A wireless patch for the monitoring of C-reactive protein in
sweat
Jiaobing Tu
1
,
Jihong Min
1
,
Yu Song
1
,
Changhao Xu
1
,
Jiahong Li
1
,
Jeff Moore
2
,
Justin
Hanson
3
,
Erin Hu
3
,
Tanyalak Parimon
4
,
Ting-Yu Wang
5
,
Elham Davoodi
1
,
Tsui-Fen Chou
5
,
Peter Chen
4
,
Jeffrey J. Hsu
3
,
Harry B. Rossiter
2
,
Wei Gao
1,*
1
Andrew and Peggy Cherng Department of Medical Engineering, Division of Engineering and
Applied Science, California Institute of Technology, Pasadena, CA 91125, USA.
2
Division of Respiratory and Critical Care Physiology and Medicine, The Lundquist Institute for
Biomedical Innovation at Harbor-UCLA Medical Center, Torrance, CA 90502, USA.
3
Division of Cardiology, University of California, Los Angeles, Los Angeles, CA, 90095, USA.
4
Department of Medicine, Women’s Guild Lung Institute, Cedars-Sinai Medical Center, Los
Angeles, CA, 90048, USA.
5
Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology,
Pasadena, CA, 91125, USA
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
of the sweat matrix. 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 gold-nanoparticle-conjugated anti-CRP
capture antibodies), as well as ionic strength, pH and temperature, for the real-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
Reprints and permissions information
is available at
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.
*
Correspondence and requests for materials
should be addressed to weigao@caltech.edu.
Author contributions
W.G., and J.T. initiated the concept and designed the overall studies; W.G. supervised the work; J.T., J.M., and Y.S. led the
experiments and collected the overall data; C.X., J.L., T.W., E.D., and T.C. contributed to sensor characterization and validation; J.M.,
J.H., E.H., T.P., P.C., J.J.H., and H.B.R. contributed to the design of the human trials and system evaluation in human subjects. All
authors contributed the data analysis and provided feedback on the manuscript.
Competing interests
The authors declare no competing interests.
Extended data
is available for this paper at
https://doi.org/10.1038/s41551-02X-XXXX-X
.
Supplementary information
The online version contains supplementary material available at
https://doi.org/10.1038/s41551-02X-
XXXX-X
.
HHS Public Access
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. 2023 October ; 7(10): 1293–1306. doi:10.1038/s41551-023-01059-5.
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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.
One-sentence editorial summary :
A wearable electrochemical patch for the real-time monitoring of the biomarker C-reactive
protein in sweat detects elevated concentrations of the protein in patients with acute or chronic
inflammation.
Inflammatory processes and immune responses are associated with a broad spectrum
of physical and mental disorders that contribute substantially to modern morbidity and
mortality globally. The top three leading causes of death worldwide, namely, ischemic heart
disease, stroke, and chronic obstructive pulmonary disease (COPD), are each characterized
by chronic inflammation
1
–
3
. Although the acute inflammatory response is a critical survival
mechanism, chronic inflammation contributes to long-term silent progression of disease
through irreversible tissue damage
4
–
6
. Delayed diagnosis and treatment of chronic diseases
impose heavy financial burdens on patients and the healthcare systems
2
,
4
. A readily
available means of monitoring inflammatory biomarkers at home could improve patient
outcomes and lower cost factors by monitoring disease progression and initiating early
treatment and intervention
7
.
Although there is no canonical standard biomarker for the measurement and prediction
of systemic chronic inflammation6, C-reactive protein (CRP), an acute-phase protein
synthesized by hepatocytes in response to a wide range of both acute and chronic stimuli,
has a close association with chronic inflammation and respective risks of mortality in several
disease states (Fig. 1a)
8
–
12
. The stable nature of CRP in plasma, the absence of circadian
variation, and its insensitivity to common medications such as corticosteroids render it
extremely attractive to clinicians as a handy means to assess a patient’s physiological
inflammatory state
13
. There is also a growing interest in exploring the effectiveness of serial
CRP measurements for therapeutic decision-making
14
,
15
.
