title: Real-time PCR microfluidic devices with concurrent electrochemical detection.
abstract: Electrochemistry-based detection methods hold great potential towards development of hand-held nucleic-acid analyses instruments. In this work, we demonstrate the implementation of in situ electrochemical (EC) detection method in a microfluidic flow-through EC-qPCR (FTEC-qPCR) device, where both the amplification of the target nucleic-acid sequence and subsequent EC detection of the PCR amplicon are realized simultaneously at selected PCR cycles in the same device. The FTEC-qPCR device utilizes methylene blue (MB), an electroactive DNA intercalator, for electrochemical signal measurements in the presence of PCR reagent components. Our EC detection method is advantageous, when compared to other existing EC methods for PCR amplicon analysis, since FTEC-qPCR does not require probe-modified electrodes, or asymmetric PCR, or solid-phase PCR. Key technical issues related to surface passivation, electrochemical measurement, PCR inhibition by metal electrode, bubble-free PCR, were investigated. By controlling the concentration of MB and the exposure of PCR mixture to the bare metal electrode, we successfully demonstrated electrochemical measurement of MB in solution-phase, symmetric PCR by amplifying a fragment of lambda phage DNA. The threshold cycle (C(t)) values for both the electrochemical and fluorescence-based assays decreased linearly with the increase of the input target quantity. The sensitivity of EC-based detection of PCR products is comparable to the sensitivity of an optical fluorescence detection system.
authors.affiliation: BioMEMS Lab, School of Mechanical and Aerospace Engineering, Nanyang Technological University, Nanyang Avenue, Singapore 639798, Singapore.
authors.forename: Teh Huey
authors.initials: TH
authors.lastname: Fang
authors.forename: Naveen
authors.initials: N
authors.lastname: Ramalingam
authors.forename: Dong
authors.initials: D
authors.lastname: Xian-Dui
authors.forename: Tan Swee
authors.initials: TS
authors.lastname: Ngin
authors.forename: Zeng
authors.initials: Z
authors.lastname: Xianting
authors.forename: Annie Tan
authors.initials: AT
authors.lastname: Lai Kuan
authors.forename: Eric Yap
authors.initials: EY
authors.lastname: Peng Huat
authors.forename: Gong
authors.initials: G
authors.lastname: Hai-Qing
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation*; D003199:Computer Systems; D004247:DNA / Q000032:analysis / Q000737:chemistry* / Q000235:genetics*; D004563:Electrochemistry / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D008904:Miniaturization; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation*; D016133:Polymerase Chain Reaction / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D004247:DNA