Palladium Hydroxide (Pearlman’s Catalyst) Doped MXene (Ti3C2Tx) Composite Modified Electrode for Selective Detection of Nicotine in Human Sweat

High concentrations of nicotine (40 to 60 mg) are more dangerous for adults who weigh about 70 kg. Herein, we developed an electrochemical transducer using an MXene (Ti3C2Tx)/palladium hydroxide-supported carbon (Pearlman’s catalyst) composite (MXene/Pd(OH)2/C) for the identification of nicotine levels in human sweat. Firstly, the MXene was doped with Pd(OH)2/C (PHC) by mechanical grinding followed by an ultrasonication process to obtain the MXene/PHC composite. Secondly, XRD, Raman, FE-SEM, EDS and E-mapping analysis were utilized to confirm the successful formation of MXene/PHC composite. Using MXene/PHC composite dispersion, an MXene/PHC composite-modified glassy carbon electrode (MXene/PHC/GCE) was prepared, which showed high sensitivity as well as selectivity towards nicotine (300 µM NIC) oxidation in 0.1 M phosphate buffer (pH = 7.4) by cyclic voltammetry (CV) and amperometry. The MXene/PHC/GCE had reduced the over potential of nicotine oxidation (about 200 mV) and also enhanced the oxidation peak current (8.9 µA) compared to bare/GCE (2.1 µA) and MXene/GCE (5.5 µA). Moreover, the optimized experimental condition was used for the quantification of NIC from 0.25 µM to 37.5 µM. The limit of detection (LOD) and sensitivity were 27 nM and 0.286 µA µM−1 cm2, respectively. The MXene/PHC/GCE was also tested in the presence of Na+, Mg2+, Ca2+, hydrogen peroxide, acetic acid, ascorbic acid, dopamine and glucose. These molecules were not interfered during NIC analysis, which indicated the good selectivity of the MXene/PHC/GCE sensor. In addition, electrochemical determination of NIC was successfully carried out in the human sweat samples collected from a tobacco smoker. The recovery percentage of NIC in the sweat sample was 97%. Finally, we concluded that the MXene/PHC composite-based sensor can be prepared for the accurate determination of NIC with high sensitivity, selectivity and stability in human sweat samples.


Introduction
Nicotine (NIC) is one of the most addictive and hazardous drugs ever. As a main alkaloid, NIC [3-(1-methyl-2-pyrrolidinyl) pyridine] is found in tobacco leaves (Nicotiana tabacum L., Solanaceae) [1]. Initially, it was used for medical applications, but later, it became well known for its use in cigarettes. Overdose of NIC may lead to an increase of blood circulation to the heart, pulse rate, hypertension and artery contraction (blood carrying vessels). Nicotine may raise the risk of heart stroke as well as contribute to arterial on the combinations of several transition metals, such Ti, Mo, V, Cr and their mixtures with N and C [37]. The unique properties of MXenes include their great electrical conductivity, good hydrophilicity, strong chemical stability, large surface area, surface tunability, possible synthesis in bulk volumes, ecofriendly traits and harmlessness [38]. MXene exhibits structural and electrical properties similar to those of other 2D materials such as graphene, metal oxides and so on. MXene has the clear benefit of high electrical conductivity, which is essential for enhancing the process of heterogeneous electron transport. One of the crucial reasons for its use in electrochemical sensors is its outstanding electroconductive feature [39]. The 2D-layered structure and distinctive surface with numerous chemical groups allowed MXene to be coupled with a wide range of functional elements or biomolecules for various analytical applications [40]. Ti 3 C 2 is the most common type of MXene, which is the ideal choice for the fabrication of electrochemical biosensors.
The MXene's surface was functionalized to enhance the sensitivity and selectivity for the determination of a particular analyte. Palladium nanoparticles (Pd NPs) are the most extensively studied among the noble metals and metal oxide nanoparticles due to their superior electrical properties and catalytic activity [41]. Furthermore, palladium may interact and strongly bind to the 2D materials (graphene) because of the development of more transmission channels and interaction states between them [42]. As a result of the oxygenated functional groups on its surface, Pd NPs can be easily functionalized on 2D materials, and can accelerate catalytic activity.
In this study, we reported an electrochemical sensor made of MXene/PHC composite for the determination of the nicotine level in a sweat sample without any pretreatment process. The mechanochemical synthesis approach was used to synthesize the MXene/PHC composite. PHC doping might have improved the stability of MXene dispersion via functional groups on the surface. Further, Raman, FE-SEM, EDX, E-mapping and XRD characterizations were carried out to confirm the formation of the MXene/PHC composite. The PHC-doped MXene (Ti 3 C 2 T x )-modified GCE was characterized by electrochemical measurements and also used to determine the NIC concentration by amperometry analysis. In a phosphate buffer solution (pH = 7.4), MXene/PHC/GCE showed remarkable electrocatalytic activity towards NIC oxidation at a lower potential compared to MXene/GCE and bare GCE. Interestingly, MXene/PHC/GCE exhibited a linear response for NIC, from 0.25 µM to 37.5 µM (Sensitivity = 0.328 µA µM −1 cm 2 ), with an LOD of 27 nM. Further studies on selectivity, repeatability and stability were carried out, which indicated that the MXene/PHC sensor can be used for the determination of NIC with excellent selectivity and stability, even in the presence of other interfering substances. Finally, the MXene/PHC-based sensor was utilized to quantify the amount of nicotine present in a human sweat sample.

