Immobilization of protein probes on biochips with brush polymer cells

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Resumo

The methods of obtaining a polymer coating from polyvinyl acetate on the surface of polyethylene terephthalate polymer substrates and subsequent production by photoinduced radical copolymerization of acrylate monomers of brush polymers have been studied. Cell matrices with numerous reactive chemical groups were formed by photolithography for subsequent immobilization of proteins. Methods of activation of carboxyl groups on brush polymers attached to the surface of polyethylene terephthalate have been tested. Immobilization of the streptavidin model protein labeled with fluorescent dye Cy3 was performed to test the activation method of carboxyl groups. A variant of immunofluorescence analysis in the format of a biological microchip was tested on the streptavidin – biotinylated immunoglobulin model. Streptavidin, immobilized in brush polymer cells, retains functionality and spatial accessibility for binding to biotinylated immunoglobulin and subsequent manifestation by antibodies fluorescently labeled with Cy5 dye, which opens up prospects for the use of biological microchips with brush polymer cells on polyethylene terephthalate substrates for immunofluorescence analysis of various protein targets.

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Sobre autores

G. Shtylev

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: chud@eimb.ru
Rússia, ul. Vavilova 32, Moscow, 119991

I. Shishkin

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: chud@eimb.ru
Rússia, ul. Vavilova 32, Moscow, 119991

V. Shershov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: chud@eimb.ru
Rússia, ul. Vavilova 32, Moscow, 119991

V. Kuznetsova

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: chud@eimb.ru
Rússia, ul. Vavilova 32, Moscow, 119991

D. Kachulyak

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: chud@eimb.ru
Rússia, ul. Vavilova 32, Moscow, 119991

V. Butvilovskaya

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: chud@eimb.ru
Rússia, ul. Vavilova 32, Moscow, 119991

A. Levashova

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: chud@eimb.ru
Rússia, ul. Vavilova 32, Moscow, 119991

V. Vasiliskov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: chud@eimb.ru
Rússia, ul. Vavilova 32, Moscow, 119991

O. Zasedateleva

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: chud@eimb.ru
Rússia, ul. Vavilova 32, Moscow, 119991

A. Chudinov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Autor responsável pela correspondência
Email: chud@eimb.ru
Rússia, ul. Vavilova 32, Moscow, 119991

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2. Fig. 1. Scheme for obtaining a biochip using photolithography (a), schematic representation of a cell made of brush polymers (b) and subsequent immobilization of streptavidin (Sav) and the Sav–Human IgG–Goat anti-Human-Сy5 ternary complex (c).

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3. Fig. 2. Fluorescent image of biochip cells in the light of Cy5 dye fluorescence after cell activation with various reagents and immobilization of Cy5-NH2 dye: 1 – cell activation with EDC/NHS in DMSO, 2 –TSTU/NHS in DMSO, 3 – HBTU/NHS in DMSO, 4 – COMU/NHS in DMSO, 5 – EDC/NHS in MES buffer.

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4. Fig. 3. Schematic diagram of the structure of fluorescent dyes.

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5. Fig. 4. Fluorescent pattern of the biochip in the light of the Cy3 dye fluorescence after immobilization of streptavidin (Sav) and streptavidin labeled with Cy3 (Sav-Cy3): 1 and 4 – Sav, 2 and 5 – Sav-Cy3, 3 – empty cells. The graph of the signal distribution along the drawn lines 2 and 5 on the fluorescent image is shown.

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6. Fig. 5. Fluorescent pattern of the biochip on the Cy5 channel with immobilized streptavidin (Sav) and streptavidin labeled with Cy3 (Sav-Cy3) after incubation with goat antibodies against human immunoglobulin labeled with biotin and Cy5 dye (Goat anti-Human IgG-Bio-Cy5). Rows 1 and 4 contain Sav, 2 and 5 – Sav-Cy3, 3 – empty cells. The graph of signal distribution along lines 4 and 5 on the fluorescent image is shown.

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7. Fig. 6. Fluorescent picture of the biochip in the light of the fluorescence of the Cy3 dye with immobilized streptavidin after incubation with human immunoglobulins labeled with biotin and the Cy3 dye (Human IgG-Bio-Cy3) – rows 1 and 3; row 2 – empty cells. The graph of the signal distribution along the drawn lines 1 and 3 on the fluorescent image is shown.

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8. Fig. 7. Fluorescent pattern of the biochip in the light of the fluorescence of the Cy5 dye with immobilized streptavidin after incubation with human immunoglobulins labeled with biotin and the Cy3 dye (Human IgG-Bio-Cy3) and development with goat antibodies against human immunoglobulins labeled with the Cy5 dye (Goat anti-Human IgG-Cy5) – rows 1 and 3; row 2 – empty cells. The graph of the signal distribution along the drawn lines 1 and 3 on the fluorescent image is shown.

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