Piezoelectric plate sensor for in situ genetic detection of hepatitis B virus in serum without DNA isolation and amplification

Mehmet Çağrı Soylu

doi:10.17918/etd-4314

Hepatitis B is a viral infection caused by Hepatitis B virus (HBV) that attacks the liver and can cause both acute and chronic disease in humans. Worldwide, two billion people are infected with the virus and about 350 million people suffer from chronic HBV infection. To diagnose chronic HB the current gold standard is quantitative real-time polymerase chain reaction (qPCR) quantifying serum viral load. However, qPCR needs expensive equipments and chemical agents. A low-cost HB viral load quantification tool will help identifying patients for treatment and save lives. A lead magnesium niobate-lead titanate (PMN-PT) piezoelectric plate sensor (PEPS) is a new type of sensor developed in Shihs laboratory. In-situ real time detection is achieved by monitoring the PEPs resonance frequency shift which is caused by binding of the target to the receptor on the PEPS surface. What makes the PEPS unique is that the shift is caused by the stress generated by the binding of the target and is inherently amplified by the polarization switching in the PMN-PT layer. 3-Mercaptopropyltrimethoxysilane (MPS) coating has previously been used on a PEPS is not sufficient for DNA detection requiring high sensitivity. Furthermore, direct detection of HB viral count in human serum in situ requires releasing the genetic material and de-hybridization of viral DNA. The goal of this thesis is first to improve the insulation coating and second to examine direct quantification of HB viral count without DNA isolation, concentration, and amplification using PMN-PT PEPS. We have shown that by coating the PEPS with 0.1% MPS and 0.5% water at pH 9 for 36 hr reduced the current by two orders of magnitude and that with such insulation the width extension mode (WEM) resonance frequency of a PEPS was stable, i.e., with <9 Hz standard deviation at a resonance frequency of 3.33 MHz for more than 24 h in a phosphate buffered saline (PBS) solution. The surface of the coating was also smooth, optimal for PEPS binding stress induced detection resonance shift. In situ detection of viral DNA spiked in simulated serum in one single test was accomplished with a flow system where the viral DNA was first denatured in a reservoir at 95°C, followed by fast cooling in narrow tubing in a cooling medium to allow the denatured DNA to be detected by the PEPS in the detection cell at 55°C. PEPS has demonstrated 30 copies/ml analytical sensitivity in such in situ viral DNA detection. By examining viral DNA detection at extremely low concentrations such as 10-100 copies/ml with different volumes, we estimated that one copy of viral DNA caused a WEM detection resonance frequency shift of -Delta_f = 1.7 Hz per copy. In situ detection of HB viruses in simulated serum in one single test was achieved by adding 10% sodium dodecyl sulfate (SDS) in the simulated serum samples spiked with HB viral particles where the viral particles were lysed, DNA released and denatured at 95°C for 10 min in the reservoir. Results indicate that using such schemes PEPS could in situ detect the genetic signature of HB viral particles in simulated serum in a single test in 40 min with 300 copies/ml sensitivity.

Piezoelectric plate sensor for in situ genetic detection of hepatitis B virus in serum without DNA isolation and amplification

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Piezoelectric plate sensor for in situ genetic detection of hepatitis B virus in serum without DNA isolation and amplification

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