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. 2025 Jul 8;10(28):30944–30957. doi: 10.1021/acsomega.5c03662

Sustainable Paper-Derived Laser-Induced Graphene Electrochemical Platform for Ultra-Sensitive Diazepam Detection in Forensic Investigations

Kasrin Saisahas †,‡, Asamee Soleh †,‡, Kritsada Samoson †,‡, Kiattisak Promsuwan †,‡,§, Jenjira Saichanapan †,‡, Sangay Wangchuk §,∥,⊥,#, Warakorn Limbut †,‡,§,⊥,*
PMCID: PMC12290644  PMID: 40727721

Abstract

The use of benzodiazepines like diazepam (DZ) in drug-facilitated crimes necessitates the development of rapid, sensitive and portable detection methods. This study presents a sustainable, paper-derived laser-induced graphene (paper-LIG) electrochemical platform for ultrasensitive DZ detection in forensic investigations. The sensor was fabricated using a cellulose paper substrate treated with sodium tetraborate to enhance thermal stability, enabling efficient graphene conversion via optimized laser scribing. The physicochemical properties of paper-LIG were characterized using field emission scanning electron microscopy, energy-dispersive X-ray spectrometry, Raman spectroscopy, Fourier-transform infrared spectrometry, and X-ray photoelectron spectroscopy. Additionally, the effects of working and auxiliary electrode sizes on electrochemical performance were evaluated. The resulting paper-LIG electrode exhibited excellent electrochemical performance, enabling direct DZ detection in beverage samples without complex pretreatment. Using an optimized voltammetric technique, the sensor achieved a wide linear range (1–1000 μmol L–1) and a low detection limit (0.4 μmol L–1). This platform demonstrated high selectivity, reproducibility (RSD 2.2%), and recovery (96.4–102.5%), confirming its reliability for forensic applications. Its low cost, scalability, and eco-friendliness make this sensor a promising tool for rapid on-site drug screening.

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1. Introduction

Electrochemical sensors are cost-effective, straightforward, and highly sensitive analytical tools, known for their excellent repeatability and potential for miniaturization. Graphene has emerged as a leading electrode material, owing to its exceptional properties and widespread application in the detection of analytes relevant to food safety, pharmaceuticals, biomedicine, environmental monitoring, and forensic science. Various graphene synthesis methods, such as exfoliation, chemical vapor deposition, and chemical-based synthesis, have been explored, but despite their effectiveness, these methods are often complex, resource-intensive, and hinder the scalability and broader practical use of graphene in real-world electrochemical applications.

In 2014, researchers expanded the field of graphene-based materials by demonstrating that CO2 laser irradiation on flexible polyimide could produce a conductive, porous graphene material known as laser-induced graphene (LIG). This breakthrough offered a simple method of producing conductive paths and electrode configurations on flexible substrates, unlocking new applications in actuators, sensors, and energy storage devices. − Recently, LIG structures have been fabricated on synthetic polymers, wood, cloth, xylan, leaves, mushroom biomass, cork, food, lignin- and cellulose-based biopolymers, phenolic resins, and paper. Biobased substrates, particularly cellulose, have received special attention, driving the development of paper-based LIG devices. With rising concerns about resource scarcity and e-waste pollution, interest in eco-friendly, flexible devices made from renewable resources has intensified. , Paper from cellulose stands out as a promising material for use in cost-effective, disposable electronic devices.

The first paper-based LIG sensor was scribed by CO2 laser on paperboard, producing a highly porous 3D graphene that was suitable for diverse electroanalytical applications. Ataide et al. used a CO2 laser on graphite-drawn office paper to fabricate paper-based electrochemical analytical devices, achieving high sensitivity for furosemide detection. Edberg et al. screen-printed cellulose and lignin inks on flexible substrates, while Claro et al. used nanocellulose for LIG-based conductive inks. Kulyk et al. synthesized paper-LIG devices using xylan biopolymers on filter papers for low-cost, eco-friendly sensors. To ensure stable, reproducible conductive tracks on thin paper substrates, the use of flame-retardant treatments is essential, allowing localized graphitization without burning. Pinheiro et al. used flame retardants to help produce robust LIG patterns for wearable electronics, incorporating wax-printing to enhance graphitization and enable low-resistance tracks. These innovations demonstrated the capacity of LIG fabrication techniques to produce sustainable, paper-based analytical devices through simple laser treatment of high-density papers or flame retardant-treated fibers. These studies also demonstrated that the large surface area and electron transfer kinetics of paper-based LIG electrodes increased sensor sensitivity and lowered detection limits for quantitative analysis.

Furthermore, Wirojsaengthong et al. demonstrated that, compared to larger LIG electrodes, smaller LIG electrodes on polyimide exhibited a higher electroactive surface area, rougher surface texture, greater proportion of edge planes to basal planes, and increased concentration of oxygen-containing functional groups. These features enhance the electroactive area, electron transfer kinetics and electrochemical reversibility. Consequently, systematic electrochemical studies of miniaturized paper-LIG electrodes are essential, as electrode size and shape may impact electrochemical properties due to surface micro/nanostructure, confined mass transfer, surface area, and chemical composition. ,

In this study, we fabricated laser-induced graphene (LIG) from chromatography paper to produce a three-electrode platform, and investigated the effects of paper-LIG electrode diameter on electrochemical performance. The paper-LIG electrode was utilized to detect and quantify diazepam (DZ), a benzodiazepine drug that has a powerful impact on the nervous system, especially when combined with alcohol, when it can lead to memory loss, hallucinations, and cognitive impairment. Due to these effects, DZ has been misused in criminal activities, emphasizing the need for effective detection methods, particularly in cases involving drunk or sedated victims. , Portable detection of DZ in beverages could directly support law enforcement efforts at crime scenes, offering a highly valuable tool for forensic analysis. The synthesis of paper-LIG and the electrochemical measurements developed here are both simple and rapid, while the sensors themselves are low-cost, disposable, and environmentally friendly, making them well-suited for real-world applications in sustainable electrochemical sensing.

