WO2009074937A1

WO2009074937A1 - New labels for surface-enhanced resonant raman spectroscopy

Info

Publication number: WO2009074937A1
Application number: PCT/IB2008/055132
Authority: WO
Inventors: Sieglinde Neerken; Kristiane A. Schmidt; Gerhardus W. Lucassen; Emile J. K. Verstegen
Original assignee: Koninklijke Philips Electronics NV
Current assignee: Koninklijke Philips NV
Priority date: 2007-12-10
Filing date: 2008-12-08
Publication date: 2009-06-18
Anticipated expiration: 2010-06-10

Abstract

The present invention is related to the use of an oligonucleotide labelled with a label suitable for SERRS, wherein the label has a negative charge and the oligonucleotide is not modified to compensate the negative charge.

Description

New labels for Surface-enhanced resonant Raman spectroscopy

FIELD OF THE INVENTION

The present invention relates to labels for use in Surface-enhanced (resonant) Raman (SE(R)RS) and methods for their use.

BACKGROUND OF THE INVENTION

Surface-enhanced (resonant) Raman spectroscopy (SE(R)RS) is a technique for sensitive and selective detection and identification of molecules adsorbed at a roughened metal surface. Raman scattering can be enhanced by several orders of magnitude. In SERRS it is possible to combine the sensitivity of molecular resonance (by a specific dye) with the sensitivity of surface-enhanced Raman scattering (SERS) so that very low concentrations of an analyte can be measured. The technique can e.g. be applied in molecular diagnostics to identify deoxyribonucleic acid (DNA) of pathogenic bacteria or proteins involved in infectious diseases. In this area a rapid, specific and highly sensitive identification is crucial for effective treatment. Optical methods, especially fluorescence spectroscopy, are widely used to identify certain biomolecules. SERRS has the unique feature that the scattered light consists of sharp, molecule-specific vibrational bands which makes discrimination of multiple analytes possible. Various articles are published on DNA identification by Surface- Enhanced (Resonant) Raman Spectroscopy [e.g. Isola et al. (1998) Anal. Chem. 70, 1352- 1356; Deckert et al. (1998) Anal. Chem. 70, 2646-2650; Graham et al. (2000) Biopolymers 57, 85-91; Faulds et al. (2004) Anal. Chem. 76, 592-598].

The presence of a certain targeted DNA sequence can be measured via the SERRS signal of a SERRS active label attached to the DNA sequence. Simultaneous detection of multiple labels (and thereby multiple DNA sequences) in solution by SERRS has been demonstrated. Multiplexing of 6 labels has recently been performed (Faulds et al. "Highly multiplexed detection of labelled oligonucleotides using surface enhanced resonance Raman scattering (SERRS)" submitted).

To obtain resonance enhancement of the labels, the excitation wavelength needs to overlap with the electronic absorption band of the label. For surface enhancement the label needs to adsorb at a roughened metal surface. Aggregated colloidal particles have been found to be practical examples of roughened metal surfaces. In solution, these colloidal particles carry a certain charge. Typically, citrate or EDTA-reduced silver colloids are used, which are negatively charged.

Currently, the common understanding is that a positively charged label will easily adsorb at the silver surface to ensure surface enhancement. Negatively charged labels, however, will not adsorb at the surface due to electrostatic repulsion and are therefore considered inappropriate for use as such as SERRS labels

When a negatively charged label is used in SERRS, a modification is introduced in the attached nucleic acid probe to create an area of positive charge, which then enables adsorption of the label onto the metal surface. Propargylamine modification of DNA has been introduced to get the negatively charged dyes HEX, TET, FAM to the surface of the metallic colloidal particles (Faulds et al, (2005) Analyst 130, 1125-1131, US 6,127,120). The use of negatively charged dyes in SERRS accordingly requires tailor made synthesis of labelled DNA probes with additional 5-(l-propargylamino)-2'-deoxyuridine nucleotides.

Faulds et al. (2005) in Talanta 67, 667-671 describe the use of unmodified DNA labelled with Bodipy TR-X, a compound without an ionic charge which carries a thiophene function considered to promote surface binding.

For multiplexing, the choice of commercially available positively charged labels that not only adsorb without any modification at the negatively charged metal surface but also have similar absorption spectra, so as to meet the resonance condition upon single wavelength excitation, is limited.

Particular and preferred aspects of the invention are set out in the accompanying independent and dependent claims. Features from the dependent claims may be combined with features of the independent claims and with features of other dependent claims as appropriate and not merely as explicitly set out in the claims.

SUMMARY FO THE INVENTION

The present invention relates to dyes suitable for SERRS which, despite their negative charge, are suitable as direct labels of oligonucleotides. It has been established that the oligonucleotides need not contain a positive charge to ensure adequate signal detection of a negatively charged label. This opens up the number of dyes/labels which can be used in SERRS multiplexing experiments.

