Development of biological microchips on an aluminum support with cells made of brush polymers

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Abstract

A method has been developed for manufacturing biological microchips on an aluminum substrate with hydrophilic cells from brush copolymers with the formation of a matrix of cells using photolithography. The surface of aluminum substrates was previously coated with a thin, durable, moderately hydrophobic layer of cross-linked polymer to prevent contact with the aluminum surface of the components used in the analysis of nucleic acids. Aluminum biochip substrates have high thermal conductivity and low heat capacity, which is important for the development of methods for multiplex PCR analysis on a chip. Oligonucleotide probes were covalently immobilized in the cells of the biochip. The preservation of the hybridization activity of the immobilized DNA probes was demonstrated in a hybridization analysis with a synthetic DNA target representing a section of the sequence of the 7th exon of the human ABO gene. The developed methods can be used in the development of a technology for parallel multiple rapid microanalysis of nucleic acids “lab on a chip” for the detection of human somatic and infectious diseases

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About the authors

I. Yu. Shishkin

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: chud@eimb.ru
Russian Federation, Moscow, 119991

G. F. Shtylev

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: chud@eimb.ru
Russian Federation, Moscow, 119991

V. E. Barsky

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: chud@eimb.ru
Russian Federation, Moscow, 119991

S. A. Lapa

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: chud@eimb.ru
Russian Federation, Moscow, 119991

O. A. Zasedateleva

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: chud@eimb.ru
Russian Federation, Moscow, 119991

V. E. Kuznetsova

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: chud@eimb.ru
Russian Federation, Moscow, 119991

V. E. Shershov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: chud@eimb.ru
Russian Federation, Moscow, 119991

V. A. Vasiliskov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: chud@eimb.ru
Russian Federation, Moscow, 119991

S. A. Polyakov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: chud@eimb.ru
Russian Federation, Moscow, 119991

A. S. Zasedatelev

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: chud@eimb.ru
Russian Federation, Moscow, 119991

A. V. Chudinov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Author for correspondence.
Email: chud@eimb.ru
Russian Federation, Moscow, 119991

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Supplementary files

Supplementary Files
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2. Fig. 1. Scheme of amination and fluorescent marking of the surface of an aluminum substrate.

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3. Fig. 2. Structures of fluorescent dyes Cy5-pNP, Cy5-NH₂ and Cy3-pNP.

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4. Fig. 3. Micrograph of an aluminum substrate in the light of Cy5 dye fluorescence with an exposure of 100 ms after surface modification with 3-aminopropyltriethoxysilane (APTES) and labeling with Cy5 fluorescent dye. A graph of the signal distribution along the drawn line in the fluorescence image is shown.

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5. Fig. 4. Schematic diagram of the arrangement of cells on the biochip substrate after UV polymerization through a photomask.

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6. Fig. 5. Scheme for obtaining cells from brush polymers on the surface of an aluminum substrate (a), activation of carboxyl groups (b), fluorescent labeling of carboxyl groups (c), immobilization of oligonucleotide probes (d) and hybridization with target DNA (d).

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7. Fig. 6. Microscopic image of brush polymer cells on the surface of an aluminum substrate in reflected visible light. The cells are 200 × 200 μm in size with a pitch of 600 μm.

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8. Fig. 7. Fluorescence pattern of brush polymer cells on the surface of an aluminum substrate in the light of Cy5 dye fluorescence with an exposure of 10 ms after activation of carboxyl groups and attachment of Cy5-NH₂ dye. A graph of the signal distribution along the drawn line on the fluorescence image is shown.

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9. Fig. 8. Fluorescence pattern of the chip cells in the Cy3 channel with immobilized DNA probes with microdroplet application of solutions containing various chaotropic reagents. Exposure time 100 ms. Carbonate buffer, pH 9.0, 5% glycerol. 1 – 10% formamide; 2 – 20% formamide; 3 – 10% DMSO; 4 – 20% DMSO; 5 – 0.01% Tween 20; 6 – 0.1% Tween 20; 7 – without additives.

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10. Fig. 9. Fluorescence pattern of the chip cells on the Cy3 channel with immobilized DNA probes with microdroplet application of solutions containing different concentrations of glycerol. Exposure time 100 ms. Carbonate buffer, pH 9.0, 10% formamide. 1 – empty cells; 2 – 5% glycerol; 3 – 10% glycerol; 4 – 20% glycerol.

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11. Fig. 10. Fluorescence pattern of the chip cells in the Cy3 channel after immobilization of oligonucleotide probes labeled with Cy3. The graph of the signal distribution along the drawn line in the fluorescence image of row 2 for probe No. 269 is shown.

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12. Fig. 11. Fluorescence pattern in the Cy5 channel of brush polymer cells on the surface of an aluminum substrate after hybridization with a target labeled with Cy5 dye. The graphs of signal distribution along the drawn line in the fluorescence image of row 1 of probe #268 and row 2 of probe #269 are shown.

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