Developing signal amplification strategies for sensitive detection of low abundance biomolecules
thesisposted on 2022-03-28, 02:25 authored by Jie Lu
Developing sensitive and rapid assays to quantify trace amounts of target biomolecules is vital to diagnosing specific diseases, understanding mechanisms of disease pathogenesis, tailoring effective treatments, and monitoring therapeutic outcomes. Current luminescent bioassays have significant limitations in dealing with background noise (auto-fluorescence), and concentration-induced self-quenching and non-specific binding of the luminescent probe, and are ineffective for rapid detection of low abundance biomolecules. This thesis explores a suit of novel luminescence-based techniques for ultrasensitive target-specific detection of biomolecules either immobilized on cell surface or suspended in a solution. Techniques of time-resolved detection after pulsed excitation and upconversion detection by infrared excitation have been employed to effectively suppress the auto-fluorescence; lanthanide materials are used to avoid concentration quenching so that nanoparticles highly doped with hundreds to thousands of emitters have been introduced to essentially amplify the signal strength; a target recycling technique has been further developed to enhance the detection sensitivity of DNA bead assays; a simple but robust bio-/nano-conjugation strategy for SUPER dots has been developed to improve the binding efficiency and specificity; luminescence biosensing of multiple analytes has been extended beyond the current spectral multiplexing to harness a new dimension-temporal multiplexing based on multiple selectable luminescence lifetimes of new-generation luminescent bioprobes. The research program reported herein has involved four parallel research projects with four key technologies successfully demonstrated: 1). Resolving and quantifying trace levels of surface antigens immobilized on rare-event cells in background-free condition by using lanthanides-dye-rich luminescence nanoparticles(chapter 2); 2). Exploring the bio-conjugation method of lanthanide upconversion SUPER dots probes (chapter 3); 3). Achieving improved sensitivity of DNA detection in flow cytometry by using the Exonuclease III assisted target recycling technique (chapter 4); 4). Expanding the multiplexing capacity through successful application of lifetime-encoded luminescent nanocrystals and microspheres to enable rapid detection of multiple pathogen DNAs (chapter 5). This thesis adopts the “thesis-by-publication” approach, and is presented by a traditional style introduction chapter but appended by a 1st-authored review article, one traditional conclusion chapter, and four result chapters of five journal publications (two published in Analytical Chemistry as 1st author, one manuscript waiting for IP protection clearance as 1st author, and one in Nature Communications as 2nd author and one in Nature Photonics as a co-author).