WO2001017670A1

WO2001017670A1 - Matrices of probes and their preparation

Info

Publication number: WO2001017670A1
Application number: PCT/IL1999/000496
Authority: WO
Inventors: Jean-Paul Lellouche; Robert S. Marks
Original assignee: Ben-Gurion University Of The Negev
Priority date: 1999-09-09
Filing date: 1999-09-09
Publication date: 2001-03-15
Also published as: AU5646899A; EP1218098A1

Abstract

A matrix for a plurality of probe species, each of which is a first member of a pair-forming group, for binding of a target which is the second member of the pair-forming group, wherein each probe species is confined to a separate and predetermined location in the matrix. The matrix comprises a conductive solid support, on which there is immobilized a conductive film, comprising a polymer or co-polymer composed of electropolymerizable monomers. The conductive film has an inner layer (7), which is in direct contact with the solid support, and comprises probe-free monomers, and an outer layer extending (8) therefrom, which comprises probe-linked monomers.

Description

MATRICES OF PROBES AND THEIR PREPARATION

FIELD OF THE INVENTION

This invention relates to matrices of probes on solid supports capable of binding targets present in a sample, methods for their preparation and methods for the detection of targets in a sample using these matrices.

BACKGROUND OF THE INVENTION

It is well known that biological molecules such as nucleic acids, polysaccharides, proteins, glycoproteins and lectins are capable of binding to other recognition molecules with a very high specificity. Most techniques for the detection and monitoring of a biological analyte termed "target" molecule in a sample take advantage of this ability of a target molecule to specifically bind to a second molecule termed "probe ".

The detection and monitoring of target molecules is often required in the clinical analysis of physiological fluids, for example, in detecting infectious pathogens, an elicited immune response, an oligonucleotide indicating infection or a genetic mutation, and the like. Such monitoring is also necessary in various other clinical, diagnostic and experimental modalities such as epitope mapping, the development of chemical analogs, sequencing by hybridization, as well as the synthesis of biomacromolecules by combinatorial techniques. A large number of these techniques, are based on the binding of the target biological molecule to a probe immobilized on a solid support. In these "solid support based detection techniques " a number of different probes are immobilized, each probe being immobilized at a predetermined location on the matrix. Binding of a target molecule to a particular location on the matrix identifies the target molecule as being capable of binding to the probe immobilized at that location. Attempts have been made to increase the density of the probe arrays so as to accommodate a large number of different probes in a relatively small area. This is advantageous for example, when screening samples containing a large number of different probes so as to accommodate a large number of different targets.

In one method, starter DNA oligonucleotides were attached to glass slides (Southern, E.M. Nuc. Acids. Res., 22: 1368-1373, 1994). In subsequent synthetic steps, these oligonucleotides were elongated by presenting nucleotides to defined areas on the slides. After the synthesis is complete, labeled complementary probes were hybridized to the target DNA on the slide. Similarly, arrays of DNA probes were synthesized on aminated polypropylene film using a controlled photodeprotection chemistry and photoprotected N-acyl-deoxynucleoside phosphoramidites (Matson, R., Anal. Biochem., 224: 110-116, 1995). Arrays of peptides have been synthesized on cellulose sheets (Frank, R., Tetrahedron, 48:9217-9232, 1992). Methods which do not include direct synthesis on the support have also been developed, which involve the attachment of PCR products to silylated glass slides (Schena, M., PNAS, 93: 10614-10619, 1996). Thus, attempts have been made to increase the density of DNA or peptide probes on the matrix by mmiatiirizing the size of the individual sites in the array.

The probes may be arranged in any desired array on the solid support using a variety of techniques including: use of light to direct the combinatorial chemical synthesis of biopolymers on a solid support; -> -

embedding of DNA sequences on a gel coated chip (Edginton Bio/Technology, 12:468-471, 1994; Yershov et al., PNAS, 93:4913-4918, 1996); micropatterning lipid bilayers onto solid supports (Groves et al., Science, 275:651-653, 1997); in situ synthesis of oligonucleotides using a synthetic mask; as well as deposition of probes on porous sheets such as nitrocellulose sheets.

One method of immobilizing oligonucleotides onto a solid support is by the electrochemically directed synthesis of oligonucleotides on a solid support (Livache et al, Synthetic Metals, 71:2143-2146, 1995). This publication describes a manner of copolymerizing pyrrole, and pyrrole covalently linked to oligonucleotides via a spacer, giving rise to a solid copolymer film on the support formed by an oligonucleotide linked pyrrole chain. Such a construction has the disadvantage of a rapid loss of the stability of the immobilized polymeric film present on the electrodes. It would have been highly desirable to provide a matrix of probes contained in a conductive polymer film which would show good chemical and physical stability.

The matrix of oligonucleotides according to the publication of Livache et al, is prepared on a solid support, comprising a two-dimensional array of electrodes, wherein each electrode can be activated individually. Preparing the matrix entails exposing the solid support to a first solution of nucleotide-linked pyrroles, and then activating a particular electrode, so that pyrroles undergo functionalized electropolymerization on this electrode and are thus immobilized on the electrode. The matrix is then thoroughly rinsed, and the process is successively repeated, each time electropolymerizing a different nucleotide-linked pyrrole onto a different electrode. Obviously, preparation of a matrix containing hundreds of different probes is extremely laborious, time consuming and expensive. There is therefore a need in the art for a method for producing a matrix of probes which is efficient, rapid and inexpensive, and in which the matrix of probes show good chemical and physical stability.

