WO2010133849A1

WO2010133849A1 - Detection of target analyte by signal amplification

Info

Publication number: WO2010133849A1
Application number: PCT/GB2010/001017
Authority: WO
Inventors: Karen Faulds; Duncyn Graham; Jim Reid
Original assignee: Renishaw Diagnostics Limited
Priority date: 2009-05-20
Filing date: 2010-05-20
Publication date: 2010-11-25
Also published as: GB0908633D0

Abstract

The present invention relates to a method of detecting a target analyte, such as a nucleic acid by way of binding to a reagent and detection of the target/reagent complex using an amplified SE(R)RS signal.

Description

DETECTION OF TARGET ANALYTE BY SIGNAL AMPLIFICATION

Field of the Invention

Background to the Invention

Many different types of methods for detecting analytes, such as nucleic acids are described in the literature. Some of these are based on the generation of nucleic acid duplexes through hybridisation of a probe nucleic acid to a target nucleic acid and subsequent detection of the duplex. These techniques can be divided into two broad categories : direct detection using techniques such as Southern blotting for DNA detection and Northern blotting for RNA detection; and indirect techniques such as sandwich hybridisation techniques.

Nevertheless, sensitivity of hybridisation techniques can be dependent on a number of factors and in particular the amount of target in a sample being tested. In order to increase sensitivity, hybridisation techniques often employ an amplification step. This amplification can be of the target itself or of the signal generated through hybridisation.

The present invention is generally concerned with a sandwich type hybridisation technique, employing a signal amplification step. Such a technique has been described previously, see for example US4,868,105 to Urdea, et al.

The assays described in US4,868,105 involve the use of a two-part capturing system designed to bind a target nucleic acid to a solid support, and a two-part labelling system designed to bind a detectable label to the target nucleic and to be detected or quantitated. The two-part capture system involves the use of capture probes bound to a solid support and capture extender molecules that hybridize both to a segment of the capture probes and to a segment of the target nucleic acid. The two-part labelling system involves the use of label extender molecules that hybridise to a segment of the polynucleotide analyte, and labelled probes that hybridise to the label extender molecules and contain or bind to a detectable label. The disclosure of this patent is hereby incorporated by way of reference.

Alkaline phosphatase-oligonucleotide conjugates may be used as the signal- generating component of such hybridisation assays. Adding an appropriate substrate, e.g., an enzyme-triggered dioxetane phosphate (Schapp et al. (1987) Tet. Lett. 28: 1159-1162 and EP 0254051 yields a detectable chemiluminescent signal. However, the background noise level may not be ideal in such assays due, in part, to the heterogenous population of alkaline phosphatase molecules available for conjugation, which contributes to non-specific binding of labelled probes. Low signal- to-noise ratios may also result from the preparation of alkaline phosphatase-labelled probes by conjugation of oligonucleotides to the enzyme under conditions that permit conjugation to reactive sites in or near the active site of the enzyme, thereby reducing the alkaline phosphatase specific enzyme activity.

Unwanted background noise may result from the use of alkaline phosphatase-oligonucleotide conjugate prepared under conditions where conjugates are formed at various sites on the enzyme, including at the enzyme active site. This source of heterogeneity in the enzyme-probe conjugate population results in the label probe with less than ideal specific enzyme activity.

The lack of a homogeneous population of detectably labelled oligonucleotide probes having high specific activity of detection may limit the sensitivity and the precision of typical nucleic acid hybridisation assays.

Another problem associated with sandwich hybridisation assays involves the removal of labelled probes which do not hybridise to the target.

It is an object of the present invention to obviate and/or mitigate at least one of the aforementioned disadvantages. Summary of the Invention

In a first aspect there is provided a method for the detection of a target analyte, comprising: a) contacting a sample potentially comprising the target analyte with a reagent capable of specifically binding to any target analyte present in the sample, under conditions whereby binding of the reagent and the target analyte occurs when the target analyte is present; b) and a plurality of signal generating probes, each signal generating probe comprising one or more signal generating means; and c) detecting an amplified signal from said plurality of signal generating probes obtainable when the reagent is complexed with the target analyte, wherein the amplified signal is detected by way of SE(R)RS.

It is to be appreciated that the signal generating probes may be a component of the reagent and linked, bound, complexed or otherwise associated with the reagent. Alternatively, the plurality of signal generating probes may be capable of binding to the target/reagent complex in some manner. The signal generating probes, may for example, be capable of binding the reagent, or may be capable of binding the complex formed between the target and the reagent.

Many target and reagent combinations can be envisaged, but the invention finds general application in relation to biological complexes such as protein/protein complexes, protein/antibody complexes, protein/nucleic acid complexes, nucleic acid/nucleic acid complexes and the like. The rest of the specification focuses on nucleic acid/nucleic acid complexes, but this should not be construed as limiting.

