiol bond to the nanoparticle surface. The nanoparticles are dissolved in phosphate buffer (3.9 mM phosphate, pH 7) with 0.1% Tween 20, 5 mM MgC, 7.7 mM NaCl. Under these conditions, the nanoparticles are stable without addition of oligonucleotides with cationic modification and show a red color. To a reaction volume of 17.7 μΙ were added in the upper row, R1 and R3, as nucleic acid DNA sequence 7 (2 μΙ a DNA concentration of 1 μΜ), in the lower row R2 and R4 was instead 2 μΙ water added as a negative control. Since sequences 4 and 7 have a high degree of complementarity with one another, the added DNA can hybridize to the oligonucleotides on the nanoparticle surface and thus block them for further hybridizations. After an incubation time of 10 minutes, each reaction vessel ZNA were added with the sequence 6 as an oligonucleotide with cationic modification (each 0.3 μΙ of a 10 μΜ ZNA solution, final concentration thus 150 nM ZNA), which is highly complementary to the sequence 4 of the nanoparticles has-bound oligonucleotides. In the top row, R1 and R3, with nucleic acid, the ZNA can no longer hybridize to the nanoparticle-bound oligonucleotides since they are occupied by the nucleic acid. There is thus no discoloration or measurable change. In the lower row, R2 and R4, however, a clear decolorization of the nanoparticle solution and thus a measurable change in the optical property of the solution is observable, since the oligonucleotides with cationic modification hybridize well to the nanoparticle-bound oligonucleotides. The image was taken 10 minutes after the reagents were mixed together, ie the nanoparticles and the ZNA.

Example 3 In Example 3, a method according to the third preferred embodiment was used. In each case, the nanoparticles in the solution carry at least one oligonucleotide with a known nucleotide sequence. Furthermore, the solution becomes oligonucleotides with cationic modification with a known nucleotide sequence added. Furthermore, a nucleic acid to be detected may initially be free in solution. The nucleotide sequence of the nucleic acid to be detected here comprises a first partial region which is at least partially complementary to the oligonucleotides on the nanoparticles and a preferably adjacent second partial region which is at least partially complementary to the oligonucleotides with cationic modification.

This example can be used to determine how well the nucleic acid is complementary to the known sequence on the nanoparticle and the known sequence of the cationic modification oligonucleotide. With high complementarity, both the oligonucleotide with cationic modification and the oligonucleotide on the nanoparticle hybridize with the nucleic acid and a measurable change is produced. Fig. 7 shows a photographic representation of reaction vessels R1 to R3 in a multiwell plate, total volume per reaction vessel 20 μΙ. Each well R1 to R3 contains 40 pM gold nanoparticles 2 with a diameter of 60 nm (supplied by BBI), each nanoparticle carries about 1000 oligonucleotides with sequence 12, which have been functionalized via a thiol bond to the nanoparticle surface. The nanoparticles 2 are dissolved in phosphate buffer (3.9 mM phosphate, pH 7) with 0.1% Tween 20, 12 mM MgCte, 7.7 mM NaCl. Under these conditions, nanoparticles 2 are stable and show a red color. To a reaction volume of 19.4 μΙ were added to the reaction vessels R2 and R3 as nucleic acid DNA with the sequence 13 (0.3 μΙ a DNA concentration of 1 μΜ), in the reaction vessel R1 instead of 0.3μΙ water as a negative control added. After an incubation time of 5 minutes, the reaction vessels R1 and R3 were added ZNA with the sequence 6 as oligonucleotide with cationic modification (each 0.3μΙ a 10 μΜ ZNA solution, final concentration thus 150 nM ZNA), which is highly complementary to a portion of the Sequence 13 of the nucleic acid to be detected, but has a low complementarity with sequence 12 of the nanoparticle-bound oligonucleotides. In the reaction vessel R2 instead of the ZNA 0.3 μΙ water was added. After a further 10 minutes incubation time, the photo was taken in FIG. Just In the first reaction vessel from the left, a decolorization of the nanoparticle solution and thus a measurable change, since only there via the sequence 13 of the nucleic acid to be detected, the oligonucleotides with cationic modification with sequence 6 with the nanoparticles or attached thereto Oligonukjeotiden with sequence 12 can be.

Example 4: Real-time PCR

Examples 4.1 to 4.3 for preferred uses of the nanoparticles and / or the cationically modified nucleic acids or use of preferred methods according to the invention in the context of a real-time PCR are described below. All real-time PCR reactions in the following examples were performed in a Roche Light Cycler 1.5 with software version 4.1.1.21 in plastic capillaries from Genaxxon. For a particular explanation of the technical advantages of the invention, the invention is shown in a comparison with conventional methods according to the prior art.

Example 4.1: State of the Art: Real-Time PCR with Intercalating Dye For an indirect detection of a nucleic acid, a polymerase chain reaction (PCR) was carried out. First, the functionality of the primer system according to the prior art was tested: here, the nucleic acid is amplified with sequence 7 by the primer oligonucleotides with sequence 8 and 9 and the PCR amplicon by an intercalating dye (Sybr-Green I Nucleic Acid Gel Stain, 0.000x concentrate, Roche), which shows increasing fluorescence as the amount of double-stranded DNA in the sample increases. The forward primer with sequence 8 carries no cationic modification, whereas the reverse primer with sequence 9 carries a cationic modification with three cationic units (ZNA3) at the 5 'end. The cationic modification in this case is at the 5 'end of the primer and not at the 3' end so as not to prevent elongation by the polymerase attaching to the 3 'end of the primer. Each reaction consists of 9 μΙ Mastermix and 1 μΙ nucleic acid solution or water. 9 μΙ Mastermix for a reaction are listed as follows in Table 1:

Table 1

The PCR protocol consists of 40 cycles of 5 seconds 90 ° C, 15 seconds 60 ° C and 15 seconds 72 ° C. Fluorescence detection occurs after every 72 ° C step. 8 shows real-time PCR curves of the 530 nm fluorescence channel of three different starting concentrations of the nucleic acid with sequence 7 (present as oligonucleotide) and a negative control without nucleic acid as target sequence. The curves show the typical course for such real-time PCR: First, the detected fluorescence remains essentially unchanged in the first PCR cycles. Although (depending on whether or not nucleic acid is included as starting material in the sample), new DNA double strands are generated as amplicon in each cycle, but the amount is not sufficient here to raise the fluorescence caused thereby beyond the measurable basic limit and to detect. For the sample in which 1 .mu.l of a 1 pM concentration of the nucleic acid with sequence 7 was used as the starting material (solid line), the fluorescence rises sharply from about the 20th cycle to then (from about cycle 27) into saturation proceed. This rise point is of great significance for the initial concentration of the nucleic acid with Sequence 7, because a sample with 10-fold lower initial concentration increases only from about the 24th cycle on (dashed line), for a 100-fold lower initial concentration, it increases from approximately the 28th cycle on (dotted line). Without nucleic acid with sequence 7 as starting material, no increase can be seen until the 30th cycle (dotted dashed line). The software of the instrument determines the so-called Ct value (Cycle Threshold for Threshold Cycle) or Cp value (English Crossing Point), which describes the cycle at which the fluorescence for the first time rises significantly above the background fluorescence , This can then be used in a sample of unknown starting concentration of the nucleic acid, typically by comparison with a known standard concentration series for quantifying the starting concentration of the nucleic acid in the sample.