Characterizations
A field-emission scanning electron microscope (FE-SEM, JSM IT800 (JEOL, Tokyo, Japan)) was used to examine the surface morphology of the MXene and MXene/PHC composite. XPLORE-30 (High Wycombe, Oxford, UK) was used to capture energy dispersive X-ray spectra (EDS) and elemental mapping (E-map) images. A 532 nm laser excitation was used to acquire all Raman spectra (XPLORA plus (Horiba, Kyoto, Japan)). X-ray diffraction (XRD) data was collected by the D8-Advance XRD instrument (BRUKER, Billerica, MS, USA). HPLC experiments were carried out using an i-series LC-2050C 3D (SHIMADZU, Kyoto, Japan) and methanol-ammonium acetate mobile phase was used. A pH meter (LH-10-(Lab Holic, Chennai, India)) fitted with the combination electrode (reference glass electrode) was used to measure all pH readings. All electrochemical studies, including electrochemical impedance spectroscopy (EIS), amperometry (i-t curve) and cyclic voltammetry (CV) were carried out on a CHI 760E workstation (CH Instruments, Austin, TX, USA) with a three-electrode configuration. The electrochemical cell contains a reference electrode (Ag/AgCl soaked in 3 M KCl), a working electrode (GCE with a 0.07 cm 2 working area) and a counter electrode (Pt wire).

Preparation of MAX Phase (Ti 3 AlC 2 ) and MXene (Ti 3 C 2 T x )
The MAX phase was prepared as reported elsewhere (Ti 3 AlC 2 ) [43]. In a nutshell, the stoichiometric ratio of 3:1:2 of titanium, aluminum and graphite precursors were combined and mechanically mixed well in a ball-mill for 360 min. After that, a high temperature split-tube furnace with inert environment (N 2 ) was used to anneal the milled mixture for 120 min at 1100 • C. Finally, Ti 3 AlC 2 (MAX powder) was collected. Later, the Al layer was selectively etched out to obtain MXene powder. For this purpose, 1 g of MAX powder was treated with hydrofluoric acid (HF 48%) for 24 h at room temperature. Furthermore, to produce MXene powder, the resultant dispersion was diluted with distilled water and centrifuged (at 5000× g rpm for 10 min and repeated this process for 18-20 times) to neutralize the pH of the supernatant. Finally, the obtained MXene precipitate was dried at 100 • C in a hot air-oven for few days.

Preparation of MXene/PHC Composite
To develop the MXene/PHC composite, MXene powder (5 mg) and palladium hydroxidesupported carbon (1:1 ratio) (Pearlman's catalyst) (5 mg) were mixed and grinded with the help of a mortar and pestle for 20 min to make fine particle composite. This mixture was transferred to a suitable container and ultrasonicated for 20 min at RT with 1 mL of diluted nafion (Nf) (a mixture of 1 mL distilled water (DI H 2 O) + 1 mL ethanol + 10 µL nafion (5 wt.%)), as shown in Figure 1. The MXene/PHC composite dispersion was again diluted with DI water at a 1:50 ratio (composite: DI water).

