Bioanalytical developments and applications to drinking water safety

我的研究项目旨在推进水中微生物和化学污染物的分析检测与表征，为安全饮用水和健康保护提供科学依据。本研究的重点是开发新的技术和检测方法，以检测无法通过传统方法检测的特异性活但不可培养（VBNC）病原体。水和食品的微生物污染是一个主要的公共卫生问题，但很难确定疫情爆发的原因。一个主要的挑战是许多细菌细胞可以在各种环境条件下进入VBNC状态。超过50种人类病原体可以存在于VBNC状态，但目前的检测方法不能直接检测到VBNC细菌。虽然隐藏在检测之外，但VBNC病原体具有传染性。为了检测病原菌，分析技术必须能够识别特异性的病原菌，并具有足够的检测灵敏度。为了应对第一个挑战，我们将开发一种新技术来生成能够特异性识别和结合VBNC病原体（如大肠杆菌O157:H7和空肠弯曲杆菌）的DNA适体分子。我们将生产VBNC细胞，并以其为靶标，选择与细胞表面分子特异性结合的适体。我们将引入修饰的核酸来增强多样性，并结合酶切来提高严格性，这将导致有效地选择理想的适体。我们将对所选适配体的稳定性、结合亲和力和特异性进行表征。利用这项新技术，我们将生产出高度稳定的针对VBNC细菌的适体，从而实现现场检测分析的发展。我们将通过将选定的适体纳入新的信号放大技术，并通过增强荧光探针和纳米材料的检测信号，开发针对特定VBNC病原体的超灵敏检测方法。我们将设计一对适体探针来识别和结合目标VBNC病原体，从而使该结合事件启动dnazyme催化的信号扩增过程。放大的信号，可检测的荧光，将使检测特定的VBNC病原体存在于痕量水平。等温扩增反应使用DNAzyme而不是蛋白酶作为催化剂。所有用于分析的DNA试剂都比蛋白质具有更好的稳定性。新的检测方法对现场检测VBNC病原菌具有潜在的实用价值。这些检测将用于研究经过不同处理过程收集的水源水和饮用水中的VBNC病原体，为最终消除饮用水中的病原体做出贡献。分析技术和适体也可应用于生物化学研究、临床诊断和药物开发。

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.