BIOSENSORS AND BIODETECTION: METHODS AND PROTOCOLS, VOL 2: ELECTROCHEMICAL, BIOELECTRONIC, PIEZOELECTRIC, CELLULAR AND MOLECULAR BIOSENSORS, 2ND EDITION, cilt.1572, ss.327-348, 2017 (Diğer Kurumların Hakemli Dergileri)
We have examined in situ detection of single-nucleotide KRAS mutations in urine using a (Pb(Mg1/3Nb2/3)O-3)(0.65)(PbTiO3)(0.35) (PMN-PT) piezoelectric plate sensor (PEPS) coated with a 17-nucleotide (nt) locked nucleic acid (LNA) probe DNA complementary to the KRAS mutation without DNA isolation and amplification. In situ mutant (MT) DNA in urine in a wild type (WT) background was carried out at a flow rate of 4 mL/min and at 63 degrees C with the PEPS vertically situated at the center of the flow. Both the temperature and the impingement flow force discriminated the wild type. Under these conditions PEPS was shown to specifically detect KRAS MT in situ within 30 min with an analytical sensitivity of 60 copies/mL in a clinically relevant background of WT with concentrations 1000-fold greater than that of MT without DNA isolation, amplification, or labeling. For validation, detection was performed in a mixture of blue MT fluorescent reporter microspheres (FRMs) (MT FRMs) that bound to only the captured MT, and orange WTFRMs that bound to only the captured WT. The captured blue MTFRMs still outnumbered the orange WT FRMs by a factor of 4-1 even though WT was 1000-fold of MT in urine, illustrating the specificity of the point mutation detection.