WO2004037422A1

WO2004037422A1 - Device for dispensing chemical species on surfaces

Info

Publication number: WO2004037422A1
Application number: PCT/EP2003/011955
Authority: WO
Inventors: Bernard Mandrand; Michel Guy
Original assignee: Apibio Sas
Priority date: 2002-10-28
Filing date: 2003-10-28
Publication date: 2004-05-06
Also published as: AU2003282054A1

Abstract

The present invention relates to a device for dispensing chemical species onto substrates for use in generating a biological and/or biochemical assay plate for detecting the presence of mobile target reactants that bind to probe reactants immobilized on said assay plate, said device comprising a chamber for holding said liquid species, a channel having a first end communicating with said chamber and a second end through which said chemical species is delivered when said device is brought into contact with said substrates and a wire being located in the channel. The object to provide a device for dispensing chemical species onto substrates that is easier to handle and has a more reliable operation than devices according to the state of the art is solved according to the present invention by a device where said wire is connected with a drop weight.

Description

DEVICE FOR DISPENSING CHEMICAL SPECIES ON SURFACES

Field of the invention

The present invention relates to a device for dispensing chemical species on surfaces for use in the manufacture of a substrate for a biological and/or biochemical assay for detecting the presence of mobile target reactants that bind to reactants immobilized on said substrate, said device comprising a chamber for holding said probe reactant a channel having a first end communicating with said chamber and a second end through which said probe reactant exits when said device is brought into contact with said surface and a wire being located in the channel.

Background of the invention

US 5,837,859 describes a process for preparing an electrically conductive copolymer by copolymerizing a monomer by applying an electric potential or electric current which results in the formation of an electrically conductive polymer when polymerized.

US 5,551,487 discloses an apparatus for dispensing probe reactants onto substrates with a chamber for holding said probe reactants. The chamber is connected to a channel having a cylindrical member therein, said cylindrical member including an enlarged region that extends into said chamber so as to prevent said cylindrical member from leaving said channel. When the cylindrical member is pressed on a surface while the cartridge assembly is lowered on a substrate, the enlarged region of the cylindrical member is lifted and allows liquid to flow from said chamber through said channel on said substrate.

When the cartridge is lifted from the substrate, the flow of liquid moves the cylindrical member in the direction of the flow and thereby closes said channel with the enlarged region. If the cylindrical member is disabled to freely move along the channel, the force applied by the liquid flow might be insufficient to close the channel with said enlarged region. This problem might occur if, for example the cylindrical member is distorted or if the liquid becomes solidified. In these cases, the liquid contained in the chamber will constantly flow out of the channel.

It is an object of the present invention to provide a device for dispensing chemical species onto substrates that is easier to handle and has a more reliable operation than devices according to the state of the art.

It is another object of the present invention to provide a device that is able to deliver droplets of the chemical species with a uniform size.

It is still a further object of the invention to provide a method for the electrically induced in-situ synthesis of polymers on substrates.

These and other objects of the present invention will become apparent to those skilled in the art from the following detailed description of the invention and the accompanying drawings. Summary of the Invention

The present invention comprises a device for dispensing chemical species on surfaces for use in the manufacture of a substrate for a biological and/or biochemical assay for detecting the presence of mobile target reactants that bind to reactants immobilized on a surface of said surface, said device comprising a chamber for holding a liquid comprising said chemical species, a channel having a first end communicating with said chamber and a second for delivering the liquid when said device is brought into contact with said surface and a wire being located in the channel wherein said wire is connected with a drop weight.

The term "chemical species" denotes a chemical entity which is able to generate alone or after a chemical reaction at the locus on the surface where it is applied a new chemical entity termed as "probe" or "probe reactant" which can interact with a target molecule (or reactant).

Said drop weight forces the wire in a downward direction. If said wire is not longer in contact with a surface, said drop weight causes the wire to stick out of the channel. This measure guarantees that said wire permanently sticks out of the channel when said wire is not in contact with a surface. When said wire is brought in contact with a surface, a small droplet of the liquid is released. When the cartridge is lifted from the substrate, said wire avoids intrusion of air into the channel.

For a better understanding of the invention the terms as used herein are defined as follows: The term "surface" as used herein refers to any kind of surface, no matter what kind of material is used or what shape the surface has. These surfaces, for example microplates, slides etc. are comprised within any kind of device or receptacle to hold a substrate, for example a cuvette or the like. The term "substrate for a biological and/or biochemical assay" is used for any kind of substrate that holds a reactant, which is able to perform a biological or biochemical reaction. "Assay" as used herein means any chemical, biological or physical interactions between the reactants on the surface of a substrate and target molecules. The result of the interaction is detectable and yields quantitative and/or qualitative information on the target molecules.