The plurality of signal generating probes may be provided by way of a branched molecule wherein the signal generating probes may be present on a plurality of said branches. Example molecules include star-dendrimer like molecules (see for example "The Concise encyclopedia of the structure of materials ed J.W. Martin, 2007 published by Elsevier - which is incorporated herein by reference), oligonucleotide - peptide conjugates, where multiple oligonucleotides may be bound to a peptide backbone using conventional techniques (for an overview see Stetsenko et al. Molecular Biology, 2000, 34, 6, 852-859), or vice versa, or oligonucleotide/oligonucleotide or peptide/peptide complexes, where multiple oligonucleotides or peptides are capable of binding to a single oligonucleotide or peptide respectively, in order to generate a branched molecule.

The rest of the specification describes in detail, the use of branched nucleic acid molecules, but this should not be construed as limiting.

It is to be understood that the signal generating means may be a molecule which itself is capable of generating a SE(R)RS signal, or a molecule which upon action, for example by another molecule, such as an enzyme, is capable of generating a SE(R)RS signal. Alternatively, the signal generating means may be in the form of said another molecule, such as an enzyme, which is capable of reacting with a substrate, so as to generate a molecule which is able to generate a SE(R)RS active signal.

In the further aspect the present invention provides a method for the detection of a target nucleic acid sequence, comprising: a) contacting a sample nucleic acid with a probe nucleic acid which probe comprises a polynucleotide sequence which is capable of hybridising to said target nucleic acid sequence, and a polynucleotide sequence which is substantially incapable of binding to said target nucleic acid sequence, under conditions whereby hybridisation of the probe to the sample nucleic acid occurs when the target nucleic acid is present; b) and a plurality of signal generating probes, each signal generating probe comprising one or more signal generating means and a nucleic acid sequence which is capable of directly or indirectly hybridising to a portion of the polynucleotide sequence of the probe nucleic acid which is substantially incapable of binding to said target nucleic acid sequence; and c) detecting an amplified signal obtainable when the probe nucleic acid is bound to the target nucleic acid and said plurality of signal generating probes, wherein the amplified signal is detected by way of SE(R)RS.

The invention provides a technique based on the use of "surface enhanced Raman scattering" (SERS) and on a modification of that principle known as SERRS (surface enhanced resonance Raman scattering). These principles are already known and well documented, and have been used before in the detection and analysis of various target materials, see for example WO97/005280 and WO01/77650). For avoidance of doubt, SE(R)RS, as used herein, refers to either or both SERS and SERRS.

Thus, by SE(R)RS is meant either surface enhanced Raman scattering or surface enhanced resonance Raman scattering. The methods of the invention may involve either form of spectroscopy, since the essential principle (the association for a Raman-active label with a Raman-active surface) is the same in each case. Preferably, the methods of the invention involve SERRS rather than SERS, since operating at the resonant frequency of the label gives increased sensitivity, in this case, the light source used.

To generate the Raman spectrum a coherent light source (e.g. a laser) tuned substantially to the maximum absorption frequency of the signal moiety being is used. (Note that this frequency may shift slightly on the association of the signal moiety with the SE(R)RS-active surface and the signal moiety and/or signal generating means, but the skilled person will be well able to tune the light source to accommodate this. Note too that the light source may be tuned to a frequency near to the signal's absorption maximum, or to a frequency at or near that of a secondary peak in the label's absorption spectrum).

By "signal moiety" is meant a molecule which is capable, of generating a detectable SE(R)RS signal when bound to a SE(R)RS active surface. A signal generating means refers to a molecule or reagent which does not substantially generate a SE(R)RS signal when bound to a SE(R)RS active surface, but upon modification, for example by action by an enzyme binding to an antibody or antibody fragment, or upon chemical derivatisation to change its structure, by way of covalent or ionic modification, results in the generation of a molecule which is capable of generating a SE(R)RS signal when bound to a SE(R)RS active surface. Hereinafter the term signal moiety may be referred to a label and can be used interchangeably.

SERRS may alternatively involve operating at the resonant frequency of the plasmons on the active surface, although in the methods of the invention it is believed to be preferable to tune to the resonant frequency of the signal moiety.

The methods of the invention may be used for the quantitative or qualitative detection of target nucleic acids. They may form part of an overall method for determining the sequence of a nucleic acid, by detecting the presence in it of several selected target nucleotides or nucleotide sequences.

The probe nucleic acid may be a single molecule which is capable of binding directly to the target nucleic acid or may take the form of a label extender probe which is capable of binding to the target nucleic acid via a first portion and trunk nucleic acid which is capable of hybridising to the label extender probe via a second portion. The trunk nucleic acid therefore comprises a first region of sequence which is capable of hybridising to the label extender probe and a second region of sequence which comprises a plurality of substantially identical sequences which are spaced apart along the second region of sequence and which are capable of hybridising to said signal generating probes. This concept is well known in the art and is described, for example, in US4,868,1055 and US4,882,269, the contents of which are hereby incorporated by way of reference.

