Presnova Galina V, Presnov Denis E, Filippova Anna A, Tsiniaikin Ilia I, Ulyashova Mariya M, Rubtsova Maya Yu
Department of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia.
D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia.
Biosensors (Basel). 2022 Apr 9;12(4):226. doi: 10.3390/bios12040226.
Digital quantification based on counting of individual molecules is a promising approach for different biomedical applications due to its enhanced sensitivity. Here, we present a method for the digital detection of nucleic acids (DNA and RNA) on silicon microchips based on the counting of gold nanoparticles (GNPs) in DNA duplexes by scanning electron microscopy (SEM). Biotin-labeled DNA is hybridized with capture oligonucleotide probes immobilized on the microchips. Then biotin is revealed by a streptavidin-GNP conjugate followed by the detection of GNPs. Sharp images of each nanoparticle allow the visualization of hybridization results on a single-molecule level. The technique was shown to provide highly sensitive quantification of both short oligonucleotide and long double-strand DNA sequences up to 800 bp. The lowest limit of detection of 0.04 pM was determined for short 19-mer oligonucleotide. The method's applicability was demonstrated for the multiplex quantification of several β-lactamase genes responsible for the development of bacterial resistance against β-lactam antibiotics. Determination of nucleic acids is effective for both specific DNA in lysates and mRNA in transcripts. The method is also characterized by high selectivity for single-nucleotide polymorphism discrimination. The proposed principle of digital quantification is a perspective for studying the mechanisms of bacterial antibiotic resistance and bacterial response to drugs.
基于单个分子计数的数字定量由于其更高的灵敏度，是一种在不同生物医学应用中很有前景的方法。在此，我们提出一种基于扫描电子显微镜（SEM）对DNA双链体中的金纳米颗粒（GNP）进行计数，从而在硅微芯片上对核酸（DNA和RNA）进行数字检测的方法。生物素标记的DNA与固定在微芯片上的捕获寡核苷酸探针杂交。然后通过链霉亲和素-GNP共轭物揭示生物素，随后检测GNP。每个纳米颗粒的清晰图像能够在单分子水平上可视化杂交结果。该技术已被证明能对短至19聚体寡核苷酸以及长达800 bp的长双链DNA序列进行高灵敏度定量。对于短的19聚体寡核苷酸，确定的最低检测限为0.04 pM。该方法的适用性已在对几种导致细菌对β-内酰胺抗生素耐药性产生的β-内酰胺酶基因进行多重定量分析中得到证明。对核酸的测定对于裂解物中的特定DNA和转录本中的mRNA均有效。该方法还具有对单核苷酸多态性区分的高选择性。所提出的数字定量原理对于研究细菌抗生素耐药机制和细菌对药物的反应具有重要意义。