Preferably, said drop weight is located in said chamber. Since the drop weight is connected to the wire and the wire is in contact with the liquid contained in the chamber, this feature avoids the presence of any means for movement of the wire that is outside an area that contains a liquid, preferably a solution comprising said chemical species.

In a preferred embodiment, said chamber is divided into an upper part for holding said chemical species and a lower part for holding said drop weight. The chemical species is usually comprised in a liquid, for example in the form of a solution, suspension or dispersion. A clearance remains between the housing of the lower part and the drop weight so that the liquid can flow from the upper part via the lower part to said channel.

In a further preferred embodiment, the outer diameter of said drop weight and the inner diameter of said lower part of said chamber form a capillary. The effect is that the surface in this area is always wetted so that the channel is always fed with liquid.

Preferably said wire and said channel form a capillary. Said wire is preferably made of an elastic material, like plastics and the like. In effect, a capillary is formed along the whole length beginning at the top of said drop weight and ending at said second end of said channel. This long capillary provides a constant feeding of liquid from said chamber to said second end. Furthermore said capillary avoids penetration of air into said channel via said second end. Preferably, said upper part of the chamber is closed by a demountable cover. Liquid containing the chemical species can be filled in the chamber by demounting the cover. Preferably the demounted cover is mounted in a tapped hole so that the cover can be easily demounted by turning the cover.

In a preferred embodiment, said channel is mounted on a grommet and said upper part as well as at least parts of said lower part of the chamber are located in a main body of said device where said grommet and said main body are connected to each other. This feature divides said device into sections, which can be manufactured with ver few manufacturing steps. Alternatively, said channel may be directly connected to said main body of said device. In this embodiment said grommet may be replaced by a tubular extension of said main body, with other words said grommet and said main body may form an integral part.

In another preferred embodiment, a portion of said device being in contact with the liquid comprising the chemical species when it is dispensed to the surface is electrically conducting. After contact of the device and the surface, the device is lifted to create a meniscus of the liquid. By applying a voltage between the electrically conducting portion of the device and another electrode for example on the surface of the surface or assay, a current can be drawn through the liquid dispensed to the surface. The electrically conducting portion can be for example said wire or said tube.

Alternatively, the portion of said device being in contact with the liquid comprising the chemical species when the solution is dispensed to the surface is electrically isolating. The isolated portion(s) of said device is in contact with the surface. The voltage is, at this step, applied between the conducting portion of said device and the surface. The current can be drawn through the liquid dispensed to this surface. A tank comprising a liquid, preferably a solution of a chemical species might be connected to chamber to increase the capacity of the device. The chemical species are for electropolymerizable monomers or macromers (oligomers), polymers biomolecules like nucleotides, oligonucleotides, amino acids, oligopeptides, carbohydrates. It is further preferred, that biomolecules as mentioned above are already chemically linked to electropolymerizable monomers or oligomers or even polymers before being dispensed on the surface.

Because of the diameter of said device and the length of said tube it is possible to use said device in combination with so-called three-dimensional assay plates. The assay plates are, for example, glass or polymer slides or have cavities on their surface to receive said probe reactants.

A system might comprise a plurality of devices according to the invention. Preferably a plurality of devices are spatially arranged to form "cassettes" and a plurality of the cassettes are arranged to groups of cassettes.

The object of the present invention is also achieved by a method for dispensing chemical species on substrates for use in generating a biological assay plate for detecting the presence of mobile reactants that bind to the probe reactants whereby a wire mounted on a drop weight in order that the wire is forced in a downward direction and the use of a device for dispensing chemical species onto substrates for use in generating a biological assay plate for detecting the presence of mobile reactants that bind to the probe reactants.

The object of the present invention is further achieved by a method for applying an electrically conductive polymer on a surface of a substrate comprising a conductive electrode comprising the steps of dispensing a liquid comprising an electrolyte with a device according to the invention, said liquid further comprising a chemical species that is electrochemically copolymerizable on said surface, said surface comprising at least one electrode, and applying a voltage between said conducting portion of device as first electrode and said electrode on the surface of the substrate. By applying the voltage between the first electrode and the electrode on the surface of the substrate a current is drawn through the electrolyte. Monomers and comonomers on the surface and in the electrolyte respectively are herewith polymerized electrochemically. Chemical species contained in the electrolyte are electropolymerizable monomers or macromers (oligomers), polymers, biomolecules like nucleotides, oligonucleotides, amino acids, oligopeptides or carbohydrates or biomolecules linked to the corresponding monomers, oligomers or polymers, respectively. By performing the method according to the invention the application of a chemical species on the surface of a substrate like for a example a so called genechip or biochip biosensor andpolymerization of the chemical species can be performed in the same manufacturing stage.