GLOSSARY The following terms may be used throughout the specification.

Pair forming group - two molecules, usually at least one of which is from a biological source, capable of binding with high specificity to each other via non-covalent bonds. Examples of pair forming groups are: two complementary DNA strands, two complementary RNA strands, complementary DNA and RNA strands, PNA and DNA strands, an enzyme and its substrate, an antibody and its antigen (the latter may be non-biological such as TNT and its corresponding antibody), a receptor and its ligand, avidin and streptavidih a lectin and its ligand, streptavidin and biotin, an antibody and a bacterium or virion, bacteriophage, virus and the like.

Affinity binding - the non-covalent binding of high specificity between two members of a pair forming group.

Probe - a first member of a pair forming group which has been, or is to be immobilized and shows a specific binding reaction.

Target - a second member of a pair forming group which is diffusible. This teπn may refer to entities obtained from a biological or non-biological source, however, usually the term refers to biological entities. It may concern biological molecules such as protein, DNA, RNA, hormones, enzymes, receptors, ligands, polysaccharides and the like as well as molecules which are laboratory produced and which are intended to be similar to biological molecules obtained from a natural source, such as laboratory produced and synthetic peptides, or oligonucleotides, antibiotics and the like. A targeting may also refer to a complex of several molecules, to cells from unicellular or multicellular organisms, as well as to cell organelles. The target may also be non-biological entities such as various organic or non-organic molecules for example, drugs, toxic materials, contaminants, TNT and the like. The probe and target may be interchanged in various applications.

Sample - a medium, usually liquid, presumed to contain targets of interest. The target in this case serves as the analyte.

Species - one type of entity (which may be targets or probes) which is distinguished from other types of similar entities, for example, a species of probes or a species of targets is a specific sequence of DNA, a specific protein or polypeptide, one type of monoclonal antibody and the like. A specific species of entities is capable of forming a pair forming group with another species of entities.

Matrix — a predetermined spatial arrangement of probe species present on a solid support (see below), where all probes of the same species are confined to a separate, specific and predetermined location in the matrix.

Location - a specific distinct spatial area in the matrix.

Pattern - the two dimensional arrangement of probes either present in a matrix or in an array of containers, pins or pipetors. A desired pattern of probes is created for example by depositing the probes on the solid support by an array of micropipetors or ins. Region - a specific distinct spatial area in the pattern, for example, one region are probes of the same species located in an area corresponding to one pipetor.

Solid support - a surface which carries the matrix of probes.

Uniform conductive solid support - a solid support essentially all of whose surface is conductive.

Non-uniform conductive solid support - a solid support comprising discrete conductive regions separated by non-conductive regions. The conductive regions may be an array of electrodes of a grid of conductive wires.

Electropolymerizable monomers - molecules capable of polymerizing when subjected to an oxidative electropolymerization in specific conditions of voltage and current.

Probe-free layer - a mono- or multi- layer of electropolymerizable monomers not linked to probes, present directly onto a solid support, in accordance with a preferred embodiment of the invention said layer is a monolayer. The probe-free layer forms the "inner-layer" (see below).

Probe-containing layer - a layer (which may be mono- or multi- layer) of electropolymerizable monomers at least some of which being linked to probes and adsorbed either directly onto a solid support or onto a probe-free layer. The probe-containing layer forms the "outer layer" (see below).

Conductive film - a polymeric or co-polymeric film composed of electropolymerizable monomers present on the solid support. The conductive film may comprise either only said probe containing layer or both the probe free layer and the probe containing layer.

Inner layer - the layer of the conductive film which is in direct contact with the solid support.

Outer layer - the layer of the conductive film which is on said inner layer and in contact with the environment, for example with the sample.

Spacer - a chemical moiety linking a conductive film to a solid support.

Imprinting - the process of depositing probes onto a solid support so as to form a desired pattern.

Epitaxial growth - the growth on a substrate having an arrayed construct on another arranged substance that mimics the orientation of the substrate.

Imprinting - the simultaneous deposition and immobilization of a plurality of probes on a solid support.

SUMMARY OF THE INVENTION

By a first aspect, the present invention is based on the realization that matrices of probes contained in a conductive film which is present on a solid support, show improved chemical and physical properties if the probe-containing layer is not bound directly to the conductive solid support, but via an mtervening probe-free inner layer. The inner probe-free layer, enhances the mechanical and chemical stability of the matrix by a multi-podant like attachment of the probe containing films, (for example via quasi-covalent SH-gold interactions) onto the corresponding films. This construction increased strongly the mechanical and chemical stabilities of each film and, so as to eradicate probe contamination by thiol exchange inside the monolayers. In addition such constructs improve the accessibility of the targets to the probes since the targets have better accessibility to the probes as ₅ will be explained hereinafter.

Thus, by the first aspect, the present invention concerns a matrix of a plurality of probe species, each probe being a first member of a pair-foπning group for binding of a target which is the second member of the pair forming group; wherein each probe species is confined to a separate, predetermined _l o location in the matrix, the matrix comprising: a conductive solid support on which there is immobilized a conductive film comprising a polymer or co-polymer composed of electropolymerizable monomers, having an inner layer which is in direct contact with the solid support said inner layer comprises probe-free monomers, and an outer layer

₁5 extending therefrom which comprises probe-linked monomers.