Fabrication of MXene/PHC Sensor
To begin, the glassy carbon electrode (GCE of 3 mm diameter) was delicately cleaned using the standard washing process to achieve a reflective surface [43]. Next, 8 µL of the MXene/PHC dispersion was drop-casted over the GCE surface and dried in a hot air oven at 50 • C ( Figure 1). After that, MXene/PHC/GCE was slightly immersed in DI water to remove any loosely bounded particles from the modified electrode surface. In addition, the MXene/GCE (without PHC) was prepared in the same way for the examination of the samples to confirm that MXene/PHC is superior to MXene for NIC sensing.

Electrochemical Measurements
The GCE was first polished with 0.05 micron alumina powder to create a reflecting surface, and cyclic voltammetry (CV) was then performed in a solution of 0.1 M KCl with 5 mM [Fe(CN) 6 ] 4−/3− using bare GCE in the potential range of −0.2 to 0.6 V. The CV was then utilized to investigate the electrochemical behavior of NIC on bare GCE in the potential window of 0 V to +1.2 V in 0.1 M phosphate buffer at a scan rate of 50 mV s −1 (Figure 1). The voltametric responses of bare GCE, MXene/GCE, and MXene/PHC/GCE were studied in the same potential window with various concentrations of nicotine in 0.1 M phosphate buffer (pH = 7.4). The pH studies were performed in different pH buffer solutions with the addition of the same concentration (1 mM) of nicotine. The phosphate buffer (from pH 6-8), KCl/HCl mixture (pH 2), acetic acid/sodium acetate buffer (pH 4) and NH 4 Cl/ammonia solution (pH 11) were utilized in this experiment as a buffer solution with 0.1 M concentration. The linear range of nicotine determination (0.25 µM-37.5 µM) was performed by an amperometry (i-t curve) analysis. The calibration plot was made using oxidation currents and NIC concentrations (amperograms were recorded for three times (n = 3) and the average value with standard deviation are provided). The recovery studies were performed by amperometry to determine the nicotine content in real-world and standard samples, respectively.

Real Sample Collection and Extraction
The sweat sample was collected from a smoker (who smokes 5-7 cigarettes per day, Age = 30). The participant was instructed to conduct strenuous physical workouts for around 2 h. The segregated sweat was then wiped with a pure thin cotton cloth, then squeezed and transferred to a 15 mL centrifuge tube. After that, the collected sweat was centrifuged for 10 min at 10,000× g rpm to separate the supernatant, then it was stored at 4 • C for further examination ( Figure S1 in Supplementary Materials).

Raman and XRD Analysis
The Raman spectra of PHC, MXene (Ti 3 C 2 T x ) and MXene/PHC were successfully recorded. The palladium hydroxide dry basic carbon ( Figure 2, brown curve) showed two major peaks of D and G bands at~1343 cm −1 and~1591 cm −1 , respectively. The MXene ( Figure 2, red curve) displayed a broad band at around 158 cm −1 , which corresponded to the Eg (doubly degenerative vibration state) of TiO [44]. Furthermore, three low intensity bands at 399 cm −1 , 618 cm −1 and 799 cm −1 were found due to the vibrations of MXene atoms [45,46]. In addition, three more Raman bands were found at~1350 cm −1 ,~1572 cm −1 and~2689 cm −1 , which correspond to the D, G and 2D bands of graphitic carbon ( Figure 2). The D-band represents the defects of graphitic carbon and the G-band represents the vibrations of sp 2 hybridized atoms of carbon in a 2D-hexagonal lattice [47]. The I D /I G peak intensity ratios of the Raman spectra were used to quantify the degree of disorder [48]. The I D /I G ratio (0.86) was increased for the MXene/PHC composite compared to MXene (0.37), which indicated the existence of PHC in the composite. Furthermore, the Raman spectrum of the MXene/PHC composite ( Figure 2, blue curve) showed the existence of MXene/PHC-specific bands (D, G as well as 2D bands). Compared to the MXene spectrum, the D, G, and 2D bands of MXene/PHC were found to be down shifted (blue shifted) and detected at lower spectral range (~9 cm −1 ). In addition, compared to MXene, the D-band intensity was increased (from 73 to 117) and the G-band intensity was decreased (from 197 to 136) compared to the MXene/PHC composite. This study revealed that PHC might have interacted with the functional groups of MXenes and also confirmed the composite formation.
The crystalline properties of MXene and MXene/PHC composite were determined by XRD analysis (Figure 3) Figure 3 (blue curve). The 2θ values of PHC were well matched with the previously reported research articles [52,53]. This data further confirmed the successful synthesis of the MXene/PHC composite. Furthermore, after the composite formation, some of the MXene peak intensities decreased and a minor peak shift was noticed. These changes could be the result of the integration of PHC into MXene as well as variations in the lattice parameters in the composite crystal structure [54].

