Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
2001-06-14
2004-10-12
Siew, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S005000, C435S006120, C435S007100, C435S091100, C435S091200, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330, C422S050000, C422S068100, C422S076000, C422S082070, C204S165000, C205S158000
Reexamination Certificate
active
06803228
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method to produce a biochip and to a biochip, said biochip being composed particularly of biological probes grafted onto a conductive polymer.
Biological analysis devices, for example DNA chips, represent high-performance tools for the parallel analysis of a large number of genes or DNA or RNA sequences. Their operating principle is based on the hybridisation or pairing property of two strands of complementary sequences in order to reconstitute the DNA double helix. To do this, oligonucleotide probes of a known sequence, immobilised on a support substrate, are placed in the presence of targets extracted from a biological specimen under analysis, and labelled using fluorescent markers.
The hybridisation is then identified and the sequence detected by analysing the surface of the chip with a suitable marker for example to detect the sequence by fluorescence.
Very different technologies have been used to produce these probe matrices. Various immobilisation or grafting techniques of probes onto different substrates have been the subject of significant studies and industrial developments.
1. State of the Related Art
There are essentially three chemical probe addressing methods which represent different approaches to the production and use of probes for different fields of application. They consist of photochemical addressing, mechanical addressing, for example by micropipetting using a dispersion robot, and electrochemical addressing.
For example, electrochemical addressing may be used for oligonucleotide probes. To do this, individually addressed electrode matrices are produced on a glass substrate.
The biological probe immobilisation principle is based on the electropolymerisation deposition of a copolymer of pyrrole and pyrrole substituted by an oligonucleotide (Py-ODN), comprising an oligonucleotide grafted onto a pyrrole nucleus either directly, or indirectly by means of a spacer.
In order to develop massively parallel biological analysis systems, with a high capacity or active site density, it is necessary to be able to address a large number of probes.
Methods using electrochemical addressing require both a large electrode and connection matrix and a multiplexer to index each of the chip's sites electrically. In addition, in these methods, it is necessary to carry out electropolymerisation by immersing the entire chip successively in solutions of each of the Py-ODNs contained in the cell. Therefore, these methods are limited to low-density chips, i.e. comprising approximately one hundred probes, for limited and specific applications.
Other methods have been described in the prior art, advantageously replacing individual electrical addressing by mechanical addressing. However, a disadvantage remains, that of carrying out electropolymerisations in microtroughs, with a solution volume of the order of one nanolitre, for which it is necessary to delay evaporation after micropipetting of all the probes on the insert so that electropolymerisation may take place.
2. Description of the Invention
The aim of the present invention is specifically to solve the above-mentioned problems by providing a method to produce a biochip composed particularly of biological probes grafted onto a conductive polymer, said method particularly offering the advantage of only requiring the use of a single solution of a mixture of suitable proportions of pyrrole and substituted pyrrole (Py and Py-R-F or F and a reactive chemical function and R is an aliphatic or aromatic spacer group) for a single collective electrodeposition on all the microtroughs.
The method according to the invention is characterised in that it comprises the following steps:
a) structuring of a substrate so as to obtain on said substrate microtroughs comprising in their base a layer of a material capable of initiating and promoting the adhesion onto said layer of a film of a pyrrole and functionalised pyrrole copolymer by electropolymerisation,
b) collective electropolymerisation, so as to form an electropolymerised film of a pyrrole and functionalised pyrrole copolymer on the base of said microtroughs, on the layer of said material, using a pyrrole and functionalised pyrrole solution, in the presence of suitable chemical reagents for said electropolymerisation,
c) direct or indirect fixation of a biological probe onto the functionalised pyrrole, by injecting a biological probe solution, either in one or more microtroughs in the presence of chemical reagents required for the direct or indirect fixation of this biological probe onto the functionalised pyrrole.
According to the invention, the layer of material capable of initiating and promoting the adhesion of a film of a pyrrole and functionalised pyrrole copolymer by electropolymerisation onto said layer may be a metallic layer, step a) mentioned above possibly comprising a deposition step of said metallic layer onto the substrate, and a deposition step of a layer of resin or polymer onto the metallic layer and development or engraving of said layer so as to form microtroughs, wherein the base is composed at least partly of the metallic layer.
According to the invention, the metallic layer may be, for example, a layer of gold, a layer of copper or silver or aluminium.
According to the invention, the substrate may be for example a silicon insert, a glass insert or a flexible plastic support if required.
According to another embodiment of the present invention, the step a) may also comprise a treatment step of the gold layer at the base of the microtroughs in the presence of a functionalised pyrrole for example with a thiol group so as to form a monolayer of pyrrole onto said metallic layer, for example on said gold layer, at the base of said microtroughs. This monolayer is capable of initiating and promoting the adhesion of a polypyrrole film by electropolymerisation as demonstrated by R. Simon et al., J. Am. Chem. Soc., 1982, 104, 2031). This is a self-assembled monolayer SAM of a functionalised pyrrole for its adhesion onto the base of the microtroughs.
According to the invention, the functionalised pyrrole may be a pyrrole which comprises a chemical group enabling its fixation by covalent bonding with the metallic layer, and/or with the biological probe. In the case of its fixation to the metallic layer, for example to the gold layer, a functionalised pyrrole with a thiol or disulphide group may also be used.
For example, the functionalised pyrrole with a thiol group may have the following chemical formula:
wherein n may have a value ranging from 1 to 10, for example n may be equal to 6.
For a metallic aluminium probe, a functionalised pyrrole with a —COOH group may be chosen.
According to another embodiment of the present invention, the substrate may be a silicon insert and the layer capable of initiating and promoting the adhesion onto said layer of a polypyrrole film by electropolymerisation may be a layer of silane comprising an alignment of pyrrole sites. Step a) of the method according to the present invention may in this case comprise a deposition step of a layer of resin on the silicon insert, said silicon insert being coated with an SiO
2
film, and engraving of said resin layer so as to form the microtroughs wherein the base is composed at least partly of the SiO
2
film; and a microtrough treatment step by means of a functionalised silanisation agent with a pyrrole so as to fix, on the SiO
2
film, in the base of the microtroughs, the silane layer comprising an alignment of pyrrole sites.
According to the invention, the silanisation agent may be chosen in a group comprising N-(3-(trimethoxy silyl) propyl) pyrrole, or any other functionalised pyrrole with an —SiCl
3
or —Si(OMe)
3
group. The SiO
2
film may be a natural SiO
2
film present on silicon inserts.
According to the invention, irrespective of the embodiment, the resin may be a photosensitive resin, wherein masking, insolation and development are used to form the microtroughs.
According to the invention, the collective electropolymerisation in step
Caillat Patrice
Rosilio Charles
Commissariart A L'Energie Atomique
Siew Jeffrey
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