Abstract

Life-threatening bloodstream infections (BSI), such as sepsis, mainly caused by the body’s syndromic response to pathogen infection, are a major public health concern, leading to ~11 million deaths each year. The identification of sepsis presents significant challenges in the diagnostic process, as the isolation of low concentration pathogens from bodily fluids (e.g., human blood) is extremely demanding, and typically relies on time-consuming and complicated isolation processes. Therefore, this work aims to explore the potential of microfluidics as a powerful tool for rapid purification and concentration of antibiotic-resistant bacteria (ARB) from blood samples, meanwhile developing a molecular diagnostic technology by combining a recombinase polymerase amplification (RPA) with clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas), aiming to rapidly and accurately targeting the specific genetic marker or DNA sequences unique to the bacteria of interest. A novel pneumatically-regulated nano-sieve device is successfully designed and fabricated for purifying and concentrating the target bacteria, such as Methicillin-resistant Staphylococcus aureus (MRSA), by applying a three-dimensional (3-D) beads-stacked microstructure within the nano-scale channel (~200 nm in depth). Leveraging this deformable nanosystem, the captured bacteria can be retrieved from the channel by retrieving with a small volume (e.g., 30 μL) of fresh buffer solution, which makes the collected sample directly eligible for downstream analysis. An on-chip concentration factor of 15 has been achieved while dealing with a 600 μL MRSA-spiked sample under a low concentration of 100 colony forming unit (CFU)/mL. As expected, RPA/CRISPR-Cas system offers a naked-eye detection after a multiplexing purification of MRSA by nano-sieve device under a flow rate of 4 μL/min per channel. This suggests that the integration of proposed nano-sieve device and RPA/CRISPR-Cas technique enables the purification and concentration of a low-concentration bacteria sample, potentially suitable for identifying sepsis-causing pathogens from whole blood.

Library of Congress Subject Headings

Drug resistance in microorganisms; Septicemia--Prevention; Blood--Filtration; Sieves--Design and construction; Nanostructured materials

Publication Date

8-2-2023

Document Type

Dissertation

Student Type

Graduate

Degree Name

Microsystems Engineering (Ph.D.)

Department, Program, or Center

Microsystems Engineering (KGCOE)

Advisor

Ke Du

Advisor/Committee Member

Thomas Gaborski

Advisor/Committee Member

Blanca Lapizco-Encinas

Campus

RIT – Main Campus

Plan Codes

MCSE-PHD

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