FE-SEM, EDS and E-Mapping Analysis
As shown in Figure 4a, FE-SEM images of as-synthesized mixed phase MXene (Ti 3 C 2 T x ) revealed a stacked layered structure. Our synthesis process clearly produced a few-layered MXene with a thickness of 100-400 nm. It can be seen that the FE-SEM image of the MXene/PHC composite showed the successful incorporation of PHC within the MXene layers (Figure 4b,c).   Figure 5a. The existence of corresponding elements (in atomic percentages) in the MXene/PHC composite were also recorded by EDS analysis (Figure 5b).  (Figure 6b). The Rcts of each electrode were calculated from the Nyquist plots using the semicircle's diameter, which was located in the high-frequency region. Among these, MXene/PHC/GCE (0.01 Ω) showed lower Rct compared to MXene/GCE (115.3 Ω) and bare/GCE (283.5 Ω). The absence of a semicircle in the case of MXene/PHC/GCE indicated that the charge transfer resistance was substantially reduced because of the good conductivity of the electrode [56]. EIS indicated that MXene/PHC/GCE possesses excellent conductivity after the incorporation of PHC with MXene. Moreover, the MXene/PHC/GCE-modified electrode showed a higher electron transfer efficacy due to its possession of the lowest Rct value.

Electrochemical Oxidation of NIC
The cyclic voltammograms (CVs) were recorded using bare/GCE, MXene/GCE, PHC/GCE and MXene/PHC/GCE in 0.1 M phosphate buffer before and after the addition of 300 µM of NIC. Before addition of NIC, the MXene/PHC/GCE exhibited CV curve with a higher background current than MXene/GCE, PHC/GCE and bare GCE (Figure 7 (inset)). There were no oxidation or reduction peaks observed in the potential window of +0.2 V to +1.2 V. However, when NIC (300 µM) was added, the MXene/PHC/GCE exhibited a higher oxidation peak current (8.9 µA) at +0.9 V than MXene/GCE (5.5 µA), PHC/GCE (5.3 µA) and bare GCE (2.1 µA) (Figure 7). According to earlier reports, NIC begins to oxidize at a potential of~0.9 V on the modified GCE [7,29,36,57].   Figure 8 shows the electro-oxidation mechanism of NIC on MXene/PHC/GCE [58]. During a cathodic scan, no reduction peak was observed until +0.2 V. As a result, NIC oxidation is an irreversible process at MXene/PHC/GCE, which is also corroborated by previous reports [30,31]. It was clear that the NIC oxidation peak was noticed at a lower potential on the composite modified electrode (+0.9 V) compared to bare GCE (+1.1 V) (Figure 7 curves i-iv). In addition, the MXene/PHC/GCE exhibited the highest oxidation peak current for NIC compared to MXene/GCE and PHC/GCE, which indicated that the MXene/PHC composite had exhibited a greater catalytic effect towards NIC.