Accordingly, one aspect of the present invention relates to the use of probes labelled with negatively charged dyes in SERRS detection, without modifying the charge of the oligonucleotide. More specifically, the invention relates to the use of oligonucleotides labelled with a label suitable for SERRS, characterised in that the oligonucleotide consists of bases with neutral nucleosides, and the label has a negative charge. Preferably the oligonucleotide consists of bases with neutral nucleosides, and the label has a negative charge

In particular embodiments the negatively charged label is a fluorescent label, more particularly a fluorescent label with an absorption maximum between 490 and 544 nm.

In further particular embodiments oligonucleotides used in the context of the invention consist of nucleotides with unmodified nucleosides, more particularly the oligonucleotide does not comprise 5-(l-propargylamino)-2'-deoxyuridine.

In further particular embodiments, a negatively charged fluorescent label as used herein is a label selected from the group consisting of FAM , Alexa Fluor 488, Dichlorofluorescein, Alexa Fluor 514, TET, JOE, Eosin, Yakima, Yellow, Tetrabromosulfonefluorescein, Erythrosin, Atto 532 , HEX , Alexa Fluor 546 , Resorfurin, Atto-488, Atto-565 and Oregon Green 500. Another aspect of the invention relates to methods which involve the use of negatively charged dyes in accordance with the invention. More particularly, methods are provided for detecting one or more nucleic acid targets in a sample by surface-enhanced resonance Raman scattering (SERRS), preferably with high reproducibility and high signal intensity, making use of labeled oligonucleotides as described herein. In particular embodiments methods are provided which comprise the steps of:

(a) Contacting the sample with an oligonucleotide with a label suitable for SERRS detection, wherein:

(b) Contacting the probe with a metal surface that is preferably roughened in the presence of an aggregating agent, and

(c) Detecting the SERRS signal generated by the label on the oligonucleotide, wherein the methods are characterized in that the labelled oligonucleotide consist of bases with unmodified neutral nucleosides, and the label suitable for SERRS detection has a negative charge.

In particular embodiments methods described herein, the label suitable for SERRS detection is a fluorescent label.

In further particular embodiments methods are provided wherein the labelled oligonucleotide is a target-specific oligonucleotide.

In yet further particular embodiments of methods described herein, oligonucleotides are used which consist of nucleotides with unmodified nucleosides, more particularly, the oligonucleotide does not comprise 5-(l-propargylamino)-2'-deoxyuridine. In particular embodiments methods are provided which are methods for detecting multiple targets in a sample, wherein at least two different oligonucleotides each labelled with a different label are contacted with the sample and the detection step comprises detecting the SERRS signal generated by each of the different labels, which methods are characterized in that at least one oligonucleotide with a negatively charged label is used. More particularly, at least one of the oligonucleotides is characterised in that the oligonucleotide consists of bases with unmodified neutral nucleosides, and the label has a negative charge.

In further particular embodiment of methods described herein using more than one label, detection of the SERRS signal of the different labels is performed at one wavelength.

In particular embodiments of multiplexing methods described hereinabove, the labels used have an absorption maximum between 490 and 544 nm.

In particular embodiments methods of detection described hereinabove making use of at least one oligonucleotide with a negatively charged label as described herein, which methods are performed with at least 5 different fluorescent labels, more particularly with labels selected from the group consisting of FAM , Alexa Fluor 488, Dichlorofluorescein, Alexa Fluor 514, TET, JOE, Eosin, Yakima, Yellow, Tetrabromosulfonefluorescein, Erythrosin, Atto 532 , HEX , Alexa Fluor 546 , Resorfurin, Atto-488, Atto-565 and Oregon Green 500. In further particular embodiments at least 6, more particularly at least 7, most particularly 8, 9 or 10 different labels, optionally selected from the list described above are used.

A preferred embodiment according to the invention is a method for detecting multiple targets in a sample, wherein at least two different oligonucleotides each labelled with a different label are contacted with said sample and said detection step comprises detecting the SERRS signal generated by each of said different labels, wherein at least one of said oligonucleotides is characterised in that: the oligonucleotide consists of bases with unmodified nucleosides, and the label has a negative charge.

The above and other characteristics, features and advantages of the present invention will become apparent from the following detailed description, taken in conjunction with the accompanying Figures, which illustrate, by way of example, the principles of the invention. This description is given for the sake of example only, without limiting the scope of the invention. The reference Figures quoted below refer to the attached drawings. BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows molecular structure of a nucleoside and nucleotide (prior art).

Fig. 2 shows the SERRS spectrum of unmodified DNA labelled with negatively charged fluorescent dyes. Excitation is performed at 532 nm. Concentration of the label in the sample is 100 pM.

Fig. 3 shows absorption spectra of 5 labels known as fluorescent dyes, linked to unmodified DNA. The peak around 260 nm is due to absorption by DNA and around 500/550 nm to absorption by the dye. The resonance conditions for SERRS are met (see overlap of excitation wavelength) at 514 or 532 nm.