The target nucleic acid may be a naturally occurring DNA, RNA, mRNA, rRNA of cDNA, or a synthetic DNA, RNA, PNA or other nucleic acid analogue. Typically, it will be a naturally occurring DNA or RNA. It may be an oligonucleotide or a polynucleotide. In this document, unless the context requires otherwise, the term "nucleotide" is used to refer to either a deoxyribo- or a ribo-nucleotide or an analogue thereof; "oligonucleotide" to a nucleotide sequence of between 2 and 50 base units; and "polynucleotide" to a nucleotide sequence of 50 base units or more.

A target oligonucleotide or polynucleotide may be substantially single- or double-stranded.

The target may be a nucleic acid "unit", by which is meant a nucleotide or nucleoside or a modified nucleotide or nucleoside or a nucleotide or nucleoside analogue, or an individual nucleobase. The choice of target will depend on the purpose for which the detection method is ultimately to be used - e.g., for detecting the presence of bacteria or viruses in cells, it is likely that genomic DNA or RNA would be the most suitable target to detect.

The target nucleic acid to be detected will generally be provided in a sample. The sample may be any suitable preparation in which the target is likely to be found. In the case of medical diagnostic techniques, for instance, the sample may comprise blood (including plasma and platelet fractions), spinal fluid, mucus, sputum, semen, stool or urine. Particularly suitable samples include, e.g. 20-1000 μl of blood or 1-10 ml of mouthwash. Samples may also comprise foodstuffs and beverages, water suspected of contamination, etc. The sample may also include cells from in vitro cell culture and/or cells from tissue biopsies, for example. These lists are clearly not exhaustive.

The sample may typically be pre-treated to isolate the target and make it suitable for subsequent SE(R)RS analysis. Many methods and kits are available for pre-treating samples of various types.

In accordance with the present invention, the target nucleic acid may be captured on a surface, which surface may be a SE(R)RS active surface. The nucleic acid may be captured on the surface using conventional means known to the skilled addressee. For example, the surface may be modified to have oligonucleotide probes (e.g. capture probes) bound to the surface and the nucleic acid may be directly captured by hybridisation to these probes or by way of intermediary oligonucleoties (e.g. capture extender probes) which are capable of binding to both the surface bound capture probes and the target nucleic acid molecule to be detected.

The capturing of target nucleic acids to a surface is well known to the skilled addressee and has been described previously, as well as in detail herein, using capture probes and optionally capture extender probes. However, in one embodiment of the present invention, the target nucleic acid is adhered directly to a SE(R)RS active substrate, such as klarite® - see for example USl 1/267619. This has the advantage of simplifying the assay, as less hybridisation reactions are required and a SE(R)RS signal can be generated, once the signal generating probes are hybridised directly or indirectly to the probe nucleic acid, by virtue of being in close enough proximity to the SE(R)RS active substrate to which the target nucleic acid is adhered.

It will be appreciated that the steps may not be performed in the order listed, although it is preferred if the steps are performed in the order listed. In particular, it will be appreciated that the order in which the various nucleic acids and probes bind is not critical to the operation of the invention. It is preferred that the target nucleic acid binds to the label extender probe and capture extender probe before binding to the capture probe (i.e. that a "preincubation" step is performed) when such probes are utilised. This may optimise the signal and minimise the background.

The hybridisation assay can be performed in any possible order and combination, depending on the specific assay target (ss or ds; RNA, RNA) and whether sensitivity or specificity is most important in a particular assay.

The solution in which assay steps are performed is generally an aqueous solution. It may be any suitable buffer solution, for instance PBS pH 7.2 or similar. By "complementary sequence" is meant a sequence that is able to hybridise with a nucleic acid sequence under conditions used in the assay. By "hybridise" is meant that the sequences are able to form a stable structure together, which can be detected by hybridisation assays well known in the art. These conditions may be such that hybridisation will occur only if the sequences are at least 60% inversely identical, more preferable at least 80% inversely identical, still more preferably at least 90% inversely identical and most preferably at least 95% inversely identical. By "% inversely identical" is meant the percentage of bases (in the region being considered) that are theoretically able to form base pairs when single stranded sequences are aligned with one strand 5'-3' and the other 3'-5'. The hybridisation steps may be carried out at between 0°C and 90⁰C in the buffer described above; preferably they are carried out at between 35⁰C - 6O⁰C.

It will be appreciated that the polynucleotide may be detected at extremely low concentration. By this is meant that less than 10⁴ copies of a target polynucleotide can be detected, preferably less than 10³ copies, still more preferably 10² copies or less. Sample volumes may be between 10 nl and 10 ml.

