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

Abstract

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 (ΔF = 34 Hz/1000 E. coli cells) as compared to existing manual method (R.S.D. = 19%, n = 5 and ΔF = 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. © 2006 Elsevier B.V. All rights reserved.