The matrix of the present invention comprises a plurality of different probe species, for example, a plurality of different oligonucleotides capable of hybridizing with a plurality of complementary target oligonucleotides in a sample: a plurality of antibodies capable of binding to a plurality of target 0 antigens present in a sample and vice versa; a plurality of receptors capable of binding a plurality of target ligands in a sample, a plurality of antibodies, microorganisms and the like.

The matrix is intended to bind a plurality of target species present in a sample. The binding may be for the purpose of detection of the targets in the 5 sample; for the purpose of separation/isolation of the targets from the sample so as to obtain purified targets. The binding may also be used for other modalities which require specific binding of targets to probes, such as for the purpose of synthesis of macromolecules by combinatorial chemistry, for the purpose of sequencing by hybridization (SBA) of long DNA stretches and the like.

The matrix comprises a conductive solid support which carries an immobilized conductive film. The conductive film is composed of two distinct layers: a layer, preferably a monolayer, made of probe-free electropolymerizable monomers which is in contact with a uniform or non-uniform conductive solid support and is immobilized to the support. Onto this probe-free layer, a second outer layer of electropolymerizable monomers, some, or all of which are linked to probes is made to grow epitaxially. This construction is different from prior art constructions wherein all of the conductive film is composed of probe-linked monomers entrapped in the film and/or present on its surface. The fact that the probe-containing layer is organized due to the epitaxial growth defined by the underlaying probe-free layer increases the physical stability of the constructive film on the solid support, increases its chemical stability to solvent and chemicals and improves the accessibility of the probes to the external environment. It is preferable that the probe-free layer is a monolayer. It is also preferable that the probe-containing layer is a monolayer.

The conductive solid support on which the matrix rests may be a uniform conductive solid support, i.e. essentially the entire surface being conductive, without non-conductive regions.

Alternatively, the solid support may comprise an array of separate electrodes, or a grid of intersecting conductive wires and these two forms will hereinafter be referred to as "non-uniformly conductive solid supports" . Each probe species (for example a specific oligonucleotide), can form a complex with a specific target species (in this example, the complementary oligonucleotide). The matrix is formed in such a manner wherein each probe species is confined to a specific predetermined and known location in the matrix. Thus, determining the locations at which a target has bound, identifies the target as one capable of binding the probe at that location.

By one embodiment, the conductive solid support has a uniform or non-uniform conductive surface made of a metal such as gold, silver, tin, copper, tin dioxide, indium tin oxide present on an insulated support like glass, various plastics and silicon dixoide.

According to this embodiment, the probe-free layer is immobilized onto the solid support by thiol or disulfide links, by quasi-covalent linkage onto metal surfaces, or by using silane chemistry onto specific oxides like indium tin oxide. The probe-free layer may be attached directly to the thiol or

-disulfides, which are chemically adsorbed onto the metallic conductive surface, or alternatively may be spaced from the thiols or disulfide functions by a suitable spacer. Such a spacer, should be of a length and chemical composition, so as to allow electron transfer from the conductive solid support to the probe-free layer when subjected to an electric field. Examples of such spacers are hydrocarbon chains from 2 angstroms to longer than 20 angstroms with all kinds of functions compatible with the electrooxidation of electropolymerized monomers as well as sugar-containing peptide chains.

The monomers comprising the conductive film, those of the probe-containing layer as well as those in the probe-free layer, can be any electropolymerizable monomer, i.e. monomers which polymerize in the presence of an electric field. Examples of such monomers are pyrrole, dipyrrole, thiophene, anilines, thiopenes, furans, dimers and rrimers of pyrroles as well as others specified in WO 94/22889 incorporated herein by reference. Each layer in the matrix may be made of more than one monomer type, i.e., it may be a co-polymer comprising at least two different types of monomers, for example pyrroles and dipyrroles.

The probe-free layer, and the probe-containing layer constituting the conductive film, may have different compositions, i.e. one layer is made of one or more types of mononers and the other made of other types of monomers.

The probe is immobilized in the probe-containing layer, by producing a said layer through polymerization of probe-linked monomers. In accordance with the invention, not all of the monomer subunits comprising the probe-containing layer, need to be probe-linked, but preferably at least 10%, preferably at least 50% and most preferably at least 90% are probe-linked.

The probe containing layer grows epitaxially on the probe-free layer. This is contrary to prior art where the probes (contained in the conductive film) are not oriented but rather merely included, or entrapped in the matrix of electropolymerized film. The epitaxial growth according to the present invention improves the presentation of the probes to the target, increases the accessibility and decreases the steric hindrance.

The probes may be linked to the electropolymerizable monomers by various types of bonds, according to the nature of the probe and the monomer.

