Ti3C2Tx Coated with TiO2 Nanosheets for the Simultaneous Detection of Ascorbic Acid, Dopamine and Uric Acid

Two-dimensional MXenes have become an important material for electrochemical sensing of biomolecules due to their excellent electric properties, large surface area and hydrophilicity. However, the simultaneous detection of multiple biomolecules using MXene-based electrodes is still a challenge. Here, a simple solvothermal process was used to synthesis the Ti3C2Tx coated with TiO2 nanosheets (Ti3C2Tx@TiO2 NSs). The surface modification of TiO2 NSs on Ti3C2Tx can effectively reduce the self-accumulation of Ti3C2Tx and improve stability. Glassy carbon electrode was modified by Ti3C2Tx@TiO2 NSs (Ti3C2Tx@TiO2 NSs/GCE) and was able simultaneously to detect dopamine (DA), ascorbic acid (AA) and uric acid (UA). Under concentrations ranging from 200 to 1000 μM, 40 to 300 μM and 50 to 400 μM, the limit of detection (LOD) is 2.91 μM, 0.19 μM and 0.25 μM for AA, DA and UA, respectively. Furthermore, Ti3C2Tx@TiO2 NSs/GCE demonstrated remarkable stability and reliable reproducibility for the detection of AA/DA/UA.


