WO2004106545A1 - Methods for enhanced detection using surface sensitive techniques. - Google Patents

Abstract

Methods for the detection and analysis of specific analytes, using techniques sensitive to surface excess are described. Detection of analytes such as nucleic acids and other biopolymers is achieved through template independent polymerization, or selective degradation. Using analyte specific initiation sites for template independent polymerisation and the measurement thereof by means of a surface sensitive technique, a functional signal amplification method is realised. When combined with immobilised specific affinity sites for the analyte, detailed information with respect to the interaction between analyte and the specific affinity sites may be obtained. Applications chiefly focus on the detection of nucleic acid based reactions and the use of the methods described to characterise nucleic acid analytes. In addition the present invention contains methods for the real time monitoring of nucleic acid substrate based enzymatic activity at the surface. Examples using voltanimetric and impedimetric techniques are described.

Description

Methods for enhanced detection using surface sensitive techniques. Background to the invention

Methods for examining the behaviour and composition of biopolymers and it's application to molecular diagnostics have traditionally involved the use of different experimental techniques, each having it's specific advantages This is well exemplified by the study of nucleic acids were sequence composition and interactions are central Spectroscopy related methods for example have been used to extract thermodynamic information relating to the sequence dependent interactions between nucleic acids (Geiduschek, (1962), J Mol Biol 4, •467- ₄₈7, Spat?, et al , (1969), J Mol Biol ₄₂, 191-219, Porschke, et al (1971), J Mol Biol , 6₂, 361-381) Electrophoresis when performed in conjunction with a suitable label (e g radiolabeied nucleotides), may unravel the composition of complex macromolecules such as nucleic acids or proteins to a large extend owing to the en ymatic or chemical sequencing techniques respectively developed by Sanger and Maxam-Gilbert (Sanger, et al (1977), Proc Natl Acad Sci USA , 7 , 5₄63-5467, Maxam, et al , (1980), Meths En7ymol , 65, 499-560) yielding quite definite information regarding to particular sequence stretches) Relative infomahon may be obtained using particular en7ymes or mixtures thereof which cut DNA at predetermined sequences (restriction fragment) Specificity in the generation of nucleic acid fragments may also be obtained by use of a binary system such as ohgo/RNaseH directed degradation of RNA (Don s- eller, (1979), Nucleic Acids Res , 7, 179-192) Furthermore specific enzymatic and chemical cleavage reactions have been used to detect single nucleotide polymorphisms (e g Myers.et al , (1985), Science, 230, 1242-1246, Cotton, et al , (1989), Nucleic Acids Res , 17, 4223-4₂33) Confidence in the methods applied to nucleic acids spawned the introduction of filter hybridisation methods based on sequence selective affinity, which made it possible to retain parts of the complex information confined in a specie's genetic make-up on a piece of paper This has made genetic information portable and exploitable and easy to compare for instance to another species Methods for the analysis of proteins has moved along not to dissimilar lines, with both physical and enzymatic methods playing a crucial role in generating our current understanding, and the development of immuno - affinity assays being a major commercial application The portable format of molecular diagnosbcs is now increasingly applied to a wider range of biologically important molecular structures, in order to obtain genetic or extra-genetic molecular correlates with disease For example particular glycoforms of otherwise similar proteins may for example be responsible for different clinical conditions (e g pπon glycoprotein, Rudd, et al (2001), Biochemistry, 40, 3759- 3766), and hence the development of diagnostic methods coping with molecular diversity is highly desirable Although very useful and reliable, many of the available molecular diagnostic methods see their applications limited because of the format and/or the labelling and detection technique For example, the reverse hybridisation format frequently involves the incorporation of a label at a particular stage duπng the experimental protocol in order to visualize a specific hybridisation event In general the signal amplification or differentiation generated by the labelling system will determine the nature of the measurement technique to evaluate the test, and very often is limited to a discontinuous measurement reflecting the state of hybridised probes at some stage in the hybridisation process So whenever discontinuous, only one significant measurement per collection of specific nucleic acid probes can be made, representing only one physical or chemical condition under which probe and target interact This approach has become problematic and quite inflexible, when high resolution tests addressing small variations in a target sequence are being developed The need for specificity implies that hybridisation of the sample proceeds at a stringent temperature which is calculated from an objective matching sequence This requires a detailed knowledge and delicate balancing of the melting behaviour of each of the probes to be used in the test Hence the outcome of experiment is to this extent predetermined, and no positive discrimination of partially matching sequences is possible Therefore unexpected mutations are easily missed, and mixed populations or heterozygous individuals can be a source of insuperable complexity when designing or interpreting a particular test Similarly the production of labelled antibodies for a specific sample protein, is often a limiting factor in the development of a protein assay and the nature of this label usually only allows for discontinuous measurement, hence no further discrimination of the signal is possible Currently there is an increasing effort to miniaturize the "portable" molecular diagnostic format as well as other techniques, although in many cases the signal generated for detection remains discontinuous Nonetheless promising new technologies or approaches have for example been applied to the reverse hybridisation foπnat that focus on the measurement of physical or chemical phenomena occurring at transducer surfaces Methods for immobilizing DNA probes on a transducer surface were described by Flowers and co-workers (Flowers, et al , (1986), Fed Proc , 45(6), 1516), who used the surface of piezoelectric oscillators to serve as a earner in the reverse hybridisation assay, offering the possibility for direct detection of DNA hybridisation Similarly, use of a surface plasmon resonance device wherein the transducer surface is modified with sequence selective probes has been disclosed in U S patent ,889, 27 (Van Veen et al ,) Of particular interest is the use of electrochemical techniques to characterize the behaviour of polyelectrolyte modified surfaces As the detection involves the measurement of electncal signals it may be integrated in relatively inexpensive ways requiring little auxiliary equipment Since the pioneering work of Oyama and others (Oyama, et al , (1980), J Electroanal Chem , 11 , 271-280, (1980) Anal Chem , 52, 1192-1198) who studied the behaviour of polyelectrolyte modified surfaces in conjunction with transition metal redox complexes carrying an opposite or similar charge (e g Co(phen)₃ ^3+2* or Fe(CN)₆ ³ '*^■) the electrochemical method has been increasingly applied for studying biopolymers immobilized to transducer surfaces Selective binding of transition metal ion complexes to nucleic acids is a subject which has been thoroughly studied both by spectroscopic and electrochemical techniques (Barton, et al , (1984), J Am Chem Soc , 106, 2172-2176, Carter, et al , (1987), J Am Chem Soc 109, 7528-7530) Mikkelson et al subsequently disclosed modified electrode surfaces in conjunction with a specific redox probe to distinguish between double and single stranded nucleic acid structures (U S Pat No 5,312,527, Mikkelson et al ,) Other examples involving the use of faradayic methods (1 e those involving electron transfer) in conjunction with nucleic acid hybπdisation assays have used the electrochemical properties of the DNA bases (Napier, et al , (1997), Bioconjug Chem , 8(6), 906-913), or draw upon exogenous labels such as redox enzymes (U S Pat No 4,840,893 , Hill, et al) In close analogy with the faradayic methods applied to transducer surfaces, impedimetπc methods draw upon the same instrumentation advantages, and are very convenient Still, many of the methods descπbed above either require complex instrumentation and are very expensive to employ, encounter sensitivity problems, or see their applications limited because of the labelling or detection technique It is the aim of the current invention to provide methods for the detection of nucleic acids as well as other compounds at transducer surfaces by means of surface excess producing reactions, which unify some of the advantages encountered in traditionally different techniques It is therefore also an aim to increase the sensitivity of detection methods that draw upon surface excess

Another aim ot the invention is to provide a method for continuous measurement of binding, hybndisation, dissociation and similar processes by means of a surface sensitive technique

SUMMARY OF THE INVENTION The present invention provides methods for the detection of the presence of an analyte using template independent polymensation and or template selective degradation, the products of which are measured as a discrete change in surface excess using a surface sensitive technique

