Analysis of polymerase chain reaction amplifications through phosphate detection using an enzyme–based microbiosensor in a microfluidic device

✍ Scribed by Muhammad J. A. Shiddiky; Md. Aminur Rahman; Jang-Su Park; Yoon-Bo Shim

• Publisher: John Wiley and Sons
• Year: 2006
• Tongue: English
• Weight: 224 KB
• Volume: 27
• Category: Article
• ISSN: 0173-0835
• DOI: 10.1002/elps.200500679

✦ Synopsis

Abstract

An electrochemical method was developed for analyzing PCR amplification through the detection of inorganic phosphates (Pi). This method coupled a microchip to a nanoparticle comprising poly‐5,2'‐5',2''‐terthiophene‐3'‐carboxylic acid (poly‐TTCA)/pyruvate oxidase (PyO) modified microbiosensor. It detects Pi produced from the pyrophosphate (PPi), which is released as a byproduct of PCR. After completion of PCR, PPi is hydrolyzed to Pi by inorganic pyrophosphatase. On the microbiosensor surface, pyruvate was converted to H~2~O~2~ by PyO in the presence of Pi and oxygen, and subsequently, the anodic current of enzymatically generated H~2~O~2~ was detected at +0.5 V versus Ag/AgCl. The CE‐EC analysis was completed within 2 min in a coated channel with 75.0 mm separation length at the field strength of −200 V/cm. Excellent operation stability of poly‐TTCA/PyO was observed for a long period of analysis. The reproducibility of the analysis yielded an RSD of 3.4% (n = 22) for the peak areas and 1.8% (n = 22) for the migration times. The sensitivity of the analysis was 0.59 ± 0.01 nA/cycle with a regression coefficient of 0.971.