Fig. 4 A and B show results of multiplexing experiments with SERRS labelled probes.

In the different Figures, the same reference signs refer to the same or analogous elements.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated.

Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The following terms or definitions are provided solely to aid in the understanding of the invention. These definitions should not be construed to have a scope less than understood by a person of ordinary skill in the art. Essentially consist of bases with neutral nucleosides means that most, prferably all bases are neutral nucleosides. A molecular structure of nucleosides is provided in figure 1 .( Alberts et al, "Molecular Biology of the cell", 4^l edition , page 121) and as can be observed, nucleosides in their unmodified form bare no charge.

The term "label" or "dye", as used herein, refers to a molecule or material capable of generating a detectable signal. Labels which are SERRS active or 'suitable for SERRS' are labels which are capable of generating a SERRS spectrum when appropriately illuminated. In order to be SERRS active a dye must be polarisable upon excitation, and be able to come into close contact (distance <30 nm) with a rough metal surface (e.g. aggregated (noble) metal colloidal particles).

The term "negatively charged" as used herein when referring to a dye, relates to the fact that the charge of the dye in the buffer wherein SERRS measurements takes place is negative as determined by methods described herein.

The present invention is based on , contrary to what is reported in the literature, labels suitable for SERRS which are negatively charged, can be used for SERRS detection without the need to compensate the charge, e.g. by adding positively charged nucleosides to the probe used for detection. Multiplexing with only negative labels without modifying the probes, for example with 5-(l-propargylamino)-2'-deoxyuridine, was not possible untill now. Additionaly, the use of the labels according to the present invention furher increases the possibilities for multiplex SERRS detection as the amount of currently available positive and or neutral labels is limited.

Accordingly, one aspect of the invention relates to the use of oligonucleotides labelled with a label suitable for SERRS, wherein the label has a negative charge and wherein the oligonucleotide essentially consists of bases with unmodified neutral nucleosides. Thus this aspect relates to the use of SERRS labels with a negative charge directly linked to an unmodified probe. In particular embodiments, the invention describes the use of fluorescent labels which are known to be suitable for SERRS, as labels for oligonucleotides without further modification of the probe to compensate for the negative charge.

The charge of a dye (i.e. whether it is negative, neutral or positive) is influenced by the pH of the buffer in which the dye is present. In the context of the present invention, the relevant conditions are those of the buffer wherein the SERRS measurements take place (typically between pH 6.5 and 9.0). The charge of dye itself, i.e. not coupled to an oligonucleotide, can be determined e.g. by assaying the movement of the dye in a gel or matrix in an electric field. Upon labelling of an oligonucleotide with a dye, a functional group on the dye is used to react the dye with the oligonucleotide. This may affect the charge of the dye. Where a charged group of a dye is used to react with an oligonucleotide, the loss of a functional group and the effect of this loss on the charge of the dye is assessed either on a theoretical basis or by measuring the charge of a modified version of the dye wherein the functional group of the dye which is used for the oligonucleotide labelling, is modified. Contrary to the generally applied practice, the present invention discloses that SERRS measurements of oligonucleotide probes labelled with a negatively charged dye, more particularly a negatively charged fluorescent dye, can be performed without incorporation of positive charges into the oligonucleotide probe. Such incorporation of positive charges was typically achieved by incorporating propargylamine-modified nucleotides to an oligonucleotide probe (typically a tail of 12 nucleotides with alternating cytosine and propargylamine-modified deoxyuridine). Surprisingly, the present invention discloses that such modification is however not necessary in order to use a oligonucleotide probe with a negatively charged dye in SERRS measurement.

Particular embodiments of the invention relate to labelled oligonucleotides for use in SERRS which do not comprise nucleotides with positively charged nucleosides (such as 5-(l-propargylamino)-2'-deoxyuridine, abbreviated in the prior art as "7"), but comprise or consist of nucleotides with uncharged nucleosides. More particularly, the first 3, 6, 9 or up to 12 nucleotides adjacent to the dye are nucleotides with unmodified nucleosides. In particular embodiments, the oligonucleotide consists of unmodified A, C, T, and G nucleotides (DNA) or unmodified A, C, U and G nucleotides (RNA). The modification as referred to herein do not refer to the modification of the 5' or 3' end of the oligonucleotide which is used for coupling an oligonucleotide with a dye, but rather to the presence of additional charged nucleotides.

