WO2010046829A1 - Separation free ser(r)s assay - Google Patents

Abstract

The present invention provides a method for detecting an analyte in a sample comprising the steps of contacting said sample with at least one target specific capture probe comprising an oligonucleotide capable of specifically binding a target sequencing within said analyte, and a label that has surface-enhanced (resonance) scattering (SE(R)S)) activity, enzymatically digesting single stranded non specifically bound capture probes and detecting SER(R)S signal from double stranded capture probe-target sequence complex in the presence of a SER(R)S-active substrate. The invention further relates to a kit and a cartridge for detecting an analyte.

Description

Separation free SER(R)S assay

FIELD OF THE INVENTION

The present invention relates to a method for detecting an analyte, particularly a nucleic acid in a sample. The invention further relates to a kit and a cartridge for detecting an analyte.

BACKGROUND OF THE INVENTION

Bioassays, i.e. assays for the specific detection of bio molecules such as DNA, RNA or proteins, are usually based on specific binding of a marker molecule potentially including a label (probe), to the target molecule. Typically, the marker molecules are added to the target molecules in vast excess to shorten the assay time and additionally to increase the sensitivity of the assay.

Surface-enhanced (resonant) Raman spectroscopy (SE(R)RS) is a highly sensitive technique that is especially well suited for the simultaneous detection of multiple biomolecules. The technique allows Raman scattering to be enhanced by several orders of magnitude by making use of a rough metal surface. In SER(R)S 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 can be measured. SER(R)S has the unique feature that the scattered light consists of sharp, molecule-specific vibrational bands which makes discrimination of multiple analytes possible. Target detection by SE(R)RS spectroscopy has been carried out with solid substrate metal surfaces. Adsorption of target analytes on the surface can be slow due to the process of diffusion. From experiments with aggregated silver colloids, detection of dye labelled DNA molecules was reported at sub-nanomolar concentrations.

Various publications can be found on DNA detection by SE(R)RS spectroscopy carried out with solid substrate metal surfaces. An example is provided by Isola et al. (Isola N et al, Anal Chem 1998, 70, 1352-1356). SUMMARY OF THE INVENTION

It is an object of the present invention to provide an improved method for detecting an analyte in a sample. This object of the present invention is realized with a method for detecting an analyte in a sample comprising the steps of: Providing a sample

Contacting said sample with at least one target specific capture probe comprising an oligonucleotide capable of specifically binding a target sequence within said analyte, and a label that has surface-enhanced (resonance) scattering (SE(R)S) activity

Enzymatically digesting single stranded non specifically bound capture probes Detecting SER(R)S signal from double stranded capture probe-target sequence complex in the presence of a SERS-active substrate.

Bioassays where at least one of the reagents is bound to a fixed substrate suffer from unfavourable reaction conditions as a result of slow kinetics of target molecules reaching the surface in order to bind to the probe. In this respect, homogeneous bioassays, including assays in solution, are interesting for diagnostic purposes because of their superior kinetics, resulting in an increased sensitivity and decreased assay time. Homogeneous assays, however, also have specific challenges in terms of assay complexity as simple washing steps will not remove the excess of unbound probe.

By removing unbound probes via enzymes that specifically digest single stranded sequences this process is facilitated. The method according to the invention is separation free, meaning no additional steps are required in the assay to separate bound from unbound probes. Furthermore, no washing steps are required, so the reaction can be performed in a single tube, including the SE(R)S detection.

The SE(R)RS signal is measured in the presence of a SER(R)S-active substrate, preferably comprising metal particles, in particular silver nanoparticles.

In a preferred embodiment, the digestion enzyme is selected from the group comprising RNase I, Exonuclease I, Exonuclease T and RecJ. RNAse I is suitable for digesting single stranded RNA probes, Exonuclease I and T and RecJ are suitable for digesting DNA probes. Preferably, the analyte is amplified prior to detection. This increases the concentration of the target molecules and provides a higher signal.