Detection of the analyte is achieved by means of addressing suitable initiation sites for template independent polymensation present within the analyte or associated with the analyte Henceforth analyte specific template independent polymensation by means of suitable enzymes such as glycosyltransferases and nucleotidylexotransferases can be used to change the surface excess as measured using a surface sensitive technique

Using a surface with discrete specific affinity sites which interact with the analyte, a significant change in surface excess is observed upon presence of the analyte by means template independent polymensation using analyte specific initiation sites

The present invention further provides a functional signal amplification by means of analyte specific template independent polymensation, wherein the surface excess may be continuously measured, for example as a function of a physical or chemical gradient It is a specific advantage that such functional amplification allows for charactensation of the interaction between analyte and discrete specific affinity sites In practice this makes it possible to positively discnminate mismatches in nucleic acid sequences, or detect multiple interactions between analyte and affinity sites, without the requirement for any further steps

Additionally the present invention provides methods for the detection of an analyte using template selective degradation To this extent discrete specific affinity sites containing degradation precursors are degraded in presence of the analyte and a suitable agent catalysing template selective degradation The presence of the analyte is then detected by measunng a change in surface excess at a transducer surface Alternatively, the degradation precursors contain in addition also initiation precursors for template independent polymensation which are activated by said template selective degradation

Furtheπnore the processes of template independent polymensation and template dependent degradation may be combined to detect the presence of an analyte by measurement of the surface excess For example template selective degradation may be used to create an initiation site for template independent polymensation

Other reaction schemes combining template independent polymensation and template selective degradation are descnbcd which provide analyte specific signal amplification in an autocatalytic manner

The present invention further includes methods for the analysis of nucleotide sequences using the advantages of functional signal amplification, and it's measurement by means of a surface sensitive technique

BRIEF DESCRIPTION OF THE FIGURES

FIG IA Cyclic voltammogram of a hybπdization using a complementary oligonucleotide 1) before hybndisation 2) after hybndisation and template independent polymensation

FIG I B Cyclic voltammogram of a hybndisation using a non complementary oligonucleotide I) before hybndisation 2) after hybndisation and template independent polymensation FIG 1C Cyclic voltammogram of a hybndisation using a complementary PCR product (sample I) 1) before hybndisation, 2) after hybndisation, 3) after hybndisation and template independent polymensation FIG ID Cychc voltammogram of a hybndisation using a non-complementary PCR product (sample 2) 1) before hybndisation, 2) after hybndisation, 3) after hybndisation and template independent polymensation UG lb Bode plot showing the conductivity of a interdigitated lmpedimemc sensing device 1) before hybπdization, 2) after hybndization with complementary nucleotide and template mdependent polymerization, 3) after hybndization with non complementary oligonucleotide and template independent polymenzation FIG 2A Shows a van t'Hoff plot obtained from a equilibnum dissociation expenment involving a fully complementary oligonucleotide sequence (Series 1) and an oligonucleotide sequence with one mιsmatch(Senes 2) and a mixture of both complementary oligonucleohdes and oligonucleotides with one mismatch (Senes 3) The parameter θ represents the relative surface coverage

FIG 2B Temperature jump expenment showing the course of denaturation of electrodes hybπdized with respectively complementary oligonucleotide and a oligonucleotide sequence with a transition mismatch and amplified using DNA nucleotidylexotransferase 0 represents the relative surface coverage FIG 3A Differential dissociation plot of homogeneous and heterogeneous surfaces, obtained from equilibnum dissociation using cyclic voltammetry Homogeneous surface 25-mer (Senes 1), Heterogeneous surface containing a 25-mer and 18-mcr complementary oligonucleotide (Senes 2), Homogeneous surface containing a 18-mer complementary olιgonucleotιde(Seπes 3) FIG 3B Difference differential dissociation plot of a heterogeneous surface containing 18 and 25-mer complement Senes 1 25-mer homogeneous surface subtracted, Senes 2 18-mer homogeneous surface subtracted

FIG 4 Temperature jump expenment analyzing the outcome of a restncted length polymenzation expenment The parameter θ represents the relative surface coverage Senes 1 and 2 ACG extension, senes 3 and 4 ACT extension (for details see example IV) FIG 5 Detection of specific endonucieolytic activity of RNase H (duplicates) (1) Chimeπc probe and complementary template, (2) Chimenc probe and partially complementary template, (3) Chimeπc probe and non complementary template, (4) chimeπc probe, no template, (5) chimeπc probe and complementary template no RNase H, (6) buffer only (for details see example V) Qi and Q respectively represent the integrated surface charge before and after the expenment FIG 6 Real time monitoring of DNase I induced degradation of a transducer surface containing specific affinity sites previously hybndised with complementary DNA ohgonucleotides and subjected to template independent polymerization Senes 1 No DNase added, Senes 2 DNase added

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term analyte or specific analyte is used to descπbe a substance which is the object of detection and further contains, or is made to contain an initiation site or initiation precursor for template independent polymensation Alternatively the term is used to descπbe an analyte which contains a precursor for template dependent degradation The term analyte is also used to specify those substances which are denved from the analyte by means of a specific process, including particular reaction products further defined below

The term "nucleic acid sample" refers to a sample containing nucleic acids, which may have been subjected to a suitable punfication or extraction method known in the general art of biochemistry The term is also more widely used to designate the analyte or sample to be investigated further dcscnbcd by the terms "specific reaction product" and "deπvative thereof

The term "specific reaction product" and "denvative thereof refers to the products obtained in a specific reaction with respect to nucleic acids It compnses but is not limited to the products obtained from a specific amplification reaction (e g polymerase chain reaction, ligase chain reaction, transcnption mediated amplification, cycling probe amplification etc ), a nucleic acid hybπdization reaction to probes of known oπgin, or a reaction involving the enzymatic redistπbution of nucleic acid sequence information due to for example the activity of specific nucleases, specific chemical cleavages, or polymerase based sequencing reactions The tenn specific reaction product also encompasses reactions relating to the combination of specific reactions such as a polymerase based amplification used in conjunction with a specific nuclease reaction or sequencing reaction In addition the specific reaction product further may include reactions involving the deprotection of soluble nucleic acid probes by means of the specific activity of a DNA or RNA endo- or exonuclease, as such making the said probes suitable for template independent polymerization using for example a RNA or DNA dependent nucleotidylexotransferase Similarly, the term "specific reaction product" or "deπvative thereof also refers to the products obtained using specific endo- or exoglycosidases, glycanases and other biological or chemical agents causing selective degradation or modification of carbohydrates and related substances In analogy to nucleic acids the specific reaction products with respect to carbohydrates may also yield initiation sites for template independent polymensation using specific glycosyltransferases The term "template selective degradation" refers both to the stepwise or processive removal of monomer units of a polymer template and to the single step cleavage of a polymer template, each of them directed by the properties of said template

The term "initiation site" is used to descπbe a specific site corresponding to the substrate to which monomers may be added or removed in a template independent or template dependent manner

The term "template dependent polymensation" refers to the stepwise addition of monomer units to an initiation site (also referred to as a primer), each of them directed by the properties of a template The term "initiation precursor" is used to descnbe the initiation site which needs activation m order to become a substrate for template dependent or independent polymenzation or degradation, and preferably due to a specific event relating to the nature and composition of the said initiation precursor

The term "degradation precursor" is used to descπbe the particular entity that is prone to a degradation process This entity is contained within the analyte, results from binding of analyte to affinity sites, or is contained within the affinity sites but only activated upon binding of the analyte

The term "template independent polymenzation" refers to the stepwise or processive addition of monomer units to the initiation site without the requirement for a template

The term "functional amplification" refers to the creation of a signal amplification which retains some of the properties of the amplified target The term "structural amplification" refers to an amplification of a specific reaction product or sample which retains some of the properties of the amplified target which by amplification induces a significant change in molecular composition, thereby forming the basis for detection of a specific sample or reaction product In the present context it is used to descnbe a change in the gross macromolecular composition of a transducer surface The term "surface excess" is used to descnbe a particular surface composition, such as the pnmary excess of a polyanion, polycation or other relevant species and components associated to them and implies that a difference exists at the interface between transducer surface and bulk medium

