A Double-Deck Structure of Reduced Graphene Oxide Modified Porous Ti3C2Tx Electrode towards Ultrasensitive and Simultaneous Detection of Dopamine and Uric Acid

Considering the vital physiological functions of dopamine (DA) and uric acid (UA) and their coexistence in the biological matrix, the development of biosensing techniques for their simultaneous and sensitive detection is highly desirable for diagnostic and analytical applications. Therefore, Ti3C2Tx/rGO heterostructure with a double-deck layer was fabricated through electrochemical reduction. The rGO was modified on a porous Ti3C2Tx electrode as the biosensor for the detection of DA and UA simultaneously. Debye length was regulated by the alteration of rGO mass on the surface of the Ti3C2Tx electrode. Debye length decreased with respect to the rGO electrode modified with further rGO mass, indicating that fewer DA molecules were capable of surpassing the equilibrium double layer and reaching the surface of rGO to achieve the voltammetric response of DA. Thus, the proposed Ti3C2Tx/rGO sensor presented an excellent performance in detecting DA and UA with a wide linear range of 0.1–100 μM and 1–1000 μM and a low detection limit of 9.5 nM and 0.3 μM, respectively. Additionally, the proposed Ti3C2Tx/rGO electrode displayed good repeatability, selectivity, and proved to be available for real sample analysis.