The Cp values for the measurement of FIG. 8 are shown in the following Table 2:

Table 2

If the instrument software is given the samples with nucleic acid as the known standard concentration series, it can calculate the efficiency of the amplification reaction from the concentrations and the distances of the Cp values from one another. The values given in the table above indicate one amplification per cycle in the concentration range of 1 -100 fM of 1.858 (+ -0.00584). With this amplification per cycle and the Cp values, it is possible to estimate from the original nucleic acid concentrations (Co) which amplicon concentration prevails when the Cp value in the sample is reached this simplifies that the duplication per cycle is the same on average over all cycles. According to the equation (1)

C (Cp) = C ₀ ^■ 1.858 ^Cp (1) thus results in an amplicon concentration on reaching the Cp value in the sample of C (Cp) = 41 nM for Co = 100 fM, C (Cp) = 40 nM for Co = 10 fM, C (Cp) = 41 nM for Co = 1 fM. For the instrument, a significantly measurable change in the fluorescence over the background fluorescence is thus achieved in this system with intercalating dye as signal generator at about 40 nM amplicon concentration.

It should be noted here that the sample without nucleic acid with sequence 7 as starting material (dotted dashed line) increases significantly in the last few cycles. This is a frequently occurring effect in conventional real-time PCR methods, especially if, as here, intercalating dyes are used for detection. The reason for this are usually so-called primer dimers, ie unwanted formation of DNA double strands, which can be caused by non-specific, unwanted bonds between primers (similar or different) and are also highly amplified.

Example 4.2: Real-time PCR with nanoparticles and ZNA - without dye

For indirect detection of a nucleic acid, a polymerase chain reaction was carried out. The primer system used is basically the same as in Example 4.1, but now the previously unbound forward primer is replaced with sequence 8 by modified primer oligonucleotides with sequence 4, which has been functionalized on a gold nanoparticle. The modified primer oligonucleotide with sequence 4 comprises as a partial sequence sequence 8 (this sequence is still used as the actual primer sequence), in addition to a partial sequence of 35 adenine bases, which serves as a spacer between the particle surface and primer partial sequence and additionally two abasic modifications Spacer9 between spacer sequence and primer sequence the the overwriting of the spacer sequence by the polymerase prevented (see, for example, patent application DE 10 2013 215 168.3). Furthermore, the modified primer oligonucleotide with sequence 4 at the 5 'end contains a thiol modification, with the aid of which the oligonucleotide is bound to the surface of gold nanoparticles. The same nanoparticle-oligonucleotide conjugates are used as in Fig. 5 and Example 2, respectively, ie 60 nm diameter gold nanoparticles (supplied by BBI), each nanoparticle carrying about 1000 oligonucleotides with sequence 4. The reverse primer is the cationic modification oligonucleotide with three cationic units at the 5 'end with sequence 9 (ZNA3). The nucleic acid is amplified with sequence 7. The PCR amplicon is now not detected by a dye, but by a measurable change, which is caused by the fact that the PCR amplicon with the oligonucleotide cationic modification (here the reverse primer) with the nanoparticles is connected. Each reaction consists of 9 μM Mastermix and 1 pl nucleic acid solution or water. 9 μM master mix for one reaction are summarized in Table 3 as follows:

Table 3

 Endkonzen¬

Stick μΙ for 1

 in 10

 Concentration reaction

 ml

 Water 2

 5x Apta Taq Genotyping

 5 1 2

 Master (Roche) [x-fold]

 Nanoparticle-oligonucleotide

200 40 2

 Conjugates [pM] *

 Reverse primer sequence 9

 3,000 300 1

 [NM]

MgCl ₂ [mM] 50 5 1

Tween 20 (%) 1 0.1 1 the nanoparticle-oligonucleotide conjugates are functionalized (according to J. Hurst et al., Anal. Chem., 78 (24), 8313-8318, 2006, the contents of which are incorporated herein by reference). After functionalization and 6 washes, the nanoparticle-oligonucleotide conjugates are present in a concentration of 200 pM in phosphate buffer (5 mM phosphate, pH 7) with 0.1% Tween 20, 10 mM NaCl.

The PCR protocol consists of 40 cycles of 5 seconds 90 ° C, 15 seconds 60 ° C and 15 seconds 72 ° C. The signal detection takes place after every 72 ° C step. No dye is used in this experiment, but the same unmodified detection unit and software of the Light Cycler Instrument are used as in Examples 4.1 and 4.2 using dyes.

Since neither instrument nor software are designed for this purpose, it may happen that the samples are not recognized at the beginning of the real-time PCR with the error message "The following samples were not found during the seek process: ..." and then be skipped with the further remark "Empty Position" in the following signal detection. In these cases no detection of the measurable change is possible. This could be avoided, for example, by a change in instrument software and / or hardware, or circumvented with the use of fluorescent dyes in the background with unchanged instrument software and / or hardware (see Example 4.3). 9A shows real-time PCR curves of the 530 nm fluorescence channel of the LIGHT CYCLER instrument, although in this case no fluorescence is measured, of three different starting concentrations of nucleic acid with sequence 7 (present as oligonucleotide) and one negative control without nucleic acid as target sequence , The curves. show again a typical for Real-Time PCR typical course, although due to the very small absolute signal change (about 300 times smaller than in Example 4.1 when using a fluorescent dye), the deviations in the baseline level of the measurement visually very differentiated the different curves. The deviations in the basic level are included exact observation also in Fig. 8 and Example 4.1 in the same absolute order of magnitude to recognize, but there due to the other scaling of the vertical axis of the graph visually not noticeable. Fig. 9B shows a slightly different representation of the graph of Fig. 9A, wherein in the Light Cycler software by means of the function "Analysis - Amplification Analysis - Absolute Quantification" an altered representation has been selected, in which the base level appears more contracted to the visual 9B, as well as in the basic illustration in Fig. 9A, the detected signal initially remains substantially unchanged in the first PCR cycles pM concentration of the nucleic acid with sequence 7 as starting material (solid lines), the signal rises sharply from about the 20th cycle onwards, and then saturates.The sample with a 10-fold lower initial concentration does not increase until about the 24th cycle on (dashed line), for a 100-fold lower initial concentration, it increases approximately from the 29th cycle on (dotted line) Liche acid with sequence 7 as starting material is not recognizable until the 40th cycle (dashed dotted line). These rise points are very similar to the rise points in Fig. 8 of the dye-based example 4.1. Also, the curves are similar to each other equidistant, which proves the good quantifiability of the measurement method. The measurable change, which is detectable above a certain minimum concentration of amplicon, does not arise here as in the prior art by fluorescence of a fluorescent dye, but by the changed optical properties of the solution, which by connecting the gold nanoparticles in the solution with a sufficient number Oligonucleotides with cationic modification (here reverse primer as ZNA) is caused. The nanoparticles have their extinction maximum at a wavelength of about 530 nm in the dissolved state without binding to oligonucleotides with cationic modification (ie, for example, in the first cycles of real-time PCR before the signal change) (see, for example, also extinction spectra in FIGS. 3A to 3D ), at this wavelength the signal is also recorded in the selected mode in the Light Cycler. By binding on oligonucleotides with cationic modification, the absorbance at 530 nm decreases (see also extinction spectra in Fig. 3B). The fact that the signal in the Light Cycler increases (and does not decrease) after a certain cycle is an indication that the decreasing nanoparticle absorbance in the sample or solution at 530 nm causes more background signal (such as reflection from the capillary walls or the liquid surface) Scattering from capillaries or reaction liquid or intrinsic fluorescence of the solution) can penetrate the sample, reaches the detector and thus becomes detectable. The reduced background signal in the presence of unbound nanoparticles in the solution may also be the reason why such samples without dyes of instrument and software at the beginning of real-time PCR in the so-called "seek process" are not recognized.

