Cargando…

Multiplex Digital Quantification of β-Lactamase Genes in Antibiotic-Resistant Bacteria by Counting Gold Nanoparticle Labels on Silicon Microchips

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 nanop...

Descripción completa

Detalles Bibliográficos
Autores principales: Presnova, Galina V., Presnov, Denis E., Filippova, Anna A., Tsiniaikin, Ilia I., Ulyashova, Mariya M., Rubtsova, Maya Yu.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9024738/
https://www.ncbi.nlm.nih.gov/pubmed/35448287
http://dx.doi.org/10.3390/bios12040226
_version_ 1784690679485562880
author Presnova, Galina V.
Presnov, Denis E.
Filippova, Anna A.
Tsiniaikin, Ilia I.
Ulyashova, Mariya M.
Rubtsova, Maya Yu.
author_facet Presnova, Galina V.
Presnov, Denis E.
Filippova, Anna A.
Tsiniaikin, Ilia I.
Ulyashova, Mariya M.
Rubtsova, Maya Yu.
author_sort Presnova, Galina V.
collection PubMed
description 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.
format Online
Article
Text
id pubmed-9024738
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-90247382022-04-23 Multiplex Digital Quantification of β-Lactamase Genes in Antibiotic-Resistant Bacteria by Counting Gold Nanoparticle Labels on Silicon Microchips Presnova, Galina V. Presnov, Denis E. Filippova, Anna A. Tsiniaikin, Ilia I. Ulyashova, Mariya M. Rubtsova, Maya Yu. Biosensors (Basel) Article 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. MDPI 2022-04-09 /pmc/articles/PMC9024738/ /pubmed/35448287 http://dx.doi.org/10.3390/bios12040226 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Presnova, Galina V.
Presnov, Denis E.
Filippova, Anna A.
Tsiniaikin, Ilia I.
Ulyashova, Mariya M.
Rubtsova, Maya Yu.
Multiplex Digital Quantification of β-Lactamase Genes in Antibiotic-Resistant Bacteria by Counting Gold Nanoparticle Labels on Silicon Microchips
title Multiplex Digital Quantification of β-Lactamase Genes in Antibiotic-Resistant Bacteria by Counting Gold Nanoparticle Labels on Silicon Microchips
title_full Multiplex Digital Quantification of β-Lactamase Genes in Antibiotic-Resistant Bacteria by Counting Gold Nanoparticle Labels on Silicon Microchips
title_fullStr Multiplex Digital Quantification of β-Lactamase Genes in Antibiotic-Resistant Bacteria by Counting Gold Nanoparticle Labels on Silicon Microchips
title_full_unstemmed Multiplex Digital Quantification of β-Lactamase Genes in Antibiotic-Resistant Bacteria by Counting Gold Nanoparticle Labels on Silicon Microchips
title_short Multiplex Digital Quantification of β-Lactamase Genes in Antibiotic-Resistant Bacteria by Counting Gold Nanoparticle Labels on Silicon Microchips
title_sort multiplex digital quantification of β-lactamase genes in antibiotic-resistant bacteria by counting gold nanoparticle labels on silicon microchips
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9024738/
https://www.ncbi.nlm.nih.gov/pubmed/35448287
http://dx.doi.org/10.3390/bios12040226
work_keys_str_mv AT presnovagalinav multiplexdigitalquantificationofblactamasegenesinantibioticresistantbacteriabycountinggoldnanoparticlelabelsonsiliconmicrochips
AT presnovdenise multiplexdigitalquantificationofblactamasegenesinantibioticresistantbacteriabycountinggoldnanoparticlelabelsonsiliconmicrochips
AT filippovaannaa multiplexdigitalquantificationofblactamasegenesinantibioticresistantbacteriabycountinggoldnanoparticlelabelsonsiliconmicrochips
AT tsiniaikiniliai multiplexdigitalquantificationofblactamasegenesinantibioticresistantbacteriabycountinggoldnanoparticlelabelsonsiliconmicrochips
AT ulyashovamariyam multiplexdigitalquantificationofblactamasegenesinantibioticresistantbacteriabycountinggoldnanoparticlelabelsonsiliconmicrochips
AT rubtsovamayayu multiplexdigitalquantificationofblactamasegenesinantibioticresistantbacteriabycountinggoldnanoparticlelabelsonsiliconmicrochips