The chemical species used in the method according to the invention can be selected from the group comprising acetylene, azine, p-phenylene, p-phenylene vinylene, pyrene, thiophene, polythiophene, furane, selenophene, pyridazine, carbazole, aniline, polyaniline, pyrrole, polypyrrole, tetrahydrofurane, polytetrahydrofurane, biomolecules like nucleic acids (like DNA, RNA, LNA PLA and the like), amino acids, peptides, carbohydrates and derivatives thereof and biomolecules already linked to monomeric or oligomeric units of the aforementioned monomers/polymers. The method can be used for the in situ synthesis of for example oligo/polynucleotides by electrochemically decovering/deprotecting nucleotides being applied in a foregoing step. Furthermore it is possible to stack different layers of electrochemically polymerizable monomers and comonomers. Definitions and Abbreviations

In the following, the terms and definitions as used herein are explained for the further illustration of the present invention.

The term "to detect" or "detection" as used herein refers to a qualitative and quantitative determination and identification of target molecules in the sample.

The term "biomolecule" refers to any molecule existing in nature or artificially synthesized according to a matrix existing in nature, and comprises for example antibodies, proteins, peptides, nucleic acid sequences, i.e. polynucleofides or oligonucleotides comprising at least two deoxyribonucleotides or ribonucleotides, optionally comprising at least one modified nucleotide for example a nucleotide containing a modified base. It is understood that the terms poly- or nucleotides refer especially to any kind of naturally existing or chemically synthesized or modified DNA (including cDNA), RNA (including cRNA, mRNA), PLA, LNA and chimeras thereof. Further to genes, nucleotide polymorphisms, antisense sequences, ribozymes, expressed sequence tags, a vector, a plasmid.

The term "peptide" as used herein refers in particular to any peptide of at least two amino acids in particular a protein, a protein fragment or oligopeptide which is extracted, separated or substantially isolated or synthesized, in particular those obtained by chemical synthesis or by expression in a recombinant organism.

The term "support" denotes any solid three-dimensional body, which does not chemically or physically interact with a sample. The support may be electrically conductive or non electrically conductive. The term "interacting" as used herein means any interaction between molecular entities, comprising formation of a chemical bond (covalent or ionic), van der Waals interactions hydrogen bond interactions, adsorption phenomena and the like.

The term "complex" refers to an entity formed by at least two different molecular entities, interacting as specified above. A complex according to the invention is held together by interactions as specified above.

Short description of the drawings

Fig. 1 shows a sectional view of the device according to the invention;

Fig. 2 shows a sectional view of detail A in Fig. 1;

Fig. 3 shows a three-dimensional view of the device;

Fig.4 shows a sectional view of a support and a further embodiment of a device according to the invention for dispensing probe reactants onto surfaces;

Fig. 5 shows a sectional view of a support and another embodiment of a device according to the invention;

Fig. 6 shows an arrangement of devices according to the invention in a top view;

Fig. 7 shows a three-dimensional view of an arrangement according to Fig. 4; Fig. 8 shows a complete system for automatically dispensing probe reactants on substrates;

Fig. 9 shows a biosensor used as support, wherein figure 9a is a horizontal projection and Fig. 9b an enlarged view of the arrangement of the electrodes. Detailed description of the invention

The following discussion of the preferred embodiments of the present invention is merely exemplary in nature. Accordingly, this discussion is in no way intended to limit the scope of the invention.

Fig. 1 shows a device 1 for dispensing chemical species onto surfaces for use in generating a biological assay plate for detecting the presence of mobile reactants that bind to probe reactants generated from the chemical species. Device 1 comprises a chamber 2 for holding said chemical species. Chamber 2 is connected with a channel 3 of a tube 15. Channel 3 has a first end 4 communicating with said chamber 2 and a second end 5 delivering said chemical species when said device 1 is brought into contact with a substrate or a surface. A wire 6 is located in channel 3. Wire 6 is made of an elastic material, for example inox or a polymer (for example polyamide 6.6, teflon and the like ). Wire 6 can be moved along channel 3. As shown in Fig. 2, wire 6 sticks out of tube 15. Between wire 6 and tube 15 a gap 16 remains so that a liquid is able to pass through said gap. The size of gap 16 is very small, that is in the range of 0,009 to 0,08 mm, preferably 0,015 to 0,06 mm, most preferably 0,02 to 0,03 mm. and gap 16 forms a capillary. The capillary drains the liquid along wire 6 and prevents generation of bubbles.

