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

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 H2O2 by PyO in the presence of Pi and oxygen, and subsequently, the anodic current of enzymatically generated H2O2 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.