2006 Fiscal Year Final Research Report Summary
Development of Sensitive "Micro-immunoassay System" and Application to Determination of Cancer Marker
| Project/Area Number |
17550088
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Analytical chemistry
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| Research Institution | Doshisha University |
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Principal Investigator |
TSUKAGOSHI Kazuhiko Doshisha University, Faculty of Engineering, Professor, 工学部, 教授 (60227361)
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| Project Period (FY) |
2005 – 2006
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| Keywords | chemiluminescence / immune reaction / electrophoresis / micro-chip / protein / cancer marker / (7) (8) |
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| Research Abstract |
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
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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
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