Development of Sensitive "Micro-immunoassay System" and Application to Determination of Cancer Marker

我们开发了一种电泳-化学发光检测的芯片分析系统，该系统采用了异硫氰酸异亮醇（ILITC）（化学发光试剂标记）-微过氧化物酶（催化剂）-过氧化氢（氧化剂）和双[2-(3,6,9-三oxadecanyloxycarbony)-4-nitrophenyl]草酸酯（TDPO）（草酸试剂）-异硫氰酸荧光素（FITC）（荧光试剂标记）-过氧化氢（氧化剂）的化学发光反应。该分析系统在20 μm深、50 μm宽的微通道芯片上进行了三个过程：免疫反应（高选择性）、电泳（样品塞的形成和运输）和化学发光检测（高灵敏度）。这三种工艺被紧凑地集成到芯片上。作为第一个过程，免疫反应是用抗体固定的玻璃珠进行的。将玻璃珠与抗原（分析…More e）和已知数量的ILITC或fitc标记抗原一起放置在其中一个贮液库中，以建立竞争性免疫反应。对于电泳，作为第二个过程，免疫反应后的反应物被电泳送入交叉点，导致样品堵塞。然后将样品塞移动到另一个含有过氧化氢或tdpo -过氧化氢混合溶液的储层中。此时进行化学发光检测作为第三道工序：标记抗原与试剂混合产生化学发光。化学发光用位于储层下方的光电倍增管进行检测。本文所描述的芯片分析系统能够以高选择性和高灵敏度测定人血清白蛋白或免疫抑制酸性蛋白（IAP）作为人血清中的癌症标志物。通过免疫反应、电泳和芯片化学发光检测对IAP进行分析。IAP测定范围为1.0 × 10^<-7> ~ 5.0 × 10^<-6> M，检出限为1.0 × 10^<-7> M (S/N=3)（相关系数为0.999）。一次分析在2分钟内完成。少

We developed a chip analysis system incorporating electrophoresis-chemiluminescence detection, in which the chemiluminescence reaction of isoluminol isothiocyanato (ILITC) (chemiluminescence reagent for labeling)-microperoxidase (catalyst) - hydrogen peroxide (oxidant) as well as bis[2-(3,6,9-trioxadecanyloxycarbony)-4-nitrophenyl]oxalate (TDPO) (oxalate reagent)-fluorescein isothiocyanate (FITC) (fluorescence reagent for labeling) - hydrogen peroxide (oxidant) were adopted. The analysis system performed the following three processes on a chip having micro-channels of 20 μm deep and 50 μm width: immune reaction for high selectivity, electrophoresis for formation and transportation of the sample plug, and chemiluminescence detection for high sensitivity. The three processes were compactly integrated onto the chip. As the first process, the immune reaction was performed using an antibody-immobilized glass bead. The glass bead was placed in one of the reservoirs along with antigen (analyt … More e) and a known amount of ILITC- or FITC-labeled antigen to set up a competitive immune reaction. For electrophoresis, as the second process, the reactant after the immune reaction was fed electrophoretically into the intersection resulting in a sample plug. The sample plug was then moved into another reservoir containing hydrogen peroxide or TDPO-hydrogen peroxide mixture solution. At this point, chemiluminescence detection was performed as the third process: the labeled antigen mixed with the reagent to produce chemiluminescence. Chemiluminescence was detected with a photomultiplier tube located under the reservoir. The chip analysis system described here was capable of determining, with high selectivity and sensitivity, human serum albumin or immunosuppressive acidic protein (IAP) as a cancer marker in human serum. IAP was analyzed by immune reaction, electrophoresis, and chemiluminescence detection on the microchip. IAP was determined over the range of 1.0x10^<-7> - 5.0x10^<-6> M (with a detection limit of 1.0x10^<-7> M (S/N=3) (correlation coefficient, 0.999). One analysis was performed within 2 min. Less