Example 4.3: Real-Time PCR with Nanoparticles and ZNA - with Background Dye In Example 4.3, a polymerase chain reaction comparable to Example 4.2 was carried out for indirect detection of a nucleic acid, but now also with dye in the reaction solution. The used primer system of nanoparticle-oligonucleotide conjugates for forward primer and ZNA as reverse primer remains unchanged from that of Example 4.2. Fluorescein is used as additional dye (as fluorescein sodium salt F6377 obtained from Sigma Aldrich and initially dissolved in water). Fluorescein has spectrally very similar properties to Sybr Green I used in Example 4.1, but fluorescein shows no intercalating properties, ie it does not typically alter its fluorescence properties with increasing amplicon concentration in the sample or solution. Each reaction consists of 9 μΙ Mastermix and 1 μΙ nucleic acid solution or water. 9 μM master mix for one reaction are summarized in Table 4 as follows: Table 4

* the nanoparticle oligonucleotide conjugates are functionalized (according to J. Hurst et al., Anal. Chem., 78 (24), 8313-8318, 2006, the contents of which are incorporated herein by reference). After functionalization and 6 washes, the nanoparticle-oligonucleotide conjugates are present in a concentration of 200 pM in phosphate buffer (5 mM phosphate, pH 7) with 0.1% Tween 20, 10 mM NaCl. The PCR protocol again consists of 40 cycles of 5 seconds 90 ° C, 15 seconds 60 ° C and 15 seconds 72 ° C. The signal detection takes place after every 72 ° C step. Due to the additional dye used, in contrast to Example 4.2, there are no longer any unrecognized samples from the detection unit and software of the Light Cycler Instrument.

10 shows real-time PCR curves of the 530 nm fluorescence channel (matching the emission wavelength of fluorescein) of three different starting concentrations of the nucleic acid with sequence 7 (present as oligonucleotide) and a negative control without nucleic acid as target sequence. The curves essentially show a course typical of real-time PCR, the base level of the signal (before reaching the first significant signal increase) is now about 4 times higher than in the comparable experiment without dyes (FIG. 9A) and thus almost reaches the level which is also achieved with the intercalating dye Sybr Green (FIG. 8th). For this reason, there are no longer unrecognized samples from the detection unit and software of the Light Cycler instrument. The absolute signal change during the nucleic acid-induced significant signal increase is 5 to 6 times higher in this example than in FIG. 9A or in Example 4.2 without dye. In FIG. 10, the sample or the solution with the highest concentration of nucleic acid with sequence 7 as starting material (1 μΙ of a 1 μM solution used) first shows a significant signal increase (solid line). As in Example 4.2, at this nucleic acid concentration, the signal increases sharply starting at the 20th cycle and then goes into saturation. The sample or the solution with 10-fold lower initial concentration increases again only from about the 24th cycle on (dashed line), for a 100-fold lower initial concentration, it increases from about the 27th cycle on (dotted line). Without nucleic acid with sequence 7 as starting material, no increase can be seen until the 40th cycle (dashed dotted line). These rise points are very similar to the rise points in FIG. 8 of the fluorescence-based example 4.1 and with the rise points in FIG. 9 of the fluorescence-free example 4.2. The measurable change that is detectable above a certain minimum concentration of amplicon arises in Example 4.3 through an interaction of initially virtually unchanged fluorescence properties of the fluorescein dye used in the background of the solution and the changed optical properties of the gold nanoparticles by combination with a sufficient number of oligonucleotides with cationic modification (here in the form of a reverse primer as ZNA). Fluorescein is excited by the Light Cycler at a wavelength of 470 nm and emits fluorescence at a maximum of 530 nm, where fluorescence is also measured in the selected mode in the Light Cycler. The nanoparticles have in the dissolved state without binding to oligonucleotides with cationic modification (ie, for example, in the first cycles of real-time PCR before the signal change) their extinction maximum at a wavelength of about 530 nm and still significant extinction at 470 nm (see, for example Extinction spectra in FIGS. 3A to 3D). The nanoparticles thus reabsorb a considerable portion of the fluorescein emission at 530 nm and additionally prevent the excitation of the nanoparticles at 470 nm. The decreasing nanoparticle absorbance in the sample at the wavelength of 470 nm as well as at the wavelength of 530 nm by the Combining the nanoparticles with a sufficient number of oligonucleotides with cationic modification now leads to an increased fluorescence signal of the fluorescein and thus to a measurable change by more effective excitation and undisturbed emission of the fluorescein dyes with simultaneous lower reabsorption by the nanoparticles.

The concentration of fluorescein may be e.g. also be 10 times higher. This increases both the base level of the fluorescence signal and the absolute signal swing (data not shown here). At a significantly reduced concentration (final concentration fluorescein 0 nM), both the base level of the fluorescence signal and the absolute signal swing are significantly reduced, and it may happen at too low concentrations that some samples or solutions of instrument and software at the beginning of real Time PCR in the so-called "seek process" are again not recognized (data not shown here) As proof that the interaction of nanoparticles and ZNA is indeed responsible for the signal change, the following slightly different control experiments were carried out:

Replacement of the nanoparticle-oligonucleotide conjugates by the oligonucleotide with sequence 8 as a primer without nanoparticles (final concentration of the oligonucleotide with sequence 8 40 pM and 300 pM tested);

Replacement of the oligonucleotide with cationic modification (here ZNA) with sequence 9 by a DNA oligonucleotide without cationic modification with sequence 10 (this is substantially similar to sequence 9, but without cationic modification and slightly longer, in spite of the lack of ZNA a similar high This could be achieved in the system with Sybr-Green in Example 4.1 are successfully used as a replacement for the oligonucleotide with cationic modification with sequence 9 (data not shown here).

All control experiments showed no significant signal increase even at the highest concentration of nucleic acid with sequence 7 as starting material (1 μΙ of a 1 pM solution used) (data not shown here).