2. Experimental Section

2.1. Materials and Chemicals

During this work, ultrapure Milli-Q water laboratory grade (resistivity of 18.2 MΩ·cm) was used to prepare all solutions. Sodium tetraborate decahydrate, potassium chloride, potassium hexacyanoferrate­(III) (K3[Fe­(CN)6]), potassium hexacyanoferrate­(II) trihydrate ((K4[Fe­(CN)6])·3H2O), and diazepam were purchased from Sigma. The morphological and surface properties of paper-LIG were characterized by using scanning electron microscopy coupled with energy-dispersive X-ray spectrometry (SEM/EDX, Apreo, FEI), Fourier-transform infrared spectrometry (FTIR, EQUINOX 55, Bruker, Raman spectrometry (RAMANforce, Nanophoton, Japan)), and X-ray photoelectron spectroscopy (XPS, AXIS Ultra DLD, Kratos Analytical Ltd.). All experiments were performed at ambient temperature.

2.2. Fabrication of Paper-Laser-Induced Graphene (Paper-LIG)

Chromatography paper sheets were first cut to A4 size (297 mm × 210 mm) and chemically treated by soaking in a 0.1 mol L–1 sodium borate solution for 10 min, then air-dried at room temperature overnight. After treatment, the sheets underwent wax printing with a Xerox ColorCube wax printer, followed by heating with a hot air dryer to induce hydrophobicity throughout the paper. Graphene was induced by a computer-controlled 100 W continuous wave CO2 laser engraver (Yu Ze Laser Equipment Co., Ltd.) with a wavelength of 10.6 μm and a beam diameter of 100 μm. The effects of different laser powers and scan speeds were assessed to optimize the graphitization of the paper while avoiding fiber ablation or incomplete LIG formation. The fabrication processes are illustrated in Figure .

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Schematic overview of the paper-LIG electrode fabrication process.

The electrode pattern was designed with the Inkscape software. The working electrode (WE) was designed in a range of diameters, including 0.5, 1.0, 2.0, and 3.0 mm. The auxiliary electrode (AE) was designed in a range of sizes from 0.5 to 3.0 mm2.

2.3. Electrical Characterization of LIG

A four-point probe enabled the collection of sheet resistance (SR) data of paper-LIG electrodes fabricated in different laser operating conditions. The reported values represented the average resistance measurements obtained by maintaining probe contact with the sample for 10 s. Averaging the measurements addressed variations resulting from the laser process that caused differences between paper-LIG electrodes (n = 3) and distinct bands (n = 3) within the same paper-LIG electrode.

2.4. Electrochemical Experimental

Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements were conducted using a potentiostat (EmStat4S, PalmSens, Netherlands) operated through the PSTrace software. All measurements were conducted using fabricated paper-LIG electrodes that comprised a WE, AE, and reference electrode (RE). A 40 μL test solution was applied to fully cover all three electrodes, and CV experiments were scanned vs a LIG pseudoreference electrode. For comparison, electrochemical measurements were also performed on a commercial screen-printed graphene electrode (WE 3.0 mm diameter). EIS measurements were recorded over a frequency range of 0.05 Hz to 50 kHz, with 50 frequency points, an E dc of 0.25 V, and an E ac of 0.01 in 5.0 mmol L–1 [Fe­(CN)6]3–/4– containing 0.1 mol L–1 KCl solution.

The electrochemical measurement of DZ was performed with CV, linear sweep voltammetry (LSV), square wave voltammetry (SWV), and differential pulse voltammetry (DPV), using 0.1 mol L–1 BR buffer solutions at pH 4.00. The potential window used ranged from −0.50 to −1.50 V. The peak potential and peak current for reduction processes were used to assess the electrochemical behavior of DZ. The mass transport properties of DZ were investigated by changing the scan rate from 10 to 200 mV s–1 within a potential window of −0.50 to −1.504 V, using 1.0 mmol L–1 DZ in the presence of 0.1 mol L–1 BR (pH = 4.00).

2.5. Optimization

To achieve the optimal analytical performance with the paper-LIG sensor, several key operating parameters were systematically optimized. These parameters included the sample pH (1.00, 2.00, 3.00, 4.00, 5.00, 6.00, 7.00 and 8.00), LSV parameters of potential step and scan rate, SWV parameters of frequency, amplitude and potential step, and DPV parameters of pulse potential, pulse time, step potential and scan rate. The optimizations were conducted by changing one parameter at a time while keeping all other parameters constant. The optimal sensor condition was determined based on the highest current response observed for the detection of 1.0 mmol L–1 DZ at each setting.

2.6. Real Sample Analysis

2.6.1. Simulation of Drug Misuse Scenario

Date rape drugs are increasingly used to facilitate sexual assault due to their discrete characteristics, which enable them to be easily administered in beverages or directly to victims. Although drug-facilitated sexual assault (DFSA) is a significant concern, its incidence is challenging to quantify due to underreporting, lack of timely medical attention, and misdiagnosisespecially when alcohol is involved, which can mask the symptoms. ,

Therefore, in this study, a drug misuse scenario was simulated by adding 5 mg DZ pill to beverage samples that comprised alcoholic and nonalcoholic products available in supermarkets. The products included Smirnoff Ice (5% alcohol, 275 mL), Chum Churum (16.5% alcohol, 360 mL), Full Moon white (5% alcohol, 275 mL), Malee coco (350 mL) and Ichitan Genmaicha (500 mL). The sample was prepared for analysis using the standard addition method (SAM). The sample was diluted 5-fold, and known concentrations of DZ (10, 20, 30, and 40 μmol L–1) were added to aliquots of the sample. Each mixture was adjusted to a final volume of 1 mL using BR buffer (pH 4.00) and manually shaken to ensure homogeneity. A 30 μL aliquot of the prepared sample was then transferred to the detection zone of the paper-LIG electrode.

3. Results and Discussion

3.1. Optimization of the Paper-LIG Fabrication

Without pretreatment, paper substrates are unsuitable for laser scribing since their cellulose content burns at around 230 °C. Since laser exposure can generate temperatures exceeding 2000 °C, untreated paper will burn even at low laser power. Inorganic salt additives like phosphates and borate derivatives can be used to increase the thermal stability of paper. , In this study, Whatman chromatography paper was treated by immersion in a 0.1 mol L–1 sodium tetraborate (borax) solution for 10 min and then air-dried overnight. Wax printing technique was also used to create a hydrophobic barrier for the electrode platform. The importance of this pretreatment step is demonstrated in Figure .

