Easley Christopher J, Karlinsey James M, Bienvenue Joan M, Legendre Lindsay A, Roper Michael G, Feldman Sanford H, Hughes Molly A, Hewlett Erik L, Merkel Tod J, Ferrance Jerome P, Landers James P
Department of Chemistry, University of Virginia, Charlottesville, VA 22904, USA.
Proc Natl Acad Sci U S A. 2006 Dec 19;103(51):19272-7. doi: 10.1073/pnas.0604663103. Epub 2006 Dec 11.
We describe a microfluidic genetic analysis system that represents a previously undescribed integrated microfluidic device capable of accepting whole blood as a crude biological sample with the endpoint generation of a genetic profile. Upon loading the sample, the glass microfluidic genetic analysis system device carries out on-chip DNA purification and PCR-based amplification, followed by separation and detection in a manner that allows for microliter samples to be screened for infectious pathogens with sample-in-answer-out results in < 30 min. A single syringe pump delivers sample/reagents to the chip for nucleic acid purification from a biological sample. Elastomeric membrane valving isolates each distinct functional region of the device and, together with resistive flow, directs purified DNA and PCR reagents from the extraction domain into a 550-nl chamber for rapid target sequence PCR amplification. Repeated pressure-based injections of nanoliter aliquots of amplicon (along with the DNA sizing standard) allow electrophoretic separation and detection to provide DNA fragment size information. The presence of Bacillus anthracis (anthrax) in 750 nl of whole blood from living asymptomatic infected mice and of Bordetella pertussis in 1 microl of nasal aspirate from a patient suspected of having whooping cough are confirmed by the resultant genetic profile.
我们描述了一种微流控基因分析系统，它代表了一种此前未被描述过的集成微流控设备，该设备能够接受全血作为原始生物样本，并最终生成基因图谱。加载样本后，玻璃微流控基因分析系统设备会进行芯片上的DNA纯化和基于PCR的扩增，随后进行分离和检测，其方式能够在不到30分钟的时间内对微升样本进行感染性病原体筛查，并给出样本进结果出的结果。单个注射泵将样本/试剂输送到芯片上，用于从生物样本中纯化核酸。弹性膜阀将设备的每个不同功能区域隔离开来，并与阻力流一起，将纯化后的DNA和PCR试剂从提取区域引导到一个550纳升的腔室中，用于快速进行目标序列PCR扩增。通过基于压力的重复微量注射扩增子（以及DNA大小标准）进行电泳分离和检测，以提供DNA片段大小信息。通过所得的基因图谱证实了来自无症状感染活体小鼠的750纳升全血中存在炭疽芽孢杆菌（炭疽），以及来自一名疑似患有百日咳患者的1微升鼻腔吸出物中存在百日咳博德特氏菌。
