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In situ, amplification-free double-stranded mutation detection at 60 copies/ml with thousand-fold wild type in urine
Journal article   Open access   Peer reviewed

In situ, amplification-free double-stranded mutation detection at 60 copies/ml with thousand-fold wild type in urine

Ceyhun Kirimli, Selena Lin, Ying-Hsiu Su, Wei-Heng Shih and Wan Y. Shih
Biosensors & bioelectronics, v 119, pp 221-229
15 Nov 2018
DOI: https://doi.org/10.1016/j.bios.2018.07.062
PMID: 30142581
Featured in Collection :   UN Sustainable Development Goals @ Drexel
url
https://europepmc.org/articles/pmc6524543View
Accepted (AM)Open Access (License Unspecified),  Open

Abstract

Amplification-free mutation detection High-specificity mutation detection Isolation-free mutation detection Label-free double-stranded DNA detection Label-free mutation detection
We have investigated amplification-free in situ double-stranded mutation detection in urine in the concentration range 10–19 M – 10–16 M using piezoelectric plate sensors (PEPs). The detection was carried out in a close-loop flow with two temperature zones. The 95 °C high-temperature zone served as the reservoir where the sample was loaded and DNA de-hybridized. The heated urine was cooled flowing through a 1 m long tubing immersed in room-temperature water bath at a flow rate of 4 ml/min to reach the detection cell at the desired temperature for the detection to take place. With hepatitis B virus double mutation (HBVDM) and KRAS G12V point mutation as model double mutations, it is shown that PEPS was able to detect double-stranded HBVDM and KRAS with 70% detection efficiency or better at concentration as low as 10–19 M against single-stranded mutation detection at the same concentrations, which was validated by the following in situ fluorescent reporter microspheres (FRMs) detection as well as microscopic visualization of the FRMs bound to the captured mutant on the PEPS surface. Furthermore, the same double-stranded mutation detection efficacy was demonstrated at 10–19 M – 10–16 M in a background of 250-fold wildtype for HBVDM and 1000-fold wildtype for KRAS. Also demonstrated was detection of KRAS mutation at 10–19 M – 10–16 M of SW480 DNA fragments in urine. •Piezoelectric sensor was contained in  a flow system that de-hybridizes double-stranded DNA.•Double-stranded mutations were detected in situ without isolation or amplification.•The detection was in urine without label with 60 copies/ml sensitivity.•KRAS single mutation was  detected in 1000-fold wildtype background.•Double-stranded detection was better than 70% as effective as single-stranded detection.

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7 citations in Web of Science
7 citations in Scopus

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UN Sustainable Development Goals (SDGs)

This publication has contributed to the advancement of the following goals:

#3 Good Health and Well-Being

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Web of Science research areas
Biophysics
Biotechnology & Applied Microbiology
Chemistry, Analytical
Electrochemistry
Nanoscience & Nanotechnology
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