Instituto Interuniversitario de Investigación de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Universitat Politècnica de València, Universitat de València, Camino de Vera s/n, 46022 Valencia, Spain.
Unidad Mixta UPV-La Fe, Nanomedicine and Sensors, IIS La Fe, Valencia, Spain.
Analyst. 2022 May 17;147(10):2180-2188. doi: 10.1039/d2an00160h.
A biosensing system was developed to accurately detect a single nucleotide change in the target organism genome, integrating a selective isothermal amplification and a sensitive dendron-mediated DNA hybridization assay in the array format. The novelty arises from the coupling reactions of the dendron and its use as a crosslinker. The allele-specific probes were oligonucleotide-dendron conjugates prepared by fast and clean click-chemistry (thiol-yne reaction) and coupled onto the photo-activated surface of polycarbonate substrates (carbodiimide reaction). The output was forest-array chips with multipoint-site crosslinkers and compatible with current microarray fabrication technologies. The products of blocked recombinase polymerase amplification (blocked RPA), formed at 37 °C, were hybridized with attached probes for specific nucleotide genotyping. The developed approach exhibited sensitive recognition of DNA variants compared to chips based on linear crosslinkers (10-100 fold), showing excellent analytical performances for planar chip and fluidic formats. The methodology was successfully applied to detect the H1047R mutation in the gene (c.3140A > G) from clinical samples of human cancer tissues, the results being consistent with sequencing techniques. The colorimetric biosensing method was reliable, versatile, low cost, sensitive (detection limit genomic DNA: 0.02 ng μL), and specific (accuracy >95%).
一个生物传感系统被开发出来，以准确地检测目标生物体基因组中的单个核苷酸变化，该系统将选择性等温扩增和敏感的树突介导的 DNA 杂交分析整合在阵列格式中。新颖之处在于树突的偶联反应及其作为交联剂的用途。等位基因特异性探针是通过快速和清洁的点击化学（硫醇-炔反应）制备的寡核苷酸-树突缀合物，并偶联到聚碳酸酯基底的光激活表面上（碳二亚胺反应）。输出是具有多点交联剂的森林阵列芯片，与当前的微阵列制造技术兼容。在 37°C 下形成的被阻断的重组酶聚合酶扩增（blocked RPA）产物与附着的探针杂交，用于特定核苷酸基因分型。与基于线性交联剂的芯片相比，所开发的方法对 DNA 变体具有敏感的识别能力（10-100 倍），在平面芯片和流体格式中表现出出色的分析性能。该方法成功地应用于从人癌症组织的临床样本中检测基因（c.3140A > G）中的 H1047R 突变，结果与测序技术一致。比色生物传感方法可靠、多功能、低成本、灵敏（检测限基因组 DNA：0.02ng μL）且具有特异性（准确率>95%）。