Detecting NIC Concentration
Using CVs, the concentrations of NIC were determined using MXene/PHC/GCE in 0.1 M phosphate buffer at a scan rate of 0.05 V s −1 . The concentrations of NIC varied from 100 µM to 1 mM. As shown in Figure 9, the oxidation peak currents (I pa ) of NIC were increased linearly with the concentrations. This demonstrated the high sensitivity and stability of the MXene/PHC/GCE towards the NIC oxidation reaction. Next, to study the effect of scan rates on NIC oxidation, CVs were recorded with the varied scan rates from 10 mV s −1 to 250 mV s −1 in 0.1 M phosphate buffer with 500 µM NIC using MXene/PHC/GCE. The oxidation peak currents (I pa ) of NIC were gradually increased with respect to the square root of the scan rate, as shown in Figure 10. According to the kinetic controlled process, the oxidation peak potential (E pa ) of NIC slowly moved to the positive side. The linear equation was found to be Y = 1.5 × 10 −6 x + 2 × 10 −6 with a correlation coefficient (R 2 ) of 0.987 (Figure 10, Inset). This data confirmed that a diffusion-controlled oxidation process of NIC occurred on the MXene/PHC/GCE [59,60]. In order to find out the suitable pH for NIC analysis, CVs were recorded at different pH buffers containing 1 mM nicotine (from pH 2 to 11) using a MXene/PHC/GCE sample.  The I pa and E pa of nicotine were examined in different pH buffer solutions (pH 2-11), as shown in Figure 11a, b. It was observed that the oxidation peak potential of NIC started to move towards the negative potential when the pH of the buffer increased; it demonstrated that the electro-oxidation of NIC involves the same number of protons. Simultaneously, the I pa of NIC was higher in pH 2 and 11, while in the other pH 4 range, almost the same current responses were observed. If we look more closely, the I pa of NIC was higher in pH 7.4 than in pH 6. By considering the pH of human body fluids (pH 5.5 to pH 8), we adopted pH 7.4 to determine the NIC in human sweat. Additionally, Figure 11b displayed the relationship between the pH and E pa of NIC, which gave a slope of −67.5 mV/pH, a value that was similar to the theoretical value (−59 mV/pH) of the Nernst equation. This demonstrated that the nicotine oxidation process on the MXene/PHC/GCE was involved with the same number of electrons and protons [61].

Amperometry and Interference Studies
The amperometry technique was carried out to investigate the linear range NIC determination at +1.0 V using a MXene/PHC/GCE in 0.1 M phosphate buffer. When NIC concentrations were increased from 0.25 µM to 37.5 µM [28,57,62], MXene/PHC/GCE produced a linear current response for each addition of nicotine by amperometry (Figure 12), and the sensitivity was calculated as 0.286 µA µM −1 cm 2 . In the dynamic linear range of determination, amperometry is more accurate than CV. The obtained high sensitivity of the sensor could be attributed to the quick response by electrocatalytic oxidation of NIC by MXene/PHC/GCE [63]. The LOD of nicotine was measured as 27 nM. It should be mentioned that at the higher concentration of NIC, the steady state current began to drop because the catalyst's active regions of the modified electrode might have been saturated. The analytical performance of the proposed method is also compared with other NIC sensors. Table 1 shows the comparison of the linear range of detection, LOD and oxidation potential of NIC. It can be mentioned that our sensor's performance is relatively comparable to other reported methods. Footnotes: E pa -oxidation or anodic peak potential, I pa -oxidation or anodic peak current, To ascertain the selectivity of newly developed MXene/PHC/GCE, amperogram (i-t) was recorded with the additions of interference compounds and NIC (5 µM), as shown in Figure 13a. The commonly present interfering chemicals in sweat and their oxidation responses were examined (examples: ascorbic acid (AsA), dopamine (DA, 0.5 µM), and glucose (Glu), acetic acid (AcA), magnesium chloride (MgCl 2 ), calcium chloride (CaCl 2 ), sodium chloride (NaCl) and hydrogen peroxide (H 2 O 2 )). All interference compounds were added to 0.1 M phosphate buffer (pH 7.4) with NIC at a 1:1 ratio. It can be noted that the oxidation current of NIC was reduced by about 10% when these chemicals were coexisted with NIC, as shown in Figure 13b. Thus, it was confirmed that these studied compounds did not strongly interfere with the determination of nicotine.