In FIG. 11, under the otherwise same experimental conditions as in FIG. 10, genomic DNA was used as starting material for the PCR as the nucleic acid to be detected. In contrast to the synthetic oligonucleotides with sequence 7 used in the above experiments for amplification, this is considerably more complex since it does not consist of 83 nucleotides but of about 3 million nucleotides, which are also present as double-stranded DNA. Sequence 7 is a part of the resistance gene MecA, which is e.g. in the bacterium methicillin-resistant Staphylococcus aureus (MRSA) occurs. In contrast, this resistance gene does not occur in methicillin-sensitive Staphylococcus aureus (MSSA). The DNA was extracted from MRSA or MSSA strains and boiled for 10 minutes prior to insertion into the PCR. The curves in FIG. 11 show real-time PCR curves (according to the software analysis function "Analysis-Amplification Analysis-Absolute Quantification") with a 9 μM master mix, as was also used in Fig. 10. Nucleic acid were now genomic DNA from MRSA or MSSA were used so that the following total amounts were contained as starting material for the amplification in the respective reaction volume of 10 μΙ: solid lines: 100,000 genome copies MRSA

dashed lines: 10,000 genome copies MRSA

dotted lines: 1,000 genome copies MRSA

dotted-dashed lines 100,000 genome copies MSSA

These real-time PCR results show the following: The more MRSA genome copies present as the starting material in the reaction volume, the sooner the corresponding curves show an increase in the measured fluorescence. The nucleic acid is thus quantifiable. - Duplicate determinations with the same number of MRSA genome copies show very similar rise points in a well reproducible manner.

Samples containing only MSSA genome copies show no significant increase in fluorescence until the 40th cycle because sequence 7 does not occur in the MSSA genome and therefore the primers used can not amplify nucleic acids. This shows the specificity of the method with correspondingly specific primers.

verschieben. Dies könnte daher nur eine Verschiebung um ca. 2 Zyklen, nicht aber eine Verschiebung um 8 bis 9 Zyklen bedeuten. Die Verschiebung der Anstiegspunkte hin zu niedrigeren Zyklen lässt sich somit nur erklären, wenn das Detektionslimit, also die Amplikon-Konzentration, bei der eine messbare Veränderung entsteht niedriger ist. Eine obere Abschätzung hierfür kann erfolgen, wenn man für die Vervielfältigung pro Zyklus das theoretische Maximum von 2 annimmt und analog zu Formel (1 ) aus Beispiel 4.1 berechnet: In Fig. 12, under the otherwise same experimental conditions as in Fig. 10, the MgC end concentration was increased from 5 mM to 15 mM. The real-time PCR curves (according to software analysis function "Analysis - Amplification Analysis - Absolute Quantification") of three different starting concentrations of nucleic acid with sequence 7 (present as oligonucleotide) and a negative control without nucleic acid as the target sequence are again very similar to the curves in 10 (assignment of line type to starting concentration of the nucleic acid is the same as in Fig. 10) It is striking that the curves for the respective nucleic acid concentration are now compared with Fig. 10 with 5 mM MgCte end concentration with rise points at cycle 12, 15 and 18 now each increase by about 8-9 cycles earlier, an earlier increase is of great advantage if you want to perform a fast PCR and the rapid achievement of results is crucial., Here you can achieve a faster result without the purchase of new Hardware is required, but with the same hardware using a new reaction mix This can therefore be implemented in existing systems in a particularly efficient and cost-effective manner. The cause of an earlier rise point, despite the same initial concentration, may mean either a higher amplification efficiency, or a more sensitive detection method that detects a measurable change even at a lower amplicon level. The Amplification efficiency was already relatively high at 85.8% (multiplication per cycle was 1.858, with amplification per cycle = 1 + amplification efficiency) in Fig. 8, which showed comparable slope points to Fig. 10. For example, an increase to the maximum possible amplification per cycle of 2 from the amplification process would produce the first rise point or the Cp value in FIG. 8 for initial concentration Co = 100 fM with the same detection limit of C (Cp) = 40 nM

move. This could therefore only mean a shift of about 2 cycles, but not a shift of 8 to 9 cycles. The shift of the rise points towards lower cycles can therefore only be explained if the detection limit, ie the amplicon concentration at which a measurable change occurs, is lower. An upper estimate for this can be done by assuming the theoretical maximum of 2 for the duplication per cycle and calculating it analogously to formula (1) from example 4.1:

C (Cp) = C ₀ ^■ 2 ^c * (3)

Thus, for Co = 100 fM and Cp = 12, a new detection limit of 410 pM results for the curves shown in FIG. A duplication per cycle of <2 results in an even smaller detection limit. A detection limit of 410 pM means at a used nanoparticle concentration of 40 pM about 10 bound oligonucleotides with cationic modification per nanoparticle. The Cp values determined by the original software are listed in Table 5 for the different initial concentrations of nucleic acid used: Table 5

It is striking that the value determined by the software is always lower by about 2 Cp values than a human observer would read by eye. This may be due to the fact that the curves are steeper than the classical curves based on dyes or that the curves start more abruptly and show an almost sudden increase. This could come from a threshold behavior of the nanoparticles, which change their properties very suddenly from a certain minimum amount of bound oligonucleotides with cationic modification. In contrast, in the conventional dye-based reading of the amount of nucleic acid in the solution in Example 4.1, the fluorescence signal is relatively directly proportional to the actual nucleic acid amount in the solution (at least once the fluorescence generated by double-stranded DNA is above the basal level of the measured signal also comes out) and therefore shows in the transition region between the base level and steep rise and a phase with a slow signal rise. The instrument evaluation software may run from such curves, or averaging over multiple measurement points may affect the software's Cp value. In Fig. 12 it is noticeable that the samples without nucleic acid with sequence 7 as starting material (dotted dashed line) despite a lacking target sequence for the amplification still show a clear increase in the signal from about cycle 24. A comparable effect was already evident in the dye-based example 4.1 in FIG. 8 and is presumably to be explained in both cases with so-called primer dimers (see example 4.1). In Fig. 13, the first derivatives of melting curves of false-positive and true-positive samples taken during continuous temperature increase are shown. False-positive samples are samples which, for example, show a positive signal course because of the formation of primer dimers, even though the nucleic acid to be detected is not present in the sample at all. False-positive samples can often be distinguished by a melting curve of true-positive samples, since a melting curve can provide information about the length and composition of the amplicon, since the melting temperature depends on the length and the base composition of the double-stranded DNA. The fact that the measurable change by combining nanoparticles with oligonucleotides with cationic modification can generally be reversed by increasing the temperature has already been shown in FIG. 3C. Now, to record a melting curve, the samples amplified in Figure 12 are heated slowly after the last cycle of amplification and fluorescence is recorded during this time (Light Cycler settings: melting curve 37-95 ° C, hold time 0s, ramp rate : 0.05 ° C / s, Acquisition Mode: Continuous). After completion of the melting curve recording, the evaluation method "Analysis - Melting Curve Analysis - Tm-Calling" of the software is carried out The assignment of line type to initial concentration of nucleic acid is the same as in Fig. 0. The derivatives of the melting curves in Fig. 13 show two groups of curves: the two curves with the maximum of the derivative at about 79 ° C are the false-positive samples (ie the samples which show a positive signal course, even though the nucleic acid to be detected is not present in the sample), the remaining samples (containing the nucleic acid to be detected with sequence 7 as starting material) have the maximum of the derivative approximately at 81 ° C. False-positive samples and truly positive samples can thus be distinguished by their melting curve is about 2.5 ° C. Thus, the half-width is the corresponding maximum of the melting curve derivatives here in the inventive use according to this preferred embodiment of nanoparticles and cationically modified nucleic acids much lower than in melting curves from Example 4.1 according to the prior art with intercalating dye, where the half-width is about 9 ° C. (Data not shown). In other words, when used according to the invention in this preferred embodiment, the melting curves of nanoparticles and cationically modified nucleic acids are substantially steeper than the melting curves of Example 4.1 according to the prior art.

