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Method for producing matrices of addressed ligands on a carrier

a technology of ligands and matrices, which is applied in the direction of diaphragms, sequential/parallax process reactions, libraries, etc., can solve the problems of requiring a large number of different masks to carry out all coupling operations, and reducing the size of the si

Inactive Publication Date: 2006-02-16
COMMISSARIAT A LENERGIE ATOMIQUE ET AUX ENERGIES ALTERNATIVES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

Enables rapid, precise, and efficient deposition of ligands onto carriers, allowing for high-density matrix production with small deposit sizes and simultaneous or successive fixing of multiple ligands, reducing the need for modified carriers and individual addressable electrodes.

Problems solved by technology

This mode of photochemical addressing has the disadvantage of requiring a large number of different masks to carry out all the coupling operations.
The use of these mechanical addressing techniques has the disadvantage of having to bring the molecule to be fixed directly to the site to be addressed.
Therefore the size of the site cannot be smaller than the size of the drop of dispensed reagent.
Moreover, it is necessary to use a carrier carrying electrodes that can be individually addressed, therefore complex systems that are possibly multiplexed.

Method used

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  • Method for producing matrices of addressed ligands on a carrier
  • Method for producing matrices of addressed ligands on a carrier
  • Method for producing matrices of addressed ligands on a carrier

Examples

Experimental program
Comparison scheme
Effect test

example 1

1—Production of Carriers Carrying Oligonucleotides

[0084] The conductive carriers used are glass plates coated with a layer of chromium (for adherence) and a continuous layer of gold of 0.5 μm. This layer is connected to the “working electrode” outlet of an EGG 283 potentiostat.

[0085] Two different oligonucleotides carrying a pyrrole group at 5′ are copolymerised on these carriers. Their sequences are as follows:

pyrM5: 5′ pyr (T)10 GGAGCTGCTGGCGT 3′pyrCP: 5′ pyr (T)10 GCCTTGACGATACAGC 3′

[0086] They were synthesized using the method described by Livache et al in [5].

[0087] To fix these oligonucleotides to the carrier, a reaction medium is used containing 0.1M LiClO4, 20 mM pyrrole and 1 μM oligonucleotide carrying a pyrrole group at 5′.

[0088] This solution is added to a reservoir in polypropylene of cone shape which contains a platinum counter-electrode (CE) connected to the potentiostat. This reservoir is easily filled using a micropipette whose volume may vary from 50 to 1000...

example 2

[0102] The same operating method is followed as in example 1 to prepare matrices of pyrM5 and pyrCP oligonucleotides but using as conductive carrier a carrier in plastic material coated with indium and tin oxide (ITO).

[0103] The results obtained with these matrices for the detection of biotinylated complementary oligonucleotides are the following: [0104] oligonucleotide on pyrM5 carrier hybridised with biocompM5: 95 [0105] oligonucleotide on pyrM5 carrier hybridised with biocompCP: 5 [0106] oligonucleotide on pyrCP carrier hybridised with biocompM5: 7 [0107] oligonucleotide on pyrCP carrier hybridised with biocompCP: 105

example 3

[0108] In this example, the same operating method as in example 1 is followed to prepare a matrix of pyrM5 oligonucleotides on a carrier in gold supported by glass but as counter-electrode a platinum wire is used charged with reaction medium instead of the reservoir fitted on the inside with a platinum electrode.

[0109] As shown in FIG. 4A, the platinum wire 15 is charged with reaction medium by immersion in a reservoir 17 containing this medium. The wire carrying the drop 19 is then brought to the carrier until contact is made with the drop. The electrochemical impulse is then made. The wire is lifted away and rinsed in water. Other deposits are made in the same manner. In this way deposits of approximately 1 mm in diameter are obtained and intense fluorescence is visible when the matrix is used to conduct hybridisation of the complementary oligonucleotide. The results obtained are the following: [0110] oligonucleotide on pyrM5 carrier hybridised with biocompM5: 400 [0111] oligonuc...

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Abstract

The invention concerns a method for fabricating matrices of addressed ligands on a carrier. According to this method, an element is used such as a reservoir (1) filled with ligand and containing an electrode (3) to deposit and electrochemically fix the ligand to the conductive carrier (7). The ligand may be an oligonucleotide or a peptide, and fixing may be obtained by electropolymerisation of this oligonucleotide or peptide carrying a pyrrole group at 5′ with pyrrole.

Description

TECHNICAL FIELD [0001] The subject of the present invention is a method for producing matrices of addressed ligands on a carrier. [0002] The ligands may be natural or synthetic products having biological activity or an affinity for biological or other molecules, for example peptides, oligonucleotides, receptors or other molecules of biological interest. Matrices of this type may find numerous applications, in particular for the detection and identification of constituents in biological samples and for screening molecule libraries. Such matrices may in particular be matrices of oligonucleotide probes. PRIOR ART [0003] In the past few years several methods have been developed for producing matrices of this type. Three methodologies are known in which addressing is made either by photochemical route, or by mechanical route, or by electrochemical route. [0004] In the document by Fodor S. et al, Science, 1991, 251, pages 767-773 [1] a method is described for making a matrix of oligonucle...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C02F1/40G01N33/53B01J19/00C09D5/44C12M1/00C12N15/09C12Q1/68C40B40/06C40B40/10C40B60/14G01N37/00
CPCB01J19/0046C40B60/14B01J2219/00369B01J2219/00527B01J2219/00585B01J2219/0059B01J2219/00596B01J2219/00605B01J2219/0061B01J2219/00612B01J2219/00628B01J2219/00637B01J2219/00659B01J2219/00713B01J2219/00722B01J2219/00725C09D5/4476C40B40/06C40B40/10B01J2219/00351
Inventor LIVACHE, THIERRYLESBRE, FREDERIC
Owner COMMISSARIAT A LENERGIE ATOMIQUE ET AUX ENERGIES ALTERNATIVES