A Laser-Induced Photoelectrochemical Sensor for Natural Sweat Cu 2+ Detection

: Tracking ﬂuctuations in the Cu 2+ level in sweat is meaningful for non-invasive and real-time assessment of Cu 2+ -abnormality-related diseases and provides important diagnostic information. However, the user-unfriendly ways to obtain sweat and sweat biofouling have limited the development of this ﬁeld. Herein, we exploit a highly sensitive photoelectrochemical (PEC) sensor as a detection method, a powerful laser engraving technique for the large-scale fabrication of laser-induced graphene and In-doped CdS (LIG-In-CdS) photoelectrodes, and a hydrophilic porous polyvinyl alcohol (PVA) hydrogel for natural sweat collection for ﬁngertip touch sweat Cu 2+ monitoring. The proposed sensor has several very attractive features: (i) the LIG-In-CdS photoelectrode with high photoelectric conversion efﬁciency can be produced by a cheap 450 nm semiconductor laser system; (ii) the sensor performs Cu 2+ detection with a wide linear range of 1.28 ng/mL~5.12 µ g/mL and good selectivity; (iii) the PVA hydrogel possesses an excellent antifouling effect ability and a rapid natural sweat collection ability; and (iv) the sensor exhibits feasibility and good reliability for PEC sensing of sweat Cu 2+ . Thus, these advantages endow the proposed method with a great deal of potential for smart monitoring of heavy metals in sweat in the future.


Introduction
As an essential trace element in human beings, Cu 2+ plays a pivotal role in physiological processes [1].Abnormal uptake of Cu is associated with various diseases, such as Wilson's disease, Menkes disease, hematological abnormalities, and kidney and cardiovascular diseases [2].Consequently, it is of considerable significance to detect Cu 2+ in human biofluids.Among the several candidate biofluids, including blood, interstitial fluid, tears, urine, saliva, and sweat, sweat has attracted a lot of research interest because it can be generated on-demand at convenient locations on the body and is ideal for continuous monitoring [3].Sweat also contains many other biological biomarkers that could potentially indicate an individual's health status, so sweat analysis allows individuals to monitor their own health, early diagnosis and predictive analysis of disease, and promotes the realization of a real-time, forward-looking, and personalized medical mode [4].However, obtaining information-rich sweat usually requires physical exercise, iontophoretic stimulation, or heat treatment, which not only place higher requirements on the research environment or conditions but also may be physically impossible for some user groups, such as patients and the elderly [5,6].To address this issue, natural-sweat-based sensors that eliminate vigorous sweat stimulation methods have demonstrated immense potential for autonomous monitoring of the patient's physical condition in the resting state [7].More importantly, natural sweat produced at lower sweat rates reduces the change in the content of secreted targets due to stimulation and thus maintains a better sweat and blood correlation [8].While exercise sweat Cu 2+ monitoring has been reported in the literature [2,9,10], the development of more sensitive and user-friendly detection methods for natural sweat Cu 2+  remains an urgent need.In addition, the biofouling of electrodes is a major challenge to accurate electrochemical detection in complex biological liquids (such as serum and urine) [11].Although many targets (e.g., electrolytes, metabolites, amino acids, proteins, and hormones) in sweat are lower in content than in the blood due to the skin barrier, it is still necessary to develop fouling-resistant wearable sensors for continuous sweat analysis to avoid sweat contamination [12,13].
To detect chemical substances in sweat, electrochemical sensors are widely used because of their low cost, ease of miniaturization, and fast response times [14].In recent years, the laser engraving technique has emerged as a versatile and powerful method for the large-scale fabrication of flexible electrodes with customizable designs, and its simple, scalable, mask-free fabrication process makes it crucial for the widespread implementation of wearable sensors [15].Laser-induced graphene (LIG) possesses high electrical conductivity and unique mechanical properties [16].As a revolutionary form of electrochemical analysis, photoelectrochemical (PEC) sensing not only has the advantages of the electrochemical sensors mentioned above but also has higher sensitivity than the traditional electrochemical method due to the separation of the excitation signal and the detection signal [17,18].The laser engraving method has also been used for the preparation of photoelectrodes for sensitive PEC analysis [19][20][21].However, these photoelectrodes were all produced using a 1060 nm CO 2 laser machine, and they were not fully optimized for photoelectric performance or used for sweat analysis.A cheaper and more readily available semiconductor laser has been successfully used to prepare LIG [22,23].Therefore, studying the fabrication of high-performance photoelectrodes using the semiconductor laser on flexible substrates is vital for the development of wearable PEC sweat sensors.Furthermore, the metal/non-metal doping strategy, as an efficient method for improving the photoelectric properties of semiconductor materials [24], is worth introducing into the laser engraving photoelectrode system.
Herein, we propose an antifouling PEC sensor based on a laser-induced graphene and In-doped CdS composite (LIG-In-CdS) for natural sweat Cu 2+ detection (Figure 1).The LIG-In-CdS was produced by engraving a chitosan (CS, polymer and carbon source) membrane containing Cd 2+ (cadmium source), In 3+ (doping element), and cysteine (Cys, sulfur source) modified on a polyimide (PI) film by a 450 nm semiconductor laser system.Compared with the polyethersulfone (PES) polymer commonly used in the preparation of laser-induced photoelectrodes, CS had a better film-forming ability and the photocurrent of the fabricated LIG-CdS photoelectrode was significantly higher than that produced by PES.After the optimization of the sulfur source, the Cys concentration, the doping element, and the concentration ratio of Cd and In, large-scale LIG-In-CdS electrodes with a high photocurrent were prepared for Cu 2+ sensing.On account of the specific binding of Cu 2+ with CdS [1], the photocurrent of the LIG-In-CdS electrodes decreased gradually with the increasing concentration of Cu 2+ and exhibited a wide linear interval of 1.28 ng/mL to 5.12 µg/mL and good selectivity.Moreover, a hydrophilic porous polyvinyl alcohol (PVA) hydrogel was prepared for fingertip natural sweat collection and displayed an outstanding antifouling ability, which is very beneficial for improving the accuracy of the proposed sweat sensor.The natural sweat Cu 2+ detection value obtained by the "touchincubate-detect" protocol fell within the reported physiological level range and was close to that measured in exercise sweat, demonstrating the feasibility of the developed sensor.Additionally, its good reliability was verified by recovery studies conducted in the collected exercise sweat.