The term "surface sensitive techniques" applies to physical methods that allow one to characteπze or measure surface compositions and changes thereof, including but not limited to faradayic and lmpedunetnc techniques, which are sensitive to charge transfer, conductivity and dielectnc phenomena These methods may typically combine chemical specificity and features relating to the overall composition of a surface Mass sensitive methods which include but are not limited to resonance methods such as the quartz crystal balance, optical wave guide methods for example resonant mirror and surface plasmon resonance in which the surface itself is transducer and work function based techniques such as the Kelvin microprobe are also included It should be clear that the list of surface sensitive techniques is bound to be incomplete, and several new techniques are being developed

The term "transducer surface" relates to the integrated assembly of a surface which may be used in conjunction with one of the above mentioned surface sensitive techniques The transducer surface in general may be part of a passive transducer (e g piezoelectric transducers, optical transducers, mechanical transducers etc ) or an active transducer (e g resistance or reactance transducers) or hybnd devices that are classified according to their stimulus requirements

The terms "modified electrode" and more general "modified transducer surface" is used to specify chemically modified electrode surfaces having specificity for a specific nucleic acid sequence or proteins, or in general have a selective affinity for a particular analyte or improve the resolution of its detection Typically the modification may consist of nucleic acid sequences, or nucleic acid analogs which arc not found in nature Presently the synthesis of such probes is fully automated and many modifications incorporating chemical functions for the attachment to solid supports are available Electrode surfaces may thus be modified according to the nature of the electrode matenal Several examples relating to the controlled modification, functionahsation of electrode surfaces as well as the general background relating to the art are descnbed in "Integrated chemical systems" by Allen J Bard (A J Bard, Wiley-Interscience 1994) The term "specific affinity site" is used to descπbe a molecule or molecular property which has been conferred to an otherwise non-specific earner, thereby inducing the propensity to specifically associate with a closely defined range of molecules under particular conditions

The term "pen-electrodic space" is used to specify the space in the immediate vicinity of the electrode, the latter being defined as a conducting substance having a connection to the external measunng circuitry Apart from the solvent or electrolyte in the immediate vicinity of the electrode surface, a regular reproducible structure having different properties with respect to conductivity and or capacitance may be included into the space defining the boundaπes of a particular measurement area The term pen-electrodic space may thus be applied to both intra and inter electrode configurations, depending on whether this space involves single or multiple distinct connections to the measunng circuitry For example fibrous carbon electrodes consisting of both conductive and non conducting compounds allowing surface modification as descπbed in "Chemically modified carbon fibers"(I N Ermolenko et al Eds , VCH 1990), provide a good example of intra peπ-electrodic space Other examples belonging to this class include the patterned or random deposition of for example organo-sulfur compounds on metal electrodes resulting in the deliberate passivation of a portion of the electrode area The term pen-electrodic space also applies to regular structures allowing deliberate manipulation between at least two independently addressable electrodes One of the earliest examples of a modular peπ-electrodic space is the well known Volta-pile More recent examples include lnterdigitated electrodes which may be microscaled using conventional photolithographic procedures, or specific micro-moulding techniques combined with structure- induced metal deposition patterning (Van Gerwen, et al European patent 0876601)

The term "exogenous label" is used to specify a chemical entity which has a chemical or physical property distinct from the sample molecule to be detected and allows to detect the sample molecule Typical exogenous labels include radioactive, fluorescent, or enzyme binding sites which may become associated with the sample by means of a probe or are integrated in the probe

Fmbodiments The general concept behind the present invention, relates directly to the use of surface sensitive techniques These techniques allow for the measurement of any substance, without the need for labels, to give nse in a measurable signal by assessing parameters that are directly altered by the presence of said substance It has been found to be very advantageous to exploit this even further by using the presence of said substance as a prerequisite step to magnify the signal, which results from said substance, even further It has been found that such can be achieved in an analyte specific way by using processes that draw upon the presence of said analyte to either magnify the signal by building more substance or by doing the reverse, and destroying the signal that was already generated by the presence of said analyte The first can be achieved by template independent polymensation provided that the analyte present contains an initiation site for said template independent polymerisation The second can be achieved by template selective degradation provided that the analyte present contains an initiation site for said template selective degradation

In a first embodiment the present invention provides a method for detecting the presence of an analyte in a sample by means of modifying and measunng the surface excess wherein the said method compnses a) increasing surface excess by means of template independent polymerization or b) decreasing the surface excess by means of template selective degradation In a embodiment the present invention provides a method for detecting the presence of an analyte in a sample compnsing the following steps a) Reacting the sample with a reaction mixture that causes template independent polymensation, by means of addressing suitable initiation sites present within, or specifically associated with, the analyte b) Assessmg the template independent polymensation, by means of measuring the surface excess using a surface sensitive technique In a embodiment the present invention also provides a method for detecting the presence of an analyte in a sample compnsing the following steps a) Binding the analyte to a surface containing discrete specific affinity sites that are selective for the said analyte b) Reacting the said bound analyte with a reaction mixture that causes template independent polymensation, by means of addressing suitable initiation sites present within, or specifically associated with, the analyte c) Assessing the template independent polymensation, by means of measuring the surface excess at the surface containing said specific affinity sites using a surface sensitive technique

In an embodiment the current mvention thus provides a method for detecting specific analytes (containing initiation sites for template independent polymensation) by means of a transducer surface and template independent polymerization or template selective degradation The transducer surface may for this purpose either contain, be brought in the vicinity of, or in contact with the analyte in order to measure the surface excess at the said surface As such a tangible interaction between transducer surface and analyte as defined by the surface excess is understood to be present In order to detect an appreciable change in surface excess at the transducer surface due to the presence of the analyte, the signal is amplified in a analyte specific way by means of template independent polymensation of suitable initiation sites contained within the said analyte Such initiation sites may be present within the specific analyte (being thus a natural property or intrinsic part of the analyte), or associated with the analyte by means of an analyte specific process which couples a natural or synthetic initiation site to the said analyte The template independent polymensation process may for example involve the addition of nucleoside or sugar (and their denvatives) residues to the said initiation site by means of an enzyme capable of template independent polymerization belonging for example to the following classes the nucleotidylexotransferases, hexosyltransferases and pentosyltransferases The process of template independent polymensation then readily results in the formation of a homopolymer, copolymer, or block copolymer which may be a polynucleotide or a polysacchaπde and hence gives nse to an appreciable change in surface excess which is analyte specific and is measured at the transducer surface

When the analyte interacting with the transducer surface is a nucleic acid sample, a specific reaction product or denvative thereof, the said analyte may be detected by template independent polymenzation of the initiation sites present within or associated with the said nucleic acid sample When the nucleic acid sample involves a deoxyπbonucleic (DNA) or πbonucleic acid (RNA), initiation sites for template independent polymensation may for example be the 3'OH termini which are contained within the sample, and are readily amenable to the addition of nucleoside residues by a template independent polymensation process involving a nucleotidylexotransferase Consequently a sample containing a nbonucleic or deoxynbonucleic homo or copolymer is synthesized, this structural amplification or increase may then be detected at the transducer surface Suitable enzymes capable of performing template independent polymensation may be employed to detect samples containing DNA, RNA or both at a transducer surface by means of template independent polymensation These include but are not limited to DNA deoxyπbonucleotidylexotiansferase, polynbonucleohdc nuclcotidyltransfcrase, polynucleotide adenylyltransferase and specific tRNA nucleotidyltransferases