Example 5: Real-Time Laser PCR

Fig. 14A shows a construction suitable for carrying out a method according to the invention in a further preferred embodiment. The structure comprises a light source, which in this example is designed as a laser 11, and a two-dimensional mirror scanner 12, which can direct light from the laser 11 to a sample. In particular, the device can be designed to direct the laser beam over the mirror scanner at different times to direct the laser beam to different samples of a plurality of samples provided. In the preferred embodiment shown, samples are provided in five reaction vessels R1 to R5, which are arranged in a sample holder 13. The sample holder can have openings or windows 15, for example, through which the laser beam can pass in order to strike the respective sample or the respective reaction vessel 15.

The two-dimensional mirror scanner 12 can deflect a laser beam emitted by the laser 1 1 in two spatial dimensions. The denaturation of double-stranded DNA in the sample occurs in the structure according to the preferred embodiment shown during a laser PCR in that a laser beam is focused on a part of at least the sample. In the course of the process, the laser beam is preferably deflected in such a way that it strikes different parts of one or more samples in preferably several reaction vessels. For example, with such a device, inventive methods can be performed on a plurality of samples. For example, different samples are present in separate reaction vessels R, which are each arranged at one of a sample holder 13, so that the Samples can be irradiated through the associated opening 15 with the laser 1. The arrangement of the samples in or on the sample holder 13 is preferably carried out such that the samples can be detected by the laser 11, preferably without the laser beam touching the sample holder 13. The number of reaction vessels R which can be mounted in the sample holder is not limited to that shown in the preferred embodiment.

Fig. 14B shows another example, which is similar to the example described in Fig. 14A, wherein the laser beam is deflected by the mirror scanner 12 such that the laser beam is the reaction vessel R, in which the Probe 16 is located, cell-like starting. The path traveled by the laser beam is shown in dashed lines in the sample 16 in FIG. 14B.

By preferentially exciting only portions of the sample 16 at any point in the process, lasers 11 having a smaller power can be used. Since excitations of the nanoparticles with a duration of less than 100 microseconds may be sufficient to denature DNA with the aid of optothermically heated nanoparticles, at typical focus diameters of a laser of about 10 to 100 pm, a laser beam at a speed of about 10 to 100 m / s sample the sample, thereby leading to a denaturation of the DNA preferably at each point, which passes over the laser beam. This allows, for example, a very fast scanning even of large sample volumes. For example, complete scanning of an area of 1 cm ² takes only 128 ms at a focus diameter of 78 μm and 128 lines at a line spacing of 78 μm and a line length of 1 cm at a scanning laser beam speed of 10 m / s. For example, if the volume has a depth, ie an extension in the laser beam propagation direction, of 10 mm, a volume of 1 ml can be processed, provided that the intensity of the excitation over the entire depth is sufficiently high to denature the double-stranded DNA contained therein , This is advantageously essential shorter time than a conventional denaturation step would require by means of a global heating of the sample 16 or the solution.

With optical elements, such as e.g. For example, in a mirror scanner shown in Figs. 14A and 14B and so-called F theta lenses, good homogeneity of the focus quality and size over the entire sampled sample can be achieved. As an alternative to a continuously emitting laser, a pulsed laser or a thermal radiator can also be used. Example 5.1: Laser PCR with Cationic Modified Nucleic Acid (ZNA)

For indirect detection of a nucleic acid, a polymerase chain reaction was performed with locally heated nanoparticles. The used primer system of nanoparticle-oligonucleotide conjugates for forward primer and ZNA as reverse primer corresponds substantially unchanged to that of the above-mentioned example 4.2. The modified primer oligonucleotide with sequence 4 contains as a partial sequence the sequence 8 (this sequence is still used as the actual primer sequence), in addition a partial sequence of 35 adenine bases, which serves as a spacer between the particle surface and primer partial sequence and additionally two abasic modifications Spacer9 between spacer sequence and primer sequence, which prevents the overwriting of the spacer sequence by the polymerase (see, for example, patent application DE 102013215168.3). Furthermore, the modified primer oligonucleotide with sequence 4 at the 5 'end contains a thiol modification with the aid of which the oligonucleotide can or will be bound to the surface of a gold nanoparticle. The same nanoparticle-oligonucleotide conjugates are used as in the example shown in FIG. 5, ie, 60nm diameter gold nanoparticles (supplied by BBI), where each nanoparticle carries about 1000 oligonucleotides with sequence 4. The reverse primer is again an oligonucleotide with cationic modification, now with four cationic units at the 5 'end with sequence 14 (ZNA4). As starting material for the laser PCR with locally heated nanoparticles genomic DNA is used as the nucleic acid to be detected, as essentially already in the explanation of FIG. 11 described. Sequence 7 (which can be amplified by the present primer system) is part of the resistance gene MecA, which occurs, for example, in the bacterium Methicillin-resistant Staphylococcus aureus (MRSA). The DNA was extracted from MRSA or MSSA strains and boiled for 10 minutes before insertion into the PCR.

Each reaction consists of 18 μΙ master mix and 2 μΙ nucleic acid solution or water. 8 μΙ master mix for a reaction are assembled as follows: Table 6

^* The nanoparticle-oligonucleotide conjugates are functionalized (by J. Hurst et al., Anal. Chem., 78 (24), 8313-8318, 2006 whose relevant contents are incorporated by reference in the present disclosure). After functionalization and 6 washes, the nanoparticle-oligonucleotide conjugates are present in a concentration of 200 pM in phosphate buffer (5 mM phosphate, pH 7) with 0.1% Tween 20, 10 mM NaCl.

The amplification reaction is carried out in a total volume of 20 μΙ in 200 μΙ sample tubes or reaction vessels. It does not take place in a classical thermocycler (such as a light cycler) in which all the sample liquid is heated, but in a device for carrying out a Laser PCR, in which the nanoparticles are heated optothermally locally. As shown in FIG. 14A, the sample tubes in a sample holder 13, which in particular comprises a tempered aluminum block, are kept at a temperature of 61 ° C., which corresponds to both the annealing and the elongation temperature. The denaturation of the double-stranded DNA is induced by a laser 1. The laser 11, which serves to excite the nanoparticles, is a frequency-doubled diode-pumped Nd: YAG laser (OEM-Micro-1600, Pegasus Lasersysteme GmbH), with an output power of 1.6 W and a wavelength of 532 nm after beam expansion Focussing a factor 2.5 with a Galilean telescope with an F-theta lens (Jenoptik, focal length 100 mm) behind a mirror scanner 12 (Cambridge Technologies, Pro Series 1) into the sample tubes R1 to R5 in the sample holder 13 (focus diameter approx 25-30 pm). The mirror scanner 12 allows the focus to be moved line by line through the sample tubes, as shown in Fig. 14B, and thus to involve the entire PCR reaction volume in the opto-thermal amplification.

