Development of Dual-channel EOC Sensor for Simultaneous Detection of Multiple Food-borne pathogenes.
- 주제(키워드) EOC
- 발행기관 고려대학교 대학원
- 지도교수 백세환
- 발행년도 2015
- 학위수여년월 2015. 8
- 학위구분 석사
- 학과 대학원 바이오마이크로시스템기술협동과정
- 원문페이지 45 p
- 실제URI http://www.dcollection.net/handler/korea/000000060160
- 본문언어 영어
- 제출원본 000045841527
초록/요약
Recently, studies to measure two or more multiple analytes simultaneously by applying immunoassay technologies and use the results in preventing diseases and finding out the causes of diseases have been conducted actively. In addition, immunoassay technologies that had been implemented only in laboratories have been developed into fast immunoassay kits which can be used in fields through the introduction of membrane based chromatography technologies and have been applied to many areas. However, if many diseases are simultaneously diagnosed through fast immunoassay kits, problems such as non-specific binding (NSB) and cross-reactions induced by many analytes will appear. Therefore, the present study for a master’s thesis was intended to make immunoassay kits that can control non-specific reactions by improving the ELISA-on-a-chip (EOC) which is a fast immunoassay technology that enhances the efficiency of signal generation using enzymes. To check the concept of the immunoassay kit for simultaneous analyses of many analytes, the bacterium Staphylococcus aureus (S. aureus) that can generate false-negative signals due to cross-reactions because protein A that reacts to the fragment crystallizable (Fc) region of certain antibodies exists on its surface was selected as a detection target. In addition, bacteria (Salmonella enteritidis; S. enteritidis, Vibrio parahaemolyticus; V. parahaemolyticus) that account for approximately 45 % of all causes of food poisoning due to bacteria together with S. aureus were also selected as other analytes. To analyze multiple analytes through the improvement of the EOC, the immuno-strip was divided into dual-channels using carbon dioxide (CO2) lasers. In addition, barriers and patterns were formed on the signal generation pad so that the parallel lateral flows and the substrate solution could cross the membrane. As a method for removing cross-reactions, human immunoglobulin (IgG) was fixed on parts of the channels where S. aureus would not be measured to form NSB blocking areas. The detection sensitivities for three species of bacteria that cause food poisoning were measured using optimized dual-channel EOC and according to the results, a sensitivity of 5 × 105 CFU/mL could be identified in the case of S. aureus and a sensitivity of 1 × 104 CFU/mL could be identified in the case of V. parahaemolyticus and S. enteritidis. In addition, to improve the insufficient sensitivity to S. aureus, the lower detection limit concentration of S. aureus could be improved by approximately 20 times to 2.5 × 104 CFU/mL using poly-horseradish peroxidase (HRP) which is a nano-signal generator conjugate. The measurement as mentioned above was conducted in real sample tests including milk samples and according to the results, all of the three species of bacteria could be detected within nine hours indicating that the optimized dual-channel EOC could be applied to not only defined buffer conditions but also food samples. The dual-channel EOC studied in the presented study for a master’s dissertation has the versatility of being applicable to not only the measurement of bacteria that can cause cross-reactions but also many other diagnosis fields. The dual-channel EOC can be easily applied to areas where the measurement of multiple-biomarkers such as cancer markers and cytokines is essential by changing some conditions such as target analytes. NSBs that may occur can be easily suppressed by adjusting blocking areas. In addition, the form of immuno-strips currently remains as dual-channels can be made into the forms of triple-channels or more channels through additional improvement of strips. Cartridges for the improved forms can be implemented using 3D printing technology.
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TABLE OF CONTENTS
LIST OF FIGURES………………………………………………Ⅳ
ABBREVIATIONS……………………………………………Ⅴ
CHAPTER
1. Introduction………………………………………………………01
2. Material and Method………………………………………………05
2.1. Materials
2.2. Identification of the occurrence of antibody cross-reactions
2.3. NC membrane processing for multi analyte detection
2.4. Dual-channel EOC based simultaneous detection of multiple food poisoning bacteria
3. Result and Discussion……………………………………………13
3.1. Antibody characterization and identification of cross-reactions for immunoassays
3.2. Analytical concept of dual-channel EOC
3.3. Multi-channel formed NC membrane fabrication
3.4. Dual-channel EOC based simultaneous detection of food poisoning bacteria
3.5. Dual-channel EOC test based on real sample
4. Conclusion…………………………………………………………31
5. References…………………………………………………………34