Chamber 2 is divided into an upper part 8 of chamber 2 and a lower part 9 of chamber 2. Lower part 9 contains a drop weight 7. Said drop weight 7 is movable in direction of arrow 17 along lower part 9 of chamber 2. The outer diameter of drop weight 7 and the inner diameter of lower part 9 of chamber 2 form a capillary. The lower part of drop weight 7 has a tip 20 machined with cuts that allow the liquid comprising the chemical species to move from lower part 9 to capillary 16. By pressing the second end of channel 6 respectively tube 15 on a surface, for example said substrate, wire 6 is pushed upwards. Wire 6 then pushes the drop weight. Because of the capillary effect in channel 3 and gap 16, a liquid, containing for example the chemical species contained in upper part 8 of chamber 2, can flow out of the second end of the channel. If device 1 is moved upwards wire 6 will not be forced in the direction of drop weight 7 any more so that drop weight 7 moves in a lower direction because of its weight. The weight of drop weight 7 forces wire 6 in a downward direction, so that wire 6 always sticks out of channel 3 when wire 6 is not in contact with a surface.

Upper part 8 of chamber 2 is covered by a cover 11. Cover 11 is mounted on a main body 14 of device 1 by a tapped hole 12. Tube 15 is connected to main body 14 of device 1 by a grommet 13. Grommet 13 is firmly connected to main body 14, for example by welding or sticking. Alternatively tube 15 is directly connected to main body 14 of device 1 by welding or sticking. In this case grommet 13 as independent part is not required, instead of grommet 13 a tubular extension or the like is formed integrally with man body 14.

Usually devices 1 for dispensing probe reactants onto substrates are arranged in cassettes 21 as shown in Fig. 6 and Fig. 7. Cassettes 21 might comprise for example six devices 1 that are mounted in heads 25. The number of heads 25 resp. devices 1 can be freely selected. In the described embodiment three cassettes 21 are arranged to a group 22 of cassettes 21. Cassettes 21 are movable in an x-direction as indicated by an Arrow x in Fig. 4 and in an y direction as indicated by an arrow y in Fig. 6. Each cassette can be rotated by an angle θ. Heads 25 are movably arranged in cassettes 21 in a way that every head 25 can be moved independently from the other heads 25 in direction of z-axis. Displacement of any head 25 or inaccuracy of the size of an associated device 1 can be compensated by adjusting head 25 in z-direction. Z-axis is indicated by an arrow z in Fig. 6 and Fig. 7. By rotation and movement cassettes 21 can be moved in a desired position to fill assay plate 23 with said substrate and cassettes 21 can equalize inaccuracy for example of the surface of assay plate 23. Assay plates 23 are moved in x-direction so that by movement of cassettes 21 in x- and y-direction every point of assay plate 23 can be reached by one of the devices 1 of cassette 21. Each outer area of assay plate 23 has a region 24 forbidden to place chemical species.

Fig. 8 illustrates a system 30 for automatically dispensing chemical species on surfaces for use in generating a biological assay plate for detecting the presence of mobile reactants that bind to the probe reactants generated from the chemical species.

System 30 comprises a first shuttle 31 and a second shuttle 32 for positioning assay plates 23. First shuttle 31 carries a first tray 38 which is moveable along the longitudinal axis and the lateral axis of first shuttle 31. Movement along the longitudinal axis is indicated by arrow 34, movement along the lateral axis is indicated by arrow 35. Manipulator 33 comprises a rotary head . System 30 further comprises a store 37 for storing assay plates 23 which integrates a tray 36 which stores and unstores this plate inside the store 37. First manipulator 33 is able to gather assay plates 23 from tray 36 . After gathering an assay plate 23 from tray 36 head 33 performs an 180° rotation along z-axis so that assay plate 23 can be received by one of the two trays 38 of the system 30.