Methods
Scanning electron microscopy (SEM) images were collected on a field emission scanning electron microscope (JSM-7100F, JEOL, Japan).Elemental distributions were

Methods
Scanning electron microscopy (SEM) images were collected on a field emission scanning electron microscope (JSM-7100F, JEOL, Japan).Elemental distributions were mapped by using energy dispersive spectroscopy (EDS) as an accessory to SEM at 15 kV.The transmission electron microscopy (TEM) images were recorded by a Tecnai G2 20 S-TWIN transmission electron microscope (FEI, Hillsboro, USA) with an acceleration voltage of 200 kV.An X-ray photoelectron spectra (XPS) scan spectrum was collected on an ESCALAB 250Xi using a monochromatic Al Kα source (Thermo Fisher, Waltham, USA).Powder X-ray diffraction (XRD) patterns were collected on a Bruker D8 Advan X-ray diffractometer (Bruker, Germany).A Raman spectrum was obtained on an inVia Reflex high-resolution confocal Raman microscope equipped with a 532 nm laser (Renishaw, Wotton-under-Edge, UK).The Mott-Schottky test was performed on a CHI 660E analyzer (CH Instruments, Shanghai, China) in 0.1 M PBS (containing 0.1 M Na 2 SO 4 ).All the photocurrent measurements were performed on the CHI 660E analyzer at open circuit potentials in an electrolyte solution of 0.1 M KCl containing 0.5 M TEA by a three-electrode system including a homemade working electrode, a platinum wire counter electrode, and a saturated calomel reference electrode.A multi-functional LED system (~50 mW, 445-465 nm, Tianjin Brillante Technology Co. LTD, Tianjin, China) with an irradiation diameter of 4 mm was used as the light source.

