Detection of Mycobacterium tuberculosis purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay
Netrapal Singh,1,2 Bhawna Dahiya,1 Venkatraman Srinivasan Radhakrishnan,3 Tulika Prasad,3 Promod K Mehta1,4 1Centre for Biotechnology, Maharshi Dayanand University, Rohtak, Haryana, India; 2Institute of Synthetic Biology (iSynBio), Shenzhen Institutes of Advanced Technology, Chinese Academy of Scie...
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| Format: | Article |
| Language: | English |
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Dove Medical Press
2018-12-01
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| Series: | International Journal of Nanomedicine |
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| Online Access: | https://www.dovepress.com/detection-of-mycobacterium-tuberculosis-purified-esat-6-rv3875-by-magn-peer-reviewed-article-IJN |
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| author | Singh N Dahiya B Radhakrishnan VS Prasad T Mehta PK |
| author_facet | Singh N Dahiya B Radhakrishnan VS Prasad T Mehta PK |
| author_sort | Singh N |
| collection | DOAJ |
| description | Netrapal Singh,1,2 Bhawna Dahiya,1 Venkatraman Srinivasan Radhakrishnan,3 Tulika Prasad,3 Promod K Mehta1,4 1Centre for Biotechnology, Maharshi Dayanand University, Rohtak, Haryana, India; 2Institute of Synthetic Biology (iSynBio), Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China; 3Advanced Instrumentation Research & Facility (AIRF) and Special Centre for Nanoscience (SCNS), Jawaharlal Nehru University (JNU), New Delhi, Delhi, India; 4Microbiology Department, Central University of Haryana, Mahendergarh, India Purpose: Immuno-PCR (I-PCR), an ultrasensitive method, combines the versatility of ELISA with the exponential amplification capacity of PCR. Coupling of detection antibodies with the reporter DNA is a critical step of I-PCR. Gold nanoparticles (GNPs) and magnetic beads (MBs) are relatively easy to attach with the antibodies and DNA. Therefore, we designed MB-coupled GNP-based I-PCR (MB-GNP-I-PCR) assay for the detection of Mycobacterium tuberculosis antigen. Methods: GNPs were synthesized by chemical reduction and seed-mediated synthesis. Functionalized GNPs were prepared by coupling GNPs with the detection antibodies and reporter DNA and were characterized. Detection limit of M. tuberculosis-specific purified early secreted antigenic target-6 (ESAT-6) (Rv3875) was determined by MB-GNP-I-PCR. Results: Transmission electron microscopy revealed spherical and slightly polydispersed GNPs of ~20 and ~60 nm size. Coupling of antibodies to GNPs was indicated by a shift in absorption maxima from 524 to 534 nm, which was confirmed by transmission electron microscopy. A color reaction with ELISA and the presence of 76 bp product by PCR further validated the coupling of detection antibodies and signal DNA to the functionalized GNPs. Also, attachment of capture antibodies with MBs was confirmed by magneto-ELISA. Detection limit of purified ESAT-6 by MB-GNP-I-PCR was determined to be 10 fg/mL, 105-fold lower than analogous ELISA. Notably, no sample matrix effect was observed in the saliva samples of healthy individuals spiked with the purified ESAT-6. Conclusion: Unlike conventional I-PCR (solid format), MB-GNP-I-PCR (liquid format) is relatively simple with the reduced background signals, which can be further exploited for the clinical diagnosis of tuberculosis. Keywords: MB-GNP-I-PCR, functionalized GNPs, ELISA, LOD |
| first_indexed | 2024-12-14T13:22:15Z |
| format | Article |
| id | doaj.art-12519051a88141aaab0444e15fc9be66 |
| institution | Directory Open Access Journal |
| issn | 1178-2013 |
| language | English |
| last_indexed | 2024-12-14T13:22:15Z |
| publishDate | 2018-12-01 |
| publisher | Dove Medical Press |
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| series | International Journal of Nanomedicine |