Laser power and scan speed critically affect both the yield of cellulose graphitization and the structural integrity of the paper-LIG electrode. The laser power was varied from 5 to 10% of the full power (103.2 W), while the laser scan speed was increased from 10 to 100 mms–1 in increments of 10 mms–1. The total energy delivered per surface area in each condition was calculated and a heatmap was constructed (Figure ). The sheet resistance (SR) of each resulting paper-LIG electrode was measured to evaluate the synergistic effect of laser power and scan speed on the graphitization yield of cellulose paper. Figure displays the common logarithm (base 10) of the SR values, color-coded for better visualization. At lower scan speeds, SR typically decreases due to longer exposure times, which facilitates more effective conversion of cellulose into graphene. However, excessively low scan speeds can result in laser etching through the paper substrate, leading to impaired films (Figure S1). Conversely, if the scan speed is too fast, the conversion of cellulose into graphene becomes ineffective, as the laser exposure time is insufficient for full conversion (Figure S1). Regarding laser power, an increase in power generally leads to a decrease in SR, as higher laser intensity promotes better graphitization. However, using excessively high laser power can also impair the film, as the substrate may be damaged due to the excessive intensity (Figure S1). The optimal condition for converting cellulose into graphene films with the lowest resistivity was a laser scan speed of 70 mms–1 and a laser power of 9%, indicated by the dark red square in Figure .

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Multifactorial optimization of the laser induction of graphene from paper presented as a heatmap. Color bar, LogSR: log10­(x), where x is the sheet resistance value in Ω sq–1; IF = impaired film due to high laser intensity; NE = not effective.

3.2. Physicochemical Characterizations of Paper-LIG

To better understand the properties and composition of paper-LIG, morphological, structural, and chemical characterizations were conducted on paper-LIG prepared at 9.0 W and 70 mms–1. The surface morphology of paper-LIG was characterized using SEM imaging. Figure a displays the surface of the paper substrate, revealing the entangled network of cellulose fibers compacted into a structure with a randomly distributed pore pattern. After borax treatment, as shown in Figure S2a, the structure of the cellulose fibers remains largely unchanged, suggesting that borax does not affect the surface morphology. Following wax printing, a thin wax layer infiltrates the porous structure, partially filling the gaps between cellulose fibers and forming a hydrophobic barrier, as illustrated in Figure S2b. After the laser scribing process, the cellulose fibers underwent carbonization, transforming into a highly porous, interconnected graphene network as shown in Figure b. The breakdown of the compact fiber network resulted in a highly porous, three-dimensional framework (Figure c).

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SEM images of (a) cellulose paper (b) paper-LIG surface (c) high-magnification SEM image of the paper-LIG surface. (d) The Raman spectrum is of paper-LIG. (e) The surface of paper-LIG mapped by EDS. (f) FT-IR spectra are of (i) paper, (ii) borax treated-paper, (iii) wax-borax treated-paper, and (iv) paper-LIG. XPS analysis produced (g) a survey spectrum and high-resolution spectra of (h) C 1s and (i) B 1s.

This structural evolution significantly increased surface area and conductivity, facilitating improved electrochemical performances. Raman spectroscopy was used to confirm the conversion of cellulose into graphene. Figure d displays a Raman spectrum of paper-LIG, that exhibits three characteristics of graphene: a symmetrical D band at 1344 cm–1, a G band at 1579 cm–1 and a 2D band at 2681 cm–1. The intensity ratio of I D/I G reflects the degree of structural disorder in the sp2 carbon lattice, which scales with defect concentration. Paper-LIG exhibited an I D/I G ratio ≈ 1.0, suggesting a high defect density. These defects are useful for electrochemical sensing because they enhance electron transport by increasing the local density of states, provided that conductivity is maintained.

Figure e presents the EDS mapping results of paper-LIG, revealing the distribution of C, O, and B elements. The presence of B atoms originates from the borax treatment. During the laser scribing process, B atoms could incorporate into the LIG structure, potentially influencing its chemical composition and electrochemical properties for the better.

FT-IR analysis was performed to examine functional groups at each stage of the paper-LIG preparation. As shown in Figure f, all stages exhibited characteristic cellulose peaks (highlighted in purple) at ∼3300, ∼290, and 1000–1200 cm–1, corresponding respectively to O–H stretching, C–H stretching, and C–O–C vibrations in the polysaccharide structure. The FT-IR spectrum of borax-treated paper (blue line) remained largely unchanged compared with the original paper substrate (green line), indicating that borax treatment did not significantly alter the cellulose backbone. However, slight peak variations suggest possible interactions with hydroxyl groups. Wax printing introduced paraffin-specific peaks (highlighted in yellow) at 1463, 2850, and 2918 cm–1, corresponding to C–H stretching, and bending of −CH3 and −CH2 groups. Peaks in the 1500–1750 cm–1 range (highlighted in green) were attributed to CC aromatic ring stretching from aromatic components and dye. After the photothermal reactions that took place during the laser scribing process, the above-mentioned functional groups were no longer detected, while new functional groups of paper-LIG (black line) were indicated by the band between ∼1315 and ∼990 cm–1 (highlighted in red). The new functional groups imply the incorporation of B into the LIG framework, , that could potentially enhance its electrochemical properties.

The incorporation of B into the LIG structure was confirmed by XPS, which was used to was further characterize the surface chemical composition of paper-LIG. The survey spectrum (Figure g) showed the presence of C 1s (284 eV), B 1s (188 eV), and O 1s (532 eV). The high-resolution C 1s spectrum (Figure h) revealed six peaks: C–C (283.4 eV), sp2 carbon (284.3 eV), sp3 (285.5 eV), C–O/C–B (286.8 eV), CO (287.8 eV), and O–CO (288.9 eV). The high-resolution B 1s spectrum (Figure i) exhibited two distinct peaks at approximately 191.9 and 192.8 eV, which were assigned to B–C and B–O bonds, respectively. The incorporation of B confirmed by these results potentially improved the chemical and electrochemical properties of paper-LIG. Specifically, B-doping modifies charge distribution and improves electrocatalytic activity, making B-doped paper-LIG a promising material for electrochemical applications.