Introduction
Dopamine (DA) is a catecholamine neurotransmitter in the central nervous system which contributes to various physiological functions, including memory, stimulus-response, motion control and vasodilation. [1,2]. The abnormality of DA is clinically related to several neurological disorders, such as senile dementia, Parkinson's disease, and schizophrenia [3]. Uric acid (UA) is the major end product of purine metabolism, and an excess of UA levels may lead to serious chronic and metabolic diseases, such as gouty, hyperuricemia and kidney injury [4,5]. Considering the vital physiological functions of DA and UA and their coexistence in the biological matrix, the development of biosensing techniques for their simultaneous detection with high sensitivity is desirable for diagnostic and analytical applications [6,7].
Conventional analytical methods for the simultaneous detection of DA and UA, such as high-performance liquid chromatography (HPLC), chemiluminescene, and capillary electrophoresis, have been under development for decades [8][9][10]. As DA and UA are electrochemically active compounds, the electrochemical method has been adopted for the detection of these biomolecules with high sensitivity, simplicity and time efficiency [11][12][13]. However, the oxidation peak positions of these biomolecules are almost the same and difficult to distinguish effectively when using conventional electrodes such as glassy carbon electrodes (GCE) [14]. By using various nanomaterials modified on GCE chemically, the peak resolutions of these biomolecules have been much improved [15]. Therefore, this method has been widely adopted for the recognition of DA and UA simultaneously [16,17].
Among them, graphene has received extensive attention, due to its high surface-tovolume ratio, good electrical conductivity and high carrier mobility [18][19][20][21][22]. Kim [25]. However, since DA in human blood is usually low as 0.01-1 µM, the sensitivity of graphene-modified electrodes needs to be further improved [26]. Conventionally, using a graphene hybrid with metal (Au, Pt, Ag) nanoparticles (NPs) or carbon nanomaterial (as carbon nanotubes (CNTs)) is a common approach to increase the electrochemical activity of a modified electrode. Wang et al. synthesized novel Au NPs and reduced the graphene oxide (rGO) composite film by electrodepositing AuNPs onto the rGO surface, showing good performance in its ability to detect DA and UA with a linear range of 6.8-41.0 µM, 8.8-53.0 µM and a low detection limit of 1.4, 1.8 µM, respectively [27]. Sun et al. demonstrated a novel sensor based on graphene and Pt NPs nanocomposite by self-assembling Pt NPs onto the graphene surface, indicating its excellent performance in detecting DA and UA with a linear range of 0.03-8.13 µM, 0.05-11.9 µM and a low detection limit of 0.03, 0.05 µM, respectively [28]. Sun et al. developed a sensor based on CNTs and GO nanocomposite, exhibiting its performance in detecting DA and UA with a linear range of 5.0-500 µM, 3.0-60.0 µM and a low detection limit of 1.5, 1.0 µM, respectively [29].
Compared to the electrode modified by graphene, the detection performance of the electrode modified by graphene-based nanocomposite is improved, but it still needs to be further promoted. The interfacial binding strength of graphene-based nanocomposite and electrode may not be high either. To overcome this problem, a new and promising 2D nanomaterial with a 2D-layered structure, MXene, especially titanium carbide MXene (Ti 3 C 2 T x ), has been extensively applied as a material with a high number of electric electrodes for batteries, supercapacitors and electrochemical detection [30][31][32][33]. Due to its excellent metallicity, electrical conductivities, hydrophilic surfaces, and environmental-friendly characteristics, Ti 3 C 2 T x has been employed for the electrochemical detection of biomolecules, H 2 O 2 , and heavy metal ions [34][35][36]. Murugan et al. proposed a Ti 3 C 2 T x -modified electrode, which exhibited good performance in determining DA and UA and obtained a low detection limit of 0.06 and 0.08 µM, respectively [37]. These successful applications of Ti 3 C 2 T x in the electrochemical detection prove that Ti 3 C 2 T x is an ideal conductive matrix and improves electron transfer kinetic effectively. Particularly, the Ti-O-C covalent bonding is formed at the Ti 3 C 2 T x /rGO heterointerface via nucleophilic substitution dehydration reaction, and charge transport through the heterointerface is increased [38]. Therefore, the interfacial binding strength of Ti 3 C 2 T x /rGO heterointerface increases, resulting in an excellent electrochemical performance in detecting biomolecules. The Debye screening length, λ D , is defined as the effective thickness of the equilibrium double layer (EDL) [39]. The detection limit of biosensors is determined by λ D between the surface of sensitive nanomaterials and the electrolyte [40]. Thus, λ D can be altered effectively to obtain the low detection limit of biosensors based on the Ti 3 C 2 T x /rGO heterostructure.
In this work, we attempted to construct Ti 3 C 2 T x /rGO heterostructure with doubledeck layer through electrochemical reduction. The rGO was modified on porous Ti 3 C 2 T x electrode as the biosensor for the detection of DA and UA simultaneously. The Debye length was regulated by the alteration of rGO on the surface of Ti 3 C 2 T x electrode. As evidenced by the differential pulse voltammetry (DPV) test, this proposed Ti 3 C 2 T x /rGO sensor exhibited an excellent performance in detecting DA and UA with a linear range of 0.1-100 µM and 1-1000 µM and a low detection limit of 0.0095 and 0.3 µM, respectively. Additionally, the proposed biosensor indicated good repeatability, selectivity, and potential for real sample analysis.

Fabrication of Ti 3 C 2 T x /rGO Electrodes
The Ti 3 C 2 T x water dispersion and GO water dispersion were dispersed ultrasonically for 1 h in an ice bath. Before modification, GCE electrodes with a diameter of 3 mm were polished using a 0.05 µm alumina slurry and cleaned in deionized water and ethanol by ultrasonication. Following that, GCE was activated via repetitive potential range scanning from −1-1 V with a scan rate of 0.1 V/s in 0.5 M H 2 SO 4 . The Ti 3 C 2 T x dispersion was uniformly dropped onto the surface of the GCE and dried, followed by GO dispersion in the same way (Ti 3 C 2 T x /GO electrode). The Ti 3 C 2 T x /rGO-modified GCE was obtained through the electrochemical reduction method of immersing Ti 3 C 2 T x /GO into PBS with cyclic voltammetry (CV) sweeping in the potential range of 0.0-1.4 V at a scan rate of 0.1 V/s for 5 cycles, which was defined as the experimental group (Ti 3 C 2 T x /rGO electrode). As shown in Figure S1 (see Supplementary Materials), a large reduction peak was observed at the potential peak position of −1.23 V in the first cycle, and vanished subsequently, which referred to the electrochemical reduction process of GO to rGO. As controls, Ti 3 C 2 T xmodified GCE (Ti 3 C 2 T x electrode) and rGO modified-GCEs (rGO electrode) were also prepared using the same method.