Introduction
MXene, a type of two-dimensional (2D) material, is characterized by the formula M n+1 X n T. In this formula, M represents transition metals, X could be either carbon or nitrogen, while T refers to surface functional groups like =O, -F, and -OH [1,2], and it attracts attention as a promising material for electrochemical modification [3,4].In particular, the abundant functional groups, high specific surface area and excellent electrical properties of MXene provide promising applications for the electrochemical detection of various biomolecules, especially for dopamine (DA), ascorbic acid (Vitamin C or AA) and uric acid (UA).The stability of physiological functions can be influenced by these three important substances which coexist in body fluids [5].As a poly-hydroxyl molecule, AA plays a dual role in promoting antibody production and neutralizing the harmful impact of free radicals [6].DA, a catecholamine neurotransmitter, contributes to nervous system regulation [7].UA serves as a purine metabolite, commonly present in living organisms [8].Several common diseases are attributed to fluctuations in the concentrations of these biomolecules, including mental illness, AIDS, Parkinson's disease, skin rashes and gout [9,10].Thus, it is important to discover an appropriate approach for swiftly and precisely identifying AA, DA and UA.
Recently, the use of MXene as a modified electrode material has been shown to enable accurate detection of AA, DA and UA [11,12].However, two factors limit the application of MXene: On the one hand, the unstable multilayer structure of MXene is prone to self-accumulation, which reduces the active sites and lowers the electron transfer rate [13].On the other hand, the functional groups on the surface of MXene caused by wet etching are unable to form covalent bonds with the biomolecules, thus reducing the detection sensitivity [14].Therefore, several strategies have been developed to resolve the above problems, such as the use of intercalating agents and surface modification/functionalization [15,16].Notably, the preparation of composites (or heterojunctions) by introducing other active materials into MXenes is considered to be the best strategy.For instance, a novel MXene/DNA/Pd/Pt material was synthesized by Zheng et al., and the sensor exhibited high selectivity against AA and H 2 O 2 [17].Amara et al. fabricated a graphitic pencil which is modified by perylene diimide/MXene (Ti 3 C 2 T x ), realizing the specific detection of DA [18].However, the performance of MXene-based materials in simultaneously detecting AA/DA/UA remains unsatisfactory due to the significant concentration differences among different biomolecules [19], their similar oxidation potentials [20] and the strong electrostatic repulsion [21].TiO 2 is recognized for its considerable potential for use in the biological sector, thanks to its affordability, customizable attributes, lack of toxicity, simple preparation process, consistent quality and improved durability [22,23].
Furthermore, the nanocomposite of MXene@TiO 2 prevents the aggregation of MXene, and it improves the stability of the electrochemical sensor and achieves reliable reproducibility [24].So far, different forms of TiO 2 have been synthesized, including nanoparticles, thin films structures, nanorods and so on [25,26].In our previous work [27], it was mentioned that materials that have a high specific surface area can enable the precise detection of AA/DA/UA because of the presence of numerous active sites.Therefore, the growth of TiO 2 nanosheets on the MXene surface can be used as a means to effectively realize the simultaneous detection of biological small molecules.
Here, Ti 3 C 2 T x coated with TiO 2 nanosheets (Ti 3 C 2 T x @TiO 2 NSs) is synthesized through a simple solvothermal process.By incorporating TiO 2 nanosheets, Ti 3 C 2 T x @TiO 2 NSs, modified glassy carbon electrode (Ti 3 C 2 T x @TiO 2 NSs/GCE) enables detection of AA/DA/UA individually and simultaneously.Additionally, Ti 3 C 2 T x @TiO 2 NSs/GCE demonstrates outstanding stability and reliable reproducibility.
Figure S2a depicts the closely aligned layered structure of the pristine Ti 3 AlC 2 .As shown in Figure S2b, the Ti 3 C 2 T x exhibits a layered accordion-like structure following treatment with HF (24 h), with each lamella measuring around 20 nm thick and a layer spacing of approximately 300 nm. Figure 1a-c illustrate the comparison of samples with varying amounts of TiCl 3 .When the TiCl 3 concentration is low (Ti 3 C 2 T x @TiO 2 NSs(0.6), Figure 1a), sparse NSs can be observed on the surface of Ti 3 C 2 T x lamellae.As the amount of TiCl 3 increases (Ti 3 C 2 T x @TiO 2 NSs(0.8), Figure 1b), the density of TiO 2 NSs on the surface of Ti 3 C 2 T x lamellae gradually increases.At the maximum TiCl 3 concentration (Ti 3 C 2 T x @TiO 2 NSs(1.0), Figure 1c), highly dense NSs, and even nanospheres composed of NSs, are formed on the surface of Ti 3 C 2 T x lamellae, indicating an excess of TiCl 3 .After annealing, the morphology remains relatively unchanged (Figure 1d-f).The TEM images of Ti 3 C 2 T x @TiO 2 NSs(0.8) are presented in Figure 1g,h.Figure 1g shows abundant NSs growing on the surface of Ti 3 C 2 T x lamellae, with lengths of 30-100 nm and thicknesses of 3-5 nm.A facet spacing of 0.253 nm wide can be observed in the edge portion of the material, corresponding to the (002) crystallographic facet of Ti 3 C 2 T x .The lattice stripe spacing (0.352 nm) of the NSs is correlated to the (101) crystal plane of anatase TiO 2 [30] (Figure 1h).The SAED image of Ti 3 C 2 T x @TiO 2 NSs is demonstrated in Figure 1i (red).The diffraction circle diameters in the SAED plots are the same as those diffracted from the (101) and (103) crystal surfaces of anatase TiO 2 , while the six bright and sharp spots exhibit a six-fold symmetry, representing the crystal planes (0110), ( 1010) and (1100) of Ti 3 C 2 T x (Figure 1i, inset).
Molecules 2024, 29, x FOR PEER REVIEW 3 of 15 treatment with HF (24 h), with each lamella measuring around 20 nm thick and a layer spacing of approximately 300 nm. Figure 1a-c illustrate the comparison of samples with varying amounts of TiCl3.When the TiCl3 concentration is low (Ti3C2Tx@TiO2 NSs(0.6), Figure 1a), sparse NSs can be observed on the surface of Ti3C2Tx lamellae.As the amount of TiCl3 increases (Ti3C2Tx@TiO2 NSs(0.8), Figure 1b), the density of TiO2 NSs on the surface of Ti3C2Tx lamellae gradually increases.At the maximum TiCl3 concentration (Ti3C2Tx@TiO2 NSs(1.0), Figure 1c), highly dense NSs, and even nanospheres composed of NSs, are formed on the surface of Ti3C2Tx lamellae, indicating an excess of TiCl3.After annealing, the morphology remains relatively unchanged (Figure 1d-f).The TEM images of Ti3C2Tx@TiO2 NSs(0.8) are presented in Figure 1g,h.Figure 1g shows abundant NSs growing on the surface of Ti3C2Tx lamellae, with lengths of 30-100 nm and thicknesses of 3-5 nm.A facet spacing of 0.253 nm wide can be observed in the edge portion of the material, corresponding to the (002) crystallographic facet of Ti3C2Tx.The lattice stripe spacing (0.352 nm) of the NSs is correlated to the (101) crystal plane of anatase TiO2 [30] (Figure 1h).The SAED image of Ti3C2Tx@TiO2 NSs is demonstrated in Figure 1i (red).The diffraction circle diameters in the SAED plots are the same as those diffracted from the (101) and (103) crystal surfaces of anatase TiO2, while the six bright and sharp spots exhibit a sixfold symmetry, representing the crystal planes (0110), ( 1010) and (1100) of Ti3C2Tx (Figure 1i, inset).
Figure 2a shows the full XPS spectra of Ti3C2Tx and Ti3C2Tx@TiO2 NSs.The red curve in Figure 2a represents Ti3C2Tx, exhibiting characteristic peaks for C 1s, Ti 2p, O 1s and F 1s at 282 eV, 457 eV, 529 eV and 683 eV, respectively.The black curve in Figure 2a corresponds to Ti3C2Tx@TiO2 NSs, where the characteristic peak at 682 eV for F 1s is absent.This observation suggests that the Ti 3+ of TiCl3 has replaced the -F functional groups.The Ti 2p profile (Figure 2b) reveals the energy levels of 461.  Figure 2a shows the full XPS spectra of Ti 3 C 2 T x and Ti 3 C 2 T x @TiO 2 NSs.The red curve in Figure 2a represents Ti 3 C 2 T x , exhibiting characteristic peaks for C 1s, Ti 2p, O 1s and F 1s at 282 eV, 457 eV, 529 eV and 683 eV, respectively.The black curve in Figure 2a corresponds to Ti 3 C 2 T x @TiO 2 NSs, where the characteristic peak at 682 eV for F 1s is absent.This observation suggests that the Ti 3+ of TiCl 3 has replaced the -F functional groups.The Ti 2p profile (Figure 2b) reveals the energy levels of 461.The process of morphological evolution involved in the fabrication of Ti 3 C 2 T x @TiO 2 NSs is illustrated in Figure 3. Initially, Ti 4+ ions are generated through the reaction of Ti 3+ of Molecules 2024, 29, 2915 4 of 14 TiCl 3 with dissolved oxygen in the solution [31,32] (Equations (S1) and (S2)).Subsequently, these positively charged Ti 4+ ions uniformly adhere to the negatively charged surface of Ti 3 C 2 T x .Concurrently, Ti 4+ ions react with ethylene glycol to form titanium glycolate crystal nucleuses (Ti(OCH 2 CH 2 O) 2 ) [33] (Equation (S3)).As the solvothermal process proceeds, a portion of Ti(OCH 2 CH 2 O) 2 undergoes hydrolysis, leading to the development of a small quantity of anatase TiO 2 on the surface of Ti 3 C 2 T x .In the final annealing step, the remaining unreacted Ti(OCH 2 CH 2 O) 2 is completely transformed into anatase TiO 2 , while the carbon-containing species are entirely eliminated (Equation (S4)).The process of morphological evolution involved in the fabrication of Ti3C2Tx @Ti NSs is illustrated in Figure 3. Initially, Ti 4+ ions are generated through the reaction of T of TiCl3 with dissolved oxygen in the solution [31,32] (Equations (S1) and (S2)).Subs quently, these positively charged Ti 4+ ions uniformly adhere to the negatively charg surface of Ti3C2Tx.Concurrently, Ti 4+ ions react with ethylene glycol to form titanium gl colate crystal nucleuses (Ti(OCH2CH2O)2) [33] (Equation (S3)).As the solvothermal pr cess proceeds, a portion of Ti(OCH2CH2O)2 undergoes hydrolysis, leading to the develo ment of a small quantity of anatase TiO2 on the surface of Ti3C2Tx.In the final anneali step, the remaining unreacted Ti(OCH2CH2O)2 is completely transformed into anata TiO2, while the carbon-containing species are entirely eliminated (Equation (S4)).