It may not be essential to remove the sample and unbound label prior to capturing the specific target related signal.

If the target nucleic acid is initially multi-stranded, such as double stranded nucleic acid or triple helix, or includes a region which self-anneals, e.g. a hair-pin loop it should be denatured prior to detection. Denaturation may be carried out by any suitable means, such as heat or alteration of ionic strength or pH. It will be appreciated that denaturation may be performed after combining the sample containing the target polynucleotide with the assay reagents. Preferably it is performed after such combination. This has the advantage that the opportunities for renaturation of the target polynucleotide before contact with the assay reagents are reduced. Denaturation may conveniently be performed by heating to 95°C for 5 minutes. Many different label extender probes may be used in a single assay.

Each region of probe designed to hybridise to another nucleic acid sequence will generally be between 5 and 5000 nucleotides, preferably between 10 and 500 nucleotides, still more preferably between 15 and 50 nucleotides in length.

The sequences and number of capture extender probes which are complementary to the target polynucleotide may be selected on the basis of particular criteria. For example, if it is desired to quantify the amount of polynucleotide with a particular mutation than sequences may be chosen that only bind to polynucleotide molecules with that particular mutation under the conditions of the assay.

The various extenders probe may be conveniently prepared by known methods of oligonucleotide synthesis or by cloning and may be modified as appropriate for labelling.

A capture probe may be a polynucleotide of between 10 and 100 nucleotides in length, preferably between 15 and 50 nucleotides, more preferably between 18 and 30 nucleotides in length. The capture probe may have a "universal" sequence, such that it may be used in the detection of several different target polynucleotides.

The capture probe may be anchored on the substrate by suitable means. For example, photochemical cross-linking may be used, as described in WO 94/27137.

In a general sense the capture probe can be immobilised by methods including hydrophobic adsorption or covalent bonding directly to the substrate or after chemical modification of the surface, for example by silanisation or the application of a polymer layer. In order to promote the immobilisation of the capture probe directly on the substrate, a thin intermediate layer, for example consisting of SiO₂ can be applied as an adhesion-promoting layer.

Covalent or ionic attachment is generally preferred, but other receptor/ligand pairs, such as avidin/biotin, aptamer, antibody-haptens or recognition systems like (His)₆-tagged oligonucleotide to NTA (nitrilotriacetic acid) are also possible. Capture extender probes each comprise two single-stranded nucleic acid regions, the first nucleic acid region having a sequence between about 10 and 100 nt long which is complementary to a sequence of the target polynucleotide and the second region not being complementary to a sequence of the target polynucleotide and being less than about 500 nt long and further comprising a capture probe recognition sequence.

It will be appreciated that more than one type of target nucleic acid can be detected at once in a sample. Different specificity capture extender probes and label extender probes can be mixed, and the different target nucleic acid detected and quantified separately if labels are used that have different SE(R)RS signals and can therefore be distinguished using suitable means.

It will be appreciated that the ratio of target to capture extender will require optimisation for a particular target nucleic acid and concentration range. This may be done by altering the ratio of capture extender probe to target nucleic acid whilst using a constant target to label extender ratio. The optimisation process will lead to determining the best results in respect to the ratio of specific to non-specific binding (i.e. binding in the absence of the target polynucleotide).

The ratio of target to label extender may similarly be optimised by applying a constant target to capture extender ratio to find the optimum ratio of target to label extender.

It will be appreciated that the weight ratios, or concentrations, of ingredients, for example label extender (LE), capture extender (CE) and label probe (LP) are essentially assay specific (i.e. may depend on the nature of the sample) and target specific and must be optimised for each target.

Many SE(R)RS-active labels are already known and referred to in SE(R)RS literature. They include species containing chromophores and/or fluorophores which can be detected relatively easily using SE(R)RS. Examples of suitable SE(R)RS-active species include fluorescein dyes, such as 5- (and 6-) carboxy-4', 5'-dichloro-2', 7'-dimethoxy fluorescein, 5-carboxy-2', 4', 5', 7-tetrachlorofluorescein and 5- carboxylfluorescein, -rhodamine dyes' such as 5- (and 6-) carboxy rhodamine, 6-carboxytetramethyl rhodamine and 6- carboxyrhodamine X; phthalocyanines such as methyl, nitrosyl, sulphonyl and amino phthalocyanines; azo dyes such as those listed in C H Munro et al, Analyst (1995), 120, p993; azomethines; cyanines and xanthines such as the methyl, nitro, sulphano and amino derivatives; and succinylfluoresceins. Each of these may be substituted in any conventional manner, giving rise to a large number of useful labels. The choice of label in any given case will depend on factors such as the resonance frequency of the label, the other species present, label availability, etc.

