Protease sensor for rapid and sensitive detection of Borrelia infections

Project Summary
Lyme disease (LD) is the most common tick-borne illness in North America caused by the bacterial species
Borrelia burgdorferi (Bb). If diagnosed early, broad-spectrum antibiotics can effectively clear an infection.
Unfortunately, delays in treatment lead to more serious symptoms including arthritis, carditis, and
neuroborreliosis. The Center for Disease Control and Prevention reports 30,000 new cases of LD annually.
However, experts estimate this number is closer to 400,000. This discrepancy is mainly due to the high
frequency of misdiagnosis. Current diagnostic guidelines recommend a standard two-tier (STT) serology
approach using an enzyme-linked immunosorbent assay (ELISA) to measure antibody levels followed by a
Western blot to verify the presence of Bb antigens. These tests have limited value because antibodies can take
weeks to develop at which point the disease has progressed beyond the stage at which it is effectively treated.
In addition, Bb antibodies continue circulating for years rendering them useless to distinguish between an
active and a prior infection. In this proposal, we outline a plan to develop a sensitive solid-state biosensor that
targets an active Bb protease as a biomarker to accurately diagnose both early- and late-stage active Bb
infections. The proposed epitaxial graphene-based biosensor provides the potential for exceptionally high
sensitivity, rapid detection, and ability to function with low volumes of patient samples by combining a novel
electrical transduction platform with a selective biorecognition element. We have selected the Bb protease,
high temperature requirement A (BbHtrA), as a biomarker because it is constitutively expressed on the bacteria
surface, is also secreted, remains active across all stages of Bb infection, and contains an active site serine
residue that is reactive towards small molecule electrophiles. This allows us to design and synthesize covalent
probes consisting of short amino acid sequences attached to an electrophilic ‘warhead’ that irreversibly
modifies the active protease with high selectivity and potency. The resulting covalent probes will be attached to
the surface of a quasi-freestanding epitaxial graphene (QEG) biosensor and used to detect Bb bacteria in
patient blood and urine, as well as in test synovial fluid samples. Success of this project will address several of
the most significant challenges associated with LD and potentially allow our overall approach to be applied to
other diseases where proteases can serve as optimal biomarkers.

Grant Number: 1R21AI191151-01
NIH Institute/Center: NIH
Principal Investigator: Matthew Bogyo