Typically the binding is strong, i.e. either a covalent bond or a high affinity non-covalent bond such as exists between avidin/streptavidin and biotin. For example, where the probe is an oligonucleotide, it may be covalently linked to a pyrrole monomer, in accordance with the teaching of Livache et al. (Synthetic Metals 71:2143-2146 (1995)). Where the probe is a peptide, it can be functionalized by incorporating a terminal electropolymerizable pyrrole residue. For example, an intermediate N-substituted pyrrole residue can be synthesized by opening a suitable anhydride using an N-(2-aminoethyl) pyrrole. The pyrrole functionalized peptides can be synthesized by Fmoc Merrified solid-phase peptide synthesis with a starting glycine residue allowing cleavage using acidic or nucleophilic conditions. The probes should include either a C-terminal glycine. or glycine amide in order to bind them to the pyrrole. Antibodies, proteins, oligosaccharides and polysaccharides can also be bound to the monomers by known chemical methodologies.

By another alternative, pyrrole-modified or pyrrole-clusterized streptavidin can be used for attachment of biotinylated probes onto electrode surfaces.

In accordance with another embodiment of the invention, the solid support is made of glass functionalized by hydroxyl groups (for example by indium tin oxide) for regular functionalization by silane surface chemistry. A probe-free layer is immobilized onto the glass, by the use of silane. The immobilization through silane may be direct, or through the same types of spacers as described above.

The present invention further concerns a kit for the detection of the presence of a target in the sample wherein when a pair foiming group is formed a detectable signal is produced indicating the location in the matrix of the binding of a target to a probe. For example, the signal may be fluorescently produced by rhodamine, fluoresceine, cyanine dyes, bioluminescence or chemiluminescence-producing enzymes. Reagents capable of producing a detectable signal are included in the kit and may be, for example, a labeled antibody directed against a specific target. For example, the matrix of probes may comprise a plurality of antibody species for the detection of different cell species. In this case it is possible to detect a cell bound to a probe using a labeled antibody directed to that cell species (for example, where all the cells are from a particular microbial source and the antibody is directed against that microbe). The species of the cell is then concluded from the location on the matrix where it has bound. The kit of the invention in this example includes both the matrix and the labeled antibody, as well as any other reagents necessary for production of the signal.

The present invention further concerns a system for the detection of the presence of targets in a sample, comprising the above kit, as well as a detector, capable of detecting said signal. Examples of such detectors where the signal is optical (e.g. a dye, or a fluorescent label) is a CCD camera, preferably attached to an analog to digital converter and to a computer for further analysis of the signal. Other detectors are photomultiplier type arrays, photodiode arrays, optical fiber bundles and the like.

Alternatively, for example where the conductive support is non uniform, it is possible to detect the presence of the pair forming group, even without said reagents capable of giving a detectable signal, since the mere binding of the target to the probe modifies the local electrical properties (conductance, or capacitance) of the matrix. Thus, if each probe species is immobilized at a location which is electrically isolated from the locations of the other probe species (for example each probe species is present on a single electrode, or is present at the intersection of two conductive wires in a grid), the modified electrode identifies the probe to which the target has bound. In such a case, the system of the invention only comprises the matrix of the invention and a detector being a device capable of identifying changes in conductance/capacitance due to the formation of a complex at a specific location.

By another aspect, the present invention concerns a method for the production of a matrix of the type specified in which each of a plurality of probe species is immobilized in a separate, predetermined location in the matrix.

The method of the present invention, is based on the realization, that it is possible to produce such a matrix by simultaneously functionalizing a plurality of different probe-linked monomers arranged in a predetermined pattern on a solid support. The method of the present invention, thus obviates the need to sequentially expose the solid support to different probe-linked monomers, wherein in each exposure only one location is electrically functionalized, in order to polymerize the probe-linked monomers only in that location.

Thus, the present invention concerns a method for the production of a matrix of a plurality of probe species, each probe being a first member of a pair forming group for binding of a target which is the second member of the pair forming group; wherein each probe species is confined to a separate predetermined location in the matrix, the method comprising:

(i) providing a conductive solid support;

(ii) imprinting a pattern of electropolymerizable monomers by deposition on the solid support, at least some of the monomers being linked to the probes; said pattern defining a plurality of locations on said solid support, each location comprising monomers with a single probe species; and

(iii) passing electric current, simultaneously, through all of said locations, thereby causing polymerization of said monomers.

In accordance with the method of the present invention, the confinement of each probe species to a specific location on the solid support, is not carried out as is done by prior art techniques. In prior art techniques, confinement of the probe species in the matrix is carried out sequentially. The solid support is indiscriminently brought into contact with monomers bound to a first probe species, and the support is electrically activated at a selected location so as to induce the electropolymerization of these monomers at that location. Unreacted monomers are then removed, and the process is repeated for each probe species, so that each probe species becomes confined to a different, known location on the support. It is clear that this procedure is laborious, expensive and time consuming. In contrast to this, in accordance with the method of the present invention, the precise spatial arrangement of the probe on the solid support is produced by using imprinting techniques well known in the art, which simultaneously deposit a plurality of species of probe-linked monomers in specific separate, predetermined locations (i.e. so as to form a specific pattern) on the matrix. All species of probe-linked monomers are then made to polymerize simultaneously, while confined to their predetermined location (by an earlier deposition) by applying an electric field to the entire support.