Preparation of LIG-In-CdS
A laser engraving system (DK-5.0,Anqing Kejin Trading Co., Ltd., Anqing, China) with a semiconductor diode laser at a wavelength of 450 nm controlled by a computer was used to fabricate the photoelectrode (Figure S1a).Firstly, a PI film was cleaned using alcohol to remove surface impurities and then attached to a layer of PVC tape, which was engraved to obtain circular hole patterns (2.5 mm in diameter, Figure S1b).Then, 20 mM CdCl 2 •2.5H 2 O and 45 mM Cys were successively dissolved in a homogeneous CS solution (7.5 mg of CS dissolved in 5 mL of acetic acid solution) and the mixed solution was stirred for 1 h.Afterward, 3 µL of the mixture was drop-coated into the circular hole and the modified PI film was then placed into a vacuum oven at 50 • C for 15 min to evaporate the solvent.After peeling off the PVC tape, the PI film modified by the mixture membrane was obtained and then was scanned by the laser engraving machine according to the design pattern (the power of the machine was set at 4 W with a scan speed of 9.4 cm/s) to generate LIG-CdS (Figure S1c,d).Images of the electrodes at different stages of the laser engraving process are shown in Figure S2.For comparison, the 20 mM Cd(acac) 2 -containing PES (0.1555 g of PES dissolved in 5 mL of DMF/DMSO (v/v = 4:1)) and the 20 mM CdCl 2 and Cys-containing PVP (25 mg of PVP dissolved in 5 mL of H 2 O) membranes that are commonly used in the literature for laser engraving were also prepared on the PI film.In addition, to improve the PEC performance of the LIG-CdS electrode, the sulfur source (TAA, thiourea, Cys), the Cys concentration (15 mM, 30 mM, 45 mM, and 60 mM), the doping element (4 mM, Mn, Bi, Mo, Zn, In, P, and B), and the concentration ratio of Cd and In (10:1, 5:1, and 5:2) were optimized.It also should be mentioned that the LIG was directly produced by engraving the PI film and the LIG-In was formed by engraving the In 3+ -containing CS membrane on the PI film.

Construction of a PEC Sensor for Cu 2+ Detection
The obtained LIG-In-CdS/PI array was rinsed repeatedly in water to remove excess materials and cut into single electrodes, which were electrically connected to the Cu wire with silver paste and then sealed with epoxy glue and used as the PEC sensor.For the detection of Cu 2+ , Cu 2+ solution at a concentration ranging between 1.28 ng/mL and 5.12 µg/mL was dropped onto the LIG-In-CdS surface.After incubation for 10 min, the photocurrent responses of LIG-In-CdS/Cu 2+ were recorded.Selectivity tests were performed by comparing the PEC response of the interferents (50 µM UA, 10 µM AA, 5 mM LA, 50 µM DA, 150 µM Glu, 86 ng/mL Mg 2+ , 930 ng/mL Zn 2+ , 1.14 µg/mL Fe 3+ , and 0.36 ng/mL Cd 2+ ) on the sensor with that of 100 ng/mL Cu 2+ .

Fabrication of PVA Hydrogel Patches
The PVA hydrogel was synthesized by following a slightly modified physical mixing and thermal crosslinking reaction method [25].Firstly, 2.0 g of PVA was dissolved in 12 mL of water by stirring at 85 • C for 30 min and 9 g of sucrose was dissolved in 4 mL of water with continuous stirring and heating.Then, the sucrose solution was transferred to the PVA solution and the obtained mixture was continuously stirred at 60 • C for 3 h to form a hydrogel precursor.After cooling to room temperature, 4 g of the precursor was then poured into a Petri dish (3.5 cm in diameter) and placed in an oven at 70 • C to remove excess water and allow for crosslinking until only 2/3 of the weight of the precursor remained.Finally, film-like PVA hydrogels with a diameter of 2.5 mm were produced by a puncher and stored in 0.1 M PBS for subsequent use.

Antifouling Test
The changes in the photocurrent of the PVA-hydrogel-modified sensor before and after incubation with 10 mg/mL BSA or 10 mg/mL Lys solution for 10 min were recorded to evaluate the antifouling property of the hydrogel film.

Detection of Natural Sweat Cu 2+
The on-body sweat test was performed on a healthy subject who had fully consented to the release of the sensing results to the public.A PVA hydrogel patch was placed on the LIG-In-CdS electrode for fingertip sweat collection.Specifically, the index finger was cleaned by a wet tissue and the sweat was allowed to accumulate on the fingertip for 3 min.Then, the subject touched the hydrogel patch for different amounts of time (30 s, 60 s, 90 s, and 120 s).Right after touching the patch, the sensor was put in a humid environment for incubation with Cu 2+ for 10 min and then its photocurrent was recorded.Moreover, we also tested the Cu 2+ concentration in sweat produced by physical exercise (in the same subject) for comparison.

