Wound state monitoring by multiplexed, electrochemical, real-time, localized, inflammation-tracking nitric oxide sensor (MERLIN)

Abstract

Nitric oxide (NO) released endogenously by induced NO synthase (iNOS) in macrophages is a key regulatory biomarker for wound inflammation. Detecting NO directly on the wound bed is challenging due to its short half-life time (6 to 50 seconds), low physiological concentration (nanomolar to micromolar), and interferences in the complex wound environment. Here, we present a compliant, multiplexed, electrochemical, real-time, localized, inflammation-tracking NO sensor (MERLIN) array for in vivo spatiotemporal measurement of NO, with high sensitivity (883 ± 283 nanoamperes per micromolar per square centimeter); selectivity against nitrites (~27,900-fold), ascorbic acid (~3800-fold), and uric acid (~6900-fold); and low limit of detection (~8.00 nM). MERLIN spatiotemporally tracked NO on rat skin wounds for 7 days, and results indicated that NO peaks on day 3, in line with previously reported iNOS activity. MERLIN allows spatial mapping of the NO gradient across the wound bed, which can be used to provide diagnostic information to assist wound care.

Fig. 1.. MERLIN for wound state monitoring.

(A) Schematic of MERLIN array. e⁻, electron. (B) Photograph of conformable MERLIN array on a 7-mm–radius surface. Scale bar, 1 cm. (C) Representative cross-sectional electron microscopy image of poly-5A1N on Pt. Scale bar, 500 nm. (D) Representative cross-sectional electron microscopy of image of fluorinated xerogel on SU-8 by spray coating. Scale bar, 10 μm. (E) Representative Raman spectra of modified electrode surface. Blue, Pt; green, 5A1N; red, fluorinated xerogel; a.u. represents arbitrary units. (F) Representative Raman spectroscopy mapping of 5A1N-Pt electrode plotted at peak intensity of C=C. (G) Representative Raman spectroscopy mapping of fluorinated xerogel-5A1N-Pt electrode plotted at peak intensity of methyl group.

Fig. 2.. MERLIN in vitro calibration and sensing performance.

(A) Representative single electrode current versus time with NO solution concentration changes. Typical interferants were added, i.e., nitrite (${\text{NO}}_2^{-}$), ascorbic acid (AA), and uric acid (UA). (B) Representative calibration curve of current versus NO concentration. (C) Sensitivity summary of MERLIN toward NO (n = 343). (D) LOD summary of MERLIN (n = 343). (E) Selectivity summary of MERLIN against common interferences such as nitrite (${\text{NO}}_2^{-}$), ascorbic acid (AA), and uric acid (UA) (n = 343); raincloud plots in (A) to (C) include half violin plots showing data distribution, boxplots, and raw data.

Fig. 3.. MERLIN in vivo rat skin wound inflammation monitoring.

(A) Schematic illustration of NO sensing experiment in vivo, with sensing measurements on days 1, 3, 5, and 7 post-surgery. (B) Representative real-time monitoring of the current reading of MERLIN at different days post–wound surgery for rat 1. (C) MERLIN temporal NO measurement by each sensor with data shown as the means ± SDs. *P < 0.05 and ****P < 0.0001, based on one-way analysis of variance (ANOVA) and Tukey post hoc test.

Fig. 4.. MERLIN spatial NO sensing in vivo.

(A) Representative day 3 MERLIN NO measurement on rat skin wound in vivo. Scale bar, 1 cm. (B) NO concentration mapping readout. (C) Representative day 7 MERLIN NO measurement on rat skin wound in vivo. Scale bar, 1 cm. (D) NO concentration mapping readout.

Fig. 5.. Tissue response to MERLIN.

(A) Representative histological images of tissue harvested from rat skin wound with and (B) without MERLIN, at day 7 post-wounding. Both NO sensed wound and control wound show similar wound morphology. Black arrows show the direction of the wound surface. (C) Zoomed-in image of histological images of tissue with MERLIN NO sensing measurement. (D) Zoomed-in image of histological images of tissue without MERLIN NO sensing measurement. E, epithelium; GT, granulation tissue; A, adipose tissue (subcutaneous); scale bars, 400 μm. (E) Cell density of hematoxylin and eosin (H&E)–stained tissues does not show statistical significance based on one-way ANOVA and Tukey post hoc test, showing good biocompatibility (n = 5 rats). (F) Quantitative analysis of epithelial and granulation tissue thickness does not show statistical significance based on one-way ANOVA and Tukey post hoc test, showing good biocompatibility (n = 5 rats).