Particular embodiments of the present invention relate to the use of negatively charged fluorescent SERRS active dyes

A list of negatively charged fluorescent SERRS active dyes, and their absorption maximum, which are suitable as SERRS dyes for use according to the invention are ATTO 532 (530 nm), Alexa Fluor dyes such as Alexa 488 (494 nm), 514 (517 nm), 546 (554 nm), HEX (537 nm), JOE (520 nm), Erythrosin (530 nm), Eosin (524 nm), Dichlorofluorescein (510 nm), Resorfurin (578 nm), Tetrabromosulfonefluorescein (528 nm), FAM (494 nm), TET (519 nm), Yakima Yellow (526 nm) Atto-488, Atto-565 and Oregon Green 500 (Table 1). Table 1. Negatively charged fluorescent dyes

In particular embodiments, the invention relates to the use of negatively charged SERRS active labels having an absorption maximum between 490 and 544 nm of which the electronic absorption band overlaps with and excitation wavelength of 514 or 532 nm. A preferred list hereof includes TET, Yakima Yellow, FAM, JOE, HEX, Atto 532, Atto 565, Atto 488, Alexa Fluor 488 and Alexa Fluor 514.

As indicated above, particular embodiments of the invention relate to the use of SERRS active fluorescent dyes. Fluorescent dyes are available from different companies (Invitrogen, Molecular Probes, Atto-Tech etc.).

Methods for coupling labels or dyes to oligonucleotides are known in the art. Oligonucleotides coupled to SERRS active fluorescent dyes are commercially available from e.g. Oswel (UK), Eurogentec (Belgium), IBA (Germany) and other companies. Another aspect of the present invention relates to methods for detecting a nucleic acid target in a sample by surface-enhanced resonance Raman scattering (SERRS) with high reproducibility and high signal intensity. In these methods a sample is contacted with a target-specific oligonucleotide probe with a label suitable for SERRS detection, which is negatively charged as described above. The methods of the present invention involve oligonucleotide probes which carry nucleosides which in sum have a neutral charge, and which carry a SERRS active label with a negative charge. In particular embodiments, the oligonucleotide probes consist of bases with neutral nucleosides. In further particular embodiments, the oligonucleotide probes used in these methods consist of nucleotides with unmodified nucleosides. Most particularly the probes do not comprise 5-(l-propargylamino)- 2'-deoxyuridine. Particular embodiments of the methods of the invention make use of fluorescent SERRS active dyes which are negatively charged.

In particular embodiments methods according to the invention comprise the steps of contacting a sample with an oligonucleotide probe labelled as described herein, contacting the (bound or unbound) labelled oligonucleotide probe with a preferably roughened metal surface, and detecting the SERRS signal generated by the label on the probe.

An important application of the methods of the present invention is multiplexing, as the invention provides a wider variety of labels suitable for detection at the same wavelength.

Accordingly in particular embodiments, methods are provided for detecting multiple targets in a sample, wherein at least two different oligonucleotide probes each labelled with a different label are contacted with a sample and the detection step comprises detecting the SERRS signal generated by each of the different fluorescent labels. In these multiplexing methods, at least one of the oligonucleotide probes is labelled with a negatively charged SERRS active label and consists of bases with neutral nucleosides. In further particular embodiments two or more of the probes comprise a SERRS label with a negative charge (whereby the oligonucleotide probe itself consists of bases with neutral nucleosides). Preferably, the SERRS active dyes are selected from the group comprising ATTO 532 (530 nm), Alexa Fluor dyes such as Alexa 488 (494 nm), 514 (517 nm), 546 (554 nm), HEX (537 nm), JOE (520 nm), Erythrosin (530 nm), Eosin (524 nm), Dichlorofluorescein (510 nm), Resorfurin (578 nm), Tetrabromosulfonefluorescein (528 nm), FAM (494 nm), TET (519 nm), Yakima Yellow (526 nm) Atto-488, Atto-565 and Oregon Green 500 (Table 1). In further particular embodiments a combination of probes with negatively charged SERRS active dyes and positively or neutrally charged SERRS active dyes are used so as to obtain an optimal number of different dyes (and thus targets) which can be detected simultaneously.

Other combinations of commercially available dyes are envisaged which enable a wide range of multiplexing without special design of the labels. The methods of the present invention allow a very low limit of detection (20 pM).

One of the well-known advantages of SERRS over the commonly used fluorescence detection is exactly that it allows the simultaneous differential detection of a number of different labels, due to the much sharper (vibrational) spectral features of SERRS as compared to (electronic) broad features in fluorescence. A further advantage of multiplexing using SERRS is that with well-chosen labels, only a single excitation wavelength can be used, whereas in fluorescence for each label a different excitation wavelength needs to be used. Using the methods and tools of the present invention which allow the use of negatively labelled dyes without the need to modify the probes, multiplexing of an assay can be significantly increased.

Suitable methods for detecting SERRS signals are known to the skilled person. The detection of dyes attached to DNA is described in extenso in e.g. the above-cited publications of Faulds et al. The present invention provides for a general principle of detecting DNA using probes labelled with a SERRS active dye. Typically, methods for detecting a target involve the use of a target-specific probe. In particular embodiments a labelled oligonucleotide probe is used which hybridises specifically with a sequence of the target. Alternatively indirect detection methods can be used wherein the labelled oligonucleotide is a molecule which competes with the target for the binding to a probe. In further particular embodiments the oligonucleotide is a probe which hybridises specifically with a sequence within an oligonucleotide, which oligonucleotide further also comprises a sequence capable of hybridising specifically with the target. These are non-limiting examples of detection principles which can be used in the context of the present invention.