Characterizations of LIG-In-CdS
At present, there are PEC sensors that use laser engraving technology to generate photoelectrodes, which are usually obtained by laser engraving the metal-ion-containing polymer (e.g., PES) modified on the electrode surface using a 1060 nm CO 2 laser [19,26].In addition, PVP and CS are often used as hydrophilic thin-film surface modifications in laser engraving [23,27].Herein, the PEC properties of LIG-CdS produced by using different polymers (PES, PVP, and CS) were compared.As displayed in Figure 2a, the photocurrent response of LIG-CdS (CS) was significantly greater than that of LIG-CdS (PES) and LIG-CdS (PVP).Although we used the same cadmium source (Cd(acac) 2 ) as that reported in the literature, and PES was also used as both a carbon and a sulfur source [19], the photocurrent of the obtained CdS was not satisfactory, probably because the 450 nm laser (~300 USD) used here did not produce as much energy as the 1060 nm laser (~20000 USD, Epilog Laser) used in the reference and because the conversion of carbon precursors to LIG originates from the photothermal effects for the IR laser, the photochemical effects for the UV laser, and the combination of photothermal and photochemical effects for the visible laser [16].Moreover, the laser-induced formation of CdS in LIG should involve the reduction of sulfur at high valence states to S 2− by the carbothermic reaction, which happens at high temperatures [19].CS, as a natural polysaccharide biopolymer with good adhesion, an excellent film-forming ability, good biocompatibility, non-toxicity, and a low cost [28], can not only serve as a carbon source to increase the conductivity of the engraved photoelectrode but can also be used to obtain LIG-CdS with uniform morphology and an excellent photocurrent under a 450 nm laser.Therefore, this simple laser engraving method with low instrumentation requirements has a great deal of potential in the preparation of high-performance flexible photoelectrodes.Then, to obtain a better LIG-CdS electrode, the sulfur source and the sulfur concentration were optimized.The optimal sulfur source was found to be Cys (Figure 2b), and the optimal sulfur concentration was found to be 45 mM (Figure S3a).In addition, metal/non-metal doping has been reported to be a very effective strategy to improve the photocatalytic properties of CdS [29][30][31][32].We added different doped ions to the precursor solution to obtain Mn, Bi, Mo, Zn, In, P, and B-doped CdS, and the highest photocurrent of LIG-In-CdS indicated that In was the optimal doping element (Figure 2c).Further experiments showed that the optimal concentration ratio of Cd and In was 5:1 (Figure S3b).To sum up, the best conditions for laser engraving LIG-In-CdS are: CS as the polymer, 20 mM CdCl 2 as the cadmium source, 45 mM Cys as the sulfur source, and 4 mM In as the doping element.
were optimized.The optimal sulfur source was found to be Cys (Figure 2b), and the optimal sulfur concentration was found to be 45 mM (Figure S3a).In addition, metal/nonmetal doping has been reported to be a very effective strategy to improve the photocatalytic properties of CdS [29][30][31][32].We added different doped ions to the precursor solution to obtain Mn, Bi, Mo, Zn, In, P, and B-doped CdS, and the highest photocurrent of LIG-In-CdS indicated that In was the optimal doping element (Figure 2c).Further experiments showed that the optimal concentration ratio of Cd and In was 5:1 (Figure S3b).To sum up, the best conditions for laser engraving LIG-In-CdS are: CS as the polymer, 20 mM CdCl2 as the cadmium source, 45 mM Cys as the sulfur source, and 4 mM In as the doping element.The surface morphologies of the synthesized electrodes were characterized by SEM and TEM.For the LIG produced by the laser carbonization of PI film at a high temperature [28], its 3D ordered and porous morphology and lamellar structure were visible in the SEM image (Figure S4a) and the TEM image (Figure S4d), respectively.After laser engraving the In 3+ -containing CS membrane, many In-based particles were distributed on the surface of the LIG (Figure S4b,e).The EDS spectrum of the LIG-In electrode (Figure S5a) demonstrated the presence of C, O, and In, which further confirmed the growth of In on the surface of the 3D porous graphene.Similarly, the SEM (Figure S4c), TEM (Figure S4f), and EDS (Figure S5b) images indicated the formation of CdS nanoparticles on the Cd 2+ and Cys-containing CS film.Moreover, as can be seen in the SEM image of LIG-In-CdS (Figure 3a), many nanoparticles existed in the LIG framework; however, the differences between the In-based particles and the CdS nanoparticles could not be identified.Fortunately, LIG-In and LIG-CdS can be clearly observed in the TEM image (Figure 3b).The EDS maps also reveal the successful coupling of LIG-In and LIG-CdS within the LIG-In-CdS (Figure 3c). Figure 3d shows a cross-sectional SEM image of the obtained LIG-In-CdS, revealing the three-layer structure of the PI film, the PI tape, and the LIG-In-CdS.The thickness of the LIG-In-CdS layer was ~60 µm.The surface morphologies of the synthesized electrodes were characterized by SEM and TEM.For the LIG produced by the laser carbonization of PI film at