The imprinting, may be carried out, for example by using a fixed array of containers, corresponding in arrangement to a fixed array of pins, or pipetors, wherein each container holds a solution of electropolymerizable monomers linked to a single probe species. Each pin or pipetor is simultaneously loaded with a small volume of its corresponding solution. The array of pins, or pipetors is then transferred to the solid substrate, and each drop of the solution is deposited in a precise location onto the solid support. Thus monomer-linked probes which were present in a specific position in the array, for example, in one container corresponding to one pipetor, are then deposited in a specific, known location on the solid support. The location of each probe-linked monomer species on the solid support thus corresponds to its location in the array of containers. The electropolymerizable probe is deposited onto the solid support, or onto a layer present on the solid support as will be explained hereinafter, so that it essentially remains at its place of deposition. All of the locations on which the probe-containing monomers are deposited are then simultaneously functionalized by electropolymerization induced by an electrical field.

The solid support may be uniformly conductive, or non-uniformly conductive as described above. In the latter case, each probe species, should be deposited on a separate electro-conductive region, for example, directly on the electrode or on the grid. In the case of a uniform conductive solid support electric current is passed through all the support simultaneously. In the case of a non-uniform conductive solid support all the conductive regions (i.e., array of electrodes or grid of conductive wires ) are functionalized simultaneously. It is preferable, in accordance with the method of the present invention, that between steps (i) and (ii) above, to add a step of depositing on said solid support, a layer, preferably a monolayer of probe-free monomers capable of electropolymerization. Said layer is immobilized onto the conductive solid support as described above, and provides the layer on which the probe-containing polymer is to be epitaxially grown. Where the solid support is non-uniform, the probe-free monolayer should be present on the conductive regions of the support. Onto said probe-free layer, said pattern of probe-linked electropolymerizable monomers as described above is deposited. Upon fimctionalization by an oxidative electropolymerization induced by an electric field, the probe-linked monomers polymerize so as to epitaxially grow from the probe-free layer and give rise to a probe-contaming layer on the probe-free layer which is in contact with the solid support.

The solid support, electropolymerizable probe-free monomers, the electropolymerizable probe-containing monomers, the . manner of the formulation of the layers on the support, the spacers, the manner of probe binding to the monomers are as described above in connection with the matrix.

The present invention further concerns a method for detecting the presence of a plurality of target species in a sample, by binding to a plurality of probe species, the probes being a first member of a pair-forming group, and the target being a second member of a pair- forming group, comprising the steps of:

(i) providing a matrix of a plurality of probe species as described above, wherein each probe species is confined to a separate predetermined location in the matrix; (ii) contacting the matrix with the sample under conditions allowing specific binding of the probe to the target; (iii) deteπnining in which locations on the matrix a pair-forming group is formed, said location identifying probe target bound thereby indicating the specific target thereto.

In the method of the present invention, the sample is presented to the matrix of the invention under conditions allowing binding between targets and probes. Examples of samples are blood samples, urine samples, food samples, water samples, etc. At this stage, various manipulations can be carried out in order to increase the specificity or affinity of said binding, such as rinsing, competitive binding as carried out in various immunoassays, raising the temperature in order to anneal imperfect complementary nucleic acid sequences and the like.

Once target binding to the probes has been completed, it is possible to determine the locations to which various targets have bound. This may be carried out by using reagents for producing a detectable signal including binding of a target to a probe at a particular location, and optionally also a detector for detecting the signal as described above, in connection with the kit and the system of the invention.

The identification of each target species relies on the fact that each probe species is confined to a separate, known, location in the matrix. Thus the location at which a target binds to the matrix identifies the target as one capable of binding to the probe confined to that location.

The method of detection in accordance with the present invention may be used for a variety of purposes wherein detection of more than one target species is necessary such as for multiplexing detection (for example of a plurality of infections, microorganisms in a sample), sequencing by hybridization, epitope mapping, synthesis of macromolecules by combinatorial chemistry, determining DNA/RNA sequences antigen/antigens or protein linkages. The present invention will now bo illustrated with, reference to some non specific drawings and examples.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carried out in practice, a preferred embodiment will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

Fig. 1 shows a perspective (A) and side (B) views of a matrix on a solid support according to one embodiment of the invention; Fig. 2. shows the preparation of thiolated peptide probes;

Figs. 3A and 3B show pyrrole containing probe synthesis and then e lectrop o ym eriza tio ;

Fig. 4 shows the synthesis of a pyrrole-phosphoramidite building block; Fig. 5 shows a phospholipid bilayer matrix on a glass support.

Fig. 6 shows the formation of a probe-free layer of silane on a silica support.

Fig. 7 shows an imprinter, used to deposit an array of probes on a solid support.

EXAMPLES

Example I Preparation of a matrix of probes on a non-uniform metallic solid support

Reference is now made to Fig. 1. A wafer 1 is used as a solid support. An array 2 of microelectrodes 3a, 3b, 3c, etc. is furmed on the support by sputtering a 0.1 μm layer of Cr onto the water 4, followed by sputtering of 0.1 μm-0.5 μm of gold 5. The array 2 of the electrodes 3. connections to the pads and the contact pads are etched in the gold layer by photolithography or proper wet chemistry. A 0.5 μm Si0₂ passivation layer 6 is then deposited on the wafer. The Si0₂ is removed from on top of the gold electrodes using a second photolithography step or dry chemistry.

