A wireless patch for the monitoring of C-reactive protein in sweat

Abstract

The quantification of protein biomarkers in blood at picomolar-level sensitivity requires labour-intensive incubation and washing steps. Sensing proteins in sweat, which would allow for point-of-care monitoring, is hindered by the typically large interpersonal and intrapersonal variations in its composition. Here we report the design and performance of a wearable and wireless patch for the real-time electrochemical detection of the inflammatory biomarker C-reactive (CRP) protein in sweat. The device integrates iontophoretic sweat extraction, microfluidic channels for sweat sampling and for reagent routing and replacement, and a graphene-based sensor array for quantifying CRP (via an electrode functionalized with anti-CRP capture antibodies-conjugated gold nanoparticles), ionic strength, pH and temperature for the real-time calibration of the CRP sensor. In patients with chronic obstructive pulmonary disease, with active or past infections or who had heart failure, the elevated concentrations of CRP measured via the patch correlated well with the protein's levels in serum. Wearable biosensors for the real-time sensitive analysis of inflammatory proteins in sweat may facilitate the management of chronic diseases.

Fig. 1 |. Wearable electrochemical nanobiosensor for automatic, non-invasive, and wireless inflammation monitoring.

a, Circulating C-reactive protein (CRP), released from inflammatory responses, is closely related to various chronic and acute health conditions and could be secreted via the sweat gland. COPD, chronic obstructive pulmonary disease. b, Schematic of the skin-interfaced multimodal wearable nanobiosensor that contains an iontophoretic module for localized sweat extraction on-demand, a microfluidic module for automated sweat sampling and reagent routing, and a flexible laser-engraved graphene (LEG) multimodal sensor array for multiplexed sensing of sweat CRP, pH, temperature, and ionic strength. PI, polyimide; carbagel, carbachol hydrogel; PET/M-tape, polyethylene terephthalate/medical tape; IP, iontophoresis. c,d, Optical images of a disposable microfluidic graphene sensor patch (c) and a vertical stack assembly of the fully integrated wireless wearable system (d). Scale bars, 0.5 cm. e, The mechanism of in situ microfluidic sweat CRP analysis that involves fully-automatic sweat sampling, reagent routing, and detection. AuNPs, gold nanoparticles; cAb, capture antibody; dAb, detector antibody; SWV, square wave voltammetry; TH, thionine; LTH, leuco thionine.

Fig. 2 |. Materials and electrochemical characterizations of the LEG-AuNPs CRP sensor.

a, Schematic of the one-step electrochemical sandwich CRP immunosensor. PS-R, Polystreptavidin R. b, Scanning electron microscope (SEM) image of the mesoporous LEG electrode. Scale bar, 100 μm. c, Transmission electron microscopy (TEM) image of AuNPs-decorated graphene flakes. Scale bar, 50 nm. d, Raman spectra of LEG electrode and AuNPs-decorated LEG electrode. e, X-ray photoelectron spectra of the LEG after the deposition of AuNPs, thiol-based self-assembled monolayer (SAM), and cAb immobilization. f, DPV voltammograms of a sensing electrode in a 0.1 M KCl solution containing 5.0 mM K₄Fe(CN)₆/K₃Fe(CN)₆ (1:1) after each surface-modification step: bare LEG, deposition of AuNPs, SAM modification, carboxylic acid group activation with N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide/N-hydroxysulfosuccinimide (EDC/Sulfo-NHS), and cAb immobilization followed by bovine serum albumin (BSA) blocking. g, Hydrodynamic sizes of the PEGylated AuNPs after each conjugation step by dynamic light scattering: PS-R immobilization, biotinylated dAb binding, and redox molecule TH conjugation followed by BSA deactivation. h, TEM image of the dispersed dAb-loaded AuNPs with protein corona shells. Scale bar, 10 nm. i,j, SWV voltammograms (i) and the corresponding calibration plot (j) of the CRP sensors in 1X PBS (pH 7.4) with 0–20 ng mL⁻¹ CRP and 1% BSA. Error bars represent the s.d. of the mean from 3 sensors. k, Selectivity of the CRP sensor to potential interferences in sweat. Error bars represent the s.d. of the mean from 3 sensors. i, Validation of the CRP sensor in human sweat samples (n=13 biological replicates) and saliva samples (n=6 biological replicates) with ELISA. The Pearson correlation coefficient was acquired through linear regression.

