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Rapid label-free visual detection of KRAS mutations using peptide nucleic acid and unmodified gold nanoparticles

机译：使用肽核酸和未修饰的金纳米颗粒快速无标记地目测KRAS突变

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Colorectal cancer (CRC) is among the most commonly diagnosed cancers worldwide. Point mutations on the Kisten rat sarcoma viral oncogene homologue (KRAS) have been identified as a crucial diagnostic and prognostic biomarker for response to cancer therapy targeting the epidermal growth factor receptor. A rapid label-free colorimetric assay for detection of point mutations in codon 12 and 13 of the KRAS gene using peptide nucleic acid (PNA) and unmodified gold nanoparticles (AuNPs) was presented in this study. Charge neutral PNA induces the AuNPs aggregation in the absence of complementary DNA; when the complementary DNA is present, PNA–DNA hybridization retains the dispersion of AuNPs as the negative charges of the DNA strand adsorbed on the AuNPs surface ensure sufficient charge repulsion. Based on this principle, the developed assay was able to detect the specific sequence in the presence of at least 20 times the amount of interference oligonucleotides, and the limit of detection was low as DNA/PNA ratio of 0.02. The PNA induced colorimetric changes in the presence of the complementary DNA at different ratios of DNA/PNA yielded a standard curve with a linear coefficient of R2 = 0.9371. Furthermore, this assay was validated in order that it could be used to detect the seven known mutant alleles in codon 12 and 13 of the KRAS gene by the naked eye with higher concentration of PNA. This simple colorimetric assay is rapid, sensitive and easily performed at room temperature without additional stringent conditions, which promises the method great potential utility for clinical sample screening and point-of-care testing.
机译：结肠直肠癌（CRC）是世界上最常见的诊断癌症之一。已确定Kisten大鼠肉瘤病毒致癌基因同源物（KRAS）上的点突变是针对靶向表皮生长因子受体的癌症治疗的重要诊断和预后生物标志物。在这项研究中提出了一种快速的无标记比色测定法，用于使用肽核酸（PNA）和未修饰的金纳米粒子（AuNPs）检测KRAS基因的密码子12和13中的点突变。电荷中性PNA在不存在互补DNA的情况下诱导AuNPs聚集;当存在互补DNA时，PNA-DNA杂交可保留AuNP的分散性，因为吸附在AuNPs表面的DNA链的负电荷可确保足够的排斥力。基于该原理，开发的测定法能够在干扰寡核苷酸的量至少为20倍的情况下检测特定序列，并且DNA / PNA之比为0.02时检测限较低。在不同DNA / PNA比率下，互补DNA存在下，PNA诱导的比色变化产生一条线性系数为 R 2 = 0.9371。此外，为了通过肉眼用较高浓度的PNA可以检测KRAS基因密码12和13中的七个已知突变等位基因，对该方法进行了验证。这种简单的比色测定法快速，灵敏并且在室温下容易进行，而无需额外的严格条件，这保证了该方法在临床样品筛选和即时检验中具有巨大的潜在用途。

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