Song Linan, Ahn Soohyoun, Walt David R
Department of Chemistry, Tufts University, Medford, MA 02155, USA.
Anal Chem. 2006 Feb 15;78(4):1023-33. doi: 10.1021/ac051417w.
We report a multiplexed high-density DNA array capable of rapid, sensitive, and reliable identification of potential biological warfare agents. An optical fiber bundle containing 6000 individual 3.1-mum-diameter fibers was chemically etched to yield microwells and used as the substrate for the array. Eighteen different 50-mer single-stranded DNA probes were covalently attached to 3.1-mum microspheres. Probe sequences were designed for Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella melitensis, Clostridium botulinum, Vaccinia virus, and one biological warfare agent (BWA) simulant, Bacillus thuringiensis kurstaki. The microspheres were distributed into the microwells to form a randomized multiplexed high-density DNA array. A detection limit of 10 fM in a 50-microL sample volume was achieved within 30 min of hybridization for B. anthracis, Y. pestis, Vaccinia virus, and B. thuringiensis kurstaki. We used both specific responses of probes upon hybridization to complementary targets as well as response patterns of the multiplexed array to identify BWAs with high accuracy. We demonstrated the application of this multiplexed high-density DNA array for parallel identification of target BWAs in spiked sewage samples after PCR amplification. The array's miniaturized feature size, fabrication flexibility, reusability, and high reproducibility may enable this array platform to be integrated into a highly sensitive, specific, and reliable portable instrument for in situ BWA detection.
我们报道了一种能够快速、灵敏且可靠地鉴定潜在生物战剂的多重高密度DNA阵列。将一束包含6000根直径为3.1微米的单根光纤进行化学蚀刻以产生微孔，并用作该阵列的基质。18种不同的50聚体单链DNA探针共价连接到3.1微米的微球上。探针序列针对炭疽芽孢杆菌、鼠疫耶尔森菌、土拉弗朗西斯菌、羊布鲁氏菌、肉毒梭菌、痘苗病毒以及一种生物战剂模拟物苏云金芽孢杆菌以色列亚种进行设计。微球被分布到微孔中以形成随机化的多重高密度DNA阵列。对于炭疽芽孢杆菌、鼠疫耶尔森菌、痘苗病毒和苏云金芽孢杆菌以色列亚种，在杂交30分钟内，50微升样品体积中的检测限达到10飞摩尔。我们利用探针与互补靶标杂交后的特异性反应以及多重阵列的反应模式来高精度地鉴定生物战剂。我们展示了这种多重高密度DNA阵列在PCR扩增后对加标污水样品中目标生物战剂进行平行鉴定的应用。该阵列的小型化特征尺寸、制造灵活性、可重复使用性和高重现性可能使该阵列平台能够集成到一种用于现场生物战剂检测的高度灵敏、特异且可靠的便携式仪器中。