The use of oligonucleotides in methods described hereinabove is characterised in that there is no need to modify these oligonucleotides by introduction of a positive charge to ensure interaction of the label which is a SERRS active dye with a SERRS active surface. This implies that probes can be designed such that only the ability to ensure detection of the target and the ability to attach a label need to be taken into account. In particular embodiments, the labelled oligonucleotide consists of bases with unmodified neutral nucleosides. As detailed above, if a (charged) functional group of a nucleoside is used for attaching the label, this need not be taken into account in the context of the present invention (as it is no longer present in the labelled oligonucleotide).

Labelled oligonucleotides used in the context of the present invention are oligonucleotides carrying one or more labels. These labels may be identical (e.g. for signal enhancement) or different (e.g. to allow double detection of the signal). In particular embodiments, the oligonucleotides carry one label.

The concentration of the oligonucleotide (and the dye where single-labelled oligonucleotides are used) in methods of the invention can vary, and will typically (but not necessarily) depend on the concentration of analyte to be detected. Since SERRS is particularly suited for measuring low concentrations of analyte (e.g. lower than 1 nM), according to a particular embodiment of the invention, the labelled oligonucleotide/SERRS active dye is used at a concentration lower than 1x10 ⁹ M.

Specific reagents suitable for use in methods involving SERRS detection are described in the art. More particularly, different conditions preparing metallic colloidal solutions and different buffer systems for performing SERRS measurements are described in the art.

Buffers used for SERRS generally have a pH between 7 and 9 and include a polyamine as aggregating agent. Exemplary buffers used in SERRS are Tris/HCl buffers between pH 7 and 9.

Methods of the present invention are suitable for the detection of different types of nucleic acids, for instance DNA such as a gene, viral DNA, bacterial DNA, fungal DNA, mammalian DNA, or DNA fragments. Methods of the present invention are similarly suitable for the detection of analytes which are RNA such as viral RNA, mRNA, rRNA. The analyte can also be cDNA, oligonucleotides, or synthetic DNA, RNA, PNA, synthetic oligonucleotides, modified oligonucleotides or other nucleic acid analogues. An analyte may comprise single-stranded and double-stranded nucleic acids. An analyte may, prior to detection, be subjected to manipulations such as digestion with restriction enzymes, copying by means of nucleic acid polymerases (e.g. PCR), shearing or sonication thus allowing fragmentation to occur.

The term "sample" is used in the context of the present invention in a broad sense and is intended to include a wide range of biological materials as well as compositions derived or extracted from such biological materials. The sample may be any suitable preparation in which the analyte is to be detected. The sample may comprise, for instance, a body tissue or fluid such as but not limited to blood (including plasma and platelet fractions), spinal fluid, mucus, sputum, saliva, semen, stool or urine or any fraction thereof. Exemplary samples include whole blood, red blood cells, white blood cells, buffy coat, hair, nails and cuticle material, swabs, including but not limited to buccal swabs, throat swabs, vaginal swabs, urethral swabs, cervical swabs, rectal swabs, lesion swabs, abcess swabs, nasopharyngeal swabs, and the like, lymphatic fluid, amniotic fluid, cerebrospinal fluid, peritoneal effusions, pleural effusions, fluid from cysts, synovial fluid, vitreous humor, aqueous humor, bursa fluid, eye washes, eye aspirates, plasma, serum, pulmonary lavage, lung aspirates, biopsy material of any tissue in the body. The skilled artisan will appreciate that lysates, extracts, or material obtained from any of the above exemplary biological samples are also considered as samples. Tissue culture cells, including explanted material, primary cells, secondary cell lines, and the like, as well as lysates, extracts, supernatants or materials obtained from any cells, tissues or organs, are also within the meaning of the term biological sample as used herein. Samples comprising microorganisms and viruses are also envisaged in the context of analyte detection using the methods of the invention. Materials obtained from forensic settings are also within the intended meaning of the term sample. Samples may also comprise foodstuffs and beverages, water suspected of contamination, etc. These lists are not intended to be exhaustive.

Detection by surface-enhanced spectroscopies such as surface-enhanced resonance Raman spectroscopy (SERRS) is based on the strong enhancement of Raman scattering observed for analytes adsorbed onto an appropriate 'SERRS active surface'. Typically, the surface is a noble (Au, Ag, Cu) or alkali (Li, Na, K) metal surface. Generally, the SERRS active surface is provided as an aggregation of metal colloid particles, which typically results in enhancements for SERRS of greater than 10⁸-10¹² of the Raman scattering. The colloidal suspension of metal particles is for example a colloidal suspension of silver particles, such as citrate reduced silver particles.