Alternatively initiation sites for template independent polymensation may be specifically associated with the said nucleic acid sample, specific reaction product or denvative thereof This may for example compnse the hybndisation of suitable initiation sites to the said sample, by means of exploiting the propensity of nucleic acids to form stable and specific hydrogen bonds with ohgonucleotides of complementary or partially complementary sequences Such ohgonucleotides may contain initiation sites for template independent polymensation, or have to them attached initiation sites for template independent polymensation tor example by means of covalent modification, further hybndisation, or enzymatic modification in a analyte specific way These initiation sites may as before consist of nucleoside residues, and subjected to template independent polymerisation by means of using a suitable nucleotidylexotransferase, in order to detect the said sample at the transducer surface It will be obvious, that multiple initiation sites may be attached to the analyte by exploiting sequence specific association schemes and hence increase the degree of structural amplification upon template independent polymensation The associated initiation sites may however also consist of oligosacchandes which may for example be attached to a oligonucleotide having a degree of specificity for the said nucleic acid sample Such oligosacchandes may then serve as initiation sites for template independent polymensation by means of particular hexosyltransferases or pentosyltransferases For example the initiation site may consist of a number of (N,N-alpha/beta-glucosyl) residues, which equal or are above the minimum length (e g typically 7) to serve as a non reducing end initiation site for template independent polymensation, by for example UDP-glucose- glycogen-glucosyltransferase (preferentially devoid of glycogenin glucosyl transferase activity) which may act in concert with a branching enzyme (e g the alpha(l,6 branching enzyme) E C 24 1 18) Alternatively the associated initiation site may be a specific ohgosacchande (e g galactosyl-galactosyl-xylosyl) which may lead to the formation of more complex polysacchandes such as hepaπn or hepaπn sulphate (incorporating D-glucuronic and N-acetylglucosamine resιdues(see for example hnd, et al , (1993) J Biol Chem , 268, 20705-20708)) by means of template independent polymensation using suitable glycosyltransferases It may be readily apparent that unlike for nucleic acids, there are numerous types of initiation sites, substrates and enzymes which may be used for the template independent synthesis of polysacchandes, and the concomitant detection of a specific analyte at a transducer surface, all of these are thus considered to be in the scope of the present invention

In another embodiment the specific analyte may be a protein, carbohydrate, a collection thereof, or a specific reaction product or deπvative of said analyte, which contains initiation sites for template independent polymensation Such initiation sites for example may be naturally present on specific types of proteins such as glycoproteins as a result of post translational modification, or on proteoglycans, the latter being more complex structures Oligo or polysacchandes present on such specific proteins may hence be used as initiation sites for template independent polymensation by means of a suitable hexosyltransferases (or combination thereof), or pentosyltransferases in order to detect a change in surface excess at a transducer surface in an analyte specific way For example high mannose glycoproteins may be detected using specific glycosyltransferases capable of utilising mannose residues as an initiation site for template independent polymensatioa Similarly proteins having a natural tendency to associate with nucleic acid sequences (the latter being initiation sites for template independent polymensation) may be detected at a transducer surface by means of template independent polymensation using a suitable nucleotidylexotransferase

In still another embodiment the specific analyte is a protein, carbohydrate,a collection thereof, or a specific reaction product or denvative of said analyte which may be treated to contain associated initiation sites for template independent polymensation in order to measure a change in surface excess at the transducer surface in a analyte specific way For this purpose ohgosacchande, or oligonucleotide initiation sites may be_assocιated with the analyte of interest or to another protein or peptide having a specific affinity for the said protein sample (e g reporter) Henceforth suitable ohgosacchande initiation sites may be directly associated with the analyte For example this may be achieved by means of forming an θ-lιnkage to accessible senne or threonine residues, a reaction which may be catalysed by O-N-acetyl-D-glucosamine transferase (EC 2 5 1 7), or using specific lectins and their conjugates (having initiation sites for template independent polymensation), and other methods known in the art Alternatively similar methodologies may be used to couple ohgosacchande or oligonucleotide initiation sites to the said analyte or reporter by means of functional linkers In another embodiment the analyte (e g a nucleic acid or protein) may be specifically immobilised to a surface containing discrete specific affinity sites for the said analyte Said surface may be a transducer surface or a surface which may be interrogated by means of measunng the surface excess at the transducer surface Discrete specific affinity sites which immobilize the analyte in a specific manner include sequence specific oligo or polynucleotides and denvatives thereof, natural nucleotide sequences, antibodies, peptides, lectins and so on, all of which may be conferred to the said surface in order to specifically immobilize the said analyte Said immobilized analyte may then be detected by measunng the surface excess at a transducer surface according to the methods descnbed in the current invention Similarly the said surface may contain discrete specific affinity sites which specifically interact with a product of template independent polymensation, in order to detect the presence of an analyte by measunng the surface excess at a transducer surface In a preferred embodiment, the cuιτent invention provides a method for detecting the interaction between a analyte preferably a nucleic acid sample, a specific reaction product or a deπvative thereof, and a surface by means of template independent polymenzation of the said nucleic acid sample, and the concomitant detection of the surface excess at a transducer surface The reaction relating to the nucleic acids, involves the interaction (e g hybndisation) of a sample nucleic acid sequence or a specific reaction product with complementary or partially complementary oligonucleotide probes unmobilized to a surface which may for example be the transducer surface The oligonucleotide may be DNA, RNA, mixtures of both, or chemical analogs of nucleic acids and their mixtures The said oligonucleotide probes or denvatives thereof arc preferably immobilized to or brought to the vicinity of the surface according to methods known in the art When the surface sensitive technique used involves a electrochemical detection method, the probe is preferably immobilized to the electrode surface or peπ-electrodic space The said immobilized nucleic acid probes are selected such that they carry no initiation sites for template independent polymerization, typically the probes are unmobilized using a 3' modification for chemisorption or covalent attachment to the surface Following hybπdization with the said sample nucleic acid or specific reaction producζ the surface is incubated with an enzyme capable of performing template independent polymenzation Depending on the nature of the said nucleic acid sample, initiation sites for template independent polymensation may be contained within the nucleic acid sample by means of for example 3'OH ends acting as initiation sites for DNA or RNA nucleotidylexotransferases (e g DNA nucleotidylexotransferase (E C 277 31), polynbonucleotide nucleotidyltransferase (E C 27 7 8), polynbonucleotide adenylyltransferase, and specific tRNA nucleotidyltransferases (E C 2 7 7 56)) Alternatively initiation sites for template independent polymensation may be associated with the nucleic acid sample by means of specific affinity sites other than these contained on the said surface, providing thereby analyte specific initiation sites for the template independent synthesis of for example a polysacchaπde or polynucleotide catalysed by suitable hexosyltransferases, pentosyltransferases or said DNA or RNA nucleotidylexotransferases Henceforth the interaction between nucleic acid sample and immobilised discrete specific affinity sites may be detected by means of template independent polymensation and the concomitant change in surface excess measured at the transducer surface

Alternatively the nucleic acid sample may be immobilized to the said surface by means of incorporating specific chemical functionalities mto the sample allowing a stable interaction with this surface For instance such funchonalisation may involve the incorporation ot biotin or modified nucleotides such as phosphorothioates dunng nucleic acid amplification This allows the sample to be probed with specific probes havmg initiation sites for template independent polymenzation Furtheπnore said nucleic acid sample or specific reaction product may be immobilised by means of oligonucleotide sequences which are complementary to specific oligonucleotide probes, or suitable affinity sites immobilised at the said surface at one end, and have a sample specific sequence at the other end It will be apparent that for anyone skilled in the art, several methods exist or may be devised to immobilize a nucleic acid sample to a surface in order to be analysed and detected according to the methods descnbed in the current invention

