Bioanalytical developments and applications to drinking water safety

饮用水安全生物分析的发展和应用

• 批准号: RGPIN-2017-05857
• 负责人: Li, XingFang
• 金额: $ 7.72万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=710572
• 关键词: Bioanalytical; developments; applications; drinking; water

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.

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和表征，为安全饮用水和健康保护提供科学依据。这项拟议的研究重点是开发针对特定活但不可培养（VBNC）病原体的新技术和检测方法，这些病原体无法通过传统方法检测到。 水和食物的微生物污染是一个主要的公共卫生问题，但爆发的原因很难确定。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 种人类病原体可以以 VBNC 状态存在，但目前的检测方法无法直接检测 VBNC 细菌。 VBNC 病原体虽然不易被发现，但具有传染性。 为了检测 VBNC 病原体，分析技术必须能够识别特定的 VBNC 病原体并对其检测具有足够的灵敏度。为了应对第一个挑战，我们将开发一种新技术来生成DNA适体分子，该分子可以特异性识别并结合VBNC病原体，例如大肠杆菌O157:H7和空肠弯曲菌。我们将生产VBNC细胞，并用它们作为靶标来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以增强多样性，并结合酶消化以提高严格性，这将导致有效选择所需的适体。我们将表征所选适体的稳定性、结合亲和力和特异性。借助新技术，我们将生产针对 VBNC 细菌的高度稳定的适配体，从而实现现场检测分析的开发。 我们将通过将选定的适体纳入新的信号放大技术并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并结合目标VBNC病原体，这样这种结合事件将启动DNAzyme催化的信号放大过程。放大的信号可通过荧光检测，从而能够检测痕量水平存在的特定 VBNC 病原体。等温扩增反应使用 DNAzyme（而不是蛋白酶）作为催化剂。用于测定的所有 DNA 试剂都比蛋白质具有更好的稳定性。新的检测方法对于 VBNC 病原体的现场检测可能有用。这些检测将用于研究源水和经过不同处理过程收集的饮用水中的VBNC病原体，有助于实现消除饮用水中病原体的最终目标。分析技术和适体还可应用于生化研究、临床诊断和药物开发。