At present, circulating CRP levels are clinically assessed in specific laboratories that rely
on invasive blood draws from patients (Supplementary Table 1). Commercial point-of-care
CRP monitors are still bulky in size and cannot reach picomolar-level sensitivity to assess
CRP levels in non-invasively accessible alternative biofluids such as sweat and saliva
(Supplementary Table 2). A faster, sensitive, non-invasive, and user-friendly approach,
accessible to not only clinicians but also patients and caregivers, could unleash the full
potential of inflammatory biomarker monitoring for clinical management beyond hospital
settings.
Recent advances in flexible electronics and digital health have transformed conventional
laboratory tests into remote wearable molecular sensing that enables real-time monitoring
of physiological biomarkers
16
–
24
. Sweat contains abundant biochemical molecules ranging
from electrolytes and metabolites, to large proteins
25
,
26
, and importantly, it is readily
accessible by non-invasive techniques (Fig. 1a). However, currently reported wearable
biosensors are largely restricted to the detection of a limited selection of biomarkers
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such as electrolytes and metabolites at μM or greater concentrations via ion-selective and
enzymatic sensors or direct oxidation/reduction
16
,
20
,
27
–
40
. The majority of clinically relevant
protein biomarkers including CRP are present at nM to pM levels in blood while the
anticipated levels of proteins in sweat are expected to be much lower than in blood
26
.
Despite recent efforts in the development of wearable bioaffinity biosensors for trace-level
biomarkers such as cortisol, the accurate and in situ detection of sweat protein biomarkers
remains a major challenge due to their extremely low concentrations (pM level) and the
large interpersonal and intrapersonal variations in sweat compositions
41
–
44
. The detection
of protein biomarkers usually requires integrating bioaffinity receptors such as antibodies
and aptamers
43
,
45
. However, such techniques typically require lengthy target incubation,
labour-intensive washing steps, and the addition of redox solutions for signal transduction.
Thus, there is a strong desire for a wearable biosensing technology that allows automatic in
situ monitoring of ultra-low-level circulating proteins at home and in community settings.
In this work, we report a wireless wearable nanobiosensor, InflaStat, for non-invasive
personalized inflammatory status monitoring (Fig. 1b–e). It consists of an autonomous
iontophoresis module for on-demand and controlled sweat extraction, a sweat gland-
powered skin-interfaced microfluidic module that capitalizes on sweat flow to achieve fully
automated protein and detector antibody capturing, subsequent washing, and picomolar-
level electrochemical detection on the skin, and a flexible nanoengineered multiplexed
sensor array for in situ sweat inflammatory biomarker analysis. The use of gold
nanoparticles (AuNPs)-decorated mass-producible laser-engraved graphene (LEG) enables
highly sensitive and efficient electrochemical detection of trace-level sweat CRP in situ on
the skin. AuNPs conjugated with electroactive redox molecule thionine (TH) and detector
antibody (dAb) enable efficient electrochemical signal transduction (Signal ON) and further
signal amplification. The integrated pH, temperature, and ionic strength graphene sensors
enable real-time personalized CRP data calibration to mitigate the interpersonal sample
matrix variation-induced sensing error, and provide a more comprehensive assessment of
the inflammatory status
46
,
47
. We confirmed the presence of CRP in human sweat from
healthy subjects and identified elevated sweat CRP levels in patients with chronic and acute
inflammations associated with COPD, heart failure (HF), and active and past infections (e.g.,
COVID-19). A strong correlation between sweat and serum CRP levels was obtained in
both healthy and patient populations, indicating the utility of this technology in non-invasive
disease classification, monitoring, and management.
Results & Discussion
Design of the wearable microfluidic LEG-AuNPs biosensor.