In one embodiment, it is the particular advantage of the current invention that the analyte specific detection by means of template independent polymensation provides a functional signal amplification and allows the continuous measurement thereof at a transducer surface Hence products formed according to the methods described in the present invention may be further analysed, by subjecting the product of template independent polymensation to conditions involving temperature, hydrophobicity, ionic strength and so on, while measunng the surface excess as a function of the applied conditions In particular, the ιnteractιon(s) between the analyte and discrete specific affinity sites used to immobilise the said analyte, as well as ιnteractιon(s) between analyte and associated initiation precursors may be charactensed (subsequent to template independent polymensation) When for example a thermal gradient is applied, both kinetic and equilibnum mfoπnation relating to the said interactions may be collected by respectively performing temperature jump or equilibnum dissociation expeπments Using these appropπate methods, thermodynaπuc parameters relating to the analyte and specific affinity sites and or associated initiation precursors may hence be extracted The use of such methodology is most typically desirable when the analyte is a nucleic acid, specific reaction product or a deπvative thereof, in which case it is often the objective to detect small vanations in nucleotide sequence For example single nucleotide polymorphisms are easily detected and positively discriminated using detection based on template independent polymensation in conjunction with the equilibnum dissociation or temperature jump method

In another embodiment further information regarding the nature of the analyte may be obtained by further subjecting the products obtained using template independent polymensation to template selective degradation For this purpose specific DNA or RNA exo or endo-nucleases, specific endo or exoglycosidases, or other specific biological or chemical agents may be used This for example allows one to investigate the interaction of a specific affinity site such as a oligonucleotide probe and a nucleic acid sample by means of a selective degradation agent such as a restnction endonuclcasc Or in the case the analyte is a carbohydrate the initiation sites used for template independent polymensation may be cleaved off using a specific endo- glycosidasc or glycanasc In a embodiment the present invention provides a method for detecting the presence of an analyte in a sample compnsing a) Reacting the sample with a reaction mixture that causes template selective degradation, by means of addressing suitable degradation precursors present within the analyte, or specifically associated with, the analyte b) Assessing the template selective degradation, by means of measunng the surface excess using a surface sensitive technique In a embodiment the present invention also provides a method for detecting the presence of an analyte in a sample compnsing a) Binding the analyte to a surface containing discrete specific affinity sites that are selective for the said analyte b) Reacting the said bound analyte with a reaction mixture that causes template selective degradation, by means of addressing suitable degradation precursors present within, or specifically associated with, the analyte In one embodiment the present invention further involves a method for the detection of an analyte by means of template selective degradation and measunng a concomitant change in surface excess at a transducer surface In contrast to the detection using template independent polymensation of analyte specific initiation sites resulting in an increase in localised mass, template dependent degradation of specific affinity sites results in a decrease of localised mass and hence also may be used for analyte specific detection For this purpose specific affinity sites, having the further property of containing degradation precursors may be immobilised to a surface which may be addressed by means of a surface sensitive technique Upon reaction with a specific analyte said degradation precursors may be cleaved, degraded, or become activated for degradation Usually such degradation occurs in the presence of a specific agent catalysing the template selective degradation process In a preferred embodiment the analyte is a nucleic acid, a specific reaction product or a denvative thereof and the specific agent catalysing template selective degradation is a nucleic acid endo or exonuclease Degradation precursors having a specific affinity for the said analyte may be immobilised to a surface which may be addressed to measure the surface excess by means of surface sensitive technique Said specific affinity sites may further also contain components which influence the measured surface excess (c g polymenc tails) but have no specific affinity relationship with the said analyte, however they may be degraded or cleaved off, upon reaction with said analyte in the presence of suitable DNA or RNA nuclease or a combination of several nucleases Schemes for template selective degradation according to this method may for example compπse the analyte (in this case a deoxyπbonucleic acid) selective degradation of specific affinity sites (l e degradation precursors) immobilised at a surface detectable by means of a transducer surface which are degraded upon hybndisation with the analyte in the presence of a suitable nuclease, for example specific affinity sites consisting of deoxynbonucleic acid and having free 5'-termιnι may be degraded upon the formation of hybπd with the analyte nucleic acid in the presence of phage lambda induced nuclease, or phage T7 gene 6 exonuclease, or exonuclease III (in the case of 3 '-termini) Similarly specific affinity sites immobilised at said surface containinig nbonucleic acid moieties, may be degraded upon the hybridisation with said analyte, in the presence of RNaseH or exonuclease III In both cases the analyte nucleic acid is released upon degradation of the specific affinity site, and as such may react with other affinity sites causing recurrent hybndisation and hence additional signal increase Optionally residues contained within the said specific affinity site (e g poly πbo-A tail) which do not specifically interact with the analyte, may also be degraded upon the degradation of the specifically interacting residues, due to the activity of a terminally directed exonuclease such as poly A specific nbonuclease Other methods for the analyte selective degradation, and the detection thereof by measunng a change in surface excess, may include the use of one or more specific affinity sites to achieve a particular conformation in the presence of the said analyte and wherein one of the components participating in said conformation is subsequently cleaved by a structure specific nuclease For example several nucleases (e g lambda nuclease, the 5'nuclease of certain DNA polymerases, as well as some nucleases involved in Okazaki- fragment processing) recognise the presence of redundant smgle strands forming a branch in a otherwise double stranded nucleic acid and may cleave the single strand exonucleolytically or endonucleolytically at a junction containing the 5' or 3'end of strands engaged in a double stranded structure (Kadaba, et al , (1975), J Biol Chem , 250, 5438-5445, Setlow, et al , (1972), J Biol Chem , 247, ₂₃₂-240) Therefore at least one of the said components required to form said particular conformation may be a discrete specific affinity site immobilised at the said surface, and may be cleaved upon the activity of a structure specific nuclease when the analyte nucleic acid participates in said conformation Other components necessary to complete a specific structure may be futher specific affinity sites present in solution or immobilised on the same said surface In the case of lambda nuclease based degradation, the immobilised specific affinity site may be a oligonucleotide containing a 3' portion not complementary to the sequence of the said analyte which foπns a redundant single strand at a junction defined by the analyte hybndised to the said specific affinity site and a auxiliary analyte specific oligonucleotide When 5' nucleases for example denved from DNA polymerases are used, the method is similar except that the immobilised specific affinity sites contain a 5' portion (l e the redundant single strand) which is not complementary to the said analyte nucleic acid In both cases the conformation may be arranged as such that either the 3' or 5' end of the specific affinity sites is used to immobilise said affinity sites The combination of nucleases, degradation precursors and analytes descnbed in this particular embodunent is non-exhaustive, and further combinations are possible However the above examples, show how progressive and structural template selective degradation m an analyte specific manner may be applied for surface excess based detection within the context of the present invention

In a further embodiment, initiation sites for template independent polymensation may be produced in a analyte specific manner by means of template selective degradation of soluble or immobilised specific affinity sites interacting with the said analyte When the analyte is a nucleic acid a specific reaction product or a denvative thereof, template dependent degradation or cleavage of the specific affinity sites interacting with said analyte may be achieved according to the methods previously descπbed in the current invention (previous embodiment) As such immobilised specific affinity sites which are cleaved or degraded upon specific interaction with the analyte (and if necessary auxiliary ohgonucleotides), may subsequently present suitable termini which may serve as initiation sites for template independent polymensation For example specific affinity sites having 3' termini which are not capable to serve as initiation sites for template independent polymensation (either due to their nature, or presence of blocking groups such as dideoxy residues), may be modified by means of analyte specific cleavage or degradation to yield 3'ends which are amenable to template independent polymensation by providing suitable initiation sites This may be the case when said specific affinity sites are DNA/RNA chimera where the 3' end may be in the deoxyπbonucleoside form, and the nbonucleoside form is exposed upon selective degradation such that the latter may be used as a initiation site for template independent polymensation using polynucleotide adenylyltransferase In a similar fashion a 3'blocked end may be removed upon selective degradation yielding an initiation site for vaπous nucleotidylexotransferases Similar schemes may be devised for analyte specific detection using structure specific nucleases which cleave redundant single strands, which also cause the creation of an initiation site for template independent polymensation It may be anticipated that whenever analyte specific detection by means of template selective degradation followed by template independent polymensation is performed, the degradation may either proceed in solution or at the surface where the surface excess is measured by means of a transducer surface In another embodiment the present invention provides additional methods for the analyte specific detection of surface excess at a transducer surface by means of template independent polymensation in conjunction with template selective degradation Using the ability to control the composition of the product of a reaction involving template independent polymensation (e g a homopolymer), this product my be used to induce the degradation of specific affinity sites (1 e degradation precursors) being present at a surface, in solution or both, causing the creation of an initiation site for template independent polymensation Alternatively initiation sites may be created from the product of template independent polymensation due to a specific cleavage thereof By definition this constitutes an autocatalytic process which may be used to increase the sensitivity of the analyte detection Dependmg on the method discrete specific affinity sites having affinity for the analyte or for the product of template independent polymensation, or a mixture thereof may be immobilised to a surface in order to measure the products of said autocatalytic process by means of determining the surface excess using a surface sensitive technique

