Development of Biosensor Materials for Sensitive Detection of Viral RNA and Foodborne Pathogen

Pathogen detection for clinical diagnosis, vector surveillance and control is one of the important issues for public health and safety. Biosensing is considered as one of the promising techniques for pathogen detection due to its potential for rapid, simple, and relatively inexpensive detection. However, many biosensors still have limitations for commercialization. Development of materials and new detection methods are the ways to overcome these limitations [1, 2]. This study aims to develop rapid and sensitive material platforms for detection of the dengue viral RNA (Chapter 2 and 3) and the foodborne pathogen Escherichia coli O157:H7 (Chapter 4). Two different materials were designed and developed as candidate materials to enhance sensitivity for the viral RNA and pathogen detection. These are a functionalized graphene-based 3-dimensional structure material for detection of Viral RNA, and gold decorated polystyrene (PS) particles for detection of E. Coli O157:H7. Chapter 2 demonstrates the enhanced sensitivity of functionalized graphene oxide wrapped SiO2 particle composite (SiO2@APTES-GO) after primer immobilization for dengue DNA and RNA detection and determines selectivity and a limit of detection (LOD) below 1fM. The enhanced sensitivity is due to the increased surface area resulting from the wrapped structure of the graphene oxide (GO) layer on the silica particles, while maintaining good electrochemical properties. Chapter 3 proposes a new method for acceleration of nucleic acid hybridization through a neutralization treatment after oligonucleotide immobilization on the functionalized material. Here, the simplest structure of the functionalized silica particles was used to determine the acceleration of dengue RNA detection using sodium acetate in 90 vol % ethanol solution for a charge neutralization process. Surface charge change due to the neutralization treatment was clearly confirmed by zeta potential analysis. The oligonucleotide probe was neutralized by the treatment, which accelerated the hybridization speed and decreased the electrode incubation time from 5 hr to 30 min in low ionic strength solutions. The sensitivities of gold-polystyrene particles (Au-PS) conjugated to antibodies are explored in Chapter 4. The sensitivity depended on the surface coverage and core particle size. Among all investigated conditions, the Au-PS with 0.46 µm diameter core PS, and 10% Au surface coverage showed the best sensitivity, of 5x10^2 CFU·mL^-1 as the LOD for E. coli O157:H7 in both phosphate buffer solution and real food samples including apple juice and ground beef. The sensitivity of biosensor platform can be further enhanced by the combination with a gold reduction-signal amplification method, to reach 1x10^2 CFU·mL^-1 LOD. This optimized surface coverage with gold nanoparticles on the submicron sized particles was the key factor for the sensitivity enhancement of lateral flow strip detection