a high temperature [28], its 3D ordered and porous morphology and lamellar structure were visible in the SEM image (Figure S4a) and the TEM image (Figure S4d), respectively.After laser engraving the In 3+ -containing CS membrane, many In-based particles were distributed on the surface of the LIG (Figure S4b,e).The EDS spectrum of the LIG-In electrode (Figure S5a) demonstrated the presence of C, O, and In, which further confirmed the growth of In on the surface of the 3D porous graphene.Similarly, the SEM (Figure S4c), TEM (Figure S4f), and EDS (Figure S5b) images indicated the formation of CdS nanoparticles on the Cd 2+ and Cyscontaining CS film.Moreover, as can be seen in the SEM image of LIG-In-CdS (Figure 3a), many nanoparticles existed in the LIG framework; however, the differences between the In-based particles and the CdS nanoparticles could not be identified.Fortunately, LIG-In and LIG-CdS can be clearly observed in the TEM image (Figure 3b).The EDS maps also reveal the successful coupling of LIG-In and LIG-CdS within the LIG-In-CdS (Figure 3c). Figure 3d shows a cross-sectional SEM image of the obtained LIG-In-CdS, revealing the three-layer structure of the PI film, the PI tape, and the LIG-In-CdS.The thickness of the LIG-In-CdS layer was ~60 µm.
Additionally, XRD was applied to investigate the phase composition of the LIG-In-CdS nanocomposite synthesized by laser induction, as shown in Figure S6a.For comparison, the XRD patterns of LIG and LIG-CdS were also recorded.It is evident that all films exhibited typical diffraction peaks at ~28 • and ~43 • corresponding to the (002) and (100) crystal plane reflections of graphene [22].However, no significant In-and CdS-related reflection peaks were observed in the LIG-In-CdS pattern, which may be ascribed to the CdS and In-CdS films being very thin [33].Raman spectroscopy was used to verify the generation of CdS in the laser-induced LIG-In-CdS (Figure 4a), which not only showed three characteristic peaks of graphene at 1350 cm −1 (D band), 1585 cm −1 (G band), and 2700 cm −1 (2D band) but also possessed another two distinguishable bands of CdS located at around 295 cm −1 (1LO) and 594 cm −1 (2LO) [26].Considering that the existing form of In has yet to be characterized, we resorted to XPS to study the valence state of elements in the as-synthesized In-CdS film.From the full-survey XPS spectra of LIG-In, LIG-CdS, and LIG-In-CdS (Figure S6b), the peaks of C 1s, O 1s, Cd 2d, S 2s, S 2p, and In 3d can be observed in the corresponding spectrum, confirming the presence of In in the In-CdS film.Peaks for In 3d 5/2 and In 3d 3/2 were observed at 444 eV and 451.5 eV (Figure 4b), respectively, and their energy difference was 7.5 eV, indicating that the In was in the +3 oxidation state and perhaps existed in the form of indium oxide [34,35].
Moreover, the appearance of S 2p 1/2 and S 2p 3/2 peaks of S 2− in the S 2p scan spectrum (Figure 4c) and the emergence of a S 2− peak at ca. 225 eV in the S 2s spectrum (Figure 4d), coupled with the coexistence of the Cd 3d peak (Figure 4e), demonstrated the successful generation of CdS [19].In addition, the positive slope of the Mott-Schottky curve (Figure 4f) revealed that the LIG-In-CdS composite was an n-type semiconductor [36].Additionally, XRD was applied to investigate the phase composition of the LIG-In-CdS nanocomposite synthesized by laser induction, as shown in Figure S6a.For comparison, the XRD patterns of LIG and LIG-CdS were also recorded.It is evident that all films exhibited typical diffraction peaks at ~28° and ~43° corresponding to the (002) and (100) crystal plane reflections of graphene [22].However, no significant In-and CdSrelated reflection peaks were observed in the LIG-In-CdS pattern, which may be ascribed to the CdS and In-CdS films being very thin [33].Raman spectroscopy was used to verify the generation of CdS in the laser-induced LIG-In-CdS (Figure 4a), which not only showed three characteristic peaks of graphene at 1350 cm −1 (D band), 1585 cm −1 (G band), and 2700 cm −1 (2D band) but also possessed another two distinguishable bands of CdS located at around 295 cm −1 (1LO) and 594 cm −1 (2LO) [26].Considering that the existing form of In has yet to be characterized, we resorted to XPS to study the valence state of elements in the as-synthesized In-CdS film.From the full-survey XPS spectra of LIG-In, LIG-CdS, and LIG-In-CdS (Figure S6b), the peaks of C 1s, O 1s, Cd 2d, S 2s, S 2p, and In 3d can be observed in the corresponding spectrum, confirming the presence of In in the In-CdS film.Peaks for In 3d5/2 and In 3d3/2 were observed at 444 eV and 451.5 eV (Figure 4b), respectively, and their energy difference was 7.5 eV, indicating that the In was in the +3 oxidation state and perhaps existed in the form of indium oxide [34,35].Moreover, the appearance of S 2p1/2 and S 2p3/2 peaks of S 2− in the S 2p scan spectrum (Figure 4c) and the emergence of a S 2− peak at ca. 225 eV in the S 2s spectrum (Figure 4d), coupled with the coexistence of the Cd 3d peak (Figure 4e), demonstrated the successful generation of CdS [19].In addition, the positive slope of the Mott-Schottky curve (Figure 4f) revealed that the LIG-In-CdS composite was an n-type semiconductor [36].

