In situ, amplification-free double-stranded mutation detection at 60 copies/ml with thousand-fold wild type in urine.

We have investigated amplification-free in situ double-stranded mutation detection in urine in the concentration range 10-19 M - 10-16 M using piezoelectric plate sensors (PEPs). The detection was carried out in a close-loop flow with two temperature zones. The 95 °C high-temperature zone served as the reservoir where the sample was loaded and DNA de-hybridized. The heated urine was cooled flowing through a 1 m long tubing immersed in room-temperature water bath at a flow rate of 4 ml/min to reach the detection cell at the desired temperature for the detection to take place. With hepatitis B virus double mutation (HBVDM) and KRAS G12V point mutation as model double mutations, it is shown that PEPS was able to detect double-stranded HBVDM and KRAS with 70% detection efficiency or better at concentration as low as 10-19 M against single-stranded mutation detection at the same concentrations, which was validated by the following in situ fluorescent reporter microspheres (FRMs) detection as well as microscopic visualization of the FRMs bound to the captured mutant on the PEPS surface. Furthermore, the same double-stranded mutation detection efficacy was demonstrated at 10-19 M - 10-16 M in a background of 250-fold wildtype for HBVDM and 1000-fold wildtype for KRAS. Also demonstrated was detection of KRAS mutation at 10-19 M - 10-16 M of SW480 DNA fragments in urine.