Other examples of non-resonant labels for use in SE(R)RS include 4,4'- azobis(pyridine), 4-mercaptobenzoic acid, 1 ,2-bis(4-pyridyl)-ethane (BPE), 2- quinolinethiol, 4-mercaptophenol (MP), 4,4'-bipyridyl (BP) and trans-1 ,2-bis(4- pyridyl)ethylene (BPET).

Most preferred are labels containing a chemi-absorptive functional group, described for example in WO97/005280.

Preferred SE(R)RS-active labels are, moreover, those which possess appropriate functional groups to allow their easy attachment to nucleic acid or to a SE(R)RS-active surface. The label should clearly not contain groups likely to interfere with the target or the target binding probe.

The signal moiety or signal generating means is preferably attached to the 5' end of the signal generating probes, although to the 3" end or to an intermediate position (e.g. to a base or to a backbone sugar group) is also possible.

Two methods by way a SE(R)RS-active label may be attached to a nucleic acid include, for instance:

1. The nucleic acid is synthesised with a nucleophilic primary amino group, usually at the 5'-terminus. After deprotection it is reacted with an appropriate reactive site (e.g. an active ester site) on the label. Purification, usually by chromatography, yields the desired product. (See e.g. J. Goodchild, Bioconjugate Chem. (1990), 1 , pp165-187).

2. The label is synthesised with a chemical group (usually an alcohol) capable of undergoing phosphorous functionalisation. The active phosphorous compound is then reacted with the nucleic acid or nucleic acid unit. This reaction can be accomplished using several types of standard chemistry, as detailed for example in M J Gait, Oligonucleotide Synthesis : A Practical Approach (1984), IRL Press Oxford.

Further examples of suitable attachments, including via linking groups, appear in P Theisen et al., - Tetrahedron Letters (1992), 33, No. 35, p5033-4036; J M Prober et al., Science (1987), 233, pp336-341; D B Shealy et al., Anal. Chem. (1995), 67, pp347-251 ; and C Mackellar et al., Nuc. Acids Res. (1992), 20, pp3411- 3417.

Still further examples of ways in which a SE(R)RS-active label may be bound to a nucleic acid are known to the skilled addressee.

Similar methods may be employed to bind a signal generating means to a signal generating probe. Methods are, for example, well known in the art for attaching enzymes, such as alkaline phosphatase horseradish peroxidase, galactosidase, proteases, lipases, etc. to nucleic acid (see for example, Table 1 , below, for examples of suitable enzymes and substrates).

The enzymes may be conjugated to nucleic acid in accordance with the teaching of US5,082,780, which is hereby incorporated by reference. The SE(R)RS-active surface may be any suitable surface, usually metallic, which gives rise to enhancement of the Raman effect, of which may are known from the SE(R)RS literature. It may for instance be an etched or otherwise roughened metallic surface, a metal sol or, more preferably, an aggregation of metal colloid particles. Silver, gold or copper surfaces, especially silver, are particularly preferred for use in the present invention and again, aggregated colloid surfaces are believed to provide the best SE(R)RS effect. As mentioned above, in one preferred embodiment, the SE(R)RS-active surface may serve to capture the target nucleic acid to be detected.

The surface may be a naked metal or may comprise a metal oxide layer on a metal surface. It may include an organic coating such as of citrate or of a suitable polymer, such as polylysine or polyphenol, to increase its sorptive-capacity. A preferred surface is Klarite®.

Where the surface is colloidal, the colloid particles are preferably 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 unaggregated colloids are already known. They involve, for instance, the reduction of a metal salt (e.g. silver nitrate) with a reducing agent such as citrate, to form a stable microcrystalline suspension (see P C Lee & D. Meisel, Phys. Chem. (1982), 86, p3391). This "stock" suspension is then aggregated immediately prior to use. Suitable aggregating agents include acids (e.g. HNO₃ or ascorbic acid), polyamines (e.g. polylysine, spermine, spermidine, 1 ,4-diaminopiperazine, diethylenetriamine, B- (2-aminoethyl)-1 ,3-propanediamine, triethylenetetramine and tetraethylenepentamine) and inorganic activating ions such as Cl", I", Na⁺ or Mg⁺. To increase control over the process, all equipment used should be scrupulously clean, and reagents should be of a high grade. Since the aggregated colloids are relatively unstable to precipitation, they are ideally formed in situ in the detection sample and the SE(R)RS spectrum obtained shortly afterwards (preferably within about 15 minutes of aggregation).

Ideally, a material such as spermine or spermidine is introduced to assist control of the aggregation process. The aggregation may be carried out at the same time as, or shortly after, the surface is introduced to the signal moiety or signal generating means.

The colloid particles are preferably monodisperse in nature and can be of any size so long as they give rise to a SE(R)RS effect - generally they will be about 4 - 100 nm in diameter, preferably 25 - 50 nm, though this will depend on the type of metal.

Typically, the surface may comprise silver colloid particles, which are substantially spherical in shape and of about 20 - 50 nm maximum diameter.