Electrochemical Behaviors of Ti 3 C 2 T x @TiO 2 NSs
Figure 4a shows the electrochemical performance of Ti 3 C 2 T x @TiO 2 NSs/GCE, Ti 3 C 2 T x / GCE and GCE using CV curves in 0.1 M KCl solution with 0.5 mM K 3 [Fe (CN) 6 ].The relative peak currents, listed in decreasing order, are as follows: Ti 3 C 2 T x @TiO 2 NSs(0.8)/GCE> Ti 3 C 2 T x @TiO 2 NSs(1.0)/GCE> Ti 3 C 2 T x @TiO 2 NSs(0.6)/GCE> Ti 3 C 2 T x /GCE > GCE.As shown in Figure 4b, the impedance of the different electrodes was obtained by EIS (voltage amplitude of 10 mV, 10 −2 -10 6 Hz).The electron transfer process occurring at the electrode surface was indicated by the semicircular arc observed at higher frequencies.The charge transfer resistance (R ct ) of the electrodes can be represented by the semicircular arc diameter of the fitted plot line.Based on the Randles equivalent circuit (Figure 4b, inset), Ti 3 C 2 T x @TiO 2 NSs(0.8)/GCEexhibits the lowest charge transfer impedance (R ct = 531 Ω), followed by Ti 3 C 2 T x /GCE (868.7 Ω), Ti 3 C 2 T x @TiO 2 NSs(0.6)/GCE(608.7 Ω) and Ti 3 C 2 T x @TiO 2 NSs(1.0)/GCE(1183 Ω).Ti3C2Tx@TiO2 NSs(1.0)/GCE> Ti3C2Tx@TiO2 NSs(0.6)/GCE> GCE (Figure 4f).Since Ti3C2Tx@TiO2 NSs(0.8)/GCEshowed excellent electrochemical detection capability, it was used as the modified electrode material for the additional experiments.The oxidation kinetics of AA, DA and UA on the Ti3C2Tx@TiO2 NSs/GCE electrode were studied using CV (pH 7.4) under various scan rates (20 mV/s-100 mV/s).The peak currents of AA, DA and UA show strong linear correlation with the scan rate (Figure 5ac), while the peak potentials are gradually biased toward large overpotentials, indicating kinetic limitations in the reaction [34].As shown in Figure 5d-f, the linear correlation coefficient (R 2 ) between the oxidation current and square root of the scan rate was determined to be 0.9868, 0.9801 and 0.9700 for AA, DA and UA.Hence, it can be confirmed that the oxidation reaction of AA, DA and UA on Ti3C2Tx@TiO2 NSs/GCE was controlled by a diffusion process [35].As shown in Figure 4c, which displays the CV curves, the peak current of Ti 3 C 2 T x @TiO 2 NSs(0.8)/GCErises in a linear fashion as the scan rate increases (20-100 mV/s).Furthermore, the redox peaks of the electrodes display a good linear relationship with the square root of scan rate (the inset of Figure 4c).The active surface area of Ti 3 C 2 T x @TiO 2 NSs/GCE was calculated using the Randles-Sevcik equation: where I p represents the peak oxidation current, n is the number of electron transfers (n = 1), A is the active surface area, D is the diffusion coefficient (7.6 × 10 −6 cm 2 s −1 for [Fe(CN) 6 ] 4+ ), C* is the concentration of [Fe(CN) 6 ] 4+ and υ is the scanning rate.The active surface area of Ti 3 C 2 T x @TiO 2 NSs(0.8)/GCE was calculated as approximately 0.74 cm 2 , which is nine times greater than that of bare GCE (0.08 cm 2 ).The TiO 2 NSs grown on the surface of Ti 3 C 2 T x effectively increased the area of catalytic activity.As shown in Figure 4d, the reaction peak could not be detected by Ti 3 C 2 T x /GCE.The oxidation peak of AA is shifted to a lower reaction potential when Ti 3 C 2 T x @TiO 2 NSs/GCE is employed.The peak current, listed in descending order, is as follows: Ti 3 C 2 T x @TiO 2 NSs(0.8)/GCE> Ti 3 C 2 T x @TiO 2 Molecules 2024, 29, 2915 6 of 14 NSs(0.6)/GCE> Ti 3 C 2 T x @TiO 2 NSs(1.0)/GCE> GCE.As shown in Figure 4e, all five electrodes can detect the oxidation peak of DA, with the peak current ordered as follows: Ti 3 C 2 T x @TiO 2 NSs(0.8)/GCE> Ti 3 C 2 T x /GCE > GCE > Ti 3 C 2 T x @TiO 2 NSs(1.0)/GCE> Ti 3 C 2 T x @TiO 2 NSs(0.6)/GCE.The five oxidation peaks of UA are clearly distinguished, with the peak current ordered as follows: Ti 3 C 2 T x @TiO 2 NSs(0.8)/GCE> Ti 3 C 2 T x /GCE > Ti 3 C 2 T x @TiO 2 NSs(1.0)/GCE> Ti 3 C 2 T x @TiO 2 NSs(0.6)/GCE> GCE (Figure 4f).Since Ti 3 C 2 T x @TiO 2 NSs(0.8)/GCEshowed excellent electrochemical detection capability, it was used as the modified electrode material for the additional experiments.
The oxidation kinetics of AA, DA and UA on the Ti 3 C 2 T x @TiO 2 NSs/GCE electrode were studied using CV (pH 7.4) under various scan rates (20 mV/s-100 mV/s).The peak currents of AA, DA and UA show strong linear correlation with the scan rate (Figure 5a-c), while the peak potentials are gradually biased toward large overpotentials, indicating kinetic limitations in the reaction [34].As shown in Figure 5d-f, the linear correlation coefficient (R 2 ) between the oxidation current and square root of the scan rate was determined to be 0.9868, 0.9801 and 0.9700 for AA, DA and UA.Hence, it can be confirmed that the oxidation reaction of AA, DA and UA on Ti 3 C 2 T x @TiO 2 NSs/GCE was controlled by a diffusion process [35].The oxidation kinetics of AA, DA and UA on the Ti3C2Tx@TiO2 NSs/GCE electrode were studied using CV (pH 7.4) under various scan rates (20 mV/s-100 mV/s).The peak currents of AA, DA and UA show strong linear correlation with the scan rate (Figure 5ac), while the peak potentials are gradually biased toward large overpotentials, indicating kinetic limitations in the reaction [34].As shown in Figure 5d-f, the linear correlation coefficient (R 2 ) between the oxidation current and square root of the scan rate was determined to be 0.9868, 0.9801 and 0.9700 for AA, DA and UA.Hence, it can be confirmed that the oxidation reaction of AA, DA and UA on Ti3C2Tx@TiO2 NSs/GCE was controlled by a diffusion process [35].6a-c show thatTi 3 C 2 T x @TiO 2 NSs/GCE exhibits the individual detection of AA, DA and UA under concentrations ranging from 40 to 300 µM, 40 to 500 µM and 50 to 600 µM, respectively.Figure 6d-f show the R 2 a of AA, DA and UA as 0.9994, 0.9941 and 0.9975, respectively.The LOD for AA, DA and UA was determined to be 1.12 µM, 0.35 µM and 0.47 µM, respectively.
Figure 7a demonstrates the simultaneous determination of AA and DA through DPV on Ti 3 C 2 T x @TiO 2 NSs/GCE.A noticeable difference was observed in the peak potentials of AA (0.27 V) and DA (0.46 V).For AA (80-280 µM) and DA (10-60 µM), a direct correlation was identified between peak currents and concentrations, resulting in R 2 values of 0.9845 and 0.9922, respectively (Figure 7b,c).Similarly, Figure 7d illustrates the distinct separation of DA (0.33 V) and UA (0.54 V) oxidation peak potentials.As shown in Figure 7e,f, a linear response was observed for DA (40-400 µM) and UA (40-400 µM) with R 2 values of 0.9764 and 0.9841, respectively.Meanwhile, it can be observed that altering the concentration of a single solute resulted in a corresponding linear change in the detection current of the AA-UA (Figures S3 and S4).These results serve as evidence for the dependable detection sensitivity exhibited by Ti 3 C 2 T x @TiO 2 NSs/GCE.scan rates on Ti3C2Tx@TiO2 NSs/GCE.(d-f) The square root of the scan rates vs. peak current of AA/DA/UA.Error bars based on S/N = 3.

