Kirimli Ceyhun E, Shih Wei-Heng, Shih Wan Y
School of Biomedical Engineering, Science, and Health Systems, Drexel University, Philadelphia, PA, 10104, USA.
Department of Materials Science and Engineering, Drexel University, Philadelphia, PA, 10104, USA.
Methods Mol Biol. 2017;1572:327-348. doi: 10.1007/978-1-4939-6911-1_22.
We have examined in situ detection of single-nucleotide KRAS mutations in urine using a (Pb(MgNb)O)(PbTiO) (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 °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 WT FRMs that bound to only the captured WT. The captured blue MT FRMs 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.
我们使用涂有与KRAS突变互补的17个核苷酸（nt）锁核酸（LNA）探针DNA的（Pb（MgNb）O）（PbTiO）（PMN-PT）压电板传感器（PEPS），在不进行DNA分离和扩增的情况下，对尿液中的单核苷酸KRAS突变进行了原位检测。在野生型（WT）背景下，以4 mL/min的流速和63°C的温度对尿液中的原位突变体（MT）DNA进行检测，PEPS垂直位于流中心。温度和冲击流力都能区分野生型。在这些条件下，PEPS被证明能在30分钟内原位特异性检测KRAS MT，在临床相关的WT背景下，分析灵敏度为60拷贝/mL，WT浓度比MT高1000倍，无需DNA分离、扩增或标记。为了验证，在仅与捕获的MT结合的蓝色MT荧光报告微球（FRMs）（MT FRMs）和仅与捕获的WT结合的橙色WT FRMs的混合物中进行检测。尽管尿液中WT是MT的1000倍，但捕获的蓝色MT FRMs数量仍比橙色WT FRMs多4至1倍，说明了点突变检测的特异性。