The "association" of the signal moiety or signal generating means with the SE(R)RS-active surface will typically be by chemi-sorption of the label onto the surface, or by chemical bonding (covalent, chelating, etc.) of the label with a coating on the surface, either directly or through a linking group. The association will usually be via suitable functional groups on the label, such as charged polar groups (e.g. NH₃ ⁺ or CO₂ ), attracted to the surface or surface coating (e.g. to free amine groups in a polyamine coating). Clearly, the type of association will depend on the nature of the surface and the label in any given case - different functional groups will be attracted to a positively-charged surface, for instance, as to a negatively-charged one.

Suitable groups by which the complex may be bound to the active surface include complexing groups such as nitrogen, oxygen, sulphur and phosphorous donors; chelating groups, - bridging ligands and polymer forming ligands.

Preferred ways of optimising the attachment between surface and label are described in WO97/006280 and WO01/77650. The detection may be carried out in a solid, liquid or gaseous format. The methods of the invention are conveniently carried out in a homogeneous format, i.e., all the required reagents are added simultaneously or substantially so and a result is obtained without the need to add further reagents at any stage or subsequently to separate any component of the test from the remaining components.

Homogeneous assays have the advantage that they are less prone to contamination, and less likely themselves to contaminate the external environment, since reaction vessels do not need to be opened at any time during the assay. The direct detection of nucleic acids and nucleic acid units without amplification, as in the present invention, is not prone to contamination by the products of previous assays as can occur in amplification based assays. Nevertheless the high sensitivity required in the methods of the invention makes it desirable that they be conducted in homogeneous systems.

Known examples of homogeneous nucleic acid assays tend to be based on fluorescence spectroscopy. The present invention now makes it possible to use the potentially much simpler and less costly SE(R)RS spectroscopy to carry out similar types of assays.

In SE(R)RS the primary measurements are of the intensity of the scattered light and the wavelengths of the emissions. Neither the angle of the incident beam nor the position of the detector is critical. With flat surfaces an incident laser beam is often positioned to strike the surface at an angle of 60° with detection at either 90° or 180° to the incident beam. With colloidal suspensions detection can be at any angle to the incident beam, 90° again often being employed.

The intensity of the Raman signals needs to be measured against an intense background from the excitation beam, and for this reason the use of Raman analytes with large Stokes' shifts is an advantage. The background is primarily Raleigh scattered light and specular reflection, which can be selectively removed with high efficiency optical filters. 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. The intensity of a SE(R)RS signal can be measured using a charge coupled device (CCD), a silicon photodiode, or photomultuplier 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.

Note that the methods of the invention may involve either obtaining a full SE(R)RS spectrum across a range of wavelengths, or selecting a peak and scanning only at the wavelength of that peak (i.e. Raman "imaging").

Apparatus for obtaining and/or analysing a SE(R)RS spectrum will almost certainly include some form of data processor such as a computer.

Raman signals consist of a series of discrete spectral lines of varying intensity. The frequencies and the relative intensities of the lines are specific to the label being detected and the Raman signal is therefore a "fingerprint" of the label. If a SE(R)RS analyzer is being used selectively to detect one label out of a mixture then it will be necessary to detect the entire "fingerprint" spectrum for identification purposes. However if the analyser is being used to quantitate the detection of one or several labels, each of which has unique spectral line, then it will only be necessary to detect signal intensity at a chosen spectral line frequency or frequencies.

Once the SE(R)RS 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. A commercial SE(R)RS analyser of use in carrying out the invention would be expected to consist of the following components: a laser light source, the appropriate optics for carrying the light to the SE(R)RS active surface, a stage for mounting the sample for analysis, optics for receiving the Raman signal, a detector for converting the Raman signal into a series of intensities at certain wavelengths and a data processor for interpreting the wavelength/intensity data and providing an analytical output.

The light source, optics, detector and processor have already been referred to. The stage for mounting the sample could be designed to accommodate one or more of the following solid supports: a microscope slide or other flat surface such as cuvette, a silicon wafer or chip, a microtitre plate or a higher density array microwell plate, or a membrane.

An assay could be carried out on a solid support and the support inserted into a SE(R)RS reader for analysis. Alternatively the assay could be carried out in a separate vessel with a subsequent transfer of the assay components to the solid support for inserting into the analyser. The use of robotics to transfer solid supports - to and from a SE(R)RS analyser stage would permit the development of a high throughput system without significant operator input with samples being run and analysed automatically.