In a preferred embodiment the analyte is a nucleic acid sample, specific reaction product or a deπvative thereof which may be detected by measunng the surface excess at a transducer surface, wherein said surface excess is produced by a process involving template independent polymensation in conjunction with template selective degradation When for example the analyte is a nbonucleic acid, said analyte may be hybπdised to a sequence specific affinity site at a surface and washe Following this step the hybπdised nbonucleic acid is subjected to a reaction mixture containing polynucleotide adenylyltransferase and nbonuclease IV, the latter of which cleaves the product of template independent polymensation to yield new initiation sites for template independent polymensation The product of the reaction may be detected by means of a surface sensitive technique at a surface containing specific affinity sites for the product of template independent polymensation When the said analyte is a deoxyπbonucleic acid a similar method may be used involving DNA nucleotidylexotransferase and deoxynbonuclease IV Other methods wherein a nucleic acid may be detected using a autocatalytic reaction involving the processes of template independent polymensation and template selective degradation, but wherein reaction products remain confined to the surface where detection proceeds, may also be devised This may include the detection of a deoxyπbonucleic acid sample having an initiation site for template independent polymensation immobilised to a surface (where detection proceeds) containing discrete specific affinity sites Following specific immobilisation of the said sample, the surface may be incubated with a reaction mixture containing degradation precursors with a specific affinity for the product of template independent polymensation (said degradation precursors may thus be present in solution, alternatively they are present on the surface containing the specific affinity sites), a specific nuclease cleaving or degrading said degradation precursors upon interacting with the product of template independent polymensation and an enzyme capable of performing template independent polymensation For example, said degradation precursors may be DNA/RNA chimera which are blocked with respect to template independent polymensation, and have both DNA and RNA residues complementary to the product of template independent polymensation, said RNA residues may be degraded when interacting with said product in the presence of RNaseH, and thereby create a initiation site for template independent polymensation by means of for example DNA nucleotidylexotransferase Henceforth an appreciable analyte specific change in surface excess may be detected by means of a transducer surface using template independent polymensation in conjunction with template selective degradation when minimal quantities of the analyte are present In another embodiment analyte specific template independent polymenzation and the ability to perform continuous detection by means of transducer surface, may be used to obtain information concerning the relative sequence composition of a nucleic acid sample, specific reaction product or a deπvative thereof In particular the specific affinity sites used to immobilise the said nucleic acid sample may serve as pnmers for a DNA or RNA polymerase reaction in order to perform template dependent polymensation using said nucleic acid sample as a template Template dependent polymensation may then be earned out using different sets nucleoside tπphosphates each consisting of less than four different nucleotide bases In this manner the length of the polymeπzed fragment becomes restricted, and the extension pattern using for example 4 different sets each containing 3 different nucleoside tnphosphates becomes typical for the sequence immediately flanking the pnmer binding region Detection by means of template independent polymensation is then performed in such a way that the extension of the nucleic acid sample is more favourable than the extension of the specific affinity sites used to pπme and immobilise the nucleic acid sample, which is possible by choosing a suitable composition (e g RNA or DNA) of said specific affinity sites, template and nucleosides which may be differentiated using the relevant nucleotidylexotransferase Consequently the fragments generated by the said restπcted polymenzation may be analyzed in terms of their affinity with the surface by measunng the continuous change in surface excess, using for example equilibnum dissociation or temperature jump methods Similar methods involving restncted length polymenzation reactions may also be earned out in solution using soluble specific probes as pnmers, the product of the reaction may then be unmobilized to a transducer surface and subjected to template independent polymenzation For this particular application, the interaction of said elongated soluble nucleic acid pnmers with partially or fully complementary probes may be inferred from affinity or structural relationships, in the latter case inhibition with respect to template independent polymenzation by for example DNA nucleotidylexotransferase due to the presence of recessive ends in the unreacted pnmer-probe complex is relieved when a particular length in the restncted length polymenzation assay is exceeded It may be anticipated that the method of restncted length polymerization using soluble pnmers and predetermined nucleic acid probes will detect minor fluctuations in a target nucleic acid sequence while at the same time taking advantage of the rapid hybndization kinetics of the relatively small nucleic acid pnmers When the restncted length polymenzation proceeds from a solution based pnmer and sample a degree of amplification of the solution based reaction may be achieved by allowing several restπcted length polymerization cycles In one particular case the pnmer used in the reaction may have degradable 5' phosphate end with a flanking sequence of deoxyπbonucleoside residues approximately spanning half the sequence of the said pnmer, this region is bound by a senes of non-degradable phosphodiester bond analogues, or nbonucleoside residues succeeded with a region of nbonucleoside or deoxynbonucleoside residues constituting the 3 'end of the said pnmer When the pnmer is incubated with the sample, a 5' deoxyexonuclease (e g phage lambda induced exonuclease), a polymerase, and a restnctive length nucleoside tnphosphate mixture the elongated pnmers are dissociated from the nucleic acid sample due to the partial digestion of the annealed pnmer and the competitive annealing of an incoming new pnmer Consequently multiple restπcted length polymenzations are obtained from one sample template and as such a substantial degree of amplification may be achieved Similarly the pnmer used for the restπcted length polymenzation may contain a sequence of nbonucleoside residues which are degraded upon sequence specific association with the nucleic acid sample, when the said restncted length polymensation for example is earned out m presence of nbonuclease H Using a rather similar approach vanahons on the dideoxy sequencing method may be devised in order to be used with the methods descπbed in the current invention In this case the analyte nucleic acid may be immobilised to specific affinity sites at the surface where detection of the surface excess is possible Said specific affinity sites may serve as a pnmer for the dideoxy sequencing reaction or alternatively the immobilised analyte nucleic acid is hybπdised with a pnmer oligonucleotide which further contains initiation sites for template independent polymensation (and do not interfere with the analyte nucleic acid) The choice for using either immobilised or associated sequencing pnmers depends mainly on the nature of the template independent polymensation reaction used to detect the surface excess Preferably the products obtained after template independent polymensation are analysed using the equilibnum dissociation method as monitored by the continuous measurement of surface excess under the application of a temperature gradient The invention is further descπbed with the following examples, which are given as illustration rather than limitation of the scope of the invention

EXAMPLES Mateπals and methods

Gold electrode matenal either evaporated on silicon wafers or polymer earners were obtained from both commercial and non-commercial sources The electrodes were mounted on glass slides or plastic sheet matenal using a low temperature curable epoxy (Epotek H-54), electncal connections between the electrode matenal and the electncal winng were established using silver loaded epoxy and graphite ink, the structure was then thoroughly sealed with epoxy leaving only the desired electrode matenal exposed Structures for impedimetπc sensing involving a lnterdigitated electrode architecture were obtained from Imec (Belgium), or commercial suppliers, with lnterelectrode spacings varying between 1 and 40 μ The gold electrode surfaces were subsequently inspected and activated by potentiodynamic cycling in a three electrode cell between -0 3 Volt and 1 2 Volt (versus Hg HgSθ4 reference) until steady state voltammograms were obtained Cyclic voltammograms and other electrochemical measurements were collected using a EGG PAR 283 potentiostat galvanostat, equipped with a model 1025 FRA unit for faradayic impedance analysis Expenments involving lmpcdimetnc sensing were analyzed using a HP 4284 A LCR meter