Second shuttle 32 carries a second tray 38 for supporting assay plates 23. In the embodiment according to Fig. 6, second tray 38 is able to carry assay plates 23. Second tray 38 is able to move along longitudinal axis 34 and lateral axis 35 of second shuttle 32. System 30 further comprises a main robot 39, which comprises means 40 for receiving a plurality of devices 1. Means 40 is connected to main robot 39 by a knuckle joint 41 so that it can be rotated along the longitudinal axis 34.

System 30 further comprises an outlet for cold air 42 and a device 43 for marking of low- quality assay plates 23. Two fixed cameras 44 are arranged to control the quality of the whole process. System 30 further comprises a tampon for holding a cleaning solution 45, a methanol bath 46, camera 47 for calibration and an ultrasonic cleaning equipment 48.Main robot 39 further comprises two cameras 49 for controlling the position of assay plates 23.

In Fig. 9, biosensor 900 used as a support for carrying out the invention consists of a t- shaped support 901 made of glass-fiber or another suitable, preferably chemically inert material, for example, glass, silica, polymer (plastics). The support is covered with an insulating varnish (a black hydrophobic photoimageable epoxy resin). At the bottom of the substrate 901, 10 electric connecting means 902 are located, preferably made of gold. 8 circular spots made of gold 905' (diameter 0.3 mm) with a usable surface 905 of 0,03 mm² are arranged in the form of a circle having a diameter of 2,35 mm. The varnish covers partially the spots 905' up to where the usable surface 905 begins, i. e. the usable surface is not covered by the varnish. It is understood that the surface of the spots 905 and 905' may be the same as in the present example, but they can also vary. The spots act as electrodes and may, for example, be obtained by depositing a layer of a copolymer of formula (I) on the surface of an electrode made of platinum, gold, chromium or titanium coated with gold, or vitreous carbon, etc. These spots 905' are used as the working electrodes and are linked each independently via thin imprinted metal wires to the connection means 902. Attached to the spots are molecular electrodes for example made of an electrically conducting polymer, like polypyrrole, polythiophene etc, where the molecular entities are linked to each other via the heteroatom or via their 3 position or a mixture of both. The molecular electrodes may further comprise a redox couple like for example ferrocene and a probe molecule, like for example a polynucleotide sequence, a chelate ligand, an antigen etc. The number of the spots 905' may be any even or uneven number, but generally an even number will be preferred in order to maintain a perfect symmetry of the electrode arrangement. A central spot 906' with a usable surface 906 with 0.28 mm² is located in the center of the circle formed by the spots 905'. The surface 906 is larger than the sum of the surfaces 905 of the spots 905'. Central spot 906' is the auxiliary electrode and also connected via a connection 903 to one of the connecting means 902. The reference electrode is in the form of a "crown". The crown is formed by a plurality of interconnected smaller open circles 904' with a usable surface 904, thus forming an electrode with one single surface and also linked via a connection 903 to one of the connecting means 905. Each spot 905' is located in the center of one of these smaller open circles 904'. This "crown" 904' is situated at about 2/3 of the distance between the center of spot 906' and the circle formed by the working electrodes 905. The overall local electrode symmetry is C₂ or more specific C_2V- A reference electrode, for example a metallic Ag/AgCl or, preferably a molecular electrode more preferably a molecular electrode comprising a redox couple is deposited on the "crown" 904'. The molecular electrode comprises an electrically conducting polymer, like polypyrrole, polythiophene etc, where the molecular entities are linked to each other via the heteroatom or via their 3 position or a mixture of both.

It is understood that any other arrangement of electrodes on the surface of support can also be used for the purpose of the present invention, namely those described in unpublished DE 10332804, the disclosure of which is incorporated herein by reference. In the following the preparation and modification of the electrodes is described in detail:

Abbreviations

ECS saturated calomel electrode (reference electrode)

CP oligonucleotide for positive control with the following sequence :

5' TΓΓ TTT TTT TGC CTT GAC GAT ACA GCT A 3'

CP-biotin (CP-bio) complementary biotinylated oligonucleotide of CP with the following sequence :

5' biotin - T AGC TGT ATC GTC AAG GCA 3'

M5 oligonucleotide for negative control; with the following sequence :

5' TTT TTT TTT TTT GGA GCT GCT GGC G 3'

M5-biotin (M5-bio) complementary biotinylated oligonucleotide of M5; with the following sequence :

5' biotin-C GCC AGC AGC TCC AAA 3'

ODN oligonucleotide

TH1X TH1X is a buffer solution consisting of <13.3 g/1, Na₂HPO₄,

29,22 g/1 NaCI, 20 g/1 PEG 4000, 6.5% Tween 20, 1 g/1 gelatine, 14% DNA of herring sperm 10 mg/ml sonicated

TR: TR is a rinsing buffer consisting of:8g/l of NaCI; 0.2g/l of

KC1; 0.76 g/1 of Na₂HPO₄; 0.19 g/1 of KH₂PO₄; 0.5% of tween 20; 1 mM of EDTA.

