INTERFIBIO Research Group, Departament d'Enginyeria Química, Universitat Rovira i Virgili, 43007 Tarragona, Spain.
Labman Automation Ltd., Seamer Hill, Stokesley, North Yorkshire TS9 5NQ, U.K.
Anal Chem. 2023 Sep 26;95(38):14192-14202. doi: 10.1021/acs.analchem.3c01668. Epub 2023 Sep 15.
The detection of single nucleotide polymorphisms (SNPs) is of increasing importance in many areas including clinical diagnostics, patient stratification for pharmacogenomics, and advanced forensic analysis. In the work reported, we apply a semiautomated system for solid-phase electrochemical melting curve analysis (éMCA) for the identification of the allele present at a specific SNP site associated with an increased risk of bone fracture and predisposition to osteoporosis. Asymmetric isothermal recombinase polymerase amplification using ferrocene labeled forward primers was employed to generate single stranded redox labeled amplicons. In a first approach to demonstrate the proof of concept of combining asymmetric RPA with solid-phase éMCA, a simplified system housing a multielectrode array within a polymeric microsystem, sandwiched between two aluminum plates of a heater device, was used. Sample manipulation through the microfluidic channel was controlled by a syringe pump, and an external Ag/AgCl reference electrode was employed. Individual electrodes of the array were functionalized with four different oligonucleotide probes, each probe equivalent in design with the exception of the middle nucleotide. The isothermally generated amplicons were allowed to hybridize to the surface-tethered probes and subsequently subjected to a controlled temperature ramp, and the melting of the duplex was monitored electrochemically. A clear difference between the fully complementary and a single mismatch was observed. Having demonstrated the proof-of-concept, a device for automated éMCA with increased flexibility to house diverse electrode arrays with internal quasi-gold reference electrodes, higher resolution, and broader melting temperature range was developed and exploited for the detection of SNP hetero/homozygosity. Using the optimized conditions, the system was applied to the identification of the allele present at an osteoporosis associated SNP site, rs2741856, in 10 real fingerprick/venous blood samples, with results validated using Sanger sequencing.
单核苷酸多态性 (SNP) 的检测在许多领域变得越来越重要，包括临床诊断、药物基因组学患者分层和高级法医分析。在报告的工作中，我们应用了一种半自动固相电化学熔融曲线分析 (éMCA) 系统，用于鉴定与骨折风险增加和骨质疏松易感性相关的特定 SNP 位点存在的等位基因。使用带有二茂铁标记的正向引物的不对称等温重组聚合酶扩增生成单链氧化还原标记的扩增子。作为将不对称 RPA 与固相 éMCA 相结合的概念验证的第一个方法，使用了一种简化的系统，该系统将多电极阵列封装在聚合物微系统中，夹在加热器装置的两个铝板之间。通过微流通道的样品处理由注射器泵控制，并且使用外部 Ag/AgCl 参比电极。阵列的各个电极用四个不同的寡核苷酸探针功能化，每个探针在设计上都等效，除了中间核苷酸。等温生成的扩增子被允许与表面固定的探针杂交，随后进行受控的温度斜坡，并且电化学监测双链体的熔化。在完全互补和单个错配之间观察到明显的差异。在证明了概念验证之后，开发并利用了一种具有更高灵活性的自动 éMCA 设备，可以容纳具有内部准金参考电极、更高分辨率和更宽熔化温度范围的各种电极阵列，用于 SNP 杂合/纯合性的检测。使用优化的条件，该系统应用于鉴定与骨质疏松症相关的 SNP 位点 rs2741856 存在的等位基因，在 10 个真实的指尖/静脉血样本中，使用 Sanger 测序验证结果。