Where metal nanoparticles are used as a SERRS active surface; these may be a naked metal or may comprise a metal oxide layer on a metal surface. They may include an organic coating such as of citrate or of a suitable polymer, such as polylysine or polyphenol, to increase its adsorptive capacity. Metal colloid particles making up a SERRS active surface are typically colloidal nanoparticles aggregated in a controlled manner so as to be of a uniform and desired size and shape and as stable as possible against self-aggregation. Processes for preparing such colloids are well known (e.g. described in US Application No. 20050130163). Alternative methods of preparing nanoparticles have also been described (e.g. U.S. Pat. Nos. 6054495, 6127120, 6149868). Nanoparticles may also be obtained from commercial sources (e.g. Nanoprobes Inc., Yaphank, N.Y.; Polysciences, Inc., Warrington, Pa.). Metal particles for use in this context can be of any size as long as they give rise to a SERRS effect. Typically they have a diameter of about 4-50 nm, most particularly between 25 nm and 40 nm, depending on the type of metal. Methods for obtaining metal particles having a diameter of 40 nm have been described such as by Munro et al. (1995) Langmuir 11, 3712-3720.

Addition of colloid metal particles to a suspension of labelled oligonucleotide results in the adsorption of the label to the metal surface. Hereafter, aggregation of the colloids is typically achieved by addition of a polyamine. Suitable polyamines include monomeric or polymeric polyamines, such as spermine or spermidine, 1,4- diaminopiperazine, diethylenetriamine, N-(2-aminoethyl)-l,3-propanediamine, triethylenetetramine and tetraethylenepentamine. Alternatively, salts like NaCl can be used for aggregation.

Particular embodiments of methods of the invention involve the application of SERRS, and operating at the resonant frequency of a SERRS active label gives increased sensitivity. In this case, the light source used to generate the Raman spectrum is a coherent light source, e.g. a laser, tuned substantially to the maximum absorption frequency of the label being used. This frequency may shift slightly on association of the label with the SERRS active surface (and the analyte and/or analyte binding species), but it is within the skill of the artisan to tune the light source to accommodate this. The light source may be tuned to a frequency in the absorption band, or near to the label's absorption maximum, or to a frequency at or near that of a secondary peak in the label's absorption spectrum. Simultaneously, by tuning the frequency of the light source in the absorption band of the aggregated colloids, plasmons are excited in the colloidal aggregates that contribute to the enhancement of the electrical fields between the aggregated particles. Excitation of the dye molecules at the interstitial spaces between the colloid particles, the so-called hot spots, gives largest enhancement of the SERRS signals.

In SERRS detection typically the fingerprint spectrum is measured in order to identify each label. However, if the different labels used each have a unique spectral line, then it can be sufficient to detect signal intensity at a chosen spectral line frequency or frequencies.

Typically, the detection step in the SERRS based detection methods of the present invention are carried out using incident light from a laser, having a frequency in the visible spectrum. The exact frequency chosen will depend on the label, surface and analyte. Frequencies in the red area of the visible spectrum tend, on the whole, to give rise to better surface enhancement effects. However, it is possible to envisage situations in which other frequencies, for instance in the ultraviolet or the near-infrared ranges, might be used. The selection and, if necessary, tuning of an appropriate light source, with an appropriate frequency and power, will be well within the capabilities of one of ordinary skill in the art, particularly on referring to the available SERRS literature.

Excitation sources for use in SERRS-based detection methods include, but are not limited to, nitrogen lasers, helium-cadmium lasers, argon ion lasers, krypton ion lasers, etc. Multiple lasers can provide a wide choice of excitation lines which is critical for resonance Raman spectroscopy. According to a specific embodiment, an argon ion laser is used in a LabRam integrated instrument (Jobin Yvon) at an excitation of 514.5 nm.

Laser power, or excitation power, used in the context of the present invention can be varied, e.g. depending on the properties of the label used. According to a specific embodiment of the invention, the excitation power is between 1 and 15 mW.

An excitation beam suitable for use in the context of the present invention may be spectrally purified with a bandpass filter and may be focused on a substrate using an objective lens. The objective lens may be used to both excite the sample and to collect the Raman signal, by using a holographic beam splitter to produce a right-angle geometry for the excitation beam and the emitted Raman signal. The intensity of the Raman signal needs to be measured against an intense background from the excitation beam. The background is primarily Rayleigh scattered light and specular reflection, which can be selectively removed with high efficiency optical filters. For example, a holographic notch filter may be used to reduce Rayleigh scattered radiation.

Several objective lenses can be used in the context of the invention. According to a specific embodiment, the objective lens is a 5x, 1Ox, 2Ox, 5Ox or a 10Ox objective lens. In most set ups, the nature of the objective lens influences the excitation volume, since the focal distance, numerical aperture and working distance determine the volume irradiated. Accordingly, where objectives of 5x, 10x, and 5Ox are used, the excitation volume typically decreases from 5x to 5Ox, accordingly.