PEC Sensor Determination of Cu 2+
To track the stepwise modification process of the PEC sensor for Cu 2+ detection, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) characterization tests were carried out in 0.1 M KCl solution containing 5.0 mM [Fe(CN) 6 ] 3−/4− .As indicated in Figure 5a, the LIG electrode showed well-defined redox peaks of the [Fe(CN) 6 ] 3−/4− marker, demonstrating the good electron transfer capability of the laser-induced graphene.The redox peak currents increased and the peak-to-peak potential difference decreased as well after the modification of the LIG-In-CdS.This was because the LIG was formed by directly engraving the PI film, and the CS used to prepare the LIG-In-CdS contained many carbon atoms, which increased the conductivity and surface area of the LIG electrode after the laser sculpturing process [28].Many studies have shown that the introduction of Cu 2+ onto the CdS surface can lead to the formation of Cu x S (x = 1, 2) since the solubility of CuS (Ksp = 6.3 × 10 −36 ) or Cu 2 S (Ksp = 2.5 × 10 −48 ) is much poorer than that of CdS (Ksp = 8 × 10 −27 ) [37,38].The Cu x S may act as a deep-level trap to facilitate the recombination of photoinduced carriers [1], so the current response of the LIG-In-CdS decreased after the absorption of Cu 2+ .The corresponding EIS test results (Figure 5b) were consistent with these CV results.In addition, the EDS spectrum shown in Figure 5c proved the existence of Cu on the LIG-In-CdS electrode (Figure S7).Hence, these results confirm that the PEC Cu 2+ sensor was successfully fabricated.Then, the analytical performance of the developed method was measured in the presence of different concentrations of Cu 2+ , and the photocurrent density obtained at the open circuit potential decreased gradually as the Cu 2+ concentration increased (Figure 5d).The photocurrent difference (∆I) between the background and the Cu 2+ showed a good linear relationship with the logarithm of the Cu 2+ concentration (lgC) from 1.28 ng/mL to 5.12 µg/mL with a correlation equation of ∆I = −0.314+ 2.52 lgC (ng/mL) (Figure 5e).To achieve accurate detection of sweat Cu 2+ , AA, UA, LA, DA, Glu, Mg 2+ , Zn 2+ , Fe 3+ , and Cd 2+ , which are some of the interfering molecules and ions often present in sweat, were selected as interferents to examine the selectivity of the sensor.The obtained result (Figure 5e) reveals that the LIG-In-CdS electrode has a good anti-interference capability and is suitable for the detection of Cu 2+ in real sweat samples.

