Grants and Contributions:

Grant or Award spanning more than one fiscal year. (2017-2018 to 2022-2023)

My research program aims to advance the analytical detection and characterization of microbial and chemical contaminants in water, providing a scientific basis for safe drinking water and health protection. This proposed research focuses on the development of novel techniques and assays for specific viable but non-culturable (VBNC) pathogens that escape detection by traditional methods.

Microbial contamination of water and food is a major public health concern, but causes of outbreaks are very difficult to identify. A major challenge is that many bacterial cells can enter into a VBNC state under various environmental conditions. Over 50 human pathogens can exist in a VBNC state, but current assays cannot directly detect VBNC bacteria. Although hidden from detection, VBNC pathogens are infectious.

To detect VBNC pathogens, analytical techniques must be able to recognize the specific VBNC pathogens and have sufficient sensitivity for their detection. To confront the first challenge, we will develop a new technology to generate DNA aptamer molecules that can specifically recognize and bind to VBNC pathogens, e.g., E. coli O157:H7 and Campylobacter jejuni . We will produce VBNC cells and use them as the targets to select aptamers specifically binding to molecules on the cell surface. We will introduce modified nucleic acids to enhance the diversity and incorporate enzyme digestion to improve stringency, which will result in efficient selection of desirable aptamers. We will characterize the selected aptamers for their stability, binding affinity and specificity. With the new technology we will produce highly stable aptamers specific to target VBNC bacteria, enabling the development of on-site detection assays.

We will develop ultra-sensitive assays for specific VBNC pathogens, by incorporating the selected aptamers into new signal amplification techniques and by enhancing detection signals of fluorescence probes and nanomaterials. We will design a pair of aptamer probes to recognize and bind to the target VBNC pathogen, so that this binding event will initiate a DNAzyme-catalyzed signal amplification process. The amplified signals, detectable with fluorescence, will enable detection of specific VBNC pathogen present at trace levels. The isothermal amplification reaction uses a DNAzyme, instead of a protein enzyme, as the catalyst. All the DNA reagents for the assay have better stability than proteins. The new assays are potentially useful for on-site detection of VBNC pathogens. These assays will be used to study VBNC pathogens in source water and in drinking water collected after different processes of treatment, contributing to the ultimate goal of eliminating pathogens in drinking water. The analytical technologies and the aptamers can also be applied to biochemical research, clinical diagnosis, and drug development.