Key components of the wearable sensor are a skin-interfaced flexible, disposable,
multiplexed microfluidic biosensor patch fabricated on a polyimide (PI) substrate via
CO
2
laser engraving and a flexible printed circuit board (FPCB) for iontophoretic sweat
induction, sensor data acquisition and wireless communication (Fig. 1b and Supplementary
Fig. 1). The sensor array consists of an electrodeposited AuNPs-decorated LEG working
electrode immobilized with anti-CRP capture antibodies (cAb), a Ag/AgCl reference
electrode, an LEG counter electrode for sweat CRP capturing and electrochemical
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analysis, an LEG-based impedimetric ionic strength sensor, a LEG-polyaniline-based
potentiometric sweat pH sensor, and a strain-insensitive resistive graphene temperature
sensor (Supplementary Fig. 2). Considering that the potential users of this technology
include sedentary and immobile patients, an iontophoresis module (based on a pair of LEG
electrodes) is incorporated for on-demand delivery of cholinergic agonist carbachol from the
carbachol hydrogel (carbagel) for autonomous sweat stimulation throughout daily activities
without the need for vigorous exercise. A cost-effective and flexible microfluidic module
is assembled by stacking laser-cut medical adhesives and polyethylene terephthalate (PET)
for efficient sweat sampling (Fig. 1c). The miniaturized FPCB interfaces compactly on top
of the microfluidic sensor patch to form the fully integrated wearable system (Fig. 1d).
Powered by a small on-board lithium battery, the wearable system is able to wirelessly
communicate with a user interface via Bluetooth Low Energy (Supplementary Fig. 3).
In order to realize automatic wearable CRP detection in situ, the microfluidic module
comprises a reagent reservoir for the storage of the labeled anti-CRP dAbs-conjugated
AuNPs, a serpentine mixing channel for mixing of dAb with sweat CRP, and a detection
reservoir for the capture and quantification of sweat CRP (Fig. 1e and Supplementary Video
1). The redox molecule, TH, is used to label the nanoparticle conjugates to achieve direct
electrochemical sensing. As the autonomously induced sweat flows into the microfluidics,
the deposited dAbs conjugated AuNPs are reconstituted within the reagent reservoir (I) and
routed along with sweat through a serpentine passive mixer to facilitate the dynamic binding
between sweat CRP and dAb (II). As the mixture enters the detection reservoir, it slowly
fills the chamber before exiting via the outlet; the detection reservoir has an optimized
size to allow sufficient time for CRP-dAb to bind with anti-CRP cAb functionalized LEG-
AuNPs working electrode (III). Subsequently, a fresh sweat stream continues to refresh
the microfluidics to achieve passive label removal (IV). Square wave voltammetry (SWV)
is used to measure the amount of TH bound to the working electrode surface. Since TH
molecules are directly conjugated to CRP dAb-immobilized AuNPs, their amount bound
is directly correlated to the amount of CRP ‘sandwiched’ between cAbs at the electrode
surface and dAb-immobilized AuNPs, and consequently, the initial concentration of CRP in
solution.
Materials and electrochemical characterizations of the LEG-AuNPs immunosensor.
The functionalization process for the preparation of the CRP immunosensor is illustrated
in Fig. 2a and Supplementary Fig. 4. AuNPs are electrodeposited on the LEG surface
followed by subsequent thiol monolayer assembly with mercaptoundecanoic acid and
mercaptohexanol. As the formation of SAM layer relies on specific gold-sulfur bonding,
immersion of the sensor patch in alkanethiol solution has negligible influence on other
graphene-based electrodes (Supplementary Fig. 5). Pulsed potential-deposited AuNPs
evenly distribute throughout the mesoporous graphene structure and possess superior
electrocatalysis capability and form a large number of binding sites on the surface of
the particles for biomolecule immobilization (Fig. 2b,c and Supplementary Fig. 2b). This
substantially improves the sensitivity of the CRP sensor with little non-specific adsorption
(Supplementary Fig. 6). The formation of LEG-AuNPs composite is confirmed through the
increased ratio of the intensity of D and G bands in the Raman spectra due to the presence
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of AuNPs (Fig. 2d)
48
. The individual sensor modification steps on the LEG electrodes
are characterized with X-ray photoelectron spectroscopy (Fig. 2e and Supplementary Fig.