Characterizations
Field emission scanning electron microscope (FE-SEM QUANTA 250 FEG, FEI, Hillsboro, OR, USA) and energy dispersive spectroscopy (EDS)-mapping were applied to observe the morphology of the nanomaterials, including CNTs and AgNWs, separately, and the composite material. The surface compositions and chemical states were carried out by Raman spectroscopy (Renishaw in Via Reflex, Renishaw plc, Wotton-under-Edge, London, UK) with a laser wavelength of 532 nm and X-ray photoelectron spectroscopy (XPS, Axis Ultra DLD, Kratos Analytical, Manchester, UK), respectively. All electrochemi-cal experiments were conducted with a CHI660e electrochemical workstation (Shanghai Chenhua Co., Ltd., Shanghai, China).

Electrochemical Tests
The electrolyte was a phosphate buffer solution (PBS) which contained 137 mM NaCl, 102.7 mM KCl, 8.1 mM Na 2 HPO 4 , and 1.8 mM KH 2 PO 4 (pH ≈ 7.4). The bare GCE, Ti 3 C 2 T x , rGO, and Ti 3 C 2 T x /rGO were applied as working electrodes, which were separately immersed into PBS containing different DA concentrations, from 9.5 nM to 100 µM, and different UA concentrations, from 0.3 µM to 1000 µM, respectively, to compare their electrochemical performances. A saturated calomel electrode (SCE, Pt Hg(l)|Hg 2 Cl 2 (s)|KCl (saturated)) and a Pt electrode were applied as a reference electrode and counter electrode, respectively. CV, DPV, and electrochemical impedance spectroscopy (EIS) tests were conducted to analyze the electrochemical behavior of different concentrations of DA and UA on the GCE modified with various materials. CV curves (five cycles) were recorded from 0 to 0.5 V with scan rate of 0.1 V/s, while DPV tests were conducted from −0.2 to 0.5 V with an increment step of 4 mV, amplitude of 50 mV, and pulse period of 0.5 s. EIS was performed in 0.1 to 100 KHz on various modified electrodes with 10 mV amplitude of the AC voltage.

Characterization of Ti 3 C 2 T x /rGO Nanocomposite
The schematic diagram of the simultaneous electrochemical detection procedures of DA and UA on Ti 3 C 2 T x /rGO electrode is displayed in Figure 1a. The uniform Ti 3 C 2 T x and GO water dispersion were prepared through ultrasonication. GO dispersion was dropped and dried on the Ti 3 C 2 T x electrode to form a Ti 3 C 2 T x /GO electrode with a double-deck structure, and an electrochemical reduction process was applied using CV sweeping to obtain a Ti 3 C 2 T x /rGO electrode for DA and UA detection. Based on previous studies ( Figure 1b) [41], a pair of reversible peaks (Ox 1 , Re 1 ) can be interpreted as the two-electron oxidation of DA to o-dopaminoquinone. Meanwhile, a pair of reversible peaks (Ox 2 , Re 2 ) originated from the transformation of UA to dehydrourate. Specifically, oxidation peaks Ox 1 , Ox 2 at 0.185 V and 0.316 V were chosen as the characteristic peaks for quantitative analysis of the electrochemical behavior of DA and UA, respectively. the composite material. The surface compositions and chemical states were carried out b Raman spectroscopy (Renishaw in Via Reflex, Renishaw plc, Wotton-under-Edge, Lon don, UK) with a laser wavelength of 532 nm and X-ray photoelectron spectroscopy (XPS Axis Ultra DLD, Kratos Analytical, Manchester, UK), respectively. All electrochemical ex periments were conducted with a CHI660e electrochemical workstation (Shangha Chenhua Co., Ltd., Shanghai, China).