The uses of the invention will be clear from the above description and include the prediction and detection of medical conditions, forensic testing, the sequencing of human and animal genes and identification of security-tagged products, the "tag" comprising a target nucleic acid or nucleic acid unit. Other techniques which involve the detection of genetic markers, and in which the present invention could therefore be of use, include the detection of positive traits in foodstuff and livestock breeding and the detection of HLA specificities to permit the matching of transplant samples. Clearly, aspects of the invention may be used to detect high as well as low concentrations of a target nucleic acid or nucleic acid unit. Analysis of the genes being expressed in tissues at various stages of the cell cycle by the detection of mRNAs is a useful means of tracking gene function. Currently mRNA levels are quantified by RT-PCR which is a difficult technique. Direct quantification of mRNA levels using the present invention would allow the rapid and simultaneous monitoring of many genes in tissues leading to insights into their function.

Detection of nucleic acid from infectious agents, especially viruses and bacteria can be carried out to, for example detect early infection or to monitor any particular treatment regimen.

The present invention also extends to the provision of kits for use with the methods described herein. Such kits may comprise all or some of the necessary probe nucleic acid molecules, substrate on to which the target nucleic acid is to be bound and appropriate SERRS signal generating means, such as SERRS active dyes and/or SERRS active substrates.

Detailed Description

The present invention will now be described by way of further example and with reference to the Figures which show:

Figure 1 shows a schematic representation of a prior art assay, for background purposes;

Figure 2 shows a schematic representation of an assay in accordance with the present invention utilising alkaline phosphatase in order to act upon a substrate and generate a SERRS signal;

Figure 3 shows a schematic representation of an assay in accordance with the present invention using probes to which a dye or SERRS label is attached, such that upon binding to a SERRS active substrate, a SERRS signal can be generated; Figure 4 schematically shows an assay in accordance with the present invention, similar to that as shown in Figure 3, but where different dyes are utilised in order to generate different SERRS signals; and

Figure 5 shows a schematic representation of a further assay in accordance with the present invention.

Figure 1 schematically shows a typical sandwich hybridisation assay as known in the art. A solid surface (10), for example a bead or plate is first modified so as to bind a capture probe (12) to its surface. The capture probe serves to allow a capture extender probe (14) to bind thereto. The capture extender probe (14) has a first portion (16) which is specific to a sequence present on the capture probe and another portion (18) which is specific to the target nucleic acid (20). The capture extender probe (14) therefore binds to both the capture probe (12) and the target nucleic acid (20), thereby immobilising the target nucleic acid (20) to the surface (10).

Thereafter, a label extender probe (22) is able to bind to the target nucleic acid (20). The label extender probe (22) has a first portion (24) which is capable of specifically hybridising to the target nucleic acid (20) and another portion (26) which is capable of specifically hybridising to a trunk nucleic acid molecule (28). The trunk nucleic acid molecule (29) has a number of identical, spaced apart, repeat sequences (31 ) which are capable of specifically hybridising to branch probe nucleic acid molecules (30), such that a number of branch probe nucleic acid molecules (30) are able to specifically hybridise to the trunk nucleic acid (20), thereby forming a branched DNA molecule (40). The 3' ends of the branch probe nucleic acids (30) are functionalised such that an alkaline phosphatase molecule (42) is bound thereto and as shown in Figure 1 , a number of alkaline phosphatase molecules are able to react with an appropriate chromogenic substrate such that upon action by the alkaline phosphatase molecules, a colour change is generated and detectable. As there is more than one alkaline phosphatase molecule present, the colour change is more easily detectable. The following discussion of assays in accordance with the present invention is to be understood as non-limiting. For example, although the Figures and corresponding description show the use of capture probes and capture extender probes, this may not be necessary. For example, a target nucleic acid may be bound directly to the surface of the substrate by use of, for example, of polylysine, or alternatively a signal target specific probe could first be bound to the substrate.

Turning to Figure 2, it will be appreciated that this is similar is some respects to the assay as described with reference to Figure 1. However, rather than using a chromogenic substrate which generates a colour change upon action by the alkaline phosphatase molecule, a material (50) is used which acts as a substrate for the alkaline phosphatase and which is bound to a SE(R)RS active substrate. However, the material is incapable of generating a SE(R)RS signal until acted upon by alkaline phosphatase. An example of this is bromo-chloro-indolyl-phosphate (BCIP). The BCIP bound to a SERRS active substrate has little or no SE(R)RS spectrum, but upon action of alkaline phosphatase causes formation of a dimer which is SE(R)RS active and therefore detectable. This has been described for example in Ruan et al (Anal. Chem. 2006. 78, p3379 - 3384 and US5082780), to which the skilled reader is directed.

Although this has been described with regards to the use of alkaline phosphatase, it is easily envisaged that other enzyme/substrate combinations could be employed. Examples of other enzymes which could be used are horseradish peroxidase, galuctosidase, proteases and lipase enzymes. It will be appreciated that simple enzymic assay formats can be employed such that the product of any enzymic reaction or cleavage can easily be detected.