7). The intensity of Au4f increases substantially after the deposition of AuNPs while
N1s increases only after the cAb immobilization step, indicating successful electrode
preparation (Fig. 2e). Differential pulse voltammetry (DPV) and electrochemical impedance
spectroscopy (EIS) were used to further characterize the LEG surface electrochemically
after each modification step (Fig. 2f and Supplementary Fig. 8). The decrease in peak
current height in DPV voltammograms and increased resistance in Nyquist plots after self-
assembled monolayer (SAM) and cAb protein immobilization indicate that SAM and cAb
impede the electron transfer at the interface. This is due to the increase in surface coverage
by non-conductive species. Moreover, the negatively charged carboxylate functional groups
in the SAM layer result in the repulsion of the negatively charged redox indicator,
ferricyanide, and further reduces the electron transfer rate. Subsequent modification of the
SAM layer with EDC/NHS chemistry replaces the negatively charged carboxylate groups
with neutral NHS-ester groups. This is empirically observed as an increase in peak current
height. Such electrode fabrication processes show high batch-to-batch reproducibility as the
main processes including laser engraving, electrochemical deposition, and solution process
are all mass-producible (Supplementary Fig. 9).
In order to realize trace-level sweat CRP analysis, PEGylated AuNPs that possess large
surface area-to-volume ratio are functionalized with polystreptavidin R to increase the
loading of biotinylated-dAbs and subsequently enhance the sensitivity (Supplementary Figs.
10 and 11). One-step direct electrochemical detection is enabled by crosslinking the redox
label TH onto the carboxylate residues on the dAb-loaded AuNPs. As the TH-labelled
dAb-loaded AuNPs bind to the mesoporous graphene electrode upon CRP recognition, TH
located on the external sites of the proteins are in close proximity to the graphene surface
in each mesopores for electron transfer. Increases in hydrodynamic sizes (Fig. 2g) and the
shifts of ultraviolet-visible (UV-Vis) absorbance (Supplementary Fig. 12) of the AuNPs
conjugate after each modification step, along with the transmission electron microscope
(TEM) image of the dispersed AuNPs-dAb conjugates (Fig. 2h) confirm the successful
immobilization of the dAbs.
The performance of the CRP sensor was evaluated with SWV in CRP spiked phosphate-
buffered saline (PBS) solutions (Fig. 2i). The increases in peak current height of TH
reduction show a linear relationship with increased target concentrations (Fig. 2j). The
sensor showed an ultralow limit of detection of 8 pM, good batch-to-batch reproducibility
(Supplementary Fig. 13), and the sensing accuracy can be further enhanced by automating
the sensor preparation and modification process (e.g.,
via
automated fluid dispensing or
inkjet printing
49
). The LEG-AuNPs CRP immunosensor demonstrates high selectivity over
other potential interference proteins and hormones attributed to the sandwich assay format
(Fig. 2k and Supplementary Fig. 14). Considering interpersonal variations during the human
study, the influence of sweat pH, ionic strength, temperature, and sample volume on the
antibody-antigen binding kinetics and redox probe electron transfer rate on CRP sensing
accuracy was investigated (Supplementary Fig. 15) and mitigated by introducing suitable
calibration mechanisms. The potential variations of the Ag/AgCl pseudo-reference electrode
in the presence of varying Cl
−
concentration in the physiologically-relevant range result
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in a small shift in the peak potential but its influence on the overall peak current density
(and thus CRP quantification) is negligible (Supplementary Fig. 16). The accuracy of the
CRP sensor for biofluid analysis was validated by the laboratory gold standard enzyme-
linked immunosorbent assay (ELISA) using human sweat and saliva samples (Fig. 2l). The
disposable CRP sensors also maintained stable sensor performance over a 10-day period
when stored in PBS in the refrigerator at 4°C (Supplementary Fig. 17).
Evaluation of sweat CRP for non-invasive monitoring of systemic inflammation.