3.3. Dimensional Influence on Electrochemical Properties of Paper-LIG

3.3.1. Working Electrode Area

The electrochemical performances of paper-LIG electrodes are influenced by the size of the WE, which affects the electroactive surface area and electron transfer kinetics, ultimately impacting electrochemical sensing. Understanding these dimensional influences is crucial for optimizing sensor performance. In this study, a paper-LIG three-electrode platform was fabricated by laser scribing the WE, AE, RE and all connectors in a cellulose paper substrate to investigate the effects of electrode dimensions on electrode properties. Figure a displays photographs of paper-LIG electrodes with WEs 0.5, 1.0, 2.0, and 3.0 mm in diameter. The photographs show a distinct visual transformation of the wax-coated paper substrate from purple to black, indicating the successful conversion of cellulose to LIG. The magnified images reveal that the paper-LIG electrodes with a WE diameter of 0.5 mm were not uniform, particularly at the edges. Due to the relatively large laser spot used (∼0.1 mm), and the inherent limitation of the instrument, some areas may have received less photothermal energy than others during scribing. Furthermore, upon examining the magnified images of the WEs, their diameters were found to be 0.86, 1.32, 2.46, and 3.46 mm rather than 0.5, 1.0, 2.0, and 3.0 mm. Laser-induced expansion of the material resulted in larger WEs than intended. The changes in surface area and conductivity of the electrode could potentially affect its electrochemical behavior.

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(a) Optical photographs show all four paper-LIG three-electrode platforms constructed from a cellulose paper substrate. The working electrode diameters of the tested platforms were 0.86, 1.32, 2.46, and 3.46 mm. (b) Cyclic voltammograms of 5.0 mmol L–1 [Fe­(CN)6]3–/4– in 0.1 mol L–1 KCl produced at all four electrodes at a scan rate of 50 mV s–1; the inset shows the current density of each paper-LIG electrode. The effect of CV scan rate on the redox reaction was investigated from 10 to 200 mV s–1 at paper-LIG working electrodes of (c) 0.86 mm, (d) 1.43 mm, (e) 2.46 mm, and (f) 3.46 mm diameters. (g) Electroactive area ratios of the four paper-LIG electrodes produced at the four paper-LIG electrodes by 5.0 mmol L–1 [Fe­(CN)6]3–/4– at 40 mV s–1. (h) The peak to peak separation (ΔE p) vs scan rate from all paper-LIG electrodes.

The electrochemical behaviors of different paper-LIG electrodes were studied in a 5.0 mmol L–1 [Fe­(CN)6]3–/4– redox couple containing 0.1 mol L–1 KCl, using CV. Figure b shows how the anodic and cathodic peak current responses increased with increments in WE size, corresponding to the increasing electrode surface area. However, the current density, based on the geometrical surface area, was higher at the smaller WEs, as shown in the inset of Figure b. To better understand the surface properties of paper-LIG electrodes, the electroactive surface area (EASA) ratio was evaluated by comparing the EASA value with the geometrical surface area (A g). A g was determined using the electrode diameter (d) based on the equation A g = 0.25πd 2. The calculated A g values for WE diameters of 0.86, 1.32, 2.46, and 3.46 mm were 0.0058, 0.0137, 0.0475, and 0.0940 mm2, respectively. The ECSA of each paper-LIG electrode was calculated by varying the CV scan rate from 10 to 200 mV s–1 in 5.0 mmol L–1 [Fe­(CN)6]3–/4– containing 0.1 mol L–1 KCl (Figure c–f). The slope of the linear relationship between peak current (I pa) and the square root of the scan rate (ν1/2), was used to estimate the electroactive area (A) from the Randles–Sevcik equation: I pa = 0.446nFAC√(nFDν/RT). In this equation, I pa is the anodic current (A), n is the electron transfer number (n = 1), F is the Faraday constant, C is the concentration of the redox probe (mol cm–3), D is the diffusion coefficient of the redox probe (7.26 × 10–6 cm2 s–1 for 5.0 mmol L–1 [Fe­(CN)6]3–/4–), ν is the scan rate (V s–1), R is the gas constant (8.314 J K–1 mol–1) and T is the temperature (298 K). The calculated ECSA values for paper-LIG electrodes with diameters of 0.86, 1.32, 2.46, and 3.46 mm were 0.0257, 0.0326, 0.0691, and 0.1213 mm2, respectively. The corresponding ECSA ratios were therefore 442.43, 238.66, 145.53, and 129.09% (Table S1). The ECSA ratio of the smallest WE (d = 0.86 mm) was the highest and the ratio decreased as the WE diameter increased to 3.46 mm (Figure g). To evaluate the electron transfer tendency of the different paper-LIG electrodes, the effect of scan rate on the peak-to-peak separation (ΔE p) produced by [Fe­(CN)6]3–/4‑ was investigated. The ΔE p changed with increasing scan rate at all the paper-LIG electrodes (Figure h). Moreover, the ΔE p of the smallest paper-LIG electrode (WE = 0.86 mm) was less affected by changes in scan rate compared to the ΔE p of larger electrodes, suggesting that the smaller electrodes exhibited better electron transfer kinetics. The higher ECSA ratios of the smaller electrodes provide a more electrochemically accessible surface, which enhances electron transfer and improves sensing performances.

The electron transfer properties of paper-LIG electrodes with varying working electrode (WE) diameters were evaluated using electrochemical impedance spectroscopy (EIS) in a solution of 5.0 mmol L–1 [Fe­(CN)6]3–/4– containing 0.1 mol L–1 KCl. In the Nyquist plots, the semicircular portion in the high-frequency region corresponds to the charge transfer resistance (R ct), while the straight-line features in the low-frequency region represent the semi-infinite diffusion process. The R ct values are estimated from the equivalent circuit fitting. The result shows that the R ct value increased with increasing WE diameter from 0.86 mm to 3.46 mm (Figure S3). This trend indicates that the smallest WE exhibited the lowest R ct, indicating a faster electron transfer rate at the electrode–electrolyte interface. To quantify this behavior, the charge transfer rate constant (k s) was calculated using the equation k s = RT/n 2 F 2 R ct C, where R, T, F, and n represent the gas constant (8.314 J mol–1 K–1), absolute temperature (298 K), Faraday’s constant (96,485 C mol–1), and the number of electrons transferred, respectively; C′ is the concentration of the redox probe. The calculated k s values for WE diameters of 0.86, 1.32, 2.46, and 3.46 mm were 7.7 × 10–4, 8.5 × 10–5, 4.9 × 10–5, and 4.1 × 10–5 m s–1, respectively. Notably, the smallest WE size exhibited the highest k s value, confirming that reduced electrode dimensions enhance interfacial electron transfer kinetics, likely due to improved current density and ion accessibility.

