Parallel microfluidic surface plasmon resonance imaging arrays
Parallel microfluidic surface plasmon resonance imaging arrays
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DOI：
10.1039/b920589f
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发表时间：
2010-01-01
期刊：
影响因子：
6.1
通讯作者：
Lagally, Eric T.
中科院分区：
文献类型：
--
作者：
Ouellet, Eric;Lausted, Christopher;Lagally, Eric T.
表面等离子体共振成像（SPRi）是一种无标记技术，用于定量各种靶分子的结合亲和力和浓度。虽然SPRi能够平行测定多个配体的结合常数，但目前的商业仪器仅限于多个配体点上的单个分析物流。结合动力学的测量需要连续引入不同的分析物浓度;这样的重复实验是手动进行的，因此是时间密集型的。为了解决这些挑战，我们已经开发了一种集成的微流体阵列，使用软光刻技术进行高通量基于SPR的检测和抗体对蛋白质靶标的结合亲和力的测定。该装置由264个由微阀隔离的元件可寻址腔室组成。由此产生的700 pL腔室体积，结合用于同时询问多达六种不同分析物浓度的连续稀释网络，允许进一步加快检测时间。为了测试装置性能，将人α-凝血酶固定在传感器表面上，并将不同浓度的抗人α-凝血酶IgG注射穿过表面。平衡解离常数测定为5.0 +/- 1.9 nM，与文献中报道的值一致。还研究了在单个装置中对多个分析物的多个配体的询问，并且回收了没有交叉污染的样品。由于每个腔室都可以独立寻址，因此该阵列能够在单个实验中询问多达264种不同的固定化配体对多种分析物的结合事件。高通量蛋白质分析测量的发展是生物学和医学系统方法的关键技术。
Surface plasmon resonance imaging (SPRi) is a label-free technique used for the quantitation of binding affinities and concentrations for a wide variety of target molecules. Although SPRi is capable of determining binding constants for multiple ligands in parallel, current commercial instruments are limited to a single analyte stream on multiple ligand spots. Measurement of binding kinetics requires the serial introduction of different analyte concentrations; such repeated experiments are conducted manually and are therefore time-intensive. To address these challenges, we have developed an integrated microfluidic array using soft lithography techniques for high-throughput SPRi-based detection and determination of binding affinities of antibodies against protein targets. The device consists of 264 element-addressable chambers isolated by microvalves. The resulting 700 pL chamber volumes, combined with a serial dilution network for simultaneous interrogation of up to six different analyte concentrations, allow for further speeding detection times. To test for device performance, human alpha-thrombin was immobilized on the sensor surface and anti-human alpha-thrombin IgG was injected across the surface at different concentrations. The equilibrium dissociation constant was determined to be 5.0 +/- 1.9 nM, which agrees well with values reported in the literature. The interrogation of multiple ligands to multiple analytes in a single device was also investigated and samples were recovered with no cross-contamination. Since each chamber can be addressed independently, this array is capable of interrogating binding events from up to 264 different immobilized ligands against multiple analytes in a single experiment. The development of high-throughput protein analytic measurements is a critical technology for systems approaches to biology and medicine.