Despite the high potential of non-invasive CRP monitoring, the presence and levels of
CRP in sweat are extremely underexplored in the literature
50
. To affirm the presence of
CRP in sweat generated by iontophoresis and by vigorous exercise, we first conducted a
proteomic characterization of different types of sweat samples using bottom-up proteomic
analysis as illustrated in Fig. 3a. Using a recombinant CRP protein standard as the reference,
we identified CRP in both exercise and iontophoretic sweat samples from human subjects
(Fig. 3b, Supplementary Fig. 18, and Supplementary Table 3). In this regard, we further
evaluated the use of our LEG-AuNPs CRP sensors for the assessment of sweat CRP as
a universal, cost-effective, and non-invasive approach to monitor systemic inflammation in
various disease states (Fig. 3c and Supplementary Tables 4–6).
We investigated healthy subjects grouped according to smoking status (current, former, and
never smokers), where CRP levels in both serum and sweat were greater in current smokers
as compared with former and never smokers (Fig. 3d), consistent with previous reports on
the effect of current smoking on serum CRP
51
. However, among COPD patients, serum and
sweat CRP values were greater in former smokers than current smokers, consistent with
irreversible tissue damage and chronic inflammation in COPD patients even after smoking
cessation
52
. Monitoring sweat CRP in COPD patients may therefore be useful for following
disease progression and/or predicting exacerbation in this patient population
53
.
Chronic systemic inflammation is also related to increased risks of cardiovascular events
3
.
In a preliminary study with HF patients, our sensor results show that serum and sweat CRP
values were substantially elevated in HF patients with preserved ejection fraction (HFpEF)
but not in HF patients with reduced ejection fraction (HFrEF) (Fig. 3e), consistent with
past studies
54
–
57
. The investigation of the dynamics of sweat CRP using our technology
could potentially have high value in predicting HFpEF disease progression and clinical
outcomes
55
.
In addition to chronic infections in COPD and HF, it is well known that acute infections
(such as COVID-19) could lead to severe inflammatory responses
14
. In a pilot study, we
evaluated our sensor on hospitalized patients with active infections for two consecutive days
(Fig. 3f). Substantial increase (over 10-fold on average) in both serum and sweat CRPs was
identified in patients with active infection as compared with healthy subjects, indicating the
presence of highly elevated sweat CRP in acute inflammation.
By analysing the samples from healthy subjects and patient populations with various
inflammatory conditions using our sensor, a high correlation coefficient (
r
) of 0.844 (n=80)
between sweat and serum CRP concentrations was obtained (Fig. 3g). Such correlation
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appears to be higher than those obtained from saliva and urine samples (Supplementary
Fig. 19), suggesting the great potential of using sweat CRP for the non-invasive monitoring
of systemic inflammation toward the management of a variety of chronic and acute health
conditions.
Characterization of the multiplexed microfluidic patch for automatic immunosensing.
In order to realize accurate and automatic immunosensing
in situ
, the flexible sensor
patch was designed to have a laser-engraved microfluidic module (consisting of a reagent
reservoir, a mixing channel, and a detection reservoir) and a multiplexed LEG sensor array
(consisting of a CRP immunosensor, an ionic strength sensor, and a pH sensor) (Fig.
