Identification of genes of bacterial enzymes beta-lactamases on silicon microchips using gold nanoparticles as a labelстатья
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Дата последнего поиска статьи во внешних источниках: 27 мая 2015 г.
Аннотация:A method for identifying genes and mutations in them by hybridization analysis on silicon microchips using gold nanoparticles as a label was developed. Oligonucleotide probes were immobilized covalently on silicon microchips. DNA duplexes were formed on the surface of the microchip in a result of the interaction of complementary oligonucleotide probes and DNA labeled by biotin. The duplexes then were developed with a conjugate of streptavidin
with gold nanoparticles. The size of the gold nanoparticles was 25 3 nm. The nanoparticles on the microchip surface were identified by scanning electron microscopy (SEM) with an electron microscope SUPRA 40 (Carl Zeiss).
The method was applied for identification the genes of bacterial enzymes beta-lactamases. These enzymes cause hydrolysis of beta-lactam antibiotics and confer bacterial resistance to beta-lactam antibiotics.
A feature of the method is to record the number of DNA duplexes on a surface by the number of gold nanoparticles revealed by SEM. To calculate the number of DNA duplexes the
fragments of 1.7–30 lm2 area were used. Spots not containing immobilized probes or containing immobilized probes with noncomplementary structure were used as control spots. The registration by number of single DNA interactions on the microchip was compared with detection of biotin-labeled DNA by means of enzymatic colorimetric detection. The advantages of the method based on nanoparticles counting consist in higher sensitivity by
2.5 orders of magnitude and better signal/background ratio.