2.3.
The Use of Ti3C2Tx@TiO2 NSs/GCE for Separate and Simultaneous Detection of AA, DA and UA Figure 6 demonstrates the separate detection of AA, DA and UA on Ti3C2Tx@TiO2 NSs/GCE in 0.1 M PBS (pH 7.4) through the use of DPV. Figure 6a-c show thatTi3C2Tx@TiO2 NSs/GCE exhibits the individual detection of AA, DA and UA under concentrations ranging from 40 to 300 µM, 40 to 500 µM and 50 to 600 µM, respectively.Figure 6d-f show the R 2 a of AA, DA and UA as 0.9994, 0.9941 and 0.9975, respectively.The LOD for AA, DA and UA was determined to be 1.12 µM, 0.35 µM and 0.47 µM, respectively.Figure 7a demonstrates the simultaneous determination of AA and DA through DPV on Ti3C2Tx@TiO2 NSs/GCE.A noticeable difference was observed in the peak potentials of AA (0.27 V) and DA (0.46 V).For AA (80-280 µM) and DA (10-60 µM), a direct correlation was identified between peak currents and concentrations, resulting in R 2 values of 0.9845 and 0.9922, respectively (Figure 7b,c).Similarly, Figure 7d illustrates the distinct separation of DA (0.33 V) and UA (0.54 V) oxidation peak potentials.As shown in Figure 7e,f, a linear response was observed for DA (40-400 µM) and UA (40-400 µM) with R 2 values of 0.9764 and 0.9841, respectively.Meanwhile, it can be observed that altering the concentration of a single solute resulted in a corresponding linear change in the detection current of the AA-UA (Figures S3 and S4).These results serve as evidence for the dependable detection sensitivity exhibited by Ti3C2Tx@TiO2 NSs/GCE.
It should be noted that when AA and UA coexisted in solution, no discernible peak splitting phenomenon was observed (Figure S5a-c).At the same time, the oxidation potential was shifted higher upon the addition of AA or UA.This phenomenon is attributed to the close oxidation potential of AA and UA; the offset of the AA and UA peaks is due to the catalytic oxidation of UA by AA [36].The DPV curves of the simultaneous determination of AA, DA and UA using Ti3C2Tx@TiO2 NSs/GCE are shown in Figure 8.As depicted in Figure 8a, the oxidation peak potentials of AA, DA and UA were observed to be 0.06 V, 0.37 V and 0.55 V, respectively.Additionally, Figure 8b-d   It should be noted that when AA and UA coexisted in solution, no discernible peak splitting phenomenon was observed (Figure S5a-c).At the same time, the oxidation potential was shifted higher upon the addition of AA or UA.This phenomenon is attributed to the close oxidation potential of AA and UA; the offset of the AA and UA peaks is due to the catalytic oxidation of UA by AA [36].
The DPV curves of the simultaneous determination of AA, DA and UA using Ti 3 C 2 T x @TiO 2 NSs/GCE are shown in Figure 8.As depicted in Figure 8a, the oxidation peak potentials of AA, DA and UA were observed to be 0.06 V, 0.37 V and 0.55 V, respectively.Additionally, Figure 8b-d highlight the linear relationship between peak currents and concentrations, with observed ranges of 200-1000 µM (R 2 = 0.9875) for AA, 40-300 µM (R 2 = 0.9875) for DA and 50-400 µM (R 2 = 0.9991) for UA.The LOD was determined to be 2.91 µM, 0.19 µM and 0.25 µM for AA, DA and UA, respectively.Subsequently, this work was compared with previous work on electrochemical sensors (Table 1).There are several key advantages in our work: Firstly, our synthesis method is simple, and the preparation conditions are gentle, in contrast to the complex synthesis processes and high-temperature preparation conditions commonly found in the existing literature [37][38][39].Secondly, our use of non-precious metals is more cost-effective than the use of precious metals; even though precious metals can enhance material sensitivity, they also escalate manufacturing costs [40,41].Materials such as MXene and TiO 2 present a promising low-cost alternative.Lastly, our work demonstrates a lower Limit of Detection (LOD) for AA when using MXenebased materials.While the negative electronegativity of the MXene surface traditionally hinders AA detection, the TiO 2 synthesized via the solvothermal method effectively optimizes the surface charge of MXene, enabling the simultaneous detection of AA, DA and UA, with a particularly noteworthy LOD of 2.9 µM for AA.It is noteworthy that the inclusion of DA exhibited an ability to separate the overlapping peaks of AA and UA.The oxidation products of AA and UA absorb or electropoly-merize on the electrodes and reduce their detection sensitivity [42,43].When DA is added, the oxidation products of DA react with AA, thereby mitigating electrode contamination.Additionally, DA is recognized as a catalyst for both AA and UA [44,45].Upon the addition of DA, it underwent reactions with AA and UA to generate intermediate complexation products, effectively lowering the activation energy of AA and increasing the activation energy of UA.Consequently, the peaks corresponding to AA and UA resurfaced during simultaneous detection.Based on previous studies [40,41], simultaneous detection of AA/DA/UA faces significant challenges due to mutual interference, such as strong electrostatic interactions and overlapping oxidation potentials [36,38,53].Through the solvothermal process, Ti 3 C 2 T x @TiO 2 NSs exhibit two distinct advantages that enhance their ability to detect these molecules simultaneously.First, the replacement of -F by additional Ti 3+ ions significantly changes the surface charge of Ti 3 C 2 T x , reducing the mutual repulsion of the analytes.Secondly, a considerable number of TiO 2 NSs are grown on the Ti 3 C 2 T x lamellae surface, which decreases the ion diffusion length [54] and increases the specific surface area of the Ti 3 C 2 T x @TiO 2 NSs.Finally, the formation of Ti 3 C 2 T x and TiO 2 heterojunctions facilitates the transport of charge carriers [39].As a result, the integration of Ti 3 C 2 T x and TiO 2 allows for the creation of a highly precise and sensitive sensor capable of detecting biological small molecules with high resolution.

