Establishment of Advanced Lateral Flow Immunoassay Sensors for Quantitative Analysis of Small Molecules

Abstract

Lateral flow immunoassay (LFI) is the most widely used paper-based device for point-of-care testing, and it is used in a variety of industries for inspection and diagnosis. This inexpensive, easy to use, and portable analytical device is becoming increasingly popular since it enables easier testing and screening, particularly in resource constrained environments. However, the device possesses the following limitations: 1) it shows relatively low sensitivity on performing complicated addition or multi-step analysis; 2) it has a “signal-off” sensing system for small molecule detection. Therefore, in this study, various LFI platforms were examined and a method to quantitatively detect small molecules with high sensitivity was devised. In a fluorescence resonance energy transfer-lateral flow immunoassay (FRET-LFI), a fluorescence resonance energy transfer (FRET)-based system was designed which exploits antibody-antigen binding to detect mycotoxins more rapidly and easily than other currently available methods. In addition, I was able to effectively counteract the matrix effect in the sample by using a nitrocellulose membrane that enabled fluorescence measurement in coffee samples. In a trap-lateral flow immunoassay (trap-LFI), the system has deletion and detection zones loaded with a target-protein conjugate and an anti-mouse IgG antibody, respectively. By lowering the amount of AuNP–antibody conjugate, conjugate that is not reacted with the free analyte is completely "trapped" in the detection zone, and only the antibody conjugate associated with the free analyte can escape from the deletion zone and display a signal in the detection zone because of reaction with immobilized secondary antibodies. Furthermore, enzyme-induced signal amplification techniques have made it possible to detect small molecules with high sensitivity. In an advanced trap-lateral flow immunoassay (αtrap-LFI), a polyvinyl alcohol (PVA) tape was used, which has the delayed release property of solutions. The PVA tape, located between conjugate pad and immune reaction part, releases gold ions from the signal enhancement pad for signal enhancement after a certain time of immune reaction. Because of the delayed release effect of PVA tape, the signal enhancement step was successfully accomplished with consecutive loading of sample and enhancement solutions. In this dissertation, various LFI sensors for detection of small molecule were proposed, and it showed quantitative results with high sensitivity. These easy to use platforms have wide applications and can be employed to detect not only small molecules, but other biomarkers with high sensitivity.