| spelling | doaj.art-12519051a88141aaab0444e15fc9be662022-12-21T22:59:54ZengDove Medical PressInternational Journal of Nanomedicine1178-20132018-12-01Volume 138523853542992Detection of Mycobacterium tuberculosis purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assaySingh NDahiya BRadhakrishnan VSPrasad TMehta PKNetrapal Singh,1,2 Bhawna Dahiya,1 Venkatraman Srinivasan Radhakrishnan,3 Tulika Prasad,3 Promod K Mehta1,4 1Centre for Biotechnology, Maharshi Dayanand University, Rohtak, Haryana, India; 2Institute of Synthetic Biology (iSynBio), Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China; 3Advanced Instrumentation Research & Facility (AIRF) and Special Centre for Nanoscience (SCNS), Jawaharlal Nehru University (JNU), New Delhi, Delhi, India; 4Microbiology Department, Central University of Haryana, Mahendergarh, India Purpose: Immuno-PCR (I-PCR), an ultrasensitive method, combines the versatility of ELISA with the exponential amplification capacity of PCR. Coupling of detection antibodies with the reporter DNA is a critical step of I-PCR. Gold nanoparticles (GNPs) and magnetic beads (MBs) are relatively easy to attach with the antibodies and DNA. Therefore, we designed MB-coupled GNP-based I-PCR (MB-GNP-I-PCR) assay for the detection of Mycobacterium tuberculosis antigen. Methods: GNPs were synthesized by chemical reduction and seed-mediated synthesis. Functionalized GNPs were prepared by coupling GNPs with the detection antibodies and reporter DNA and were characterized. Detection limit of M. tuberculosis-specific purified early secreted antigenic target-6 (ESAT-6) (Rv3875) was determined by MB-GNP-I-PCR. Results: Transmission electron microscopy revealed spherical and slightly polydispersed GNPs of ~20 and ~60 nm size. Coupling of antibodies to GNPs was indicated by a shift in absorption maxima from 524 to 534 nm, which was confirmed by transmission electron microscopy. A color reaction with ELISA and the presence of 76 bp product by PCR further validated the coupling of detection antibodies and signal DNA to the functionalized GNPs. Also, attachment of capture antibodies with MBs was confirmed by magneto-ELISA. Detection limit of purified ESAT-6 by MB-GNP-I-PCR was determined to be 10 fg/mL, 105-fold lower than analogous ELISA. Notably, no sample matrix effect was observed in the saliva samples of healthy individuals spiked with the purified ESAT-6. Conclusion: Unlike conventional I-PCR (solid format), MB-GNP-I-PCR (liquid format) is relatively simple with the reduced background signals, which can be further exploited for the clinical diagnosis of tuberculosis. Keywords: MB-GNP-I-PCR, functionalized GNPs, ELISA, LODhttps://www.dovepress.com/detection-of-mycobacterium-tuberculosis-purified-esat-6-rv3875-by-magn-peer-reviewed-article-IJNMB-GNP-I-PCRFunctionalized GNPsELISALOD |
| spellingShingle | Singh N Dahiya B Radhakrishnan VS Prasad T Mehta PK Detection of Mycobacterium tuberculosis purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay International Journal of Nanomedicine MB-GNP-I-PCR Functionalized GNPs ELISA LOD |
| title | Detection of Mycobacterium tuberculosis purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay |
| title_full | Detection of Mycobacterium tuberculosis purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay |
| title_fullStr | Detection of Mycobacterium tuberculosis purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay |
| title_full_unstemmed | Detection of Mycobacterium tuberculosis purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay |
| title_short | Detection of Mycobacterium tuberculosis purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay |
| title_sort | detection of mycobacterium tuberculosis purified esat 6 rv3875 by magnetic bead coupled gold nanoparticle based immuno pcr assay |
| topic | MB-GNP-I-PCR Functionalized GNPs ELISA LOD |
| url | https://www.dovepress.com/detection-of-mycobacterium-tuberculosis-purified-esat-6-rv3875-by-magn-peer-reviewed-article-IJN |
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