Li LiuFollow

Abstract

CRISPR-Cas enabled biosensors offer great potential to be at the forefront of diagnostic medicine. This thesis focuses on the principles of CRISPR-Cas technology and its application to biosensors and virus diagnostics. It also provides a detailed discussion of the different detection methods available at this stage. The breakthrough achievements of CRISPR biosensors in nucleic acid detection are summarized. The CRISPR-Cas nano biosensors exhibit high accuracy, sensitivity, selectivity, and versatility, offering great potential for next-generation diagnostic and point-of-care devices. The prospects and future trends of CRISPR biosensors are also described. From an engineering perspective, this thesis develops and applies several methods for CRISPR-based viral detection. A nanopore array platform for high-throughput single-molecule sensing was developed. Gold nanoparticle-labeled reactions for CRISPR virus detection were used as reporter signals, demonstrating the ability to lower the limits of detection reached while maintaining CRISPR accuracy and selectivity. An electrochemical sensor platform based on CRISPR virus detection was then developed, with electrochemistry offering the advantages of simple measurement procedures, short reaction times, and adequate sensitivity and selectivity. Combined with the high selectivity of CRISPR, it makes detection more rapid and convenient. Finally, we have developed an integrated digital microfluidic chip that realizes precise quantitative detection of nucleic acids and incorporates an isothermal amplification method to greatly reduce the detection limit, providing an excellent advancement for next-generation bedside diagnostics. Digital microfluidic chips and nanofluidic chips developed in recent years offer the advantages of greater resistance to inhibition, higher sensitivity, and more precise detection. Digital microarrays, in combination with nucleic acid amplification methods, allow absolute quantitative analysis of nucleic acid targets by dispensing the target molecules into small wells or droplets. When the sample is divided into numerous aliquots, these aliquots contain no target molecules or only one molecule. The concentration of the target molecule can then be derived by counting the number of positive aliquots. This research addresses current hotspots in virus detection by developing several different virus detection platforms that not only reduce the detection limit, but also allow in-depth analysis of the physical and chemical properties of the molecules during the reaction.

Library of Congress Subject Headings

Biosensors; CRISPR (Genetics); Nanofluids; Viruses--Isolation

Publication Date

5-21-2024

Document Type

Dissertation

Student Type

Graduate

Degree Name

Microsystems Engineering (Ph.D.)

Department, Program, or Center

Microsystems Engineering

College

Kate Gleason College of Engineering

Advisor

Ke Du

Advisor/Committee Member

Mitchell R. O'Connell

Advisor/Committee Member

Chuanhua Duan

Campus

RIT – Main Campus

Plan Codes

MCSE-PHD

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