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...
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| Format: | Article |
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MDPI AG
2022-04-01
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| Series: | Biosensors |
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| Online Access: | https://www.mdpi.com/2079-6374/12/4/226 |
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| author | Galina V. Presnova Denis E. Presnov Anna A. Filippova Ilia I. Tsiniaikin Mariya M. Ulyashova Maya Yu. Rubtsova |
| author_facet | Galina V. Presnova Denis E. Presnov Anna A. Filippova Ilia I. Tsiniaikin Mariya M. Ulyashova Maya Yu. Rubtsova |
| author_sort | Galina V. Presnova |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-03-09T11:04:46Z |
| format | Article |
| id | doaj.art-035f638ff1364f38806b649ebea46eee |
| institution | Directory Open Access Journal |
| issn | 2079-6374 |
| language | English |
| last_indexed | 2024-03-09T11:04:46Z |
| publishDate | 2022-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biosensors |
| spelling | doaj.art-035f638ff1364f38806b649ebea46eee2023-12-01T00:58:11ZengMDPI AGBiosensors2079-63742022-04-0112422610.3390/bios12040226Multiplex Digital Quantification of β-Lactamase Genes in Antibiotic-Resistant Bacteria by Counting Gold Nanoparticle Labels on Silicon MicrochipsGalina V. Presnova0Denis E. Presnov1Anna A. Filippova2Ilia I. Tsiniaikin3Mariya M. Ulyashova4Maya Yu. Rubtsova5Department of Chemistry, Lomonosov Moscow State University, 119991 Moscow, RussiaD.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov Moscow State University, 119991 Moscow, RussiaDepartment of Chemistry, Lomonosov Moscow State University, 119991 Moscow, RussiaMSU Quantum Technology Centre, 119991 Moscow, RussiaDepartment of Chemistry, Lomonosov Moscow State University, 119991 Moscow, RussiaDepartment of Chemistry, Lomonosov Moscow State University, 119991 Moscow, RussiaDigital 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.https://www.mdpi.com/2079-6374/12/4/226high-sensitive digital detectionquantificationfunctionalized gold nanoparticlesmicrochipsβ-lactamasesantibiotic-resistant bacteria |
| spellingShingle | Galina V. Presnova Denis E. Presnov Anna A. Filippova Ilia I. Tsiniaikin Mariya M. Ulyashova Maya Yu. Rubtsova Multiplex Digital Quantification of β-Lactamase Genes in Antibiotic-Resistant Bacteria by Counting Gold Nanoparticle Labels on Silicon Microchips Biosensors high-sensitive digital detection quantification functionalized gold nanoparticles microchips β-lactamases antibiotic-resistant bacteria |
| 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 | high-sensitive digital detection quantification functionalized gold nanoparticles microchips β-lactamases antibiotic-resistant bacteria |
| url | https://www.mdpi.com/2079-6374/12/4/226 |
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