The surface-enhanced Raman emission signal may be detected by a Raman detector. A variety of detection units of potential use in Raman spectroscopy are known in the art and any known Raman detection unit may be used. An example of a Raman detection unit is disclosed e.g. in U.S. Pat. No. US 6002471. Other types of detectors may be used, such as a charge coupled device (CCD), with a red-enhanced intensified charge-coupled device (RE-ICCD), a silicon photodiode, or photomultiplier tubes arranged either singly or in series for cascade amplification of the signal. Photon counting electronics can be used for sensitive detection. The choice of detector will largely depend on the sensitivity of detection required to carry out a particular assay. Several devices are suitable for collecting SERRS signals, including wavelength selective mirrors, holographic optical elements for scattered light detection and fibre-optic waveguides.

Devices suitable for obtaining and/or analysing a SERRS spectrum may include some form of data processor such as a computer. Once a SERRS signal has been captured by an appropriate detector, its frequency and intensity data will typically be passed to a computer for analysis. Either the fingerprint Raman spectrum will be compared to reference spectra for identification of the detected Raman active compound or the signal intensity at the measured frequencies will be used to calculate the amount of Raman active compound detected.

Methods of the present invention include measuring the SERRS signal. The time used for detection of the SERRS spectrum has been found not to be a critical factor in ensuring low variability. Accordingly, the present invention envisages embodiments wherein the SERRS signal is measured for a time period between 0.1 and 300 seconds. According to particular embodiments, SERRS signals are measured for a time period of 100 seconds. According to other particular embodiments, SERRS signals are measured for a time period of between 0.1 and 10 seconds.

Other arrangements of the systems and methods embodying the invention will be obvious for those skilled in the art.

It is to be understood that although preferred embodiments, specific constructions and configurations, as well as materials, have been discussed herein for devices according to the present invention, various changes or modifications in form and detail may be made without departing from the scope and spirit of this invention.

Example 1 : sample preparation and measuring conditions

A sample for SERRS measurements was prepared as follows: To 20 μl of the dye-labelled probe, 20 μl of spermine was added followed by 55 μl of colloids. The sample was mixed and after 4 minutes the SERRS spectrum was measured using an excitation wavelength of 532 nm (Raman Systems Inc) or 514 nm (Jobin Yvon). Typical integration times are between 0.1 -10s. Colloidal silver particles with a particle size of 25 to 40 nm were either citrate- reduced or EDTA -reduced silver nanoparticles.

EDTA reduced silver nanoparticles were synthesized according to Heard et al. (Jounal of Colloid and Interface Science, VoI 93 (1983) 535), and the citrate-reduced silver nanoparticles were prepared following a modified Lee-Meisel protocol (Munro et al. Langmuir, VoI 11 (1995) 3712).

The colloids were diluted in a 10 mM Tris-HCl buffer, containing 0.01% Tween 20 with a pH of 7.5. For the EDTA colloids the dilution before sample preparation was 40% colloids, 60% buffer (v/v). The final concentration of EDTA colloids in the measurement was 23%. For citrate-reduced silver particles the colloidal suspension was diluted 10 times in Tris-Tween buffer (before sample preparation). For the synthesis of 500 ml colloids, 22.1 mg and 90 mg of AgNθ3 was used for EDTA colloids (Heard et al.) and citrate colloids (Munro et al.), respectively.

Spermine tetrahydrochloride

The nanoparticles were aggregated using spermine tetrahydrochloride. The final concentration in the measurements was 0.1 mM when EDTA colloids were used. In case of citrate-reduced silver nanoparticles the final concentration was 0.48 mM. Spermine tetrahydrochloride was dissolved in 10 mM Tris-HCl buffer, containing 0.01% Tween 20 with a pH of 7.5.

Example 2 detection limit

The detection limit of the dyes was further investigated upon excitation with 532 nm and 514 nm, respectively. The results of example Atto 532 are provided in Table 2 Table 2: Detection limit (pM) of unmodified oligonucleotides labelled with fluorescent dyes in SERRS upon excitation with 532 nm and 514 nm, respectively

## Limit of Detection = 3 x RMSECV [pM] determined from partial least squares regression analysis, with 2 latent variables. As indicated in the table the limit of detection was defined as LoD = 3 x RMSECV [pM] determined from partial least squares regression analysis, with 2 latent variables In the prior art LoD is determined from a signal at a concentration which is 3 times larger than the background noise. Using this type of calculation, the obtained LoD values are in the femtomolar range.

