In situ detection of transrenal gene mutations without DNA isolation and amplification using Array Piezoelectric Plate Sensor (PEPS)

Abstract

Low molecular weight (LMW) DNA fragments from distant organs can pass through kidneys and exist in urine as transrenal DNA. Transrenal DNA provides a noninvasive means to access cell-free DNAs from distant organs such as liver, pancreas, colon that are otherwise difficult to obtain. Shih and Shihs laboratory at Drexel has developed a highly sensitive piezoelectric plate sensor (PEPS) based on 8 um thick highly piezoelectric lead magnesium niobate-lead titanate (PMN-PT) freestanding layer. For detection, receptor specific to a target analyte is immobilized on the PEPS. Binding of the target analyte to the receptor on the PEPS surface decreases its resonance frequency. In-situ, label-free detection of the target analyte is achieved by monitoring the PEPS resonance frequency shift. The detection resonance frequency shift of PEPS is enhanced more than 1000 times due to the PMN-PT layers capability to change its crystalline orientation upon analyte binding. As a result, PEPS has exhibited unprecedented attomolar (aM) sensitivity in label-free detection of single-stranded DNA. The purpose of this study was to further develop PEPS as a tool for rapid (in <40 min), label-free, multiplexed and highly sensitive detection of transrenal gene mutations and gene methylations of distant organs in urine. 1762T/1764A double mutation of hepatitis B virus (HBV), a risk factor associated with hepatocellular carcinoma (HCC) and Kras point mutation at codon 12, a biomarker present in 50% of colorectal carcinomas (CRC) will be the model transrenal gene mutations in this study. To achieve the goal, a signal processing algorithm was developed to reduce noise in the resonance spectrum to increase the resolution of the detection resonance frequency shift. A flow system was implemented with temperature-controlled compartments to effectively dehybridize the naturally double-stranded target DNA so that the target DNA can be detected without the need for DNA isolation, concentration, and amplification. The use of temperature, flow speed and locked nucleic acid (LNA) were exploited to enhance the detection specificity of the mutated DNA against the wild type and an array of PEPS is used to detect multiple target DNAs simultaneously. Results indicate that PEPS could directly detect kRas point mutations in urine with 0.1 aM (60 copies/ml) sensitivity and 1:1000 mutant to wild type specificity without labeling, DNA isolation and amplification.%%%%Ph.D., Biomedical Engineering – Drexel University, 2013