Reproducibility, Interference Immunity Testing and Stability Analysis
Several potential coexisting substances were detected using the amperometric method to verify the interference resistance of Ti 3 C 2 T x @TiO 2 NSs/GCE (Figure 9a-c).The continuous addition of the interfering substance did not affect the detection current of the AA/DA/UA.Therefore, Ti 3 C 2 T x @TiO 2 NSs/GCE demonstrated excellent anti-interference capability for detecting AA, DA and UA.Five consecutive measurements were conducted in the solution containing 200 µM AA, 60 µM DA and 40 µM UA, investigating the stability of Ti 3 C 2 T x @TiO 2 NSs/GCE.The results shown in Figure 9d indicate that the peaks of the five consecutive measurements remained almost unchanged, with a calculated RSD of 0.54%.This suggests that Ti 3 C 2 T x @TiO 2 NSs/GCE exhibits good stability under testing conditions.Furthermore, a DPV test was performed after the electrode was exposed to air for 5 days to evaluate the environmental stability of Ti 3 C 2 T x @TiO 2 NSs/GCE (Figure 9e).The peak currents remained nearly constant, indicating the excellent environmental stability of Ti 3 C 2 T x @TiO 2 NSs/GCE.As shown in Figure 9f, the simultaneous detection of AA/DA/UA was utilized on five different Ti 3 C 2 T x @TiO 2 NSs/GCE electrodes in 0.1 M PBS using DPV.The oxidation peak currents of AA (200 M), DA (40 M) and UA (60 M) were not significantly altered.
the five consecutive measurements remained almost unchanged, with a calculated RSD of 0.54%.This suggests that Ti3C2Tx@TiO2 NSs/GCE exhibits good stability under testing conditions.Furthermore, a DPV test was performed after the electrode was exposed to air for 5 days to evaluate the environmental stability of Ti3C2Tx@TiO2 NSs/GCE (Figure 9e).The peak currents remained nearly constant, indicating the excellent environmental stability of Ti3C2Tx@TiO2 NSs/GCE.As shown in Figure 9f, the simultaneous detection of AA/DA/UA was utilized on five different Ti3C2Tx@TiO2 NSs/GCE electrodes in 0.1 M PBS using DPV.The oxidation peak currents of AA (200 M), DA (40 M) and UA (60 M) were not significantly altered.