In yet another embodiment, multiple analytes are detected simultaneously by using multiple specific capture probes. Indeed, one of the advantages of SE(R)RS is that it allows the simultaneous detection of different labels, allowing different targets to be detected simultaneously, often referred to as "multiplexing". In this regard it will be understood by the skilled person that the methods of the present invention can be applied to the detection of different target nucleic acids within one sample, whereby each of the target nucleic acids is detected with multiple probes with the same and/or different optical properties. It is preferred that the probes are in solution. Furthermore it is preferable to detect the SE(R)RS signal as end-point measurement.

In another embodiment of the present invention, an aggregating agent is added to the colloidal SER(R)S-active particles prior to measuring the SE(R)RS signal, preferably a polyamine, in particular spermine. The use of the above indicated additions leads to improved conditions for

SER(R)S measurements.

The object of the invention is further realized by a cartridge for use in a system for detecting an analyte in a sample, comprising a site for reacting the analyte in the sample with a specific capture probe comprising an oligonucleotide capable of specifically binding a target sequence within said analyte, and a label that has surface-enhanced (resonance) scattering (SE(R)S)) activity, and at least one different site where an enzyme capable of digesting unbound probes is stored.

Additionally, the object of the invention is realized by a kit comprising holders with - capture probe comprising an oligonucleotide capable of specifically binding a target sequence within said analyte, and a label that has surface-enhanced (resonance) scattering (SE(R)S)) activity enzyme capable of digesting the specific capture probe in single stranded form Preferably, the digestion enzyme is selected from the group comprising RNase I, Exonuclease I, Exonuclease T and RecJ.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 : Schematic representation of enzymatic degradation of unbound probes in a SER(R)S assay Fig. 2: "Bare" dye label gives only a low SER(R)S signal

Fig. 3: Separation- free SER(R)S assay employing digestion of RNA probes by RNase If

Fig. 4: Exonuclease can be used to digest probes in PCR buffer and therefore remove unbound probes after hybridization Fig. 5: Exonuclease can be used to remove unbound probes after hybridization

DETAILED DESCRIPTION OF THE INVENTION

The following terms or definitions are provided solely to aid in the understanding of the invention. Unless specifically indicated, these definitions should not be construed to have a scope less than understood by a person of ordinary skill in the art. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. Finally it is pointed out that in the present application the term "comprising" does not exclude other elements or steps, that "a" or "an" does not exclude a plurality, and that a single processor or other unit may fulfill the functions of several means. The invention resides in each and every novel characteristic feature and each and every combination of characteristic features. Moreover, reference signs in the claims shall not be construed as limiting their scope. The term "analyte", as used herein, refers to the substance to be detected and/or quantified in the methods of the present invention.

The term "label", as used herein, refers to a molecule or material capable of generating a detectable signal. Labels which are SER(R)S reactive (i.e. which are capable of generating a SER(R)S spectrum when appropriately illuminated), are also referred to herein as SER(R) S -reactive dyes. A non-limiting list of labels envisaged for use in the methods of the present invention is provided below. Preferred labels are selected from the group comprising Rhodamine 6G, Atto 520, Atto 532, Bodipy FL, Rhodamine Green, FAM, Alexa 532 and JOE.

The term "probe" as used herein, refers to an analyte specific molecule comprising a structure or sequence which is specific for the analyte to be detected. The binding of the analyte-specific probe to the analyte can be based on any type of interaction including but not limited to complementary nucleotide sequences, antigen/antibody interaction, ligand/receptor binding, enzyme/substrate interaction, etc.

In a homogenous assay for nucleic acid detection in a sample by SER(R)S, typically dye-labelled probes, i.e. oligonucleotides which are complementary to the target sequence are added in excess. The probes hybridize to the target sequence if this sequence is present in the sample. However, to determine the presence of the target, the unbound probes need to be separated from the specifically bound probes to prevent generation of unspecific background signals. In conventional methods this can be achieved by separation/washing steps, e.g. PCR with biotinylated primers, hybridization with dye labeled probes, magnetic bead separation, which is a rather complicated and time consuming process.

Without wishing to be bound by any theory, the present invention is based on the insight that none or only a very weak SER(R)S signal is obtained from free dyes or dye- labelled short oligonucleotides (1-5 nucleotides) when SER(R)S active substrates such as silver colloidal suspensions, aggregated with for example spermine are employed. However, if the same dye is coupled to a longer DNA or RNA molecule (ss or ds) SER(R)S signals can be detected at pico molar to femtomolar concentrations.