Figure 3 shows another embodiment of the present invention, whereby the branched probes (60) are end-labelled with a SERRS active dye or label (62). Figure 3a) initially shows the branched DNA molecule (6) being formed such that a number of SERRS active dyes or labels (62) are hybridised to a target nucleic acid (64). However, since there is no SERRS active surface or substrate present, little or no SERRS signal would be detectable. Nevertheless, as shown in part b) upon binding of nano particles (66) which comprise a SE(R)RS active substrate, to the SE(R)RS active dyes or labels, a SE(R)RS signal can be generated and detected. More detail of this can be found in Graham and Faulds Chem. Soc. Rev., 2008, 37, 1042-1051.

Although this embodiment shows the use of nano particles to provide the SERRS active substrate, it is possible that the substrate upon which the target nucleic acid is initially immobilised to, could itself be a SE(R)RS active substrate and in this manner, upon forming the branched DNA structure, an amplified SERRS signal would be generated.

Figure 4 shows a similar embodiment to the assay as described in relation to Figure 3, but in this instance rather than employing a trunk nucleic acid which is universally able to bind to the label extender, each trunk nucleic acid (70, 72) is specific to the target molecule (74, 76) being detected. In this manner, specifically labelled branched probe nucleic acid molecules comprising different dye moieties (78, 80) are able to bind to specific trunk nucleic acid molecules (70, 72) in order to generate different and quantifiable SE(R)RS signals. This multiplex type system allows for the possibility of carrying out a single reaction, whilst being able to detect multiple targets in a single assay.

Figure 5 shows an embodiment where the branch nucleic acid molecules (90) are unlabelled initially, but a separate SE(R)RS labelled probe nucleic acid (92) is capable of specifically hybridising to the branch nucleic acid molecules (90), in order to generate, molecules (94), capable of eliciting a detectable/discemable branched SE(R)RS labelled a SE(R)RS signal. It is understood that the act of aggregating the SE(R)RS active moieties, bound to a SE(R)RS active substrate leads to the generation of the SE(R)RS signal. This is described for example in WO99/60157.

Claims

1. A method for the detection of a target analyte, comprising: a) contacting a sample potentially comprising the target analyte with a reagent capable of specifically binding to any target analyte present in the sample, under conditions whereby binding of the reagent and the target analyte occurs when the target analyte is present; b) and a plurality of signal generating probes, each signal generating probe comprising one or more signal generating means; and c) detecting an amplified signal from said plurality of signal generating probes obtainable when the reagent is complexed with the target analyte, wherein the amplified signal is detected by way of SE(R)RS.

2. The method according to claim 1 wherein the signal generating probes are a component of the reagent and linked, bound, complexed or otherwise associated with the reagent, or the plurality of signal generating probes may be capable of binding to the target/reagent complex in some manner.

3. The method according to claim 2 wherein the signal generating probes are capable of binding the reagent, or are capable of binding the complex formed between the target and the reagent.

4. The method according to any preceding claim wherein the reagent/target complex is a biological complex such as a protein/protein complex, a protein/antibody complex, a protein/nucleic acid complex, a nucleic acid/nucleic acid complex and the like.

5. The method according to any preceding claim wherein the plurality of signal generating probes is provided by way of a branched molecule wherein the signal generating probes may be present on a plurality of said branches.

6. The method according to any preceding claim wherein the signal generating means is a molecule which itself is capable of generating a SE(R)RS signal, or a molecule which upon action, for example by another molecule, such as an enzyme, is capable of generating a SE(R)RS signal.

7. A method for the detection of a target nucleic acid sequence, comprising: a) contacting a sample nucleic acid with a probe nucleic acid which probe comprises a polynucleotide sequence which is capable of hybridising to said target nucleic acid sequence, and a polynucleotide sequence which is substantially incapable of binding to said target nucleic acid sequence, under conditions whereby hybridisation of the probe to the sample nucleic acid occurs when the target nucleic acid is present; b) and a plurality of signal generating probes, each signal generating probe comprising one or more signal generating means and a nucleic acid sequence which is capable of directly or indirectly hybridising to a portion of the polynucleotide sequence of the probe nucleic acid which is substantially incapable of binding to said target nucleic acid sequence; and c) detecting an amplified signal obtainable when the probe nucleic acid is bound to the target nucleic acid and said plurality of signal generating probes, wherein the amplified signal is detected by way of SE(R)RS.

8. The method according to any preceding claim wherein the probe is a single molecule which is capable of binding directly to the target analyte or is a label extender probe which is capable of binding to the target analyte via a first portion and trunk analyte which is capable of hydridising to the label extender probe via a second portion.

9. The method according to any preceding claim wherein the target nucleic acid is adhered directly to a SE(R)RS active substrate, such as klarite®.

10. The method according to any preceding claim wherein more than one type of target analyte can be detected at once in a single sample.

11. The method according to any preceding claim wherein the signal moiety or signal generating means is attached to the 5' end of the signal generating probes.