3.3.2. Auxiliary Electrode Area

To understand the behavior of the AE of the paper-LIG electrode, the effect of the size of the AE on the electrochemical response was evaluated. Figure S4a displays the CV results produced in an electrolyte of 5.0 mmol L–1 [Fe­(CN)6]3–/4– containing 0.1 mol L–1 KCl by paper-LIG electrodes with AE geometric areas of 1.28, 1.70, 3.20, and 4.24 mm2. Generally, the voltammograms display two pairs of peaks: the primary redox peaks around +0.30 V vs the pseudo RE, and the secondary redox peaks at approximately +0.07 V vs the pseudo-RE. The CV results show no difference in current response and peak-to-peak separation between the different electrodes (Figure S4b). A notable characteristic of the redox-probe voltammograms at these paper-LIG electrodes is the high reversibility of the primary redox reaction, indicated by the ratio of the cathodic peak current to the anodic peak current (i pc/i pa) (Figure S4c). All the paper-LIG electrodes produced high peak current ratios, indicating that the lowest AE size could provide good electrochemical performances. This finding can be explained by considering the potential (E) versus time (t) transients for different AE sizes, as shown in Figure S4d. Several key observations can be made from the AE potential transients: (i) The E AE vs t profile exhibits nonlinear behavior, differing distinctly from the E WE vs t profile, and resembling charging/discharging curves previously observed in electrochemical systems with different electrode surface area ratios. (ii) When E WE reaches its maximum value, E AE attains its minimum, and vice versa, a phenomenon influenced by charge redistribution between the working and auxiliary electrodes. (iii) E AE increases rapidly when E WE is scanned in the cathodic direction, indicating a strong coupling between the electrochemical behaviors of the two electrodes. To further evaluate the impact of AE size, the E AE values were compared for different AE sizes with a fixed WE diameter (d = 0.5 mm). When E WE was scanned between −0.40 and 0.70 V, the corresponding E AE values for AEs of 1.28, 1.70, 3.20, and 4.24 mm2 ranged from −0.87 to 0.81, −0.60 to 0.78, −0.55 to 0.69, and −0.43 to 0.31 V, respectively. In contrast, when a commercial SPCE was scanned similarly, the E AE vs t transient formed a distorted plateau with values in the −1.20 to 1.30 V range, approximately 0.60 V higher than E WE range, a trend similar to previously reported potential shifts observed with different electrode configurations. Additionally, the AE of the commercial SPCE remained within the oxygen evolution potential region for an extended duration (∼16 s at a scan rate of 50 mV s–1), potentially influencing the stability and reproducibility of the electrochemical response. These findings demonstrated that the AE of the fabricated paper-LIG electrode demonstrated a promising electrochemical performance, making the electrode a viable platform for electrochemical sensing applications.

3.4. Electrochemical Behavior of DZ

The electrochemical behavior of DZ on the paper-LIG electrode was investigated using CV within a potential range of −0.50 to −1.50 V in BR buffer (pH = 4.00) without and with 1.0 mmol DZ L–1. In Figure b, the voltammogram shows irreversible reduction peaks at −1.10 V, characteristic of the 4,5-azomethine group reduction. To investigate the electrochemical reaction mechanism of DZ at the paper-LIG electrode surface, CV was used to analyze the relationship between peak current (I p) and the square root of the scan rate (v1/2). Figure c presents the CV curves obtained from the paper-LIG electrode over a scan rate range of 10 to 200 mV s–1 in BR buffer (pH 4.00) with a fixed DZ concentration of 1.0 mmol L–1. As the scan rate increases, the reduction peak shifts toward more negative potentials, accompanied by a corresponding increase in peak current. Figure d illustrates the relationship between I pc and ν1/2 values, which gave a linear regression equation of I pc (μA) = −0.565 ν1/2 – 0.113 (R 2 = 0.995). According to the literature, a linear correlation between peak current and ν1/2 indicates a diffusion-controlled process, which is consistent with previous studies on the voltammetric determination of DZ. , The logarithmic plot of peak current (log I) versus log scan rate (log ν) followed the equation log I = 0.489 log ν – 0.216 (R 2 = 0.994). The slope of 0.49, which is close to the theoretical value of 0.5, further confirmed that DZ reduction on the paper-LIG electrode was primarily governed by diffusion.

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(a) Schematic illustration shows the proposed electrochemical mechanism of DZ reduction on the paper-LIG electrode surface. (b) CV of 1.0 mmol L–1 DZ on paper-LIG at a scan rate of 50 mV s–1. (c) CV results at different scan rates (ν = 10 to 200 mV s–1) for a paper-LIG electrode in BR buffer (pH 4.00) containing 1.0 mmol L–1 DZ. (d) The relationship between I pc and ν1/2 values, and the logarithmic plot of peak current (log I) versus scan rate (log ν). (e) DPV curves of DZ in BR buffer at various pH values were produced at the paper-LIG electrode. (f) The relationship between the current response of DZ and peak potential was influenced by the pH of the BR buffer.

To determine the number of electrons (n) involved in the reduction process, electron transfer was determined using Laviron’s equation, plotting the cathodic peak potential (E p) of DZ vs the natural logarithm of the scan rate (ln v), as shown in Figure S5. The resulting linear regression equation was E p = −0.0594 ln v – 1.273. According to Laviron’s theory, the slope of this plot (−0.0594) = 0.059/nα, where α is the charge transfer coefficient, assumed to be 0.5 for an irreversible electrochemical process. Solving for n, the number of electrons involved in the reduction of DZ was determined to be 2. Furthermore, the effect of pH on the cathodic peak potential and peak current of DZ was investigated using the paper-LIG electrode to measure 1.0 mmol L–1 of DZ in BR buffer at pH 2.00–9.00. The cathodic peak potential of DZ exhibited a negative shift with each unit increase in pH (Figure e). A linear relationship was observed in the plot of E p vs pH (Figure f) that produced a slope of 56 mV/pH, indicating that the proton-to-electron redox process occurred in equal proportions. Consistent with Honeychurch et al., the electrochemical behavior of DZ at the paper-LIG electrode was a two-electron, two-proton reduction of the 4,5-azomethine group to 4,5-dihydro-diazepam (Figure a), which confirmed the proposed reaction pathway. To achieve a trade-off between lower detection potential and higher current response, pH 4.00 was selected for further studies.