Fig. 1 shows a schematic overview. In Fig. IA a sample potentially including a target sequence 1 is contacted with complementary probes labelled with a dye 2. After hybridizing the probes with the target sequence, double stranded probe-target complexes 3 are formed as shown in Fig. IB. The excess of labelled probe is enzymatically digested, leaving single nucleotide fragments. 4. When determining the amount of SER(R)S signal, only the SER(R)S labels coupled to hybridized probes provide a detectable signal. The respective sizes of probe, label and target sequence are schematic and are not limiting to this specific size and size ratio.

Fig. 2 demonstrates experimental proof of the fact that free dye labels give no SER(R)S signal. The effect is shown for the SER(R)S dye RhodamineβG (R6G) in combination with EDTA-reduced silver colloids aggregated with spermine, using 532 nm excitation. The baseline level is generated by lOOpM R6G not coupled to a nucleotide probe in buffer. When the dye is coupled to a DNA probe, a profound increase in signal is observed.

In one embodiment, the separation- free SER(R)S assay is performed without prior amplification of the target DNA or RNA. In a second preferred embodiment, the target DNA or RNA is amplified prior to the assay, e.g. by PCR. Since it is not necessary to introduce dye or biotin labels during the amplification process, the invention can also be used in combination with unspecific PCR amplification (i.e. using one set of two primers to amplify multiple target sequences simultaneously), rolling circle amplification using random primers for amplification, or isothermal amplification methods that make use of signal amplification (e.g. LAMP).

In another embodiment, the separation-free SER(R)S assay is used in combination with multiplex nucleic acid detection. For this, multiple oligonucleotide probes, each targeting a different gene and each labelled with a different SER(R)S-active dye, are added simultaneously to the sample. After hybridization of the probes, unbound probes are digested by incubation with an enzyme specifically digesting the single stranded probes and a SER(R)S spectrum is measured. This SER(R)S spectrum contains only contributions from those probes that have hybridized to the sample, i.e. spectral decomposition of the SER(R)S signal reveals which genes are present in the sample. The present invention can be deployed with any nucleotide based probes including but not limited to DNA, and RNA probes.

Fig. 3 shows experimental results of the separation- free SER(R)S assay employing digestion of RNA probes by RNase If. An excess of dye labelled RNA probe is added to a sample containing the target DNA. The RNA probes hybridize with the target DNA, forming a double-stranded (ds) RNA-DNA chimer. After hybridization a specific enzyme, RNase If, is added and incubated with the sample. RNase If digests single-stranded (ss) unbound RNA probes, generating dye-labelled (single or oligonucleotides which do not generate a SER(R)S signal. In contrast, ss or ds DNA as well as RNA-DNA chimers are not affected by the enzyme, meaning that those RNA probes that have hybridized to the target remain intact and generate a SER(R)S signal.

If the target DNA is not present in the sample, all RNA probes will be digested by the enzyme, meaning that no SER(R)S signal can be detected. In contrast, if the target DNA is present in the sample, some of the RNA probes will hybridize to the target and thereby will be protected against digestion by the enzyme, resulting in a SER(R)S signal that is proportional to the amount of hybridized probes.

The high intensity SER(R)S signal in Fig. 3 is obtained from an RNA probe (29 bases, concentration: 200 pM) labelled covalently with R6G after hybridization with its perfect match followed by digestion (20 min at 37⁰C) with RNase If. The baseline SER(R)S signals are obtained from the same RNA probe after hybridization with a non-matching oligonucleotide followed by digestion with RNase If and from the same RNA probe without the addition of target molecules after digestion with RNase If. Experimental conditions are equimolar amounts of probe and target and the use of EDTA-reduced silver colloids aggregated with spermine, 532 nm excitation.

As can be observed in Fig. 3, the SER(R)S spectrum detected after hybridization of an RNA probe with a perfectly matching oligonucleotide is about eight times higher than the signal detected after "hybridization" with a non-matching oligonucleotide.