CV Cyclic voltammetry

Fc ferrocene

Example 1

Electrosynthesis of molecular polypyrrole/DNA on a substrate. The substrate was a biochip as described in the foregoing.

Copolymer chains comprised of a mixture of polypyrrole and nitrogen functionahzed polypyrrole and DNA sequences (CP = TTT TTT TTT TGC CTT GAC GAT ACA GCT A) were attached to the 8 metallic spots for the working electrode. The aqueous solution for electrosynthesis of the polymer chains (pH=3) contained 0.1 M of pyrrole-OH, 5 μM of pyrrole-CP and 0.7 M of LiClO₄ . A droplet of this solution (≡ 50 microlitres) is deposited on the biochip, covering the 8 working electrodes, the counter electrode and the reference electrode. A potential of + 0.8 volt measured against gold is applied on each of the spots during different times.

Example 2 Electrosynthesis of molecular polypyrrole/DNA copolymers

Electrosynthesis was realised at + 0.85 volts vs. gold from an aqueous electrolytic solution of LiClO₄ 0,7 M, composed of a mixture of pyrrole-alcohol (Ppy-OH) which is functionalised in its 3 position (PPy-OH) 0.1 M and of pyrrole, functionalised in 3 positions by CP or M5 DNA sequences (5 μM). In a first step, a droplet (50 microlitres) of the solution CP is deposited on a biochip covering 8 working electrodes, the counter electrode and the reference electrode. Four of the eight spots are simultaneously addressed under the same conditions. Identical polymeric chains of PPy-OH/PPy-CP (Qs = 20 mC • cm^"2) were formed on these four spots. On the remaining four spots, four identical polymeric "brushes" of PPy-OH/PPy-M5 (Qs = 20 mC • cm ²) are formed.

Example 3

Electrosynthesis of polypyrrole DNA Fc copolymers.

In this example, deposits (10 mC -cm^"2) are realised at +0.8 Volt vs. gold from an organic solution of propylencarbonate containing 0.08 M of pyrrole-Fc-NHP, 0,02 M of pyrrole- OH and 0.5 M of LiClO₄. The aminated DNA sequences were grafted on a biochip comprising eight molecular electrodes of PPy-OH/PPy-NHP. The DNA sequences are termed as S I (5'TCA ATC TCG GGA ATC TCA ATG TTA G3') and grafted by incubating during two hours at ambient temperature the biochip in a grafting solution (50 % phosphate buffer / 50 % acetonitrile) of SI (50 μM). The biochip was afterwards rinsed with the phosphate buffer. Before analysis, the eight spots of the biochip are neutralized with ethanolamine (hydrolysis of activated ester groups which are remaining after grafting). By way of non-limiting example electrically conductive polymers comprise polyacetylene, polyazine, poly(p-phenylene), poly(p-phenylene vinylene), polypyrene, polypyrrole, polythiophene, polyfuran, polytetrahydrofuran, polyselenophene, polypyridazine, polycarbazole, polyaniline, etc. and derivatives thereof.

Usually electrochemical copolymerization is, for example, carried out by cyclic voltammetry, by subjecting solution comprising a monomer to be polymerized to electrical potential variations which are sufficient to bring about the polymerization by a successive oxidation and reduction; since the polymer formed is conductive, the oxidation-reduction cycle may be repeated several times.

The methods of electrochemical polymerization generally used for the preparation of the ECPs, (electrically conductive polymers) such as polymerization at set current (chronopotentiometry) or at set potential (chronoamperometry) are also applicable to the method according to the invention.

The quality of the deposit may be controlled by the choice of experimental conditions: the concentration of the monomer e. g. pyrrole, thf and the like, the oligonucleotide- monomer/monomer ratio, the nature of the solvent, the electrochemical method used (cyclic voltammetry, chronoamperometry or chronopotentiometry). The copolymer obtained may accordingly have different qualities of porosity and of accessibility depending on the desired subsequent use, and if the amount of bound oligonucleotide may be modified.

By measuring the current delivered during the reaction, the electrode effectively makes it possible to monitor the progress of the polymerization reaction (for example the thickness of the polymer formed) or the progress of subsequent reactions carried out on the copolymer.

