Department of Chemistry, University of California, Berkeley, CA 94720, United States; Department of Chemistry, Ulsan National Institute of Science and Technology, Ulsan 689-798, Republic of Korea.
Department of Chemistry, University of California, Berkeley, CA 94720, United States; Department of Mechanical Engineering, Texas Tech University, Lubbock, TX 79409, United States.
Biosens Bioelectron. 2016 May 15;79:371-8. doi: 10.1016/j.bios.2015.12.068. Epub 2015 Dec 21.
A microfluidic hydrogel DNA microarray is developed to overcome the limitations of conventional planar microarrays such as low sensitivity, long overnight hybridization time, lack of a melting verification of proper hybrid, and complicated sample preparation process for genotyping of clinical samples. Unlike our previous prototype hydrogel array which can analyze only single-stranded DNA (ssDNA) targets, the device is the first of its type to allow direct multiplexed single nucleotide polymorphism (SNP) detection of human clinical samples comprising double-stranded DNA (dsDNA). This advance is made possible by incorporating a streptavidin (SA) hydrogel capture/purification element in a double T-junction at the start of the linear hydrogel array structure and fabricating ten different probe DNAs-entrapped hydrogels in microfluidic channels. The purified or unpurified polymerase chain reaction (PCR) products labeled with a fluorophore and a biotin are electrophoresed through the SA hydrogel for binding and purification. After electrophoretic washing, the fluorophore-labeled DNA strand is then thermally released for hybridization capture by its complementary probe gel element. We demonstrate the precise and rapid discrimination of the genotypes of five different clinical targets by melting curve analysis based on temperature-gradient electrophoresis within 3h, which is at least 3-fold decrease in incubation time compared to conventional microarrays. In addition, a 1.7 pg (0.024 femtomoles) limit of detection for clinical samples is achieved which is ~100-fold better sensitivity than planar microarrays.
一种微流控水凝胶 DNA 微阵列被开发出来，以克服传统平面微阵列的局限性，如低灵敏度、长的过夜杂交时间、缺乏适当杂交的熔融验证以及用于临床样本基因分型的复杂样品制备过程。与我们之前的原型水凝胶阵列不同，该设备可以分析单链 DNA（ssDNA）靶标，而是第一个允许直接对包含双链 DNA（dsDNA）的人类临床样本进行多重单核苷酸多态性（SNP）检测的设备。这一进展是通过在线性水凝胶阵列结构的双 T 型连接处结合一个链霉亲和素（SA）水凝胶捕获/纯化元件，并在微流道中制造十个不同的探针 DNA 捕获水凝胶来实现的。用荧光染料和生物素标记的经纯化或未经纯化的聚合酶链反应（PCR）产物通过 SA 水凝胶电泳进行结合和纯化。电泳洗涤后，荧光标记的 DNA 链通过互补的探针凝胶元件进行热释放以进行杂交捕获。我们通过基于温度梯度电泳的熔解曲线分析在 3 小时内精确快速地鉴别了五种不同临床靶标的基因型，与传统微阵列相比，孵育时间至少缩短了 3 倍。此外，实现了对临床样本的 1.7pg（0.024 飞摩尔）检测限，比平面微阵列的灵敏度提高了约 100 倍。
