Plastic Surgery Research Council

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Molecular Assessment of the Wound Environment with a Point-of-Care Immunoassay
Daniel Joh, BA, MEng., Roger Cason, MD, Amy Cheng, BS, Rebecca Blair, BS, Angus Hucknall, PhD, David Brown, MD, PhD, Ashutosh Chilkoti, PhD.
Duke University, Durham, NC, USA.

TITLE: Molecular Assessment of the Wound Environment with a Point-of-Care Immunoassay
PURPOSE: Diabetic foot ulcers (DFUs) affect >6.5 million Americans and are the leading cause of lower extremity amputation, with an estimated annual cost of $25 billion. Current management algorithms for DFUs rely on subjective assessments by practitioners, with minimal objective data to guide treatment. Wound care relies heavily on trial-and-error approaches, and targeted treatments are implemented as a last resort with limited efficacy. Combined, this prolongs morbidity and increases overall cost. Several investigative biomarkers found in wound fluid and blood are being actively pursued as objective metrics of nonhealing. Examples include proteases and their inhibitors, cytokines, procalcitonin, and others. However, implementation of biomarkers in clinical settings is hindered by standard assays (e.g. ELISA) that are low-throughput and costly. This approach has proven impractical for routine evaluations. Novel biomarker assessments are needed to guide treatment for chronic wounds and optimize care for these patients.
METHODS: To overcome these limitations, our goal is to develop a low-cost, multiplexed “point-of-care” test (POCT) against investigative markers of wound healing, and to demonstrate the feasibility of using this POCT in clinical settings. As a proof-of-concept, our assay will target markers of elevated protease activity (EPA), which are currently being explored as predictive markers for delayed healing. This POCT is based on a novel platform we have previously developed (the “D4” POCT) that converts the traditional sandwich immunoassay into a multiplexed, smartphone-compatible POCT (Fig. 1). The D4 is fabricated by inkjet-printing assay reagents as microarrays on nanoscale polymer brushes grown from glass chips. All reagents are stored “on-chip” within the stabilizing polymer matrix, allowing prolonged storage without refrigeration. Adding a drop of blood drives the assay to completion, and chip readout is performed with a tabletop scanner or a handheld smartphone detector (Fig. 1A).
RESULTS: In our preliminary work, we have already demonstrated a multiplexed D4 against cytokine markers and showed concordance between D4 and ELISA in patient specimens (Fig. 1B). These design principles will be applied towards developing a D4 assay against EPA markers (“EPA-D4”), specifically matrix metalloproteinases 7 and 9 (MMP-7, -9), tissue inhibitor of metalloproteinases-1 (TIMP-1), and human neutrophil elastase (HNE). Ultimately, our objective is to provide “proof-of-concept” evidence that a multiplex D4 can accurately quantify panels of known wound markers in wound fluid and blood at POC (in this case EPA markers), particularly in the hands of clinical technicians not involved in assay development.
CONCLUSIONS: Taken together, the D4 platform may offer a scalable and sustainable approach toward rapid, on-site assessment of the molecular environment of wounds. Feedback from implementing the EPA-D4 as an alpha device here will drive beta design for future studies. Importantly, the D4 platform can target any analyte for which antibody pairs are available, and is readily customizable to target other markers of wound healing with rapid turnaround as they become available.


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