Publication Date

Fall 2020

Advisor(s) - Committee Chair

Moon-Soo Kim (Director), Kevin Williams and Ajay Srivastava

Degree Program

Department of Chemistry

Degree Type

Master of Science

Abstract

Antibiotic resistance is one of the biggest public health challenges globally. To combat this threat, we have designed a method for the facile and rapid detection of antibiotic resistance gene (ARG) in bacteria. We utilized an emerging tool termed the clusters of regularly interspaced short palindromic repeats (CRISPR) in complex with deactivated CRISPR associated proteins (dCas9) on conjugated 2-dimensional graphene oxide (GO) sheets. Nuclease-deactivated Cas9 in complex with customized single guided RNA (sgRNA) was used for the target DNA recognition. The sgRNA was easy to design and we demonstrated that it is complementary to its target sequence by performing a Cas9 cleavage assay. The sgRNA was then successfully labelled with Alexa-488 dye. The GO was immobilized with anti-CRISPR Cas9 antibody and its surface morphology was characterized by Atomic Force Microscopy (AFM) to confirm the successful immobilization of the antibody to the GO. Our approach is based on the fluorescence quenching mechanism of antibody-GO in the absence and presence of target DNA. It was then validated by performing a sensitivity assay to detect the specific dsDNA within the tetM gene using the labelled-sgRNA/dcas9 ribonucleoprotein. The CRISPR complex was adsorbed onto the GO surface via the Cas9 antibody immobilized to the GO. In the absence of target DNA, fluorescence signal of the labeled sgRNA/dCas9 was quenched due to fluorescence resonance energy transfer (FRET) between Alexa fluorophore and GO when they are in close proximity. However, in the presence of target DNA, the bound dcas9/sgRNA-DNA complex was dissociated from the GO, restoring the fluorescence signal. The detection limit of the tetM gene was as low as 1 nM. This novel detection approach has the potential to be further developed and applied at the point-of-care (POC) for antibiotic resistant bacteria detection.

Disciplines

Biochemical and Biomolecular Engineering | Life Sciences | Medicine and Health Sciences

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