The following example is visualized in Fig. 4 and shows a reaction based on DNA probes. The contrast between non-digested probe (with heat inactivated Exo SAP, USB Europe GmbH, Staufen) and digested probe (incubated with Exo SAP) is significant. Peak height at 1500cm-l : 130c/s non-digested, 5c/s digested. Contrast 26:1. The results show that Exonuclease I can be used to remove unbound probes after hybridization, as they will be digested and give little SER(R)S, whereas bound probes will remain hybridized and double stranded and therefore undigested, and produce a strong SER(R)S signal. Experimental conditions: 12OnM R6G DNA probe (29 bases) was incubated 15min at 37°C with 5μl Exo SAP exonuclease from USB (either heat inactivated 15min at 80⁰C or not). All samples were diluted 10Ox in water before SER(R)S measurement leading to approximately 20OpM final probe concentration.

Fig. 5 shows that indeed, after probe hybridisation, treatment of the sample with exonuclease results in digestion of the unbound single stranded dye-labelled DNA probes whereas the ds probe targets are left intact (the exonuclease specifically digests single stranded DNA, leaving double stranded DNA undigested). There is a 2:1 contrast between the positive and the non-match samples so between bound and unbound probes. Due to the digestion dye-labelled nucleotides are released from the unbound probes. The Exo-SAP exonuclease actually cuts nucleotides and leaves a dinucleotide linked to the dye. The probes that have bound to the target are not digested, meaning that the dye -DNA molecules remain intact.

Excitation of free dye molecules (with a dinucleotide attached) and free nucleotides in colloidal suspensions aggregated with spermine does not give rise to a detectable SER(R)S signal at concentrations lower than one micromolar. However, if a dye is coupled to DNA (ss or ds) a good SER(R)S signal can be measured (down to pico molar to femtomolar concentrations). Therefore, the bound probes give a stronger SER(R)S signal than the unbound probes (see Fig 5). Experimental conditions: 15OnM R6G labeled probe was added to 5OnM PCR product, match and non-match. Hybridization was followed by digestion with exonuclease (15 min at 37°C). All samples were diluted 10Ox in water before SER(R)S measurement leading to approximately 20OpM final probe concentration. SER(R)S detection was performed with EDTA-reduced silver colloids aggregated with spermine, using 532 nm excitation."

The present method allows for the separation free, sensitive and accurate detection and/or quantification of one or more target nucleic acids in a sample. The term

"sample" is used in a broad sense herein 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 target nucleic acid 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, abscess swabs, nasopharyngeal swabs, nasal 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.

In particular embodiments of the invention, the sample is pre-treated to facilitate the detection of the sample with the detection method. For instance, typically a pre- treatment of the sample resulting in a semi- isolation or isolation of the target nucleic acid or ensuring the amplification of the target nucleic acid is envisaged. Many methods and kits are available for pre-treating samples of various types.

Particular embodiments of the methods provided herein relate to methods of detection based on SE(R)RS, and optionally involve operating at the resonant frequency of SER(R)S labels used, which gives increased sensitivity. The light source used to generate the Raman spectrum for this purpose 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 SERS-active surface and based on the binding to or incorporation in the amplicon, but the skilled person will be well able 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. SER(R)S detection may alternatively involve operating at, or close to, the resonant frequency of the plasmons on the active surface of the metal colloids. As stated above, the choice of the optimal operating frequency can easily be made by one of ordinary skill in the art based on the metal(s) and label(s) chosen. In SE(R)RS 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 the signal intensity at a chosen spectral line frequency or frequencies.

Typically, the detection step in the SE(R)RS based detection methods of the present invention is 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 and/or green 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, the near-infrared or 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 SE(R)RS literature.

Excitation sources for use in SE(R)RS-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. The laser power, or excitation power, can be varied depending, e.g., on the properties of the label used.