Repeatability and Stability Studies
The newly prepared MXene/PHC/GCE was used to investigate the oxidation of 300 µM NIC in 0.1 M phosphate buffer in order to evaluate the sensor's repeatability and analyze the precision of the modified electrode (Figure 14a). The differences in the oxidation peak current (I pa ) of NIC at +0.95 V are clearly shown in the inset of ( Figure 14a). The I pa of NIC was slightly increased at +0.95 V during the first five independent measurements of NIC. Following that, the I pa of NIC started to decrease after the sixth and seventh consecutive measurements, possibly due to material leaching out of the GCE after the fifth measurement. These results showed that the MXene/PHC composite has reasonably good stability for multiple repeated experiments.   Likewise, the stability of the MXene/PHC/GCE was investigated by oxidation of NIC in 0.1 M phosphate buffer (pH = 7.4) from the first to the seventh days (Figure 14b). For this study, the MXene/PHC/GCE was newly prepared and used to measure the oxidation current of 300 µM NIC in 0.1M phosphate buffer. After this measurement, the MXene/PHC dispersion was stored at room temperature. After 7 days of storage, the MXene/PHC dispersion was used to prepare the sensor, which showed a well-defined oxidation peak for 300 µM of NIC (inset Figure 14b). However, the oxidation peak current was decreased about 7% compared to its initial electro-catalytic activity. This study indicated that the MXene/PHC dispersion was relatively stable and could be used for the preparation of stable sensors for nicotine analysis.

Real-World Sample Analysis
A human sweat sample was collected and evaluated in order to determine the effectiveness of the MXene/PHC composite-based sensor for real-world applications. All samples were examined in triplicate measurements (n = 3) by amperometry to determine the nicotine level, and the results are displayed as the average of the three measurements. The collected sweat (supernatant) sample was diluted (sweat and DI water with 1:2 ratio) and added to the 0.1 M phosphate buffer (pH = 7.4), where the designated sensor recorded the oxidation current response. The current response was increased after each addition of a sweat sample (Figure 15). Moreover, high-performance liquid chromatography (HPLC) was used to identify nicotine in the real sample to ensure that the results produced by the MXene/PHC/GCE sensors were accurate ( Figure S2). The recovery studies for NIC were carried out on the human sweat sample ( Figure 16). Initially, 100 µL of sweat was added to the 0.1 M phosphate buffer (pH = 7.4), after that, NIC was spiked in to the solution mixture (sweat + phosphate buffer), followed by the addition of three different concentrations of NIC (1 µM, 2 µM, and 4 µM). The amperogram was recorded using the MXene/PHC/GCE at +1.0 V and the calibration curve was used to calculate the unknown concentration of NIC in the sweat sample. Table 2 depicts the estimated nicotine concentration in the human sweat sample by using amperometry and also spiked standard NIC concentrations, which demonstrated the good recovery of spiked NIC in the sweat sample (97-103%).

Conclusions
A MXene/PHC composite-based electrochemical sensor was used for the first time to determine the nicotine concentration in a human sweat sample with good specificity and sensitivity. The developed composite was comprehensively characterized by FE-SEM, EDS, E-mapping, Raman and XRD, which confirmed that the MXene/PHC composite was successfully formed with a uniform distribution of elements. Next, the MXene/PHC/GCEbased electrochemical sensor was prepared for NIC oxidation. MXene/PHC/GCE enhanced the oxidation peak current of nicotine compared to MXene/GCE and bare/GCE. It was found that NIC was oxidized electrocatalytically on the MXene/PHC composite by involving an equal quantity of protons and electrons. Thus, the newly developed MXene/PHC composite could be used to develop an effective sensor for NIC determination in various samples. In addition, MXene/PHC/GCE provided a wide linear range of NIC determination, from 0.25 µM to 37.5 µM, with LOD of 27 nM, as well as capable of sensing nicotine at physiological condition compared to other studies (Table 1). In addition, the nicotine concentration in human sweat sample was determined using the MXene/PHCbased sensor in 0.1 M phosphate buffer. Finally, we believe that the MXene/PHC-based sensor could be used to determine the NIC concentration in a human sweat sample with high selectivity.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/bios13010054/s1, Figure S1: Schematic illustration of sweat sample collection and electrochemical analysis of nicotine; Figure S2: HPLC spectra of standard nicotine (NIC) solution and real sweat sample were recorded. Reference [74] is cited in Supplementary Materials.