The gold microelectrodes in the array are simultaneously protected in one chemisorption reaction using non-functionalized octadecanethiol, nCisHs SH in ethanol, thus forming a stable and compact self-assembled monolayer 7 on each electrode. A location-specific electrochemical reduction desorption of the non-functionalized monolayer by voltage cycling between -1.0 V and -1.5 V vs Ag/AgCl/3 M KC1 in a buffered aqueous medium at pH 7.0-7.3, is followed by a fimctionalization of the same gold working microelectrode (3a) with a molecular probe attached to lipoic acid thiol moieties 8, while the other sites remain protected. A closely-packed self-assembled probe-containing layer is thus formed on the microelectrode but separated from it by a probe-free layer composed of the lipoic acid thiol moieties. This self-assembled matrix is highly stable due to the formation of polar amide-derived hydrogen bonds. A repetitive sequence of the above steps using the electronically-active matrix, provides a two- dimensional, well-defined array of site-selective and covalently-linked peptide probe pattern.

Example 2 Preparation of probe-linked electropolymerizable monomers

Several synthetic Cholera toxin B subunit peptides CTP 2, CTP 3, CTP 4 and CTP 5 are synthesized by classical Merrifield solid-phase peptide synthesis using the Boc strategy as shown in Fig. 2 and used as probes.

One embodiment of the invention uses a peptide coupling reaction with 5-tert Boc amino caproic acid 1 followed by an acidic cleavage followed by a coupling to lipoic acid 2 in order to introduce the desired disulfide moiety. After anhydrous HF/anisole final deprotection of ligands, lip CTP 2-5 is carried out.

A second embodiment uses a modification at the final peptide coupling step using 4-methylbenzylmercaptopropionic acid to introduce a free thiol function ligands mer CTP 2-5.

Example 3 Preparation of a non-uniform matrix of pyrrole-containing peptides

A wafer bearing gold electrodes is prepared as in Example 1. As shown in Figs. 3 A and 3B the array of electrodes is covered with a probe-free layer of pyrrole-thiol or disulfides in an electrochemical cell equipped with counter and reference electrodes (Pt wire and ' salt saturated calomel electrode). By sweeping the potential of all of the gold electrodes to positive values (vs SCE, cyclic voltamperometric method: -0.35 V/1.10 V) in the presence of an aqueous solution of probe-free pyrrole monomers, a probe-free layer of pyrrole becomes attached to the electrodes. The probe-free pyrrole monomers are then replaced with a pyrrole-containing peptide. The potential of an individual electrode is then swept to positive values as above. The epitaxial growth of the corresponding functionalized surface polypyrrole film on the probe-free pyrrole layer occurs at around 1 V (vs SCE). The surface confined polypyrrole film is electrically conductive and can be dedoped in order to obtain an uncharged film using a reduction step (~ - 0.3 V vs SCE). These oxidative/reductive electrochemical conditions are compatible with. nucleic acid and peptide probes.

Example 4 Synthesis of pyrrole modified oligonucleotide

A pyrrole-modified phosphoramidite (pyrrole amidite) and

1-N-aminoethylpyrrole are synthesized as shown in Fig. 4. A series of oligonucleotides is prepared using an Applied Biosystems oligonucleotide synthesizer (381 A). Tne coupling of the "pyrrole amidite " with an oligonucleotide probe is achieved using a standard cycle with a coupling time of 30 sec. and using a 0.2 M concentration of the "pyrrole amidite ". The fully deprotected oligonucleotide probes are purified on reversed phase HPLC with a gradient of acetronitrile (5 to 50%) in 25 mM triethylarnmonium acetate pH 7.0. The base composition analysis of the Py-oligonucleotide by digestion showed no alteration. This demonstrates that the 5' incorporation of the pyrrole modified nucleoside is fully compatible with oligonucleotide synthesis.

The electrochemical reactions are carried out with an EGG Princeton Applied Research Model 273 potentiostat with two platinum disks (1 cm area) as working and counter electrodes and a saturated calomel electrode (SCE) as a reference (Tacussel). The synthesis is monitored on a 8300 Schlumberger X/Y recorder. The polypyrrole films are synthesized on the Pt working electrode dipped in 3 ml of a aqueous solution containing 10 mM pyrrole (Tokyo Kasei), 0.1 M LiC10₄ (Flulca) and 0.5 μM of Py-oligonucleotide in a 5 ml conical tube.

This solution is quickly degazed with argon bubbling before use. The syntheses of polypyrrole on the support of Fig. 1 are carried out under the same conditions followed by a rinsing step. For specificity attachment studies. 5'-^J"P labelled Py-oligonucleotides are added to the solution.

Two electropolymerization methods may be used: a galvanostatic method (i = 1.2 mAcm^'") and a potentiodynamic method (potential sweeping between -0.3 and +0.85 V vs SCE at a scan rate of 50 mV/s) is stopped when the charge value gives a thickness of about 50-200 nm. The synthesis is followed by a reduction step at -0.3 V/SCE for 30 sec. to obtain dedoped polymers.

After synthesis, the support is rinsed in 10 x SSPE buffer (lx: 150 mM NaCl, 1 mM EDTA, 10 mM Phosphate pH 7.4) and 0.5% SDS for 1 hr. The supports can be stored at 4°C in 1 x SSPE for at least 2 weeks. The polypyrrole supports are first rinsed with 1 x SSPE, SDS 0.5% and

32 then hybridized in the same buffer containing 3.3 pmol of P-labelled complementary oligonucleotide targets. The hybridizations are carried out at 25°C for 3 hrs. After wshing, the bound radioactivity is determined by direct counting with Cerenkov effect in a Beckman LS 2800 or a NEN-Dupont BC 2000 counter or by autoradiography.

