Amplification-free in situ KRAS point mutation detection at 60 copies per mL in urine in a background of 1000-fold wild type.
Amplification-free in situ KRAS point mutation detection at 60 copies per mL in urine in a background of 1000-fold wild type.
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DOI：
10.1039/c5an02048d
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发表时间：
2016-02-21
期刊：
影响因子：
--
通讯作者：
Shih WY
中科院分区：
文献类型：
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作者：
Kirimli CE;Shih WH;Shih WY
我们使用涂有与 KRAS 突变互补的 17 核苷酸 (nt) 锁定核酸 (LNA) 探针 DNA 的 (Pb(Mg1/3Nb2/3)O3)0.65(PbTiO3)0.35 (PMN-PT) 压电板传感器 (PEPS) 对尿液中的单核苷酸 KRAS 突变进行原位检测。为了增强针对野生型 (WT) 的原位突变 (MT) DNA 检测特异性，检测在流速为 4 mL/min、温度为 63°C 的流中进行，PEPS 垂直位于流的中心，其中温度和流冲击力都可以区分野生型。在这种条件下，PEPS 能够在 30 分钟内以 60 拷贝/mL 的分析灵敏度在临床相关 1000 倍以上 WT 的背景下特异性原位检测 KRAS MT，无需 DNA 分离、扩增或标记。为了验证，检测后在仅与捕获的 MT 结合的蓝色 MT 荧光报告微球 (FRM) (MT FRM) 和仅与捕获的 WT 结合的橙色 WT FRM 的混合物中进行检测。显微镜检查显示，尽管 WT 是尿液中 MT 的 1000 倍，但捕获的蓝色 MT FRM 数量仍然是橙色 WT FRM 的 4 比 1 倍。最后，使用 6-PEPS 阵列演示了多重特异性突变检测，每个阵列都有一个针对 6 个密码子 12 KRAS 突变之一的探针 DNA。
We have examined in situ detection of single-nucleotide KRAS mutation in urine using a (Pb(Mg1/3Nb2/3)O3)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. To enhance in situ mutant (MT) DNA detection specificity against the wild type (WT), the detection was carried out in a flow with a flow rate of 4 mL/min and at 63°C with the PEPS vertically situated at the center of the flow in which both the temperature and the flow impingement force discriminated the wild type. Under such conditions, PEPS was shown to specifically detect KRAS MT in situ with 60 copies/mL analytical sensitivity in a background of clinically-relevant 1000-fold more WT in 30 min without DNA isolation, amplification, or labeling. For validation, the detection was followed with detection 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. Microscopic examinations showed that the captured blue MT FRMs still outnumbered the orange WT FRMs by a factor of 4 to 1 even though WT was 1000-fold of MT in urine. Finally, multiplexed specific mutation detection was demonstrated using a 6-PEPS array each with a probe DNA targeting one of the 6 codon-12 KRAS mutations.