4a). As the microfluidic module routes sweat passively on the skin, the impedimetric
ionic strength sensor automatically captures the state of the detection reservoir (reagent
flow and refreshment); the measured admittance signals show a log-linear response with
the electrolyte concentrations (Fig. 4b,c). As large interpersonal variations in electrolyte
and pH levels were observed in both exercise and chemically induced sweat samples
(Supplementary Fig. 20), high-level buffering salts were deposited with the dAbs in the
reagent reservoir to mitigate potential binding environment changes caused by sweat
composition variations (Supplementary Fig. 21). As such, this introduces an electrolyte
gradient between the detection reagent reconstituted sweat (mixture) and fresh sweat that
subsequently enters the detection reservoir. According to the numerical simulation, the
routing of sweat and detection reagents can be summarized into four steps: reconstitution
(I), incubation (II), refreshment (III), and detection (IV) (Fig. 4d,e and Supplementary Note
1). Based on the microfluidic flow test using artificial sweat (0.2X PBS) under a mean
physiological sweat rate (1.5 μL min
−1
), the admittance signal is close to zero initially
when no fluid enters the chamber during the reconstitution stage; as reconstituted, high-salt
loaded detection reagents flow into the detection chamber, admittance reaches its peak
value and gradually decreases as high-salt loaded reagents are flushed out of the detection
chamber by newly secreted sweat (Fig. 4f). Since electrolyte content in iontophoresis sweat
remains relatively stable for the same individual
28
, the admittance response plateaus after
all reagents have been refreshed by natural sweat, indicating the working electrode is ready
for electrochemical CRP detection. Further experimental flow test using fluorescent proteins
(fluorescein isothiocyanate-albumin as CRP surrogate and peridinin chlorophyll protein
as detection reagent) shows a similar trend in incubation and refreshment process as the
simulation and electrolyte flow test (Fig. 4g and Supplementary Video 2). Based on sweat
rate information collected from 24 current and former smokers with and without COPD
(Supplementary Fig. 22), flow tests with flow rates varying from 0.5 to 3.5 μL min
−1
show
similar admittance patterns with plateaus after various refreshing processes (Fig. 4h). The
gradient of admittance at different flow rates converges to zero, as pre-loaded salts and
dye are refreshed from the detection reservoir. The mean sweat volume routed during this
process before sensors readings were taken was estimated to be 21 μL based on flow rate
and admittance measurements (Fig. 4h).
The performance of CRP sensors based on this automated electrolyte monitoring mechanism
was evaluated in multiple microfluidic flow tests. SWV electrochemical measurements were
initiated during the admittance plateaus (Fig. 4i). An increased concentration (from 1 to 5 ng
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mL
−1
) led to an increased SWV peak current height while no substantial difference in CRP
sensor response was observed for the same concentration under physiologically relevant
flow rates (1, 1.5, 2.5, and 3.5 μL min
−1
) (Fig. 4i,j and Supplementary Fig. 23). Although
a higher flow rate could also result in a faster refreshment of the detection chamber and
thus a shorter incubation time for the detection antibody and CRP, the increment in CRP
signals under varying incubation time corresponding to the physiologically relevant sweat
rates (between 5 and 20 minutes) is relatively small (Supplementary Fig. 15). Although the
binding condition is pre-adjusted with deposited salts, the flow test with different initial
electrolyte concentrations (0.1X and 0.2X PBS were chosen as artificial sweat to simulate
interpersonal variations in sweat electrolyte concentrations) shows slightly decreased SWV
signals at the lower electrolyte concentration due to the influence of electrolyte levels
on the rate of TH reduction (Fig. 4k,l). Similar to
in vitro
selectivity results, no major
interferences on the CRP detection signal were observed in the flow test (Supplementary
Fig. 24). Moreover, flow tests using artificial sweat with different pH levels lead to varied
SWV signals (Supplementary Fig. 25). These results indicate that sweat rate calibration is
not necessary while additional
in situ
signal calibrations with sweat pH and electrolyte levels
are needed to mitigate the interpersonal variations on CRP detection accuracy. Compared to
previously reported passive wearable microfluidic sensors which rely on vigorous exercise
to induce sweat and cannot reach sensitivities below mM levels (Supplementary Table 7),
our technology offers an attractive fully automated microfluidic sweat induction, harvesting,
and high-accuracy quantitative analysis solution, ideally suitable for at-home monitoring of
clinically relevant trace-level biomarkers.
System integration and on-body evaluation of the wearable biosensor.
The fully integrated wearable inflammation monitoring system, InflaStat, is designed based
on vertical stack assembly of a flexible microfluidic sensor patch and an FPCB and
can be comfortably worn by the subjects (Fig. 5a). As illustrated in electronic circuit
block diagram and schematic in Fig. 5b and Supplementary Fig. 26, the FPCB is able
to perform current-controlled iontophoresis, multiplexed electrochemical measurements
(including voltammetry, impedimetry, and potentiometry), signal processing, and wireless
communication. The integrated system could also accurately obtain the dynamic responses
of the integrated LEG-based pH, ionic strength, and skin temperature sensors for real-time
CRP sensor calibration (Fig. 5c–f, Supplementary Fig. 27). The InflaStat is designed to
have good mechanical flexibility and stability toward practical usage during various physical
activities. Each individual sensor shows relatively small variations under a moderate radius
of bending curvature (5 cm) (Supplementary Fig. 28). More strain-insensitive sensor designs
can be included when necessary
58
. During on-body operation, the InflaStat can conformally
adhere to the skin through medical adhesive with
in situ
CRP sensing performed in the
microfluidics without direct sensor-skin contact.