The excitation beam 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. Detection of the SE(R)RS spectrum of one or more SE(R)RS labels as envisaged in methods described herein may be done using devices known in the art, e.g. commercially available spectrophotometers. 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. 6,002,471. 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 SE(R)RS signals, including wavelength selective mirrors, holographic optical elements for scattered light detection and fibre-optic waveguides. Various options are discussed, e.g. in WO 97/05280. The apparatus used for obtaining and/or analysing a SE(R)RS spectrum as envisaged in methods according to the invention may include some form of data processor such as a computer. Once the SE(R)RS signal has been captured by an appropriate detector, its frequency and intensity data will typically be passed on 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. Therefore, the detection can be qualitative, semi-quantitative or quantitative, when the various spectra obtained as described are compared with an internal standard chosen appropriately, as is well known by a person skilled in the art. According to the methods provided herein, the measurement of the SE(R)RS spectrum of the SE(R)RS labels is an indication of the presence and/or quantity of a target in a sample. Indeed, the intensity and the nature of the SE(R)RS spectrum detected can be correlated to the presence of a target molecule in the sample. According to particular embodiments, the methods comprise an additional step, wherein the SE(R)RS spectrum or the intensity of the SE(R)RS spectrum is correlated to the presence of the target molecule. When using quantitative correlation, a calibration curve can be made first, to assess the nature of the correlation between SE(R)RS signal and amount of target molecule. EXAMPLES

Example 1 The following non limiting table 1 shows SER(R)S dyes that are preferred for use according to the invention.

Example 2

Protocol for SER(R)S measurement

Mix probe and PCR product: 1 μl of R6G labeled DNA probe at a concentration of 3μM is added to 20 μl of 5OnM PCR product in Kapa master mix. The probe is then hybridized to the PCR product with a fast hybridization protocol: the sample is placed for 2 minutes at 95°C, followed by 2 minutes on ice. The exonuclease is then added to digest the non-hybridized probes, as it specifically digests single-stranded DNA. 5μl of ExoSAP from USB is added to the probe-PCR reaction and digestion is carried out for 15 minutes at 37°C. The sample is then diluted 100 times in water before SERRS measurement. SERRS detection is performed with EDTA-reduced silver colloids aggregated with spermine, 532 nm excitation.

Claims

CLAIMS:

1. Method for detecting an analyte in a sample comprising the steps of:

Providing a sample

Contacting said sample with at least one target specific capture probe comprising an oligonucleotide capable of specifically binding a target sequence within said analyte, and a label that has surface-enhanced (resonance) scattering (SE(R)S)) activity

Enzymatically digesting single stranded non specifically bound capture probes

Detecting SER(R)S signal from double stranded capture probe-target sequence complex in the presence of a SER(R)S-active substrate.

2. The method according to claim 1 wherein the digestion enzyme is selected from the group comprising RNase I, Exonuclease I, Exonuclease T and RecJ.

3. A method according to claim 1 and 2 wherein the analyte is amplified prior to detection.

4. A method according to any one of claims 1 to 3, wherein multiple analytes are detected simultaneously by using multiple specific capture probes.

5. A method according to any one of claims 1 to 4, wherein the probes are in solution.

6. The method according to any one of claims 1 to 5, wherein the SE(R)RS signal is detected as end-point measurement.

7. The method according to claim 5 or 6, wherein an aggregating agent is added to the SER(R)S active substrate prior to measuring the SE(R)RS signal.

8. The method according to claim 7, wherein the aggregating agent is a polyamine.

9. A cartridge for use in a system for detecting an analyte in a sample, comprising a site for reacting the analyte in the sample with a specific capture probe comprising an oligonucleotide capable of specifically binding a target sequence within said analyte, and a label that has surface-enhanced (resonance) scattering (SE(R)S)) activity, and at least one different site where an enzyme capable of digesting unbound probes is stored.

10. A cartridge according to claim 9, wherein the digestion enzyme is selected from the group comprising RNase I, Exonuclease I, Exonuclease T and RecJ.

11. A kit comprising holders comprising capture probe comprising an oligonucleotide capable of specifically binding a target sequence within said analyte, and a label that has surface-enhanced (resonance) scattering (SE(R)S)) activity enzyme capable of digesting the specific capture probe in single stranded form

12. A kit according to claim 11 wherein the digestion enzyme is selected from the group comprising RNase I, Exonuclease I, Exonuclease T and RecJ.

13. A kit according to claim 11 or 12 with additional holders comprising an aggregating agent

SER(R)S active substrate