Date of Graduation

7-2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Engineering (PhD)

Degree Level

Graduate

Department

Biological and Agricultural Engineering

Advisor/Mentor

Li, Yanbin

Committee Member

Chen, Jingyi

Second Committee Member

Kwon, Young Min

Third Committee Member

Kim, Jin-Woo

Fourth Committee Member

Tung, Chao-Hung Steve

Keywords

Avian influenza virus H5N1; Fluorescent biosensor; Portable device; QCM biosensor; Salmonella Typhimurium; Target-triggered release

Abstract

This research focused on developing biosensing method and biosensing device for rapid detection of pathogens in poultry: Salmonella Typhimurium and avian influenza virus H5N1. The first part of the dissertation reports an original research on the development of a portable biosensing device for Salmonella detection. The device was designed and constructed based on a previously developed optical biosensing method, using immuno-magnetic nanoparticles to specifically capture target bacteria, and immuno-quantum dot beads to label the target bacteria for fluorescence detection. All the actions of sample mixing, magnetic separation, and fluorescence detection were controlled automatically in a disposable microfluidic chip in the device to simplify the operation, avoid cross-contamination and reduce costs. Experiments were conducted to validate the magnetic capture efficiency and evaluate the performance of the device. The results showed that the biosensing device was able to detect S. Typhimurium with a detection limit of 5.4×10E2 CFU/mL in 1 h and minimal manual operation. The platform developed in this research has the potential to be used for detection of other bacterial pathogens in food, agriculture, and the environment.

The research described in the second part of the dissertation applied the concept of target-triggered release of cargo molecules in gold nanocages using aptamers as the biosensing element to the development of a biosensor for thrombin detection. A label-free quartz crystal microbalance (QCM) biosensor was designed and fabricated based on target-triggered release of cargo molecules in gold nanocages capped with specifically selected aptamers and was evaluated for rapid, sensitive and specific detection of thrombin. The biosensor had a linear detection range of 0.0086-86 nM with a limit of detection of 0.8 pM, and the specificity of the biosensor was confirmed by testing against other proteins at 1 µM. The detection could be completed within 1.5 h. This work provided an innovative method for sensitive sensing of small molecules on a QCM platform.

The third part of the dissertation presents a fluorescent biosensor developed for detection of avian influenza virus H5N1 in poultry based on the concept proved in thrombin detection. Aptamers against H5N1 virus were used as the target-triggered gates over gold nanocages to selectively release the rhodamine B molecules for generating fluorescence signals. The biosensor had a linear range of 2E-3 to 2E4 HAU/50 µL and 2E0 to 2E4 HAU/50 µL for AIV H5N1 in phosphate buffered saline (PBS) and chicken tracheal swab samples, respectively, and a limit of detection of 0.1 HAU/50 µL and 0.7 HAU/50 µL for AIV H5N1 in PBS and chicken tracheal swab samples, respectively. The biosensor was specific to the target H5N1 virus by testing against avian influenza virus H1N1, H2N2, H5N2, and H7N2 at 1 HAU/50 µL. Parallel tests using real-time RT-PCR were conducted to confirm the results of the developed biosensor. This biosensor has the potential for simultaneous detection of multiple viral pathogens using their specific aptamers and fluorophores with different emission wavelengths loaded in gold nanocages.

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