Example 5 A probe-free layer of silane on a silica support and a probe-linked layer containing protein probes

The formation of a probe-free layer of silane on a silica support is summarized in Fig. 6. The silica support is treated with piranha (7:3 [v/v] H₂SO₄/H₂O₂) solution to produce surface hydroxyl groups, rinsed with nanopure water and dried with nitrogen gas. The support is then silanized with 3 -glycidoxypropylfriethoxy silane for 60 mins. at 298K, to form the probe- free layer. The support is then rinsed with nanopure water and exposed to 11.6 M HC1 for 60 mins. at 0°C to encourage the formation of vicinal diols. After this treatment, the support is placed in 100 mM sodium metaperiodate in 10%o (v/v) acetic acid for 60 mins. at 298K, in order to oxidize the diols to terminal aldehyde groups on the silica surface. Finally, the support is rinsed with nanopure water and treated with an excess of glycine for 60 mins. at 298K. It is then exposed to 0.3 mM sodium cyanoborohydride for 60 mins. at 298K.

For the formation of a probe-linked layer on the probe-free silane layer, the probe-free layer is incubated for 15 mins (at 298K) with Elknan's reagent (5,5'-dithiobis-(2-nitrobenzoic acid) to generate mercaptosilane-TNB (2-nitro-5-thiobenzoic acid) conjugate. After activation with Elknan's reagent, the conjugated disulphide group is exchanged with 4% (v.v) mercaptoproprionic acid in 0.1M sodium phosphate buffer at pH 7.2, thereby releasing 2-nitro-5-thiobenzoic acid, which is monitored using a cysteine standard. Protein probes are deposited onto the probe-free silane surface so that a predeteπnined array of probe species is imprinted on the probe-free layer. The protein probes are then immobilized on the surface through the use of N-hydroxysuccinimide and N,N'-dicyclohexylcarbodiimide in dimethyl- formamide and finally the protein itself.

Example 6 Preparation of a matrix of probes on a uniform solid support

A wafer is used as a solid support. A 0.1 μm layer of Cr is sputtered onto the wafer followed by a 0.1 μm - 0.5 μm layer of gold. Probes are prepared attached to a lipoic acid disulfide. As shown in Fig. 7. individual linked probes are initially stored in separate wells 9 on a multiwell plate 10 such that the array of probes in the multiwell plate 10 corresponds to the desired array of probes to be formed on the solid support 11. The imprinter 12 consists of an array of pins or micropipets 13 identical to the aray of wells in the multiwell plate 10 . Imprinter 12 is brought into contact with the multiwell plate and each individual probe is loaded onto the corresponding pin or micropipet as indicated by the dashed lines. The imprinter 12 is then brought into contact with the solid support 11, and the probes are released from each pin or micropipet so as to form an array of probes on the solid support identical to the array of probes in the multiwell plate 10. The probe-containing layer is separated from the gold surface by a probe free layer composed of lipoic acid moieties. Depositing all of the probe species simultaneously onto the gold surface by imprinting is faster than the sequential activation of discrete electrodes. The matrix formed is highly stable due to the formation of polar amide derived hydrogen bonds. Example 7 Preparation of a uniform matrix of pyrrole containing peptides

A wafer having a uniform gold electrode is formed as in Example 6. A probe-free layer of pyrrole is formed in the electrode as in Example 3. Peptide probes linked to pyrrole residues are prepared as in Example 2. The pyrrole linked probes are simultaneously deposited onto the gold surface so that a predetermined array of probe species is imprinted on the probe-free layer. By sweeping the potential of the gold electrode to positive values (vs SCE, cyclic voltamperometric method: -0.35V/1.10V) the epitaxial growth of the probe-linked pyrrole monomers occurs on the probe-free layer so that a predetermined array of probe species is imprinted in the probe-containing layer of the matrix. These oxidative/reductive electrochemical conditions are compatible with nucleic acid and peptide probes.

Claims

CLATMS:

1. A matrix of a plurality of probe species, each probe being a first member of a pair-forming group for binding of a target which is the second member of the pair forming group; wherein each probe species is confined to a separate, predetermined location in the matrix, the matrix comprising: a conductive solid support on which there is immobilized a conductive film comprising a polymer or co-polymer composed of electropolymerizable monomers, having an inner layer which is in direct contact with the solid support and said inner layer comprises probe-free monomers, and an outer layer extending therefrom which comprises probe-linked monomers.

2. A matrix according to Claim 1, wherein the outer layer extends epitaxially from the solid support.

3. A matrix according to Claims 1 or 2, wherein said inner layer is a monolayer.

4. A matrix according to any one of Claims 1, 2 or 3, wherein the outer layer is a monolayer.

5. A matrix according to Claim 1, wherein the solid support is essentially uniformly conductive.

6. A matrix according to Claim 1, wherein the solid support is essentially non-uniformly conductive.

7. A matrix according to Claim 6, wherein the solid support is composed of an array of conductive electrodes.

8. A matrix according to Claim 6, wherein the solid support is composed of a grid of conductive wires.

9. A matrix according to Claims 1 to 8, wherein the solid support has a metallic surface.

10. A matrix according to Claim 9, wherein the solid support is selected from the group consisting of: gold, silver, copper, iron, indium tin oxide, and conductive metal oxides.

