Han Jinhee, Tan Matthew, Sudheendra Lakshmana, Weiss Robert H, Kennedy Ian M
Department of Mechanical and Aerospace Engineering, University of California, Davis, California 95616, USA.
Division of Nephrology, Department of Internal Medicine, University of California, Davis, California 95616, USA.
Nano Res. 2014 Sep 1;7(9):1302-1310. doi: 10.1007/s12274-014-0494-z.
A nanoparticle-assembled photonic crystal (PC) array was used to detect single nucleotide polymorphism (SNP). The assay platform with PC nanostructure enhanced the fluorescent signal from nanoparticle-hybridized DNA complexes due to phase matching of excitation and emission. Nanoparticles coupled with probe DNA were trapped into nanowells in an array by using an electrophoretic particle entrapment system. The PC/DNA assay platform was able to identify a 1 base pair (bp) difference in synthesized nucleotide sequences that mimicked the mutation seen in a feline model of human autosomal dominant polycystic kidney disease (PKD) with a sensitivity of 0.9 fg/mL (50 aM)-sensitivity, which corresponds to 30 oligos/array. The reliability of the PC/DNA assay platform to detect SNP in a real sample was demonstrated by using genomic DNA (gDNA) extracted from the urine and blood of two PKD wild type and three PKD positive cats. The standard curves for PKD positive (PKD) and negative (PKD) DNA were created using two feline-urine samples. An additional three urine samples were analyzed in a similar fashion and showed satisfactory agreement with the standard curve, confirming the presence of the mutation in affected urine. The limit of detection (LOD) was 0.005 ng/mL which corresponds to 6 fg per array for gDNA in urine and blood. The PC system demonstrated the ability to detect a number of genome equivalents for the PKD SNP that was very similar to the results reported with real time polymerase chain reaction (PCR). The favorable comparison with quantitative PCR suggests that the PC technology may find application well beyond the detection of the PKD SNP, into areas where a simple, cheap and portable nucleic acid analysis is desirable.
一种纳米颗粒组装的光子晶体（PC）阵列被用于检测单核苷酸多态性（SNP）。具有PC纳米结构的检测平台由于激发和发射的相位匹配增强了来自纳米颗粒杂交DNA复合物的荧光信号。通过使用电泳颗粒捕获系统，将与探针DNA偶联的纳米颗粒捕获到阵列中的纳米孔中。该PC/DNA检测平台能够识别合成核苷酸序列中1个碱基对（bp）的差异，该差异模拟了人类常染色体显性多囊肾病（PKD）猫科动物模型中出现的突变，灵敏度为0.9 fg/mL（50 aM），即每个阵列对应30个寡核苷酸。通过使用从两只PKD野生型和三只PKD阳性猫的尿液和血液中提取的基因组DNA（gDNA），证明了该PC/DNA检测平台在实际样品中检测SNP的可靠性。使用两个猫尿液样本建立了PKD阳性（PKD）和阴性（PKD）DNA的标准曲线。另外三个尿液样本以类似方式进行分析，与标准曲线显示出令人满意的一致性，证实了受影响尿液中存在突变。检测限（LOD）为0.005 ng/mL，对应于尿液和血液中gDNA每个阵列6 fg。该PC系统展示了检测PKD SNP多个基因组当量的能力，这与实时聚合酶链反应（PCR）报道的结果非常相似。与定量PCR的良好比较表明，PC技术可能在PKD SNP检测之外有更广泛的应用，进入需要简单、廉价和便携式核酸分析的领域。
