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

建立了一种荧光-化学发光芯片分析系统，（化学发光标记试剂）-微过氧化物酶（催化剂）-过氧化氢（氧化剂）以及双[2-（3，6，9-三氧癸烷氧基羰基）-4-硝基苯基]草酸酯（TDPO）采用草酸盐试剂-异硫氰酸荧光素（FITC）-过氧化氢（氧化剂）标记。该分析系统在具有20 μm深和50 μm宽微通道的芯片上执行以下三个过程：高选择性的免疫反应、形成和运输样品塞的电泳和高灵敏度的化学发光检测。这三个过程都是完全集成在芯片上的。作为第一步，使用抗体固定化玻璃珠进行免疫反应。将玻璃珠与抗原（分析物）一起沿着放置在其中一个容器中。 ...更多信息 e）和已知量的ILITC-或FITC-标记的抗原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地进料到交叉点中，导致样品塞。然后将样品塞移动到另一个含有过氧化氢或TDPO-过氧化氢混合物溶液的储存器中。此时，化学发光检测作为第三个过程进行：标记抗原与试剂混合产生化学发光。化学发光用位于储液器下方的光电倍增管检测。这里描述的芯片分析系统能够以高选择性和灵敏度测定人血清中作为癌症标志物的人血清白蛋白或免疫抑制酸性蛋白（IAP）。免疫反应、电泳、化学发光检测等方法检测IAP。在1.0 × 10 ^<-7>-5.0 × 10 ^<-6>M范围内测定IAP（检测限为1.0 × 10 ^<-7>M（S/N=3）（相关系数为0.999））。在2分钟内完成一次分析。减

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