A real-time microfluidic multiplex electrochemical loop-mediated isothermal amplification chip for differentiating bacteria

Juan Luo; Xueen Fang; Daixing Ye; Huixiang Li; Hui Chen; Song Zhang; Jilie Kong

doi:10.1016/j.bios.2014.03.073

A real-time microfluidic multiplex electrochemical loop-mediated isothermal amplification chip for differentiating bacteria

Biosens Bioelectron. 2014 Oct 15:60:84-91. doi: 10.1016/j.bios.2014.03.073. Epub 2014 Apr 8.

Authors

Juan Luo¹, Xueen Fang¹, Daixing Ye¹, Huixiang Li¹, Hui Chen¹, Song Zhang¹, Jilie Kong²

Affiliations

¹ Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, PR China.
² Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, PR China. Electronic address: jlkong@fudan.edu.cn.

PMID: 24787122
DOI: 10.1016/j.bios.2014.03.073

Abstract

This report shows that loop-mediated isothermal amplification (LAMP) of nucleic acid can be integrated on a laser etched indium tin oxide (ITO) electrode-based multiplex microfluidic chip for real-time quantitative differentiation of bacteria; we call this technique microfluidic multiplex electrochemical LAMP (μME-LAMP) system. Three important acute upper respiratory tract infections (URTI) related bacteria, namely Mycobacterium tuberculosis (MTB), Haemophilus influenza (HIN), and Klebsiella pneumonia (KPN) were chosen for this study. We monitored the amplification process by measuring and analyzing the electrochemical signal of methylene blue (MB) through eight etched ITO electrochemical reactors. The results indicated that this assay with the ability of analyzing multiple genes qualitatively and quantitatively is highly specific, operationally simple, and cost/time effective. It exhibits high sensitivity with detection limits of 28, 17, and 16 copies μL(-1) for MTB, HIN, and KPN, respectively. The whole differentiation can be finished in a short time of 45 min, which has the potential to apply in clinical diagnosis.

Keywords: Electrochemistry; ITO electrode; Loop mediated isothermal amplification (LAMP); Microfluidic chip; Multiple.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Bacteria / genetics*
Bacteria / isolation & purification*
Biosensing Techniques / instrumentation
Computer Systems
Conductometry / instrumentation*
DNA, Bacterial / analysis
DNA, Bacterial / genetics*
Equipment Design
Equipment Failure Analysis
Microfluidic Analytical Techniques / instrumentation*
Nucleic Acid Amplification Techniques / instrumentation*
Oligonucleotide Array Sequence Analysis / instrumentation*
Reproducibility of Results
Sensitivity and Specificity

Substances

DNA, Bacterial