Apparatus
An electrochemical workstation was used to obtain the electrochemical properties of the modified electrode.The model of the electrochemical workstation is CHI, 660E (Dalian, China).The surface morphology of the material was obtained by scanning electron microscopy (SEM) using the Nova 230, FEI (Hillsboro, OR, USA) model.The ultrastructure of the material was obtained by transmission electron microscopy, model Tecnai G2 F30 S-TWIN, FEI (USA).The high-resolution electron microscopy was obtained using JEM-2010 (HRTEM, JEOL Japan Electronics Co., Ltd, Akishima-shi in Japan).The structure and phase of the materials were analyzed using X-ray powder diffraction (XRD), model TTRIII, Rigaku (Tokyo, Japan) with Cu Kα radiation (λ = 1.5406Å) and X-ray photoelectron spectroscopy (XPS), using the VG Multilab 2009 (Manchester, UK) model.

Synthesis of Ti 3 C 2 T x @TiO 2 NSs
Scheme 1 shows the preparation process of Ti 3 C 2 T x @TiO 2 NSs.Ti 3 C 2 T x was obtained by the one-step wet etching method [55].The Ti 3 AlC 2 powder (1 g) was added gradually to a solution of HF (40 mL, 40 wt%) and allowed to react at 35 • C for 24 h with stirring at a speed of 500 rpm.The etched substance was rinsed with deionized water (DI) until the pH reached above 6.0.High-speed centrifugation (300 rpm, 3 min) allows the powder to be separated from the DI.Finally, the gelatinous Ti 3 C 2 T x was dehydrated under vacuum freezing conditions (−80 • C, 2 h).A solution was prepared by mixing 30 mL of EG with an equal volume (k mL) of TiCl 3 and DI (k = 0.6, 0.8, 1.0) and stirring it for approximately 30 min.Subsequently, 20 mg of as-prepared Ti 3 C 2 T x was added to the solution and sonicated for 30 min to create a homogeneous suspension.The suspension was moved to a stainless steel autoclave and subjected to heat treatment at 150 • C for 12 h before being cooled to room temperature.The precipitates were washed with ethyl alcohol, and dried for 2 days.Finally, the precipitates were annealed at 350 • C in flowing Ar for 2 h.The obtained granular powders were marked as Ti 3 C 2 T x @TiO 2 NSs(0.6),Ti 3 C 2 T x @TiO 2 NSs(0.8), and Ti 3 C 2 T x @TiO 2 NSs(1.0),respectively.model TTRIII, Rigaku (Tokyo, Japan) with Cu Kα radiation (λ = 1.5406Å) and X-ray photoelectron spectroscopy (XPS), using the VG Multilab 2009 (Manchester, UK) model.

Synthesis of Ti3C2Tx@TiO2 NSs
Scheme 1 shows the preparation process of Ti3C2Tx@TiO2 NSs.Ti3C2Tx was obtained by the one-step wet etching method [55].The Ti3AlC2 powder (1 g) was added gradually to a solution of HF (40 mL, 40 wt%) and allowed to react at 35 °C for 24 h with stirring at a speed of 500 rpm.The etched substance was rinsed with deionized water (DI) until the pH reached above 6.0.High-speed centrifugation (300 rpm, 3 min) allows the powder to be separated from the DI.Finally, the gelatinous Ti3C2Tx was dehydrated under vacuum freezing conditions (−80 °C, 2 h).A solution was prepared by mixing 30 mL of EG with an equal volume (k mL) of TiCl3 and DI (k = 0.6, 0.8, 1.0) and stirring it for approximately 30 min.Subsequently, 20 mg of as-prepared Ti3C2Tx was added to the solution and sonicated for 30 min to create a homogeneous suspension.The suspension was moved to a stainless steel autoclave and subjected to heat treatment at 150 °C for 12 h before being cooled to room temperature.The precipitates were washed with ethyl alcohol, and dried for 2 days.Finally, the precipitates were annealed at 350 °C in flowing Ar for 2 h.The obtained granular powders were marked as Ti3C2Tx@TiO2 NSs(0.6),Ti3C2Tx@TiO2 NSs(0.8), and Ti3C2Tx@TiO2 NSs(1.0),respectively.Scheme 1.The synthesis process of Ti3C2Tx@TiO2 NSs.

