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.