On-chip pressure injection for integration of infrared-mediated DNA amplification with electrophoretic separation
On-chip pressure injection for integration of infrared-mediated DNA amplification with electrophoretic separation
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DOI：
10.1039/b600039h
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发表时间：
2006-01-01
期刊：
影响因子：
6.1
通讯作者：
Landers, JP
中科院分区：
文献类型：
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作者：
Easley, CJ;Karlinsey, JM;Landers, JP
聚（二甲基硅氧烷）（PDMS）膜阀用于PDMS-玻璃混合微器件上的隔膜泵，以耦合红外介导的DNA扩增与电泳分离的产品在一个单一的设备。通过基于芯片的隔膜泵将在非接触式红外（IR）介导的聚合酶链式反应（PCR）期间产生的特异性扩增产物注入电泳分离通道。通道尺寸被设计用于通过优先流动路径的注射塞成形，这有助于使塞宽度最小化。样品的无偏进样可以在短至190 ms内实现，从而将电动进样所需的时间减少了两个数量级。此外，使用层流或偏置层流加载促进样品堆积，以与PCR扩增的样品一起共注射水或低离子强度DNA标记物溶液沿着。在有效长度仅为3.0cm的分离通道中，实现了pUC-18 DNA标记溶液的80-和102-bp片段的完全基线分辨率（Res = 2.11），257-和267-bp片段部分分辨率（Res = 0.56）。该分辨率被认为对于许多PCR扩增子分离是足够的，具有分离时间短的附加优点-通常在< 120 s内完成。减少PCR室周围的玻璃量减少了DNA扩增时间，进一步提高了分析速度，加热和冷却速率分别高达13.4和-6.4 ℃ s（-1）。随着每个步骤的时间要求大大降低，可以在单个设备上无缝耦合IR介导的扩增，样品注射和分离/检测来自1000个起始拷贝的鼠伤寒沙门氏菌DNA的invA基因的278-bp片段，这相当于12分钟，代表了迄今为止实现的最快的PCR-ME整合。
Poly(dimethylsiloxane) (PDMS) membrane valves were utilized for diaphragm pumping on a PDMS-glass hybrid microdevice in order to couple infrared-mediated DNA amplification with electrophoretic separation of the products in a single device. Specific amplification products created during non-contact, infrared (IR) mediated polymerase chain reaction (PCR) were injected via chip-based diaphragm pumping into an electrophoretic separation channel. Channel dimensions were designed for injection plug shaping via preferential flow paths, which aided in minimizing the plug widths. Unbiased injection of sample could be achieved in as little as 190 ms, decreasing the time required with electrokinetic injection by two orders of magnitude. Additionally, sample stacking was promoted using laminar or biased-laminar loading to co-inject either water or low ionic strength DNA marker solution along with the PCR-amplified sample. Complete baseline resolution (Res = 2.11) of the 80- and 102-bp fragments of pUC-18 DNA marker solution was achieved, with partially resolved 257- and 267-bp fragments (Res = 0.56), in a separation channel having an effective length of only 3.0 cm. This resolution was deemed adequate for many PCR amplicon separations, with the added advantage of short separation time-typically complete in < 120 s. Decreasing the amount of glass surrounding the PCR chamber reduced the DNA amplification time, yielding a further enhancement in analysis speed, with heating and cooling rates as high as 13.4 and -6.4 degrees C s(-1), respectively. With the time requirements greatly reduced for each step, it was possible to seamlessly couple IR-mediated amplification, sample injection, and separation/detection of a 278-bp fragment from the invA gene of,1000 starting copies of Salmonella typhimurium DNA in similar to 12 min on a single device, representing the fastest PCR-ME integration achieved to date.