Clinical on-body evaluation of the wearable system was performed on healthy subjects
(involving both never smokers and current smokers) as well as patients with COPD and
post-COVID-19 infection (Fig. 5g–l and Supplementary Fig. 29–31). During the on-body
trials, the wearable system laminates conformally on the subject’s arm, chemically induces
and analyzes sweat (Supplementary Video 3 and Supplementary Fig. 32), and acquires
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inflammatory biomarker information non-invasively and wirelessly (Fig. 5g). The obtained
sensor data can be displayed on a custom developed mobile app in real-time (Fig. 5h).
In
situ
pH, temperature, and CRP sensor readings are acquired after the ionic strength sensor
indicate full refreshment of the detection reservoir (Fig. 5h–l). It should be noted that the
TH’s reduction peak for the CRP sensor appears at a slightly shifted potential given the
variations in sweat pH (Supplementary Fig. 33). The CRP concentration was converted
in the mobile app based on the obtained SWV voltammogram and the corresponding
real-time obtained ionic strength, pH, and temperature values (Supplementary Fig. 34
and Supplementary Note 2). As expected, an elevated CRP level was observed from the
current smokers as compared with the never smokers in healthy subjects. The CRP levels
in the COPD patients and post-COVID subjects were substantially greater than those of
non-smoking healthy subjects, suggesting the promise of using the InflaStat in practical
non-invasive systemic inflammation monitoring and disease management applications.
In
vitro
analysis of sweat and serum from post-COVID subjects corroborate the on-body
observation that patients who experienced moderate symptoms during COVID may still
present a low-grade inflammation post COVID episode as indicated by the slightly elevated
CRP levels (Supplementary Fig. 35 and Supplementary Table 8). It should be noted that
similar as serum, sweat CRP levels remained stable during the test period (Supplementary
Fig. 36) and no substantial variations were observed for chemically-induced sweat samples
at different body locations (Supplementary Fig. 37).
Conclusion
We developed a fully integrated wearable biosensor patch for real-time, non-invasive
inflammatory biomarker monitoring through automatic
in situ
microfluidic analysis. The
wearable sensor is capable of autonomous sweat extraction, harvesting, biomarker analysis,
and wireless data transmission in sedentary individuals on-demand across daily human
activities. In contrast to previous wearable technologies for monitoring biomarkers and our
previously reported LEG-based sensors which typically detects metabolites at μM or higher
level
27
,
59
, this technology realizes highly sensitive detection of ultra-low-level inflammatory
proteins
in situ
with a 6 orders-of-magnitude (picomolar level) improvement in sensitivity
through a holistic combination of 1) a nanoengineered immunosensor highly sensitive and
selective CRP analysis, 2) a microfluidic module for automatic sweat extraction, sampling,
reagent routing and refreshing, 3) and a multiplexed graphene sensor array for real-time data
acquisition and sensor calibration. The operation principle proposed herein can be readily
adapted to survey a broad array of inflammatory biomarkers (e.g., cytokines) and beyond.
We assessed the elevation of sweat CRP in healthy subjects and patients with various health
conditions (e.g., COPD, HF, and active and past infections) for the monitoring of chronic
and acute systemic inflammation and reported a high correlation between sweat and serum
CRP levels. In practice, the spot checking of CRP every several hours is sufficient to
monitor active infections and immune responses. The disposable point-of-care CRP sensor
patch design with a reusable wearable electronic system serves the purpose of immediate,
non-invasive, on-the-skin assessment of circulating CRP at any given time. When necessary,
dynamic and automatic wearable CRP sensing could be realized by incorporating capillary
bursting valves
60
and CRP sensor arrays into a single disposable sensor patch. It is also
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