11. A matrix according to Claim 10, wherein the conductive film is immobilized to the solid support by thiol, disulfides, thioureas, sclenoureas, xanthates and thioxanthates.

12. A matrix according to Claims 1 to 8, wherein the solid support has a ₅ surface made of conductive glass functionalized by hydroxyl groups.

13. A matrix according to Claim 12, wherein the conductive film is immobilized to the glass by silane type chemistry.

14. A matrix according to Claims 1-13, wherein the electropolymerizable monomers are selected from the group consisting of: pyrrole, dipyrrole, ιo thiophene, aniline and a combination of mixed or uniform oligomers comprising at least five monomer units.

15. A matrix according to Claims 1-13, wherein the inner layer is distanced from the solid support by a spacer molecule which does not impede transfer of electric current from the solid support to the monolayer.

₁5 16. A matrix according to Claim 15, wherein the spacer is selected from the group consisting of hydrocarbon linkers and sugar-containing peptide chains.

17. A kit for the detection of the presence of a target species in a sample, comprising the matrix of any one of Claims 1 to 16 and reagents for 0 producing a detectable signal upon formation of a pair-forming group.

18. A system for the detection of the presence of a target species in a sample, comprising the matrix of any one of Claims 1 to 16 and a detector capable of detecting the formation of a pair- forming group.

19. A system according to Claim 18, comprising the matrix of Claims 7 5 or 8 and a detector capable of reading localized changes in capacitance or conductance.

20. A system for the detection of the presence of a target species in a sample, comprising the kit of Claim 17 and a photo detector capable of reading said detectable signal.

21. A method for the production of a matrix of a plurality of probe species, each probe being a first member of a pair forming group for binding of a target which is the second member of the pair forming group; wherein each probe species is confined to a separate predetermined location in the matrix, the method comprising:

(i) providing a conductive solid support;

(ii) imprinting a pattern of electropolymerizable monomers by deposition on the solid support, at least some of the monomers being linked to the probes; said pattern defining a plurality of locations on said solid support, each location comprising monomers with a single probe species; and (iii) passing electric current, simultaneously, through all of said locations, thereby causing polymerization of said monomers.

22. A method for the production of a matrix of a plurality of probe species, each probe being a first member of a pair forming group for binding of a target which is the second member of the pair forming group; wherein each probe species is confined to a separate predetermined location in the matrix, the method comprising:

(i) providing a conductive solid support; (ii) immobilizing on said solid support an inner layer of electropolymerizable probe-free monomers; (iii) imprinting a pattern of electropolymerizable monomers on said inner layer, at least some of the monomers being linked to the probes; said pattern defining a plurality of locations on said solid support, each location comprising monomers with a single probe species; (iv) passing electric current, simultaneously, through all of said locations, thereby causing polymerization of said monomers.

23. A method according to Claim 22, wherein the inner layer of electropolymerizable probe-free monomers is a monolayer.

24. A method according to Claim 21 or 22, wherein said solid support is essentially uniformly conductive.

25. A method according to Claim 21, wherein the solid support is essentially non-uniformly conductive and wherein deposition of the electropolymerizable probe-containing monomers is carried out essentially only on conductive regions of the solid support.

26. A method according to Claim 22, wherein the solid support is essentially non-uniformly conductive and wherein deposition of the electropolymerizable probe-free monomers and subsequent deposition of probe-containing monomers is carried out essentially only on the conductive regions of the solid support.

27. A method according to Claims 25 or 26, wherein the solid support is composed of an array of conductive electrodes.

28. A method according to Claims 25 or 26, wherein the solid support is composed of a grid of conductive wires.

29. A method according to Claim 21 or 22, wherein the solid support is metallic.

30. A method according to Claim 28, wherein the solid support is selected from the group consisting of: gold, silver, copper, iron, indium tin oxide, or any conductive metal oxides.

31. A method according to Claim 30, wherein the electropolymerizable monomers are immobilized to the solid support by thiol, disulfides, thioureas, sclenoureas, xanthates or thioxanthates.

32. A method according to Claim 21 or 22, wherein the solid support has a surface made of conductive glass functionalized by hydroxyl groups.

33. A method according to Claim 32, wherein the conductive film is immobilized to the glass by silane type chemistry.

34. A method according to Claims 21 or 22, wherein the electropolymerizable monomers are selected from the group consisting of: pyrrole, dipyrrole, thiophene, aniline and a combination of mixed or uniform oligomers comprising at least five monomer units.

35. A method according to Claim 21 or 22, wherein the monolayer is distanced from the solid support by a spacer, which does not impede electric transfer from its solid support to the electropolymerize monomers.

36. A method according to Claims 21 or 22, wherein the spacer is selected from the group consisting of: hydrocarbon linker and sugar controlling peptide chains.

37. A method for detecting the presence of a plurality of target species in the sample, by binding to a plurahty of probe species, the probes being a first member of a pa -forrning group, and the target being a second member of a pair-foiming group, comprising the steps of:

(i) providing a matrix according to any one of Claims 1 to 16; (ii) contacting the matrix with the sample under conditions allowing specific binding of the probe to the target; and

(iii) determining in which locations on the matrix a pa -foπning group is formed, said location identifying probe target bound thereby indicating the specific target thereto.