Oligonucleotide probes and synthetic complements were purchased from Eurogentec (Belgium) and used without further puπfication All chemicals used were analytical grade or better, CofPhen)₃ ^{+ 3+} was prepared according to the method as descπbed by Dollnnore and Gillard (Dollimore, J Chem Soc Dalton, 1973, 933- 940 ) and modifications thereof Modification of the gold electrodes was earned out taking advantage of the well known gold sulfur interaction, which will be readily apparent to the person skilled in the art Bπefly 3' or 5' thiol labelled ohgonucleotides are dissolved in a high salt acidic buffer (e g 1M KII₂Pθ pH 4 5) to a final concentration of 3μM, further containing 2μM 3-mercaptoproρylmethyldιmethoxysιlane (ABCR, Germany) Freshly cleaned electrode surfaces are then fully covered with the modification solution, and left to react for 90 minutes at room temperature Subsequently the electrode surfaces are nnsed with distilled water and reacted with a solution containing 1 M KH₂PO_< and 20 μM 3-mercaptopropylmethyldimethoxysilane for a further 30 minutes at room temperature The electrodes are then finalized by immersing the surfaces in a nucleic acid hybndisation buffer (e g 200mM Na₂Sθ₄, lOmM Tπs-S0₄) at 73°C for 10 minutes, and stored in the refhgirator in the measurement buffer until use A similar approach was used for the modification of the impedimetnc sensing structures Modifications were thoroughly checked by cyclic voltammetry or impedance spectroscopy pnor to use, confinnmg successful functionalization of the surfaces Electrochemical measurements typically were performed in a low ionic strength buffer containing a affinity redox complex (e g 5mM NaC10 , 5mM Tπs HCI0₄ pH 8 and 40 to 80 μM Co(phen)₃ ^2+/3+) Typically cyclic voltammetry was used to asses the surface excess and cycling generally is performed between 03V and - 03V versus

Alternatively faradayic impedance is recorded in the same buffer as above and the signal is analyzed in terms of a pseudo-capacity reflecting the surface excess of the electroactive species, the charge transfer resistance or the total impedance Analysis of the conductivity relating to experiments earned out with impedimetnc sensing structures was inferred from the collected bode plots Bnefly the conductivity was taken at the frequencies between respectively the double layer relaxation and dielectnc relaxation frequencies, which are bound respectively by the low and high frequency capacities Also impedimetnc measurements were earned out in buffers of low ionic strength or in distilled water

Example I

Amplification of nucleic acid hybndization signal by DNA nucleotidylexotransferase, using electrochemical or impedimetnc detection Sequence selective electrode surfaces, or impedimetnc sensing structures using nucleic acid probes with a 3' modification group such as a thiol, are prepared according to the procedure as descnbed in mateπals and methods section The response of the modified electrode is checked and hybndised with a sample nucleic acid for example, a oligonucleotide complement (25-mer complementary sequence (5 nM) and 18-mer non complementary sequence (10 nM) Fιg I A, or 18-mer non complementary sequence (10 nM) alone Fig 1 B), or a polymerase chain reaction product (sample 1 Fig 1 C, sample 2 Fig 1 D), in a suitable hybndisation solution (e g 200mM Na₂S0₄ and 10 mM Tns-HCl pH 8 5) for about 60 minutes at a temperature roughly corresponding to 08 T_m (T _m is the anticipated melting temperature in that solution of the oligonuclcotidc/targct duplex, corresponding to a 50% transition of the double to single stranded conformation) Following hybndisation the electrode may be measured, or is directly incubated with the DNA nucleotidylexotransferase reaction mixture (eg 30 Units μl ' DNA nucleotidylexotransferase, 5 mM CoCl₂ 2 mM dTTP, 200mM potassium cacodylate, ₂5 mM Tns-HCl pH 66 and 1 25 mg ml ' BSA ) preferably for about 10 to 60 minutes Homopolymenc or copolymer tails of varying composition may be obtained by using a suitable metal activation factor (Kato et al , (1967), J Biol Chem 242, 2780-2789) in the presence of the relevant nucleoside tπphosphates In this way the 3' end of the hybπdized nucleic acid sample is extended, creating a surface excess which may be addressed using for example cyclic voltammetry in the presence of affinity redox probes, or measurement of the surface or localised conductivity

Details relating to polymerase chain reaction product Target amplified Human Immunodeficiency Virus (HIV) reverse transcπptase fragment, 571 base-pairs

The target was amplified at Innogenetics Research Labs according to methods known to the art, for convenience one phosphorylated pnmer was used allowing the generation of single stranded amphcon by means of phage lambda exonuclease Targets further are used as received and diluted in the hybndisation buffer (1/10) Different samples (sample 1 and sample 2) are used corresponding to vanations in the target sequence, and measured with respect to the nucleic acid probe at the electrode surface

Sequence probe 5' ATA GAG GAA CTG AGA C

(Partial) sequence sample 1 3' TAT CTC CTT GAC I CT G

(Partial) sequence sample 2 3' TAT CTC CTC AAC TCT T

Example II

Detection of mismatches using continuous electrochemical momtonng of surface excess, induced by analyte specific template independent polymensation

Sequence selective electrodes (using a 25-mer oligonucleotide having a 3'- thiol moiety) are prepared as descnbed in the matenal and methods section Following modification the surfaces are hybπdised under low stnngency conditions with sample ohgonucleotides which are either fully or partially complementary to the sequence immobilized at the electrode surface (see below) The electrodes are subsequently subjected to template independent polymensation by DNA nucleotidylexotransferase usmg thymidme residues After a bπef πnsing step in hybndization solution the electrodes are analyzed using either the equilibrium dissociation or temperature jump method The response of the electrodes to thermal equilibnum dissociation (figure 2A) or a temperature jump (at 52 8°C) (figure 2B) are reconstructed from the integrated cuπents as obtained by continuous cyclic voltammetry

Sample ohgonucleotides used

Fully complementary 5' TAG ΛTG CTC GCΛ ACC ACT ΛTC CΛG T Single mismatch 5' TAG ATG CTC GCA CCC ACT ATC CAG T Example m

Heterogeneous surfaces

Sequence selective electrode surfaces are prepared as descπbed in the matenals and methods section The response of the electrode is checked for modification, and challenged with sample or a specific nucleic acid reaction product In the present example electrode surfaces are incubated with respectively 1 -mer and 25-mer complementary ohgonucleotides and mixtures thereof Following hybndisation, the electrode surface is subjected to template independent polymensation using DNA nucleotidylexotransferase Subsequently a temperature gradient (for equilibnum dissociation, see Fig 3A and 3B) or jump is applied to the transducer surface, while the surface excess is continuously monitored using for example cyclic voltammetry Sample ohgonucleotides used

25-mer 5' TAG ATG CTC GCA ACC ACT ATC CAG T

18-mer 5 ' TC GCA ACC ACT ATC CAG T

Example IV Determination of sequence dependent restπcted polymenzation length using dynamic electrochemical monitoπng of surface excess

Nucleic acid probe modified electrode surfaces are challenged with a nucleic acid pnmer which satisfies particular pruning cntena known by those skilled in the art Complementary nucleotides are incorporated using a polymerase such as DNA polymerase I klenow fragment in presence of a restπcting nucleotide mixture (0 ImM each dN 1 P, 004 U μl ' Klenow fragment, 50 mM I ns-HCl pH 7 5, 1 OniM MgCl₂) In the present example only 3 of the four nucleotides are used, and as such polymenzation is terminated when no matching nucleotide is present When the four possible combinations of the 3 nucleotides are used in conjunction with a pπmcr of a primary specificity a particular extension pattern is obtained Alternatively the extension reaction is earned out in solution in which a nucleic acid sample is present, and the extended pnmers are hybndized to the electrode surface Following extension and hybndization the nucleic acid product is amplified by template independent polymerization, when DNA is used this is readily accomplished with DNA nucleotidylexotransferase The extension length is subsequently analyzed by voltammetnc analysis of the thermally induced dissociation behaviour The sequence outcomes are outlined below, and the temperature jump responses for the positive reactions are given in Fig 4 Note that due to the limited length of the pnmer the extensions in presence of ATG and CTG react weakly due to the lower melting temperature and the properties of DNA nucleotidylexotransferase, which has decreased affininity for regressive ends as well as steric factors The latter properties may be used to direct the outcome of the reaction and hence simplify the analysis

