Title Page
Contents
LIST OF ABBREVIATION 7
요약 12
Abstract 13
1. INTRODUCTION 15
1.1. Research Background 15
1.2. Related Research 16
1.3. Research Purpose 21
2. CONCEPTS AND THEORY 23
2.1. Device Concept and Smartphone Application 23
2.2. Lateral flow immunoassay 27
2.3. Horseradish peroxidase for Enzyme-Labeled Conjugates in LFIA 31
2.4. DAB staining 31
3. MATERIAL AND EXPERIMENTAL 33
3.1. Materials and reagents 33
3.2. Bacteria preparation 34
3.3. Design and fabrication of the device 34
3.4. Application Development 38
3.5. Principle of Performing Automated Multistep LFIA 46
3.6. Preparation of bacteria target for Detection of Bacteria from Contaminated Lettuce 48
3.7. Detection of Specificity 48
3.8. Sensor Stability 49
4. RESULT AND DISCUSSION 50
4.1. Calibration Curve using Smartphone Application 50
4.2. Detection Specificity 52
4.3. Sensor Stability 53
4.4. Detection of Bacteria from Contaminated Lettuce 55
5. CONCLUSIONS 58
6. REFERENCES 60
PUBLICATIONS 76
Table 1. Comparison of the related research that using the different method for detection of foodborne pathogens or bacteria. 17
Table 2. The developed point-of-care testing device using smartphone 20
Table 3. The quantified signal intensity of the test line obtained from the stability. 55
Table 4. Comparison between the number of bacteria inoculated from counting colonies and the number of bacteria inoculated from the device performing multistep... 56
Figure 1. Schematic diagram of the electronic components used for making the automatic device. 23
Figure 2. Direction of rotation of the rotary pad and reagent flow direction. 24
Figure 3. Screenshot of the designer editor part of MIT App Inventor. 25
Figure 4. Screenshot of the block editor part of MIT App Inventor. 25
Figure 5. Screenshot of the connect function of MIT App Inventor. 26
Figure 6. Screenshot of the build function of MIT App Inventor. 26
Figure 7. Screenshot of icon of application that made by MIT App Inventor. 27
Figure 8. Schematic representation of a typical LFA strip. 28
Figure 9. The schematic representation of test result obtained from LFIA strip. 29
Figure 10. (a) sandwich format lateral flow assay. (b) competitive format lateral flow assay. 30
Figure 11. The process of the HRP-catalyzed oxidation reaction of DAB by H₂O₂. 32
Figure 12. Schematic of the operating platform and the disposable rotary devices showing the cover box and the electronic box with the rotary device placed inside. 35
Figure 13. A jigs inside the cover box to position the smartphone. 36
Figure 14. The rotary device (a) Photo of the rotary device with the NC membrane, glass fiber pads, and absorbent pads installed in it. (b) Exploded view of... 37
Figure 15. Photo of the operating platform showing the assembly of the electronic box (a) without the rotary device. Red arrows show the location of the key... 37
Figure 16. Screenshot of the first page of the application; the user can select the Setting and Run, Calibration, and Calculation menus. 38
Figure 17. Screenshot of the Setting and Run, which is a page used for inputting flow durations. 39
Figure 18. Screenshots of the first page of the Calibration screen, show the image of the NC strip, and the user can specify the known concentration. 41
Figure 19. Screenshots of the second page of the Calibration screen, show the Calibration curve. 42
Figure 20. Screenshots of the last page of the Calibration screen, show a Linear fit curve that shows the slope and the equation of the curve. 43
Figure 21. Screenshot of the Calculation, showing the image of the test result of an unknown sample, and the calculated bacteria concentration based on the... 45
Figure 22. Schematics of each step of strip-based multistep immunoassay showing (a) loading the reagent for assay on the rotary pad, (b) bacteria capturing step,... 47
Figure 23. Diagram of performing Automated Multistep LFIA. 47
Figure 24. Schematics of each step of performing the detection of Bacteria from Contaminated Lettuce 49
Figure 25. (a) Photos showing the signal generation on the NC strip of bacteria concentration 5 × 10⁰ to 5 × 10⁷ CFU/mL. TL and CL are the test line and... 51
Figure 26. Photos of the detection result of different bacteria; (a) E. coli O157:H7, (b) S. aureus, (c) S. Typhimurium, and (d) Nagative contol. 52
Figure 27. The bar graph of the detection intensity. 53
Figure 28. Result of detecting 5 × 10⁷ CFU/mL E. coli O157:H7 on the NC membrane (a) immediately after immobilizing antibodies, (b) 5 days after, and (c) 10... 54
Figure 29. Graph that showing the quantified signal intensity of the test line obtained from the stability. 54