3.5. DZ Determination by Different Techniques

In this study DZ was determined by LSV, SWV, and DPV using the paper-LIG electrode. Each technique was optimized by adjusting key parameters, to enhance the current response of the electrochemical signal. The optimized conditions were then used to evaluate the determination of DZ by each technique with the paper-LIG electrode.

In LSV, the potential applied to the working electrode is linearly swept over time, as shown in the LSV waveform in Figure a-i. The current response produced indicates the electrochemical reactions at the electrode surface. The determination of DZ using LSV with the paper-LIG electrode was significantly influenced by the potential step and scan rate. Figure S6a displays the effect of potential steps of 1.0, 2.0, 3.0, 4.0, and 5.0 mV on the current response of 0.5 mmol L–1 DZ, using a constant scan rate of 50 mV s–1. The highest current signal was obtained with a potential step of 1 mV. Therefore, a potential step of 1 mV was applied and the effect of scan rate was evaluated from 20 to 100 mV s–1 (Figure S6b). While the current signal increased with increments of scan rate, the peak was also broader at higher scan rates. Consequently, a scan rate of 100 mV s–1 with a potential step of 1 mV was selected as the optimal condition of LSV for comparison with the other techniques.

6.

6

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(a) Voltammetric techniques: potential waveforms are shown from (i) linear sweep voltammetry (LSV); (ii) square wave voltammetry (SWV); and (iii) differential pulse voltammetry (DPV). (b) Voltametric response curves and (c) peak currents and corresponding peak potentials of DZ at the paper-LIG electrode using different voltametric techniques.

SWV minimizes non-Faradaic contributions and measures Faradaic currents by applying a staircase potential ramp with square-shaped pulses at each step. These pulses are applied alternately in anodic and cathodic directions, completing a single potential cycle, as illustrated in Figure a-ii. The determination of DZ by SWV was highly dependent on the applied frequency, amplitude and potential step. The effect of frequency was studied from 1 to 40 Hz, using a constant pulse amplitude of 50 mV and potential step of 5 mV. The current signal was highest at 25 Hz, and frequencies higher than 25 Hz resulted in a decreased signal (Figure S6c). A frequency of 25 Hz was then fixed, and the effect of pulse amplitude was evaluated from 10 to 150 mV. The current signal increased with pulse amplitude up to 90 mV and then decreased (Figure S6d). The potential step was varied between 1 and 18 mV. A larger potential step reduced analysis time, with the highest current signal achieved at a potential step of 12 mV (Figure S6e). Thus, the optimal SWV condition was a frequency of 25 Hz, pulse amplitude of 90 mV, and potential step of 12 mV.

DPV enhances the current response while minimizing charging effects during background current formation. In DPV, the base potential is carefully selected to avoid Faradaic reactions and is incrementally increased between pulses, with the current measured at both the beginning (I 1) and end (I 2) of each pulse. The fast decay of capacitive current compared to Faradaic current allows for nearly complete elimination of capacitive effects, as shown in the DPV waveform in Figure a-iii. The effect of pulse potential (E pulse) was studied from 20 to 160 mV. The highest current response was produced at 120 mV (Figure S6f). The effect of pulse duration (t pulse) was evaluated from 10 to 50 ms. From 10 to 20 ms, the current response increased. Above 20 ms the current response declined, making 20 ms the optimal pulse duration (Figure S6g). The effect of potential step (E step) was tested between 10 and 35 mV. The highest current was observed at E step = 25 mV (Figure S6h). Finally, the scan rate, which determines the pulse period in DPV, was varied from 10 to 70 mV s–1. The peak current was observed at a scan rate of 50 mV s–1 (Figure S6i). Therefore, the optimal DPV condition was an E pulse of 120 mV, t pulse of 20 ms, E step of 25 mV, and a scan rate of 50 mV s–1.

In conclusion, the optimization of these three voltammetric techniques was necessary to enhance the sensitivity and selectivity of the electrochemical sensor. By carefully adjusting key parameters of each technique, the reliable determination of DZ could be achieved. When the voltammetric performances of the three techniques were compared (Figure b), DPV was seen to provide the highest current response (−17.06 μA) at the lowest peak potential (−1.05 V) (Figure c), and therefore outperformed LSV and SWV. The reduced capacitive current and enhanced Faradaic signal of DPV were key factors in its superiority.

3.6. Electrochemical Sensing of Diazepam

The quantification of DZ was performed by DPV in a potential range of −0.70 to −1.40 V (vs pseudo-Ag/AgCl) in the optimized condition. The selected potential range corresponds to the irreversible reduction of diazepam, which involves the azomethine group. DPV curves were obtained by successive additions of standard DZ solutions. Figure a shows well-defined peaks at −1.00 V whose current intensity increases with DZ concentration. The analytical curve for DZ (Figure b) was constructed using the peak currents obtained from the DPV curve. Two linear regions were observed in the analytical curve: the first linear region was in the concentration range from 1.0 to 100 μmol L–1 and the second linear region was from 100 to 1000 μmol L–1. The linear regions gave the following linear regression equations: y = (−0.092 ± 0.003)x – (0.6 ± 0.1); R 2 = 0.995 and y = (−0.0166 ± 0.0006)x – (8.9 ± 0.4); R 2 = 0.994, respectively. It should be noted that the presence of two linear ranges was attributed to the concentration-dependent mass transport behavior of diazepam. At lower concentrations, diffusion proceeds efficiently, resulting in a high sensitivity. However, at higher concentrations, the diffusion of the analyte toward the electrode becomes hindered, leading to slower mass transport and a reduced slope. This phenomenon also causes a shift in peak potential, as the electrochemical system applies increased potential to re-establish the current transfer process. The limit of detection (LOD) was calculated from the equation LOD = 3 × SDblank/S, where SDblank is the standard deviation of the blank measurements in triplicate (n = 3) and S is the slope of the analytical curve. The limit of quantification (LOQ) was calculated as LOQ = 3.3 × LOD. The LOD and LOQ values for DZ were 0.4 and 1.2 μmol L–1, respectively. In addition to its broad linear range and low detection limit, the paper-LIG sensor exhibited high sensitivity (15.86 μA μmol–1 L cm2) and excellent resolution, with the ability to discriminate diazepam concentration changes as small as 1.0 μmol L–1. These attributes underscore the sensor’s potential for reliable quantification in real-world forensic applications, where both trace detection and concentration discrimination are essential.

7.