After placing the sample tubes in the preheated aluminum block at 61 ° C, an initial one-time thermalization time of 30 s takes place, followed by the first denaturation cycle. For each sample tube R1 to R5, 500 lines at a distance of approximately 14 pm at a line speed in the sample tube of approximately 4 m / s with the focus are traversed for this purpose. This corresponds to one cycle in the first sample tube. Subsequently, all other sample tubes are run in sequence, so that each sample tube has undergone a cycle. After a waiting period of 7 seconds, the next cycle is started. This is repeated 80 times for each sample tube. In the sample holder 13 are located on two opposite sides (the laser-facing and the laser-facing side) to each sample tube each have a window 15 and a recess 15 to couple the laser 11 in the sample tube and order on the laser -abgewandten page using a photodiode to perform a transmission measurement. That is, it can be determined during each cycle to each sample tube how much intensity of the laser 11 is transmitted through the sample 16 or in other words how much laser light the sample 16 absorbs. The laser 11 thus serves to locally heat the nanoparticles and thus to denature the double-stranded DNA (amplicon) on the nanoparticles and also to determine the measurable change (the physical or optical property of the solution) by combining oligonucleotides with cationic modification (here Reverse primer) with the nanoparticles, which here carry oligonucleotides as forward primer.

Fig. 15A shows real-time laser PCR curves measured by a change in the absorbance (in arbitrary units) of the sample 16 and the solution at the central wavelength of the laser (532 nm), respectively. The measured data shown were averaged over 5 measuring points each. 2 μΙ genomic DNA from MRSA or as negative control water were used as nucleic acid, so that the following total amounts were contained as starting material for the amplification in the respective reaction volume of 20 μΙ: solid lines: 2,000,000 genome copies MRSA

dashed lines: 200,000 genome copies MRSA

dotted lines: 20,000 genome copies MRSA

dotted-dashed lines: no genome copies, only water as negative control

It is observed that the absorption of all samples initially increases. The cause of this initial increase can be observed over wide ranges of the visible spectrum and can be deduced from measurements at at least two different wavelengths by choosing one wavelength to be slightly blue-shifted and the other slightly red-shifted to the absorption maximum of the nanoparticles. Since the initial increase in absorption is approximately the same at both wavelengths, but a shift in the nanoparticle absorption leads to contrasting changes in the absorption at the two wavelengths chosen, the initial increase in absorption can, in two-color measurement, be due to the desired effect of the absorption maximum shift Nanoparticles can be distinguished. From a certain Time or a certain number of cycles (both variables are correlated over the cycle duration - in this case 7s -), the absorption decreases downwards (ie the absorption decreases). The decreasing absorption is a consequence of the aggregating nanoparticles, which are at least partially bound by the resulting amplicon with the ZNA primers. The higher the amount of target DNA originally used, the sooner the absorption curve will break. A quantitative statement about the amount of DNA used can be made, for example, by determining the time of maximum absorption for each curve. This can be achieved, for example, by forming the first derivative of the curves from FIG. 15A according to time and determining their point of intersection with the time axis (preferably with decreasing derivative).

The derivative curves of the curves of Fig. 15A are shown in Fig. 15B. The time of maximum absorption, which can be regarded as equivalent to the Cp value in the classical qPCR, is thus obtained for 2,000,000 genome copies MRSA a period of 2.6 minutes, for 200,000 genome copies MRSA a period of 3.2 minutes and for 20,000 genome copies MRSA a period of 3.5 minutes. It should be noted that even the negative control at a later time (here 4.8 minutes) shows a break point, which is probably due to the non-specific formation of primer dimers.

Example 5.2: Laser PCR with DNA

The curves in Fig. 16A show real-time laser PCR curves, specifically, the change in the absorbance of the sample at the wavelength of the laser (532 nm) in arbitrary units, with the measurements being averaged over every 5 measurement points.

The measurement performed is essentially identical to the measurement described in Example 5.1. While in Example 5.1 an oligonucleotide with cationic modification or a ZNA with sequence 14 was used as a reverse primer, in Example 5.2 for comparison purposes, a DNA oligonucleotide without cationic modification with sequence 10 as a reverse primer used. Sequence O is substantially similar to Sequence 14, but Sequence 10 has no cationic modification and is instead somewhat longer to achieve a similar high melting point despite the lack of cationic modification. The DNA oligonucleotide without cationic modification with sequence 10 could be successfully used in the system with Sybr-Green in Example 4.1 as a replacement for the oligonucleotide with cationic modification with sequence 9, which differs only by one cationic unit of sequence 14 (data not here shown). The concentration of this oligonucleotide with the sequence 10 is also 300 nM. The control experiment in FIG. 16A, in contrast to the measurement with reverse primer with cationic modification from Example 5.1, does not show such a significant absorption kinking in any of the starting amounts of target DNA used, as can be observed in Example 5.1. The representation of the curves to the target DNA amounts used corresponds to the representation chosen in FIG. 15A. Without oligonucleotides with cationic modification or in other words only with oligonucleotides without cationic modification as primer, the amplificate or the originally used target DNA can not be reliably detected, in contrast to the methods according to preferred embodiments of the present invention.

The curves in Fig. 16B show real-time laser PCR curves, specifically, the change in absorbance of the sample at the wavelength of the laser (532 nm) in arbitrary units. In each case over 5 measuring points were averaged. The measurement is essentially identical to the measurement in Example 5.1, but here, too, the reverse primer was replaced by a DNA oligonucleotide without cationic modification with sequence 10, instead of an oligonucleotide with cationic modification or ZNA) as in Example 5.1 Sequence 14 to use.

The concentration of this oligonucleotide with the sequence 10 is now only 20 nM. In addition, however, second nanoparticles were added to the reaction. The final concentration of the second nanoparticles in the total volume of the reaction is while 20 pM. The second nanoparticles were prepared and stored essentially identically as the first nanoparticles from Table 6, but they do not carry on the surface the oligonucleotide with sequence 4, which serves as a forward primer in a partial sequence, but the oligonucleotide with sequence 12, the in a partial sequence preferably serves as a reverse primer. At least the sequence-12 oligonucleotide can hybridize to the partial sequence that results when the forward primer on the first nanoparticles is elongated upon presentation of the target sequence 7 (as is also found in MRSA) by a polymerase during an amplification reaction. However, since this partial sequence can be occupied by this or by an amplicon strand when using higher concentrations of reverse primer with sequence 10 and thus may not be available for hybridization with the second nanoparticles, the reverse primer with sequence 10 in very low concentrations (here 20 nM) can be used to achieve asymmetric amplification. Upon reaching a certain minimum amount of forward primers elongated by the polymerase on the first nanoparticles, the first nanoparticles can thus be linked by hybridization to the second nanoparticles by the amplicon. This, too, may cause the absorbance to bend, as shown in Fig. 16B. The representation of the curves to the target DNA amounts used corresponds to the representation in FIG. 15A.