According to a preferred embodiment of the process in accordance with the invention in one or other of its variants, it additionally comprises the elongation of an oligonucleotide chain in several successive steps, each of these steps consisting of the binding of one or more nucleotides, or oligonucleotides.

Elongation of the oligonucleotide is carried out at the surface of the support by assembling the protected monomers, starting with at least one nucleotide or oligonucleotide bound to the surface of the electrically conductive polymer. The standard methods for the chemical synthesis of nucleic acids may be used in the implementation of this embodiment.

The supports in accordance with the invention additionally allow the oligonucleotide to be elongated electrochemically, by using variations in the electrode potential in order to carry out the protection, deprotection and condensation reactions of the growing polymer chain, for example, by depositing a layer of a copolymer of formula (I) on the surface of an electrode made of platinum, gold, chromium or titanium coated with gold, or vitreous carbon, etc.

The device of the present invention allows further the device synthesis of oligonucleotides (and also peptides) directly on the electrical copolymer by electrochemical deprotection in situ. In general, the assembly of a nucleotide on a growing polynucleotide chain on a support uses a series of reactions which involve protecting groups in order to direct the reaction onto a given function and to prevent it at another. In accordance with the present invention, the protecting group for the growing oligonucleotide chain may be removed locally by an electrochemical reaction, thereby making it possible to add a nucleotide to the chosen position.

Additional advantages follow from this possibility of carrying out, on the support by using the device according to the invention in accordance with the invention, an oligonucleotide synthesis in situ. Indeed, in this case, it is possible to synthesize in situ and in parallel the set of oligonucleotides which will be arranged on the grid, instead of independently synthesizing oligonucleotides bearing a pyrrole arm and then carrying out successive copolymerizations. This makes it possible to envisage the industrial production of matrices of several thousand microsurfaces.

Figure 4 shows a sectional view of another embodiment of a device 40 for dispensing probe reactants onto surfaces according to the present invention. On the surface 48 of substrate 49, a plurality of metallic electrodes 45, 46 is arranged. The substrate may be of any material suitable for the intended purpose like for example glass, silicon, plastics or mixtures thereof and the like. The electrodes 45 are usually arranged in the form of an array as described above with reference to Fig. 9. Electrode(s) 46 serve as working electrodes and electrodes 45 are used as reference electrodes.

Tube 41 (corresponds to tube 15 in Fig. 1) is made of an electrically conducting material that does not react chemically with an electrolyte 44 being dispensed on the surface, for example stainless steel, steel with chromium plated surface, electrically conducting plastics or the like. Tube 41 is connected with a power source (not shown) and used as a counterelectrode. An electrolyte 44 is dispensed through an outlet 42 (corresponds to reference number 5 of said device 1 as described above with reference to Figs. 1 to 3) of device 40. The electrode 45, is connected with said power source and used as a reference- electrode. The electrode 46 is used as a working electrode. The elements being in contact with the surface, e.g. wire 43 (corresponds to wire 6 in Fig. 1) is made for example of isolating material for example plastics or glass or conductive material like stainless steel.. An electrically conducting contact between surface and device 40 is only provided through electrolyte 44. A short-circuit is generated between wire 43 and electrode 46 when being in contact. Similar, a short circuit might occurr between tube 41 and one of the electrodes 45, 46. When a short circuit is generated, no electrical current flows through electrolyte 44, so that the above mentioned electrochemical reaction is not carried out. In order to avoid this, voltage is applied between device 40 and electrodes 46 only after device 40 is taken off from surface 48.

When a voltage is applied between the two electrodes, an electrical current flows through tube 41 via electrolyte 44 to electrodes 46. The current causes the electrochemical copolymerization of the chemical species contained in electrolyte 44 as described above. An electrically conducting polymer is separated on surface 48 of substrate 49.

Fig. 5 shows another embodiment of a device 50 (corresponds to device 40 in Fig. 4). Tube 51 is divided in an upper part 51a and a lower part 51b. Upper part 51a is made of conducting material as for example stainless steel or the like. Electrolyte 44 is dispensed through an outlet 52 (corresponds to outlet 42 in Fig. 4). Wire 53 (corresponds to wire 43 in Fig. 4) as well as lower part 51 b of Tube 51 (corresponds to tube 41 in Fig. 4) are made of an isolated material, for example polymer, ceramics, optical fiber etc. As shown in Fig. 5, tube 51 as well as wire 53 are brought in contact with surface 48.