Antifouling Test and Natural Sweat Cu 2+ Detection
Sweat is a complex biological fluid that may cause photoelectrode passivation and thus affect the test results.The PVA hydrogel used in this study for natural sweat collection has been reported to have antifouling properties [39].Therefore, we coated the

Antifouling Test and Natural Sweat Cu 2+ Detection
Sweat is a complex biological fluid that may cause photoelectrode passivation and thus affect the test results.The PVA hydrogel used in this study for natural sweat collection has been reported to have antifouling properties [39].Therefore, we coated the LIG-In-CdS surface with the prepared hydrogel and dropped H 2 O droplets onto it, as illustrated in the inset of Figure 6a.The droplets were quasi-spherical on the LIG-In-CdS surface, while they tended to be flattened on the LIG-In-CdS/hydrogel substrate, which proved that the hydrogel had excellent hydrophilicity [40].Additionally, the PVA hydrogel had little influence on the detection of Cu 2+ by the fabricated sensor (Figure 6a) because Cu 2+ can pass through the hydrogel and react with the LIG-In-CdS underneath.Then, we evaluated the antifouling ability of the hydrogel using a high concentration of proteins.The negligible photocurrent signal changes after incubation with 10 mg/mL BSA or 10 mg/mL Lys confirmed its outstanding antifouling performance (Figure 6b).In contrast, in the absence of the hydrogel, BSA and Lys had a significant influence on the photocurrent of the sensor.A tightly bound hydration layer can form on the hydrophilic PVA hydrogel when it is in contact with water, which acts as a physical and energetic barrier that resists the adsorption of fouling molecules [41].This may account for the good fouling resistance of the hydrogel.Fingers are suitable sites for natural sweat collection due to the high density of eccrine sweat glands (~400 glands cm −2 ) and the high sweat rates [8].Moreover, the accurate detection of Cu 2+ in human sweat is extremely critical for on-site and noninvasive assessment of Cu 2+ abnormalities, because these abnormalities are closely associated with hematological, kidney, and cardiovascular diseases and Wilson's disease [9].Therefore, a touch-based fingertip sweat Cu 2+ sensor was developed.Its sensing process consists of three steps: the collection of sweat by touching the PVA hydrogel membrane (covering the LIG-In-CdS sensor) (inset of Figure 6c); the reaction between the sweat Cu 2+ and the LIG-In-CdS; and the detection of the photocurrent of the LIG-In-CdS/CuxS.As indicated Fingers are suitable sites for natural sweat collection due to the high density of eccrine sweat glands (~400 glands cm −2 ) and the high sweat rates [8].Moreover, the accurate detection of Cu 2+ in human sweat is extremely critical for on-site and noninvasive assessment of Cu 2+ abnormalities, because these abnormalities are closely associated with hematological, kidney, and cardiovascular diseases and Wilson's disease [9].Therefore, a touch-based fingertip sweat Cu 2+ sensor was developed.Its sensing process consists of three steps: the collection of sweat by touching the PVA hydrogel membrane (covering the LIG-In-CdS sensor) (inset of Figure 6c); the reaction between the sweat Cu 2+ and the LIG-In-CdS; and the detection of the photocurrent of the LIG-In-CdS/Cu x S. As indicated in Figure 6c, the ∆I steadily increased upon increasing the touching time from 30 to 90 s and leveled off with longer touching times.Thus, the optimal touching time was determined to be 90 s.This short collection time is due to the ability of the hydrophilic porous PVA hydrogel to collect sweat [42,43].The Cu 2+ content in the touch sweat calculated from the photocurrent test results (Figure 6d) was 197 ng/mL, which is in agreement with the physiological values in sweat (100-1000 µg/L) reported in the literature [10].Further, we also collected exercise sweat from the same subject and measured its Cu 2+ concentration.The obtained result of 222 ng/mL was close to that in the natural touch sweat.Finally, a standard addition method was applied for exercise sweat Cu 2+ quantification, as reported in Table 1.The good reliability of the developed method was successfully validated by the 98.1-102.9%recovery rates.

Conclusions
In this study, we successfully developed a PEC sensor for natural sweat Cu 2+ monitoring without external stimulation of sweating.This sensor was constructed by integrating an LIG-In-CdS photoelectrode and a hydrophilic PVA hydrogel, which were prepared by a powerful laser engraving technique and a simple physical mixing and thermal crosslinking reaction method, respectively.Moreover, the laser engraving conditions, including the sulfur source, the Cys concentration, the doping element, and the concentration ratio of Cd and In, were optimized.As a result, the optimally fabricated LIG-In-CdS exhibited a high photocurrent and achieved highly sensitive and selective detection of Cu 2+ .Additionally, the PVA hydrogel displayed good antifouling activity and collected a sufficient amount of fingertip sweat in a short time.In addition, based on the "touch-incubate-detect" protocol, the fabricated sensor showed feasibility and good reliability for sweat Cu 2+ analysis.The speed and simplicity of the touch-based fingertip sweat assay, and the elimination of sweat fouling, should lead to frequent self-testing of physiological Cu 2+ levels and enhanced patient compliance, resulting in improved management of related diseases.To this end, further studies should be devoted to combining the constructed PEC sensor with near-field communication to achieve battery-free and wireless PEC sensing.