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(a) DPV voltammograms of different DZ concentrations (1–1000 μmol L–1) obtained at the paper-LIG electrode in BR buffer at pH 4.00. (b) The analytical curve obtained by plotting the cathodic peak current as a function of DZ concentration. All experiments were conducted in the optimized DPV condition: E pulse = 120 mV, t pulse = 20 ms, E step = 25 mV, and scan rate = 50 mV s–1. (c) Reproducibility of paper-LIG electrode. (d) Interference and (e) selectivity test on DZ sensing using the paper-LIG electrode; Note: APZ is alprazolam; CLZ is clonazepam; LZ is lorazepam; KET is ketamine and MA is methamphetamine.

The analytical performances of our paper-based LIG sensor for DZ detection were compared with those of reported voltammetric methods (Table ). Key factors such as electrode type, electrochemical technique, sample type, linear concentration range, and LOD were considered. Our sensor offers a broader linear detection range along with advantages of scalable manufacturing, low cost, disposability, eco-friendliness, and portability, making it well-suited for on-site and field applications.

1. Comparison of Analytical Parameters for DZ Detection Using Electrochemical Approaches Reported in the Literature .

sensor technique sample application linear range (μmol L–1) LOD (μmol L–1) refs
unmodified SPCE DPV beverage 24.94–1000.91 6.32
PEI-LSG SWV beverage 2.5–25.0 and 25.0–100.0 0.66
wearable glove sensor/MWCNTs/PEDOT:PSS SWV beverage 0.5–10.0 and 10.0–100.0 0.06
Si@GNRs/EμPAD CV urine 0.0035–3.50 0.0015
AgNDs/GNs/GCE DPV DZ tablet, injection and plasma 0.1–1.0 and 1.0–20.0 0.086
C60-CNTs/IL/GCE DPV urine and human blood serum 0.3–50.0 and 50.0–700.0 0.087
BiPPGE DPV DZ tablet and urine 1.4–16.7 1.1
paper-LIG DPV beverage samples 1–100 and 100–1000 0.36 this work
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a

Legend: screen-printed carbon electrode (SPCE), laser-scribed graphene (PEI-LSG), multiwalled carbon nanotubes (MWCNTs), poly­(3,4-ethylenedioxythiophene)/poly­(styrenesulfonate)­(PEDOT:PSS), silica coated gold nanorods (Si@GNRs), paper-based device (EμPAD/ePAD), Ag nanodendrimers (AgNDs), graphene nanosheets (GNs), glassy carbon electrode (GCE), carbon nanotubes (C60-CNTs), ionic liquid (IL), bismuth modified pretreated pencil graphite electrode (BiPPGE), differential pulse voltammetry (DPV), square wave voltammetry (SWV), cyclic voltammetry (CV), diazepam (DZ).

The reproducibility of the paper-LIG electrode fabrication was evaluated using ten electrodes produced in the same condition of laser scribing. These electrodes were used to measure DZ at a concentration of 0.1 mmol L–1, resulting in a relative standard deviation (RSD) of 2.2% for the current response (Figures c and S7). According to the AOAC guidelines, which recommends an RSD of up to 8% for 100 mg L–1 (equivalent to 0.1 mmol L–1 or 28.5 mg L–1 of DZ), these results demonstrate excellent reproducibility.

The effects of potential interferents commonly present in commercial beverage samples were investigated on the response of 50.0 μmol L–1 DZ at the paper-LIG electrode. Interfering species included Ca2+, K+, Mg2+, Na+, Zn2+, Cl–, CO3 2–, SO4 2–, ascorbic acid, citric acid, fructose, glucose, sucrose, and ethanol. As shown in Figures d and S8a, no significant interference was observed in the presence of 100-fold concentrations of Ca2+, K+, Mg2+, Zn2+, CO3 2–, SO4 2– and citric acid, 150-fold concentrations of Na+, Cl–, fructose, glucose, and sucrose, and 200-fold concentration of AA. Additionally, the paper-LIG electrode demonstrated tolerance to ethanol concentrations of up to 10% for DZ detection.

The selectivity of the paper-LIG electrode was an important factor in its potential application for analyzing real samples. Other benzodiazepines such as alprazolam (APZ), clonazepam (CLZ), and lorazepam (LZ), have similar chemical structures and electrochemical properties, which often lead to overlapping oxidation or reduction signals. However, the negligible interference observed in Figures e and S8b showed that the paper-LIG electrode could effectively distinguish DZ from these structurally related compounds. This selectivity may be attributable to different responses of the analytes to operating conditions (such as pH and DPV parameters), differences in electrochemical reactions at the electrode surface, or differences in redox behavior at specific redox potentials. Additionally, ketamine (KET) and methamphetamine (MA), showed no significant interference, confirming the ability of the electrode to distinguish DZ in complex samples. The excellent selectivity of the paper-LIG electrode, combined with its scalability, low cost, and portability, makes it a promising platform for routine drug screening in beverages and forensic investigations.

3.7. Real Samples and Accuracy Test

The practical applicability of the proposed sensor was validated by detecting DZ in both alcoholic and nonalcoholic beverages, including Smirnoff Ice (5% alcohol, 275 mL), Chum Churum (16.5% alcohol, 360 mL), Full Moon white (5% alcohol, 275 mL), Malee coco (350 mL) and Ichitan Genmaicha (500 mL). To simulate real-case scenarios of drug-facilitated crimes, a single tablet containing 5.0 mg of DZ was added to each sample as shown in Video S1. Electroanalysis required no pretreatment steps, and DZ was quantified in the cited samples using the SAM, which eliminates the need for calibration curves as it accounts for matrix effects. The standard addition curves for each beverage are presented in Figure S9. Table summarizes the data obtained from these analyses. Using the SAM, the DZ concentration found in beverage samples closely matched the calculated values, with recoveries ranging from 96.4 to 99.0% and errors within ±3.58%. These results demonstrated the high precision and reliability of the method across different sample matrices. Moreover, the results obtained using the electrochemical sensor were compared with those obtained from a UV–visible spectrophotometric method (described in the Supporting Information, including Figure S10), as summarized in Table . No significant differences were observed between the two methods, thereby confirming the accuracy and reliability of the paper-LIG sensor for diazepam detection in real beverage samples. Furthermore, the recovery test results in Table S2 showed excellent agreement. The recovery data fell within the expected range of 90–107% for this concentration level. Overall, these findings underscored the reliability and accuracy of quantifying the target analyte by DPV with the paper-LIG electrode.

