Date of Award
8-16-2024
Document Type
Open Access Dissertation
Department
Chemical Engineering
First Advisor
Chang Liu
Abstract
Nanopore detection technology enables nanometer-scale single-molecule detection, yielding valuable insights into analyte molecules' properties such as size, shape, charge, and concentration. Due to its label-free nature, high sensitivity, and real-time capabilities, it finds widespread application in genomics, proteomics, drug discovery, and environmental monitoring. Click chemistry, encompassing the attachment of a probe or substrate to a biomolecule, termed bioconjugation, features the copper-catalyzed azide-alkyne cycloaddition as a quintessential reaction, forming a 5-membered heteroatom ring. This study harnesses this reaction to craft single-strand DNA probes for nanopore testing, leveraging copper's catalytic properties for signal amplification. Host-guest reaction, a noncovalent bond between molecules, specifically between a macrocyclic compound as a host and a guest molecule. This study exploits host-guest reaction between click-modified DNA probes and cucurbit[6]uril to generate characteristic nanopore signals, significantly enhancing specificity of protein detection.
In this study, the developed assay was applied to test pathogen antigens in diseases. Diagnosing pediatric tuberculosis (TB) presents significant challenges compared to diagnosing in adults. The primary difficulty lies in traditional diagnostic methods, which is less sensitive in children compared to adults due to the lower bacillary load in their respiratory secretions. Additionally, obtaining respiratory samples from children, especially younger ones, can be challenging and may require invasive procedures such as gastric aspiration or induced sputum collection. In this study, the nanopore assay tests the Mycobacterium tuberculosis (Mtb) virulence factor ESAT-6/CFP-10 antigen complex in blood samples for TB in children. It showed superior sensitivity with confirmed TB compared to clinical “gold standard” diagnostic technologies and filled the diagnostic gap for children with unconfirmed TB, where these traditional technologies fell short. We envision that, in combination with automated sample processing and portable nanopore devices, this methodology will offer a powerful tool to support the diagnosis of TB in children.
Inspired by TB study, we introduced different DNA probes and generated four distinct nanopore signals, applied to sepsis pathogen identification. Our proof-of-concept preliminary findings demonstrate that assigning different probes that can generate distinct characteristic nanopore signals can reliably achieve simultaneous identification of multiple pathogens causing sepsis. Furthermore, leveraging the amplification effect of the click reaction enables ultra-sensitive quantitative analysis of the pathogen antigens. This research presents a promising detection method with significant potential for the concurrent identification of multiple sepsis pathogens, thereby providing valuable support for sepsis diagnosis. Moreover, we explored diblock synthetic polyelectrolytes as flexible and cost-effective nanopore sensing probes compared to DNA probes. Efficient translocations of polyelectrolytes through nanopores offer insights into enhancing probe selection and flexibility for nanopore sensing applications.
Rights
© 2024, Xiaoqin Wang
Recommended Citation
Wang, X.(2024). A Click Chemistry Amplified Nanopore Assay for High Performance Testing of Protein Biomarkers in Diseases. (Doctoral dissertation). Retrieved from https://scholarcommons.sc.edu/etd/7826