Fabrication of the Ti3C2Tx@TiO2 NSs Electrode
Scheme 1.The synthesis process of Ti 3 C 2 T x @TiO 2 NSs.
3.4.Fabrication of the Ti 3 C 2 T x @TiO 2 NSs Electrode Aluminum oxide powder with particle sizes of 1.0, 0.3 and 0.05 µm was first placed on a nylon cloth for polishing the glassy carbon electrode (GCE).The electrodes were then washed continuously for 5 min using acetone, ethanol and deionized water (DI), respectively, until it was ensured that a smooth electrode surface was obtained.Then, 10 mg of Ti 3 C 2 T x @TiO 2 NSs was dispersed in 10 mL of DI using the ultrasonic method.Next, 3 µL of the aforementioned Ti 3 C 2 T x @TiO 2 NSs solution was dropped on the GCE surface and allowed to dry under nitrogen atmosphere to form a flat film.For comparison, the preparation process of the remaining electrodes was performed in a similar way.

Electrochemical Measurement
The main purpose of this work is to discuss the electrochemical detection performance of modified electrodes for AA/DA/UA in human physiological situations.Therefore, pH 7.4 was chosen as the detection solution condition in this work.The pH of the solution was regulated by PBS.GCE or Ti 3 C 2 T x /GCE or Ti 3 C 2 T x @TiO 2 NSs/GCE served as the working electrode.Ag/AgCl was used as the reference electrode.Platinum (Pt) served as the counter electrode.The electrochemical detection capability was evaluated using differential pulse voltammetry (DPV).Furthermore, the time-amperometric method was used to assess the ability to resist interferences.The interferents included NaCl, KCl, K 2 SO 4 , Na 2 SO 4 , glucose, glutamate, glycine and lysine.

Conclusions
In this work, Ti 3 C 2 T x coated with TiO 2 nanosheets was synthesized using a simple solvothermal process.The Ti 3 C 2 T x @TiO 2 NSs-modified GCE has been proven to enable the simultaneous detection of AA at concentrations ranging from 200 to 1000 µM, DA at concentrations ranging from 40 to 300 µM, and UA at concentrations ranging from 50 to 400 µM.The LOD achieved for these analytes is 2.91 µM (AA), 0.19 µM (DA) and 0.25 µM (UA).Furthermore, the Ti 3 C 2 T x @TiO 2 /GCE exhibits excellent resistance to interference, good repeatability and high selectivity.Two key advantages of the Ti 3 C 2 T x @TiO 2 are demonstrated to enhance their capability for simultaneous detection.Firstly, the substitution of -F ions with additional Ti 3+ ions leading to a negative to neutral surface charge change of Ti 3 C 2 T x , thereby reducing electrostatic repulsion.Secondly, the growth of a mass of TiO 2 nanosheets on the surface of Ti 3 C 2 T x significantly increases the specific surface area and charge transport properties of the composite material.This work provides a novel approach for AA, DA and UA recognition in MXene-based composites.In future electrochemical detection, researchers may utilize targeted nanomaterial modifications between MXene lamellae to achieve precise detection of various analytes.This approach not only mitigates the self-accumulation of MXene but also increase the specific active sites, which is conducive to improving the selectivity and sensitivity of MXene-based materials.
Figure2ashows the full XPS spectra of Ti3C2Tx and Ti3C2Tx@TiO2 NSs.The red curve in Figure2arepresents Ti3C2Tx, exhibiting characteristic peaks for C 1s, Ti 2p, O 1s and F 1s at 282 eV, 457 eV, 529 eV and 683 eV, respectively.The black curve in Figure2acorresponds to Ti3C2Tx@TiO2 NSs, where the characteristic peak at 682 eV for F 1s is absent.This observation suggests that the Ti 3+ of TiCl3 has replaced the -F functional groups.The Ti 2p profile (Figure2b) reveals the energy levels of 461.2 and 457.2 eV, corresponding to Ti 2p1/2 and Ti 2p3/2, which indicate the formation of anatase TiO2.Concerning the C 1s profile
Figure2ashows the full XPS spectra of Ti 3 C 2 T x and Ti 3 C 2 T x @TiO 2 NSs.The red curve in Figure2arepresents Ti 3 C 2 T x , exhibiting characteristic peaks for C 1s, Ti 2p, O 1s and F 1s at 282 eV, 457 eV, 529 eV and 683 eV, respectively.The black curve in Figure2acorresponds to Ti 3 C 2 T x @TiO 2 NSs, where the characteristic peak at 682 eV for F 1s is absent.This observation suggests that the Ti 3+ of TiCl 3 has replaced the -F functional groups.The Ti 2p profile (Figure2b) reveals the energy levels of 461.2 and 457.2 eV, corresponding to Ti 2p 1/2 and Ti 2p 3/2 , which indicate the formation of anatase TiO 2 .Concerning the C 1s profile (Figure 2c), characteristic peaks occur at 285.8, 283.2 and 281.7 eV, corresponding to O-C=O, C-O, and C-C, respectively.Furthermore, the O 1s profile (Figure 2d) shows peaks at 529.5 and 527.3 eV that represent C-O and Ti-O bonds in TiO 2 , respectively.These findings provide evidence for the disappearance of -F functional groups and the concomitant generation of TiO 2 on the surface of Ti 3 C 2 T x .The process of morphological evolution involved in the fabrication of Ti 3 C 2 T x @TiO 2 NSs is illustrated in Figure3.Initially, Ti 4+ ions are generated through the reaction of Ti 3+ of

