Flow analysis coupled with PQC/DNA biosensor for assay of E. coli based on detecting DNA products from PCR amplification.
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 0.2M NaCl in TE buffer (10mM Tris, 1mM 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 (DeltaF=34 Hz/1000 E. coli cells) as compared to existing manual method (R.S.D.=19%, n=5 and DeltaF=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.
Sun
Hui
H
Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China.
Zhang
Youyu
Y
Fung
Yingsing
Y
D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D015169:Colony Count, Microbial / Q000295:instrumentation* / Q000379:methods; D004269:DNA, Bacterial / Q000032:analysis*; D004563:Electrochemistry / Q000295:instrumentation* / Q000379:methods; D004784:Environmental Monitoring / Q000295:instrumentation; D004867:Equipment Design; D019544:Equipment Failure Analysis; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification*; D017022:Flow Injection Analysis / Q000295:instrumentation* / Q000379:methods; D016133:Polymerase Chain Reaction / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014871:Water Microbiology; D014876:Water Pollution / Q000032:analysis
D004269:DNA, Bacterial