Figure 1 .
Figure 1.Schematic illustration of the laser engraving fabrication process of a PEC sensor for natural sweat Cu 2+ detection.

Figure 1 .
Figure 1.Schematic illustration of the laser engraving fabrication process of a PEC sensor for natural sweat Cu 2+ detection.

Figure 2 .
Figure 2. Comparison of the photocurrents of CdS produced by using different polymers (a) or different sulfur sources (b, CS as the polymer) in laser engraving.(c) Optimization of doped elements for laser-induced CdS photoelectrodes (CS as the polymer, Cys as the sulfur source).The cadmium source for PES was 20 mM Cd(acac)2; PES itself acted as the sulfur source.All other cadmium sources were 20 mM CdCl2 and all other sulfur sources had a concentration of 30 mM.

Figure 2 .
Figure 2. Comparison of the photocurrents of CdS produced by using different polymers (a) or different sulfur sources (b, CS as the polymer) in laser engraving.(c) Optimization of doped elements for laser-induced CdS photoelectrodes (CS as the polymer, Cys as the sulfur source).The cadmium source for PES was 20 mM Cd(acac) 2 ; PES itself acted as the sulfur source.All other cadmium sources were 20 mM CdCl 2 and all other sulfur sources had a concentration of 30 mM.

Chemosensors 2022 , 13 Figure 3 .
Figure 3. SEM images of (a) the surface and (d) the cross-section; (b) a TEM image; and (c) elemental (C, O, S, Cd, and In) EDS mapping images of the fabricated LIG-In-CdS electrode.The inset of (a) is the enlarged image.

Figure 3 . 13 Figure 4 .
Figure 3. SEM images of (a) the surface and (d) the cross-section; (b) a TEM image; and (c) elemental (C, O, S, Cd, and In) EDS mapping images of the fabricated LIG-In-CdS electrode.The inset of (a) is the enlarged image.Chemosensors 2022, 10, x FOR PEER REVIEW 8 of 13

13 Figure 6 .
Figure 6.(a) Comparison of the results of detection of 100 ng/mL Cu 2+ by LIG-In-CdS before and after incubation with hydrogel.Inset: photos of the PVA hydrogel and behaviors of two H2O droplets on the LIG-In-CdS and LIG-In-CdS/hydrogel substrates.(b) Comparison of the photocurrent responses of the LIG-In-CdS and LIG-In-CdS/hydrogel substrates before (black curve) and after being incubated with 10 mg/mL BSA (red curve) or 10 mg/mL Lys (blue curve) for 10 min.(c) Optimization of the touching time for the fingertip on the LIG-In-CdS/hydrogel substrate in the Cu 2+ sensing procedure.The inset is the corresponding picture of the fingertip touching the substrate.(d) The photocurrent responses of the LIG-In-CdS/hydrogel substrate to Cu 2+ in touch sweat and exercise sweat.

Figure 6 .
Figure 6.(a) Comparison of the results of detection of 100 ng/mL Cu 2+ by LIG-In-CdS before and after incubation with hydrogel.Inset: photos of the PVA hydrogel and behaviors of two H 2 O droplets on the LIG-In-CdS and LIG-In-CdS/hydrogel substrates.(b) Comparison of the photocurrent responses of the LIG-In-CdS and LIG-In-CdS/hydrogel substrates before (black curve) and after being incubated with 10 mg/mL BSA (red curve) or 10 mg/mL Lys (blue curve) for 10 min.(c) Optimization of the touching time for the fingertip on the LIG-In-CdS/hydrogel substrate in the Cu 2+ sensing procedure.The inset is the corresponding picture of the fingertip touching the substrate.(d) The photocurrent responses of the LIG-In-CdS/hydrogel substrate to Cu 2+ in touch sweat and exercise sweat.

:
Photo of the laser engraving machine; Figure S2: Images of the laser engraving process for the preparation of LIG-In-CdS; Figure S3: Optimization of the Cys concentration and the concentration ratio of Cd and In; Figure S4: SEM and TEM images of laser-induced electrodes; Figure S5: Elemental EDS mapping images of LIG-In and LIG-CdS; Figure S6: XRD patterns and XPS survey spectra of laser-induced electrodes; and Figure S7: Elemental EDS mapping images of LIG-In-CdS/Cu 2+ .

Table 1 .
Recovery experiment on Cu 2+ in exercise sweat samples.