The kinking of the absorption and thus the detection of the amplificate and / or the target DNA originally used with the method according to Example 5.2, however, depending on the target DNA amount used, only after about 7-10 minutes and thus significantly later than when using a method according to the invention according to Example 5.1 (see FIG. 15A), in which the detection was carried out by means of oligonucleotides with cationic modification. In Example 5.1, absorption of the absorption was observed after about 2.6 to 3.5 minutes.

Also, the kinking of the absorption in Fig. 16B is not so pronounced and therefore can not be interpreted and measured with equal reliability. This is therefore another clear indication that a Detection of a nucleic acid with a method according to the invention, ie in particular using nanoparticles and nucleic acids with cationic modification, can be done much faster, more accurate and more reliable than with other methods.

Example 6: Real-time PCR with ZNA primers and DNA primers and nanoparticle-bound oligonucleotides as a specific sample

For indirect detection of a nucleic acid, a polymerase chain reaction was carried out. The primer system used in this example consists of ZNA as a forward primer (Sequence 15, see Appendix) and DNA as a reverse primer (Sequence 16, see Appendix). Both primers are initially not associated with nanoparticles. Nanoparticles functionalized with oligonucleotides with sequence 17 (see Appendix) and used as hybridization probes serve for the detection of the nucleic acid. The oligonucleotide with sequence 17 contains a specific partial sequence B which can hybridize to a part elongated by the polymerase on the ZNA forward primer if the originally used target DNA has been constructed accordingly. In other words, the specific partial sequence B of the oligonucleotide functionalized on the nanoparticle is substantially at least partially identical to the nucleic acid to be detected. In addition, sequence 17 contains a partial sequence of 35 adenine bases, which serves as a spacer between the particle surface and specific partial sequence B, further contains the modified oligonucleotide with sequence 17 at the 5 'end of a thiol modification, with the aid of the oligonucleotide on the Surface of gold nanoparticles is bound and further contains sequence 17 at the 3 'end of a ddC modification (dideoxycytidine), which serves as a terminating modification, thus preventing the elongation by the polymerase. Gold nanoparticles of 60 nm diameter (supplied by BBI) are used, each nanoparticle carries about 1000 oligonucleotides with sequence 17. Fluorescein is used as additional dye. Each reaction comprises 9 μM master mix and 1 μM nucleic acid solution (Sequence 7 oligonucleotide) or water. 9 μΙ master mix for a reaction are assembled as follows: Table 7

the nanoparticle-oligonucleotide conjugates are functionalized (according to J. Hurst et al., Anal. Chem., 78 (24), 8313-8318, 2006, the contents of which are incorporated herein by reference). After functionalization and 6 washes, the nanoparticle-oligonucleotide conjugates are present in a concentration of 200 pM in phosphate buffer (5 mM phosphate, pH 7) with 0.1% Tween 20, 10 mM NaCl.

The PCR protocol consists of 50 cycles of 5 seconds 90 ° C, 15 seconds 58 ° C and 15 seconds 72 ° C. The signal detection takes place after every 58 ° C step.

Fig. 17 shows real-time PCR curves of the 530 nm fluorescence channel (matching the emission wavelength of fluorescein) of four different ones Starting concentrations of the nucleic acid with sequence 7 (present as oligonucleotide) and a negative control without nucleic acid as the target sequence. The curves are essentially very similar to the curves in FIG. 12. As the starting concentration of nucleic acid with sequence 7 increases, the number of cycles decreases, after which an increase in the signal can be observed or measured.

The curves in FIG. 17 have the following starting concentration of the nucleic acid (oligonucleotide with sequence 7): solid line: 1 fM

dashed line: 100 aM

dotted line: 10 aM

solid line with triangles: 1 aM

dotted-dashed line: negative control without sequence 7

Striking in this example is that the curve of the negative control without nucleic acid to be detected (dotted-dashed line) now shows no nonspecific, unwanted kinking as the negative control in Fig. 12. This is a consequence of the specific detection format. In the PCR and laser PCR examples described above, both primers (ZNA primer and nanoparticle-bound primer) were also used to generate a measurable signal by combining nanoparticles and cationically modified nucleic acids with sufficient amount of amplicon. In the example 6 described here, although the ZNA with sequence 15 serves both as a primer and for generating a measurable signal, the oligonucleotides on the nanoparticles serve in this case only to generate a measurable signal, but not as a primer. In order to prevent them from being able to serve as primers or to be elongated, they have a terminating ddC modification at the 3 'end which prevents elongation by the polymerase.

The formation of primer dimers in this case does not lead to a combination of nanoparticles and cationically modified nucleic acids and thus not to a measurable change. The specific detection is possible in that the nucleic acid to be detected with sequence 7 read from 3 'to 5' comprises the partial sequences C, B and A. Partial sequence A contains the sequence 16 of the reverse primer, partial sequence B is identical to the partial sequence B of the modified oligonucleotide which is functionalized on the nanoparticles, and partial sequence C contains a sequence which is complementary to the forward primer with sequence 15. Since the forward primer is present in excess compared to the reverse primer, after a corresponding number of cycles an excess arises at the substrand of the amplicon which results from the elongation of the forward primer with cationic modification.

The elongated part then contains the partial sequence B ', which is complementary to the partial sequence B of the modified oligonucleotide on the nanoparticle. Thus, it may lead to a combination of nanoparticles and cationically modified nucleic acids and thus to a measurable change in the physical property of the solution. For example, the excess of the forward primer with ZNA may be necessary to obtain a sufficient number of unoccupied partial strands of the amplicon to which partial sequence B of the modified oligonucleotide can hybridize to the nanoparticle. For example, without the excess, there could be too much competition between partial sequence B of the modified oligonucleotide on the nanoparticle and the complementary strand of the amplicon resulting from the elongation of the reverse primer. In this way, the connection between nanoparticles and cationically modified nucleic acid and thus the measurable change could be suppressed or prevented. By using the specific and terminated sequence 17 on the nanoparticles, which does not serve as a primer in the PCR, even lower initial concentrations of the nucleic acid to be detected with sequence 7 can be distinguished from the negative control without sequence 7. In Fig. 12, for example, the initial concentration of 1 fM could be clearly distinguished from the negative control.

In FIG. 17, ie with a method according to the invention according to the preferred embodiment described in Example 6, moreover, a 1000-fold lower initial concentration (1 aM) than are detected in comparison to FIG. 12. As can be seen in Fig. 17, even with such a low initial concentration, reliable discrimination is possible because the negative control shows no kinking.