When carrying out the method according to the present invention, in a first step, electrolyte 44 comprising e.g. a polymerizable monomer like pyrrole and the like as described above is applied onto the surface 48 i. e. on the electrode 45, 46 of substrate 49 via outlet 42 ,52. The amount of electrolyte 44 is determined in such a way that both electrodes 45, 46 are covered by the electrolyte 44.

In effect, device 1 is used not only to apply a compound of monomers onto the surface of a biosensor but also to polymerize the compound in the same manufacturing stage.

Claims

1. Device (1) for dispensing chemical species onto a surface for use in the manufacture of a substrate for a biological and/or biochemical assay for detecting the presence of mobile target reactants interacting with probe reactants immobilized on said surface, said device comprising

1.1) a chamber (2) for holding a liquid comprising the chemical species

1.2) a channel (3) having a first end (4) communicating with said chamber (2) and a second end (5) for delivering the liquid comprising the chemical species when said device (1) is brought in contact with said surface

1.3) a wire (6) being located in the channel (3),

characterized in that said wire (6) is connected to a drop weight (7).

2. Device according to claim 1, characterized in that said drop weight (7) is located in said chamber (2).

3. Device according to anyone of the preceding claims, characterized in that said chamber (2) is divided in an upper part (8) for holding the liquid comprising the probe reactant and a lower part (9) for holding said drop weight (7).

4. Device according to anyone of the preceding claims, characterized in that the outer diameter of the drop weight (7) and the inner diameter of the lower part (9) of said chamber (2) form a capillary.

5. Device according to anyone of the preceding claims, characterized in that said wire (6) and said channel (3) form a capillary.

6. Device according to anyone of the preceding claims, characterized in that said wire is made of an elastic material.

7. Device according to anyone of the preceding claims, characterized in that said upper part (8) of said chamber (2) is closed by a demountable cover (11).

8. Device according to anyone of the preceding claims, characterized in that said demountable cover (11) is mounted in a tapped hole (12).

9. Device according to anyone of the preceding claims, characterized in that said channel (3) is mounted on a grommet (13) and that said upper part (8) as well as at least parts of said lower part (9) of said chamber (2) are located in the main body (14) of said device (1) where said grommet (13) and said main body (14) being connected to each other.

10. Device according to anyone of claims 1 to 8, characterized in that said channel (3) is connected to said main body (14).

11. Device according to anyone of the preceding claims, characterized in that a portion of said device being in contact with the liquid comprising the chemical species when the liquid is dispensed to the surface is electrically conducting.

12. Devices according to claim 11, characterized in that the electrically conducting portion is the wire (6).

13. Device according to claim 11 or 12, characterized in that the electrically conducting portion is the tube (15).

14. Device according to anyone of claims 1 to 10, characterized in that the portion of said device being in contact with the liquid comprising the chemical species when the liquid is dispensed to the surface is electrically isolating.

15. Device according to anyone of the preceding claims characterized in that a tank comprising the liquid comprising the chemical species is connected to chamber (2).

l ό.Device according to claim 15, characterized in that the chemical species are electropolymerizable monomers or macromers, polymers and biomolecules like nucleotides, oligonucleotides, amino acids, oligopeptides, carbohydrates.

17. Device according to claim 16, characterized in that the biomolecules are linked to the electropolymerizable monomers or macromers.

18. System comprising a plurality of the devices according to anyone of the preceding claims.

19. Method for applying an electrically conductive polymer on a surface of a substrate comprising a conductive electrode comprising the steps of - dispensing a liquid comprising an electrolyte with a device according to claims 11 to

13, said liquid further comprising a chemical species that can be electrochemically copolymerized on said surface, said surface comprising at least one electrode, - applying a voltage between said conducting portion of device (1) as counter electrode and said electrode on the surface of the substrate when the device (1) is taken off until the creation of a meniscus between device and the surface

20. Method according to claim 19, wherein the chemical species is selected from acetylene, azine, p-phenylene, p-phenylene vinylene, pyrene, thiophene, polythiophene, furane, selenophene, pyridazine, carbazole, aniline, polyaniline, pyrrole, polypyrrole, tetrahydrofurane, polytetrahydrofurane, biomolecules like nucleotides, oligonucleotides, amino acids, oligopeptides, carbohydrates and derivatives thereof.

21. Use of a device (1) or a system according to anyone of the preceding claims for dispensing probe reactants onto substrates for use in generating a biological assay plate for detecting the presence of mobile reactants that bind to the probe reactants.