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

建立了一种芯片分析系统，该系统采用异硫氰酸异鲁米酚(化学发光标记剂)-微量过氧化物酶(催化剂)-过氧化氢(氧化剂)和bis[2-(3，6，9-trioxadecanyloxycarbony)-4-nitrophenyl]oxalate(草酸盐试剂)-异硫氰酸荧光素(荧光标记剂)-过氧化氢(氧化剂)的化学发光反应。该分析系统在一个深度为20μm、宽度为50μm的芯片上进行了以下三个过程：免疫反应用于高选择性，电泳法用于样品塞的形成和运输，化学发光检测用于高灵敏度。这三种工艺被紧密地集成到芯片上。作为第一个过程，免疫反应是使用抗体固定的玻璃微珠进行的。将玻璃珠与抗原(分析…)一起放置在其中一个储存库中更多的e)和已知数量的ILITC或FITC标记的抗原，以建立竞争性免疫反应。对于电泳法，作为第二道工序，将免疫反应后的反应物以电泳法送入交叉点，形成样品塞。然后将样品塞移动到另一个装有过氧化氢或TDPO-过氧化氢混合溶液的储存罐中。在这一点上，化学发光检测作为第三个过程：标记的抗原与试剂混合产生化学发光。化学发光检测用位于蓄水池下方的光电倍增管。本文介绍的芯片分析系统能够高选择性和高灵敏度地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。在芯片上进行免疫反应、电泳法和化学发光法检测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。较少

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