2. Results of DZ Detection in Both Alcoholic and Non-Alcoholic Beverage Samples Using the Developed Sensor and Standard Reference Method.

        concentration (μmol L–1)
    
sample DZ tablet (mg/tablet) dilution (fold) sample volume (mL) calculated detected ref recovered (%) error (%)
S1 5 5 275 63.9 62 ± 2 60.0 97.1 2.9
S2 5 5 360 48.8 50 ± 3 48.1 99.0 –2.0
S3 5 5 275 63.9 58 ± 3 57.9 96.4 3.6
S4 5 5 350 50.2 49 ± 3 48.7 98.1 1.9
S5 5 5 500 35.1 34 ± 3 33.2 97.5 –2.5
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a

Ref: UV-visible spectrophotometric method.

4. Conclusions

In this study, a sustainable electrochemical sensing platform was successfully developed using laser-induced graphene (paper-LIG) fabricated on sodium tetraborate-treated cellulose paper. The pretreatment improved the thermal stability of the paper, enabling efficient graphitization by CO2 laser scribing. The fabricated paper-LIG electrode exhibited favorable surface morphology, electrical conductivity, and chemical composition, as confirmed through comprehensive physicochemical analyses. Systematic evaluations of the working and auxiliary electrode dimensions revealed their significant influence on electrochemical behavior, particularly on the electron transfer kinetics and electroactive surface area. These insights are crucial for the rational design of miniaturized paper-based sensors. The optimized paper-LIG electrode enabled highly sensitive, selective, and reproducible detection of diazepam (DZ). Using differential pulse voltammetry (DPV), the sensor achieved a wide linear range (1.0–1000 μmol L–1), a low detection limit of 0.4 μmol L–1, and high analytical sensitivity of 15.86 μA μmol–1 L cm2. The sensor also demonstrated excellent recovery (96.4–102.5%) in complex beverage matrices, good tolerance to common interferents, and strong selectivity over structurally related benzodiazepines. These results confirm the reliability of the developed platform and its potential as an eco-friendly, low-cost, and scalable tool for rapid on-site forensic screening of diazepam.

Supplementary Material

ao5c03662_si_001.pdf (1.3MB, pdf)
ao5c03662_si_002.mp4 (2.2MB, mp4)

Acknowledgments

This research was supported by a Postdoctoral Fellowship from Prince of Songkla University. For the use of instruments, apparatus, and their financial support, the authors gratefully acknowledge the Center of Excellence for Trace Analysis and Biosensors (TAB-CoE), Center of Excellence for Innovation in Chemistry (PERCH–CIC), the Forensic Science Innovation and Service Center, the Talent Management Project, the Division of Health and Applied Sciences, and the Division of Physical Science, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, Thailand. The authors would also like to thank Thomas Duncan Coyne, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, Thailand, for assistance with the English text.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c03662.

  • Optical photographs of 10 × 10 mm2 samples of laser-induced graphene produced on borax-treated wax-coated paper with different laser powers (5–10 W) and scan speeds (10–100 mm s–1); paper substrates treat with borax and wax printing paper substrates; Nyquist plots of paper-LIG electrode at difference working electrode diameters of 0.86, 1.32, 2.46, and 3.46 mm in 5.0 mmol L–1 [Fe (CN)6]3–/4– containing 0.10 mol L–1 KCl; cyclic voltammograms were produced at a scan rate of 50 mV s–1 in 5.0 mmol L–1 [Fe (CN)6]3–/4– containing 0.10 mol L–1 KCl at paper-LIG electrodes with auxiliary electrodes (AE) designed with areas of 0.50, 1.0, 2.0, and 3.0 mm2. The actual AE areas are listed in the figure. The current response and peak-to-peak separation no paper-LIG electrodes with AEs of different areas. The cathodic peak current to anodic peak current ratios on paper-LIG electrodes with AEs of different areas. Potential (E) versus time (t) transients for the 0.86 mm WE and AEs of 0.5, 1.0, 2.0, and 3.0 mm2, and a commercial SPCE; Cathodic peak potential versus ln scan rate and the corresponding linear regressions; Optimization of LSV parameters: effect of step potential (E step), effect of scan rate. Optimization of SWV parameters: effect of frequency (Hz), effect of amplitude, effect of step potential. Optimization of DPV parameters: effect of pulse potential (E pulse), effect of pulse time (t pulse), effect of step potential, effect of scan rate; reproducibility test: DPV responses of DZ (0.1 mmol L–1) on ten paper-LIG electrodes; Interferents test: DPV responses of DZ (50.0 μmol L–1) with interfering species of 100-fold Ca2+, K+, Mg2+, Zn2+, CO3 2–, SO4 2– and citric acid, 150-fold concentrations of Na+, Cl–, fructose, glucose, and sucrose, and 200-fold concentration of AA. Selectivity test: DPV responses of DZ (50.0 μmol L–1) with 50.0 μmol L–1 of alprazolam (APZ), clonazepam (CLZ), lorazepam (LZ), ketamine (KET) and methamphetamine (MA); standard addition curve of the Smirnoff Ice, Chum Churum, Full Moon white, Malee coco and Ichitan Genmaicha samples using paper-LIG; spectrophotometric method procedure; absorbance spectrum of standard DZ with calibration curves showing the relationship between absorbance intensity (at 320 nm) and DZ concentration; working electrode geometrical and electroactive areas, and electroactive area ratios of paper-LIG electrodes, and the recoveries of diazepam spiked in beverage samples (PDF)

  • Simulate real-case scenarios of drug-facilitated crimes, a single tablet containing 5.0 mg of DZ was added to each sample (MP4)

K.S.: conducted conceptualization, methodology, formal analysis and investigation, validation, data curation, original draft preparation, and review and editing of the manuscript; A.S.: performed conceptualization, methodology, formal analysis and investigation, validation, data curation, original draft preparation, review and editing of the manuscript; K.S.: contributed to conceptualization and discussion and reviewed the manuscript before submission; K.P. contributed to conceptualization and discussion and review and editing of the manuscript; J.S.: contributed to conceptualization and discussion and review and editing of the manuscript; S.W.: contributed to conceptualization and discussion and review and editing of the manuscript; W.L.: undertook conceptualization, methodology, investigation, validation, data curation, original draft preparation, review and editing of the manuscript and supervision.

The authors declare no competing financial interest.

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