Molecules 2024 ,
29,  x FOR PEER REVIEW 4 of (Figure2c), characteristic peaks occur at 285.8, 283.2 and 281.7 eV, corresponding to C=O, C-O, and C-C, respectively.Furthermore, the O 1s profile (Figure2d) shows pea at 529.5 and 527.3 eV that represent C-O and Ti-O bonds in TiO2, respectively.These fin ings provide evidence for the disappearance of -F functional groups and the concomita generation of TiO2 on the surface of Ti3C2Tx.

Figure 3 .
Figure 3. Growth mechanism of TiO 2 nanosheet on Ti 3 C 2 T x surface.

Figure 5 .
Figure 5. CV curves recorded in (a) 100 µM AA, (b) 100 µM DA and (c) 100 µM UA, with increasing scan rates on Ti 3 C 2 T x @TiO 2 NSs/GCE.(d-f) The square root of the scan rates vs. peak current of AA/DA/UA.Error bars based on S/N = 3.

2. 3 .
Figure6demonstrates the separate detection of AA, DA and UA on Ti 3 C 2 T x @TiO 2 NSs/GCE in 0.1 M PBS (pH 7.4) through the use of DPV.Figure6a-cshow thatTi 3 C 2 T x @TiO 2 NSs/GCE exhibits the individual detection of AA, DA and UA under concentrations ranging from 40 to 300 µM, 40 to 500 µM and 50 to 600 µM, respectively.Figure6d-fshow the R 2 a of AA, DA and UA as 0.9994, 0.9941 and 0.9975, respectively.The LOD for AA, DA and UA was determined to be 1.12 µM, 0.35 µM and 0.47 µM, respectively.Figure7ademonstrates the simultaneous determination of AA and DA through DPV on Ti 3 C 2 T x @TiO 2 NSs/GCE.A noticeable difference was observed in the peak potentials of AA (0.27 V) and DA (0.46 V).For AA (80-280 µM) and DA (10-60 µM), a direct correlation was identified between peak currents and concentrations, resulting in R 2 values of 0.9845 and 0.9922, respectively (Figure7b,c).Similarly, Figure7dillustrates the distinct separation of DA (0.33 V) and UA (0.54 V) oxidation peak potentials.As shown in Figure7e,f, a linear

Figure
Figure6demonstrates the separate detection of AA, DA and UA on Ti 3 C 2 T x @TiO 2 NSs/GCE in 0.1 M PBS (pH 7.4) through the use of DPV.Figure6a-cshow thatTi 3 C 2 T x @TiO 2 NSs/GCE exhibits the individual detection of AA, DA and UA under concentrations ranging from 40 to 300 µM, 40 to 500 µM and 50 to 600 µM, respectively.Figure6d-fshow the R 2 a of AA, DA and UA as 0.9994, 0.9941 and 0.9975, respectively.The LOD for AA, DA and UA was determined to be 1.12 µM, 0.35 µM and 0.47 µM, respectively.Figure7ademonstrates the simultaneous determination of AA and DA through DPV on Ti 3 C 2 T x @TiO 2 NSs/GCE.A noticeable difference was observed in the peak potentials of AA (0.27 V) and DA (0.46 V).For AA (80-280 µM) and DA (10-60 µM), a direct correlation was identified between peak currents and concentrations, resulting in R 2 values of 0.9845 and 0.9922, respectively (Figure7b,c).Similarly, Figure7dillustrates the distinct separation of DA (0.33 V) and UA (0.54 V) oxidation peak potentials.As shown in Figure7e,f, a linear

Figure 7 .
Figure 7. DPV curves based on different concentrations when detecting (a) AA and DA and (d) DA and UA on Ti3C2Tx@TiO2 NSs/GCE in 0.1 M PBS (pH 7.4).(b,c) Calibration curves for the current vs. concentrations of AA and UA, (e,f) calibration curves for the current vs. concentrations of DA and UA.Error bars based on S/N = 3.

Figure 7 .
Figure 7. DPV curves based on different concentrations when detecting (a) AA and DA and (d) DA and UA on Ti 3 C 2 T x @TiO 2 NSs/GCE in 0.1 M PBS (pH 7.4).(b,c) Calibration curves for the current vs. concentrations of AA and UA, (e,f) calibration curves for the current vs. concentrations of DA and UA.Error bars based on S/N = 3.

Table 1 .
The performance of sensors for the electrochemical simultaneous detection of AA, DA and UA has been reported recently.ElectrodeLinear Range (µM) LOD (µM)

Table 1 .
The performance of sensors for the electrochemical simultaneous detection of AA, DA and UA has been reported recently.