Fig. 3 |. Evaluation of sweat CRP for non-invasive monitoring of systemic inflammation in healthy and patient populations.

a, Schematic of proteomic analysis of human sweat using the liquid chromatography-mass spectrometry (LC-MS/MS). b, Chromatograms of the recombinant CRP reference peptide GYSIFSYATKR, iontophoresis-extracted and exercise sweat samples from human subjects. c, Schematic of the non-invasive inflammation monitoring in various health conditions with the LEG-AuNPs CRP sensor. d, Box-and-whisker plot of CRP levels in iontophoresis-extracted sweat and serum samples from subjects with COPD (n=10 biological replicates) and without COPD (n=24 biological replicates). The subjects are classified into five subgroups: current smokers with COPD (n=6 biological replicates) or without COPD (n=10 biological replicates), former smokers with COPD (n=4 biological replicates) and without COPD (n=9 biological replicates), and never smokers without COPD (n=5 biological replicates). The bottom whisker represents the minima; the top whisker represents the maxima; and the square in the box represents the mean. e, Box-and-whisker plot of CRP levels in sweat and serum samples from healthy subjects (n=7 biological replicates), patients with heart failure with reduced ejection fraction (HFrEF) (n=7 biological replicates), and patients with heart failure with preserved ejection fraction (HFpEF) (n=9 biological replicates). The bottom whisker represents the minima; the top whisker represents the maxima; the square in the box represents the mean. f, Box-and-whisker plot of CRP levels in sweat and serum samples from 3 patients with active infection on two consequent days (n=3 biological replicates). The bottom whisker represents the minima; the top whisker represents the maxima; the square in the box represents the mean. Dotted lines represent the mean values of the sweat and serum CRP levels for healthy subjects. g, Correlation of serum and sweat CRP levels. The correlation coefficient r was acquired through Pearson’s correlation analysis (n=80, p< .00001).

Fig. 4 |. Multiplexed microfluidic patch for automatic immunosensing.

a, Illustration of the multiplexed sensor array for automatic immunosensing. b,c, Admittance responses (b) and the corresponding calibration plot (c) of the impedimetric ionic strength sensor in NaCl solutions. Error bars represent the s.d. of the mean from 3 sensors. d, Simulated CRP-dAb concentration changes on the working electrode over time. Red dot in the inset image indicates the location of the concentration change plot. e, Simulated CRP-dAb concentrations colormaps showing phases of automatic sweat sampling and reagents routing toward in situ CRP detection: reconstitution (I), incubation (II), refreshment (III), and detection (IV). Scale bar, 200 μm. f,g, Admittance changes of the LEG ionic strength sensor (f) and optical images (g) during the four stages (I–IV) of automatic CRP sensing process in a laboratory flow test using artificial sweat (0.2X PBS) at a flow rate of 1.5 μL min⁻¹. Yellow fluorescein isothiocyanate (FITC)-albumin fluorescent label was used to imitate the flow of sweat CRP and red Peridinin Chlorophyll Protein Complex (PerCP) was used in place of dAb-loaded AuNPs. Scale bar, 200 μm. h, Admittance responses of the ionic strength sensor in artificial sweat (0.2X PBS) at different flow rates from 0.5 to 3.5 μL min⁻¹. i–l, Influence of the flow rates (i,j) and ionic strengths (k,l) on microfluidic automatic CRP sensing. Solid and dotted lines represent tests performed in 1 and 5 ng mL⁻¹ CRP, respectively.

Fig. 5 |. On-body evaluation of the multiplexed wearable patch toward non-invasive automatic inflammation monitoring.

a, Image of a fully integrated wearable sensor on the arm of a human subject. Scale bar, 1 cm. b, Block diagram of the electronic system of the InflaStat. c–f, Calibration plots obtained using the wearable system from the CRP (c), ionic strength (d), pH (e) and temperature (f) sensors. Error bars represent the s.d. of the mean from 3 sensors. g, Photograph of a subject wearing the sensor patch during a clinical study. h, Custom mobile application for real-time data acquisition and display toward inflammation tracking. i–l, On-body multiplexed physicochemical analysis and CRP analysis with real-time sensor calibrations using the wearable sensor from a healthy never smoker (i), a healthy smoker (j), a COPD patient (k) and a post-COVID subject (l).