Bezugszeicheniiste

2 nanoparticles

 4 nucleic acid (attached to the nanoparticle)

 6 cationically modified nucleic acid / cationically modified

 oligonucleotide

 6a hybridization area

6b cationic section

 6c cationic unit

 8 dye

 10 (to be detected) nucleic acid

 1 light source or laser

13 sample holder

 15 windows or recesses

 16 sample

 R, R1 - R8 reaction vessel

 11 Intensity of incident excitation light

T1, T2 intensity of tran emitted light

F1, F2 Intensity of fluorescence emission

attachment

Sequence list (Sequence sequence each from 5 'to 3'): Sequence 1: 5 Thiol - AAAAATCATAGCGTCATTATTCCAGGAA (SEQ ID No. 1) Sequence 2: Z2- TTCCTG G AATAATG ACG CTATG A (SEQ ID No. 2)

Sequence 3: Z2-CCAATCTAACTTCGACAT (SEQ ID No. 3)

Sequence 4: 5'thiol - AAAAAAAAAAAAAAAAAAAAAAAAAAA

/ sp9 // sp9 / AG ATG G TATG TG G AAG TTAG ATTG G

(SEQ ID No. 4, SEQ ID No. 5)

 Sequence 5: Z4-GTTCGCATCAAACGGAAAAT (SEQ ID No. 6)

Sequence 6: Z4-CCAATCTAACTTCGACAT (SEQ ID No. 7)

Sequence 7:

 TCAATATGTATGCTTTGGTCTTTCTGCATTCCTGGAATAATGACGCTATGATCC CAATCTAACTTCCACATACCATCTTCTTT

(SEQ ID No. 8)

 Sequence 8: AGATGGTATGTGGAAGTTAGATTGG (SEQ ID No. 9)

Sequence 9: Z3-CCTG GATATAATG ACG CTATG A (SEQ ID No. 0)

Sequence 10: GCATTCCTGGAATAATGACGCTATGA (SEQ ID No. 11)

Sequence 11: 5'-thiol-AAAAAAAAAAAAAAAAAAAAAAAAAAA

/ sp9 // sp9 / GCAGAGCAAGGACTGATACA

(SEQ ID No. 12, SEQ ID No. 13)

 Sequence 12: 5'thiol - AAAAAAAAAAAAAAAAAAAAAAAAAAA TTCCTG G AATAATG ACG CTATG A (SEQ ID No. 14)

Sequence 13:

 AAAGAAGATGGTATGTGGAAGTTAGATTGGGATCATAGCGTCATTATTC CAGGAATGCAGAAAGACCAAAGCATACATATTGA

 (SEQ ID No. 15)

Sequence 14: Z4-CTGGAATAATGACGCTATGA (SEQ ID No. 16)

Sequence 15: Z4-AGATGGTATGTGGAAGTTAGATTGG (SEQ ID NO.

17)

Sequence 16: ATGTATGCTTTGGTCTTTCTGC (SEQ ID No. 18) Sequence 17: 5'-thiol-AAAAAAAAAAAAAAAAAAAAAAAAAAA TTCCTGGAATAATGACGCTATGA_ddC (SEQ ID No. 19)

Z2 represents two cationic units in the ZNA modification

Z3 represents three cationic units in the ZNA modification

Z4 represents four cationic units in the ZNA modification

/ sp9 / is an abasic modification of Spacer9

ddC is a ddC modification (dideoxycytidine)

Claims

Expectations 1. Use of at least two nanoparticles (2) in combination with a cationically modified nucleic acid (6) for detecting a nucleic acid (10) to be detected in a solution. 2. Use of a cationically modified nucleic acid (6) in combination with at least two nanoparticles (2) for detecting a nucleic acid (10) to be detected in a solution. 3. Method for detecting a nucleic acid (10) to be detected in a solution, comprising the steps: Providing at least two nanoparticles (2) in the solution; Providing a cationically modified nucleic acid (6) in the solution; Determining a change in at least one physical property of the solution, wherein the physical property of the solution is at least partially changed by the at least partial binding of the at least two nanoparticles (2) to the cationically modified nucleic acid (6) and wherein the nucleic acid (10) to be detected is in the Solution that at least partially promotes or at least partially inhibits binding of the at least two nanoparticles (2) to the cationically modified nucleic acid (6). 4. The method according to claim 3, wherein at least one nucleic acid (4) is bound to at least one of the nanoparticles (2). 5. The method according to claim 4, wherein the nucleic acid (4) bound to the at least one nanoparticle (2) comprises at least one oligonucleotide (4). 6. The method according to claim 5, wherein the cationically modified nucleic acid (6) is at least partially complementary to one to be detected in the solution is nucleic acid (10) and/or wherein the at least one oligonucleotide (4) is at least partially complementary to the nucleic acid (10) to be detected in the solution. 7. The method according to claim 5, wherein: the at least one oligonucleotide (4) is at least partially complementary to a first nucleotide sequence section of a nucleic acid (10) to be detected in the solution, and the cationically modified nucleic acid (6) is at least partially complementary to a second nucleotide sequence section of the nucleic acid (10) to be detected in the solution; where preferably: the first nucleotide sequence section is formed on a first strand of the nucleic acid (10) to be detected, and the second nucleotide sequence section is formed on a second strand of the nucleic acid (10) to be detected that is different from the first strand. 8. The method according to any one of claims 5 to 7, wherein at least part of the at least one oligonucleotide (4) and / or at least part of the cationically modified nucleic acid (6) is a primer. 9. The method according to any one of claims 6 to 8, wherein the method is used to determine the concentration of the nucleic acid (10) to be detected in the solution and / or the method comprises a duplication of the nucleic acid (10) to be detected. 10. The method according to any one of claims 6 to 9, wherein the detection of the nucleic acid (10) to be detected in the solution takes place during and / or after an amplification of the nucleic acid (10) to be detected by means of a polymerase chain reaction. 1 . Method according to claim 9 or 10, wherein the nucleic acid (10) to be detected in the solution is multiplied by means of a polymerase chain reaction, in which case a cycle in the polymerase chain reaction preferably comprises the steps of denaturation, annealing and elongation and the steps are preferably repeated several times . Method according to one of claims 3 to 11, wherein an environment of the excited nanoparticles (2) is locally heated by stimulating at least some of the nanoparticles (2). 13. The method according to any one of claims 3 to 12, wherein the at least one physical property of the solution changes when at least some of the nanoparticles (2) are connected to the at least one cationically modified nucleic acid (6), the at least one property of Solution preferably comprises an optical property, and wherein the optical Property is in particular an extinction and/or a scattering and/or an absorption. 14. The method according to any one of claims 6 to 13, wherein the physical property of the solution is measured at different temperatures of the solution. 15. Kit for detecting a nucleic acid (10) to be detected in a solution, comprising: - at least one cationically modified nucleic acid (6); and at least two nanoparticles (2); wherein the at least two nanoparticles (2) are designed to at least partially change the physical property of the solution by at least partially binding the at least two nanoparticles (2) to the cationically modified nucleic acid (6) and wherein the nucleic acid (10) to be detected is in the Solution that at least partially promotes or at least partially inhibits binding of the at least two nanoparticles (2) to the cationically modified nucleic acid (6).