Example 3: Use of negatively charged label in SERRS (figure 2)

An oligonucleotide consisting of 25 unmodified nucleotides (i.e. with neutral nucleosides), was directly labelled with the negatively charged FAM dye (absorption maximum at 494 nm). The FAM labelled oligonucleotide was assayed as SERRS label. 250 pM SERRS probe was mixed with citrate reduced Ag colloids (prepared from a modified Lee-Meisel procedure, Munro et al. (1995) Langmuir 11, 3712-3720 ) and aggregated using spermine. Using a Raman Systems 3000HR spectrometer with an excitation wavelength of 532 nm, a strong SERRS signal was obtained (Figure 2). Similar experiments were performed with the following labels; Atto-488, Atto-532, Atto-565, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 546, HEX, JOE, Oregon Green 500, TET, Yakima Yellow. Corresponding signals are shown in figure 2 (background levels are shifted with 350 counts/sec).

This demonstrates that a negatively charged label can be used directly for SERRS detection, without modification of the probe to compensate for the charge of the label.

Example 4 : Multiplexing using negative SERRS labels (figure 3)

Selection of SERRS spectra (background corrected) for 6 negatively charged dye-labeled DNA probes (-30 DNA bases) also used in a multiplex experiment. No modification was added to enhance surface adsorption on negatively charged colloidal surface. Final concentration of the probes: 100 pM.

Each of the labels was linked to an oligonucleotide, with no modifications apart from the reaction with the dye (IBA GmbH, Gδttingen, Germany) with a length of 25- 30 basepairs. SERRS measurements were performed using citrate-reduced silver colloids. Spermine was used as an aggregating agent. Results are shown in figure 3. Example 5 : multiplexing negative SERRS dyes with coventional dyes Figure 4A:

SERRS spectra of 9 labels used in a multiplexing experiment. Spectra are background corrected, normalized to acquisition times (counts/sec) and offset for visual clarity. The labels are indicated at the right, 6 negatively charged labels were used and are indicated in italic bold. Sample preparation: Different mixtures with at most 3 labels present were prepared from solutions where each label had a final concentration adjusted to give lOOOcounts /sec. No modification was added to enhance surface adsorption on negatively charged colloidal surface. To 20 μl of the mixture, 20 μl of spermine was added followed by 55 μl of colloids. The sample was mixed and after 4 minutes the SERRS spectrum was measured. Experimental conditions: 532nm, 1 ImW excitation laser, integration times between 1 and 15sec.

Figure 4B:

Example for 6 background corrected spectra from a multiplexing experiment. The composition of the samples is indicated at the right. Negatively charged labels are indicated in italic -bold.

This shows that the negatively charged labels give SERRS without the use of modification added to enhance surface adsorption on negatively charged colloidal surface, and that SERRS multiplexing using the negative label gives good results.

Claims

CLAIMS:

1. Use of an oligonucleotide labelled with a label suitable for SERRS in SERRS detection, characterised in that: the oligonucleotide consists of bases with unmodified neutral nucleosides, and the label has a negative charge.

2. The use according to claim 1, wherein the label is a fluorescent label.

3. The use according to claim 2, wherein the fluorescent label has an absorption maximum between 490 and 544 nm.

4. The use according to claim 2, wherein the fluorescent label is selected from the group consisting of FAM , Alexa Fluor 488, Dichloro fluorescein, Alexa Fluor 514, TET, JOE, Eosin, Yakima, Yellow, Tetrabromosulfonefluorescein, Erythrosin, Atto 532 , HEX , Alexa Fluor 546 , Resorfurin, Atto-488, Atto-565 and Oregon Green 500.

5. A method for detecting a nucleic acid target in a sample by surface-enhanced resonance Raman scattering (SERRS), the method comprising the steps of:

(a) Contacting the sample with an oligonucleotide with a label suitable for SERRS detection, wherein: the oligonucleotide consists of bases with unmodified neutral nucleosides, and the label has a negative charge,

(b) Contacting said probe with a metal surface in the presence of an aggregating agent, and

(c) Detecting the SERRS signal generated by said label on said oligonucleotide.

6. The method according to claim 5, wherein the label is a fluorescent label.

7. The method according to claim 5 or 6, wherein the oligonucleotide is a target- specific oligonucleotide.

8. The method according to claim 5, which is a method for detecting multiple targets in a sample, wherein at least two different oligonucleotides each labelled with a different label are contacted with said sample and said detection step comprises detecting the SERRS signal generated by each of said different labels, wherein at least one of said oligonucleotides is characterised in that: the oligonucleotide consists of bases with unmodified neutral nucleosides, and the label has a negative charge.

9. The method of claim 8, which comprises detecting the SERRS signal of said different labels at one wavelength.

10. The method of claim 8 or 9, which comprises using at least 5 different fluorescent labels.

11. The method of any one of claims 5 to 10, wherein the labels are selected from the group comprising FAM , Alexa Fluor 488, Dichlorofluorescein, Alexa Fluor 514, TET, JOE, Eosin, Yakima, Yellow, Tetrabromosulfonefluorescein, Erythrosin, Atto 532 , HEX , Alexa Fluor 546 , Resorfurin, Atto-488, Atto-565 and Oregon Green 500.

12. The method of any one of claims 5 to 11, wherein the aggregating agent comprises a polyamine.