Flow analysis coupled with PQC/DNA biosensor for assay of E-coli based on detecting DNA products from PCR amplification

摘要

A flow-through PQC/DNA biosensor system is developed by combining sequential flow polymerase chain reaction (PCR) products denaturing prior to piezoelectric quartz crystal (PQC) detection via hybridization of ssDNA. The PQC/DNA biosensor is fabricated based on complex formation of neutravidin/biotinylated probe in 0.2 M NaCl in TE buffer (10 mM Tris, 1 mM EDTA, pH 7.5). Results show that the coating fabricated provides a desirable quality with satisfactory performance. Its application for Escherichia coli detection under controlled flow at 0.02 mL/min for denaturing PCR products and 10 mL/min for transferring solution between reactors and delivering samples to detector to reduce rehybridization leads to significant improvement in repeatability (R.S.D. < 6%, n = 5) and sensitivity (Delta F= 34 Hz/1000 E. coli cells) as compared to existing manual method (R.S.D. = 19%, n = 5 and Delta F = 26 Hz/1000 E. coli cells, respectively). Down to 23 E. coli cells are detected, satisfying the HKEPD requirements for E. coli count in beach water. (c) 2006 Elsevier B.V. All rights reserved.