Electrode(Au)-SH-Spacer-ATC TAC GAG CGT TGG TGA TAG CTC A5' 5 pnmer 5'TAG ATG CTC GCA3'

ATG extension A3'

CTG extension

ACG extension ACC AC3 '

ACT extension ACC ACT ATC A3 '

10 Example V

Detection of template dependent RNase H activity, using template independent polymenzation In order to demonstrate the activity of a specific endonuclease, sample sequences are incubated in conjunction with chimeπc probes and RNase H Bπefly 0,1 μM sample sequence and 0,5 μM chimenc probe are incubated in plastic tubes in the presence of RNase H in a buffer (e g Tπs HC1 pH 7,5) containing 8 to 10 mM MgCl₂ at

15 37°C Following incubation the reactions are stopped by adding EDTA up to a concentration which equals or is greater than the MgCl₂ concentration and addmg Na₂S0₄ to a final concentration of 200 mM (these steps roughly double the oπginal volume) The reactions are subsequently heated to 75°C for 5 minutes, and put on ice until further analysis Following the incubation the reaction products are hybndized under low stπngency conditions to a transducer surface (e g a gold electrode) which is modified with probes which are partially or fully

20 complementary to the chimeπc probe, and are not viable to template independent polymerization (e g carrying a 3' thiol moiety) The transducer surfaces are now subjected to template independent polymenzation using DNA nucleotidylexotransferase The sequences corresponding to the legend in Tig 5 are

Chimenc probe 5' TAG ATG CTC GCA ACC acu aug gAG T

25 Complementary template 3' ATC TAC GAG CGT TGG TGA TAC CTC A

Partially complementary template 3' ATC TAC GAG CGT TGG TGA TAG GT C A Non complementary template 3' AGC GGC GAC GTG ACA CT1 CGA G

Upper case deoxyπbonucleoside residue, Lower case nbonucleoside residue Example VI

Real time momtonng of nuclease activity

Sequence selective electrode surfaces having 3'thιol moiety are prepared as descπbed in the mateπals and methods section Subsequently complementary deoxyohgonucleotide is immobilized to the electrode surfaces and the obtained surface is subjected to template mdependent polymenzation using DNA nucleotidylexotransferase The obtamed electrode surface may now be examined for the activity of a particular nuclease In this example the electrode is incubated with DNase I (2 Units / 100 μl), in a buffer containing 10 mM MgCI₂, 50 mM Tns-HCl pH 76 and 5 mM Fe(CN)6³ '* l"^e electrode surface is subsequently continuously cycled in a conventional 3 electrode cell between 05 V and -07 V versus Ag/AgCI not compensated for IR drop thermostattcd at 37°C (see Fig 6)

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Claims

1 A method for detecting the presence of an analyte in a sample by means of modifying and measunng the surface excess wherein the said method compnses a) increasing surface excess by means of template independent polymenzation or b) decreasing the surface excess by means of template selective degradation

2 A method according to claim 1 compnsing a) Reacting the sample with a reaction mixture that causes template independent polymensation, by means of addressing suitable initiation sites present within, or specifically associated with, the analyte b) Assessing the template independent polymensation, by means of measunng the surface excess using a surface sensitive technique

3 A method according to claims I and 2 compnsing a) Binding the analyte to a surface containing discrete specific affinity sites that are selective for the said analyte b) Reacting the said bound analyte with a reaction mixture that causes template independent polymensation, by means of addressing suitable initiation sites present within, or specifically associated with, the analyte c) Assessing the template independent polymensation, by means of measunng the surface excess at the surface containing said specific affinity sites using a surface sensitive technique

A A method according to claims 1 or 2 wherein the template independent polymensation products compπse a functional amplification, and wherein the surface excess is determined continuously as a function of a physical or chemical gradient

5 A method according to claims 2 or 3 wherein the analyte is a nucleic acid, or a specific reaction product or a denvative thereof, and wherein said affinity sites are oligonucleotide probes or chemical analogues thereof 6 A method according to any of claims 1 to 5 wherein said template independent polymensation compnses reactions catalysed by glycosyltransferases or nucleotidylexotransferases

7 A method according to any of claims I to 5 wherein said suitable initiation site constitutes a free 3' OH group or a nonreducing N-OH end

₈ A method according to any of claims 1 to 7 wherein said initiation sites for template independent polymensation are created by a template selective degradation

9 A method according to any of claims 2 to 8 wherein the said template independent polymensation proceeds from initiation sites present on said analyte

10 A method according to any of claims 2 to 8 wherein the said template independent polymensation proceeds from initiation sites present on said specific binding sites

11 A method according to any of claims 9 to 10 wherein said template selective degradation results in a free 3' OH group

12 A method according to any of claims 1 to 11 wherein said template independent polymensation yields products that result in secondary initiation sites for template independent polymensation

13 A method according to claim 12 wherein said secondary initiation sites arc created through a branching enzyme

1₄ A method according to claim 12 wherein said secondary initiation sites are created after binding of said products of template independent polymensation to discrete affinity sites which are selective for said products of template independent polymensation

15 A method according to claim 14 wherein said secondary initiation sites for template independent polymensation are created by a template selective degradation 16 A method according to claim 1 compnsing a) Reacting the sample with a reaction mixture that causes template selective degradation, by means of addressing suitable degradation precursors present within the analyte, or specifically associated with, the analyte b) Assessing the template selective degradation, by means of measunng the surface excess using a surface sensitive technique

17 A method according to claim 16 comprising a) Binding the analyte to a surface containing discrete specific affinity sites that are selective for the said analyte b) Reacting the said bound analyte with a reaction mixture that causes template selective degradation, by means of addressing suitable degradation precursors present within, or specifically associated with, the analyte c) Assessing the template selective degradation, by means of measunng the surface excess at the surface containing said specific affinity sites using a surface sensitive technique

18 A method according to claims 16 or 17 wherein the outcome of template selective degradation is measured as a function of a physical or chemical gradient

19 A method according to any of claims 16 to 18 wherein the analyte is a nucleic acid, or a specific reaction product or a denvative thereof, and wherem said associated degradation precursor is a oligonucleotide probe or a chemical analogue thereof

20 A method according to any of claims 16 to 19 wherein said template selective degradation results in a secondary initiation site for a secondary template selective degradation

21 A method according to any of clauns 16 to 20 wherein the said template selective degradation proceeds from initiation sites present on said analyte 22 A method according to any of claims 16 to 20 wherein the said template selective degradation proceeds from initiation sites present on said specific binding sites or on said associated degradation precursor

23 A method according to any of claims 16 to 22 wherein said template selective degradation compnses a endonucieolytic cleavage, and said secondary template selective degradation compnses a exonucleolyhc degradation

24 A method accordmg to any of claims 16 to 23 wherem said template selective degradation compnses a endonucieolytic cleavage that creates a free 5' phosphate group that is an initiation site for the said secondary template selective degradation by lambda exonuclease

25 A method according to any of claims 8 to 24 wherein said selective degradation is catalysed by lambda exonuclease, T7 gene 6 product, DNA polymerase, Rnase H, or exonuclease 3 or a combination thereof

26 A method according to any of claims I to 25 wherein said surface sensitive technique compnses the use of faradayic techniques or impedimetnc techniques

27 A method according to any of claims 2 to 15 and 17 to 26 wherein said discrete specific affinity sites are immobilised directly unto the surface of the electrodes used for said faradayic or impedimetnc techniques or in the peπ-electrodic spaces