Electrochemical Tests
The electrolyte was a phosphate buffer solution (PBS) which contained 137 mM NaC 102.7 mM KCl, 8.1 mM Na2HPO4, and 1.8 mM KH2PO4 (pH ≈ 7.4). The bare GCE, Ti3C2Tx rGO, and Ti3C2Tx/rGO were applied as working electrodes, which were separately im mersed into PBS containing different DA concentrations, from 9.5 nM to 100 μM, and dif ferent UA concentrations, from 0.3 μM to 1000 μM, respectively, to compare their electro chemical performances. A saturated calomel electrode (SCE, Pt Hg(l)|Hg2Cl2 (s)|KCl (sat urated)) and a Pt electrode were applied as a reference electrode and counter electrode respectively. CV, DPV, and electrochemical impedance spectroscopy (EIS) tests were con ducted to analyze the electrochemical behavior of different concentrations of DA and UA on the GCE modified with various materials. CV curves (five cycles) were recorded from 0 to 0.5 V with scan rate of 0.1 V/s, while DPV tests were conducted from −0.2 to 0.5 V with an increment step of 4 mV, amplitude of 50 mV, and pulse period of 0.5 s. EIS was performed in 0.1 to 100 KHz on various modified electrodes with 10 mV amplitude of the AC voltage

Characterization of Ti3C2Tx/rGO Nanocomposite
The schematic diagram of the simultaneous electrochemical detection procedures o DA and UA on Ti3C2Tx/rGO electrode is displayed in Figure 1a. The uniform Ti3C2Tx and GO water dispersion were prepared through ultrasonication. GO dispersion was dropped and dried on the Ti3C2Tx electrode to form a Ti3C2Tx/GO electrode with a double-dec structure, and an electrochemical reduction process was applied using CV sweeping t obtain a Ti3C2Tx/rGO electrode for DA and UA detection. Based on previous studies (Fig  ure 1b) [41], a pair of reversible peaks (Ox1, Re1) can be interpreted as the two-electron oxidation of DA to o-dopaminoquinone. Meanwhile, a pair of reversible peaks (Ox2, Re2 originated from the transformation of UA to dehydrourate. Specifically, oxidation peak Ox1, Ox2 at 0.185 V and 0.316 V were chosen as the characteristic peaks for quantitativ analysis of the electrochemical behavior of DA and UA, respectively.  According to the morphologies of Ti 3 C 2 T x shown in Figure 2a, Ti 3 C 2 T x was well distributed on the surface of GCE, and a porous electrode with good electrical conductivity was formed. Figure 2b is an enlarged view of Figure 2a, and the corresponding EDS mapping of C, Ti, F, and O are shown. The results indicate the two-dimensional layered sheet-like structures of Ti 3 C 2 T x with good flatness. Figure 2c displays the morphologies of Ti 3 C 2 T x /rGO, exhibiting the rough surface of rGO with random wrinkles and the layered structures of Ti 3 C 2 T x . Figure 2d is an enlarged view of Figure 2c, and the corresponding EDS mapping of C, Ti, F, and O are shown. The morphology revealed the recovery of rGO film to the surface of Ti 3 C 2 T x . According to the morphologies of Ti3C2Tx shown in Figure 2a, Ti3C2Tx was well distributed on the surface of GCE, and a porous electrode with good electrical conductivity was formed. Figure 2b is an enlarged view of Figure 2a, and the corresponding EDS mapping of C, Ti, F, and O are shown. The results indicate the two-dimensional layered sheetlike structures of Ti3C2Tx with good flatness. Figure 2c displays the morphologies of Ti3C2Tx/rGO, exhibiting the rough surface of rGO with random wrinkles and the layered structures of Ti3C2Tx. Figure 2d is an enlarged view of Figure 2c, and the corresponding EDS mapping of C, Ti, F, and O are shown. The morphology revealed the recovery of rGO film to the surface of Ti3C2Tx. The Raman spectra of rGO, Ti3C2Tx and Ti3C2Tx/rGO are shown in Figure 2e. Three main peaks of rGO, namely the D band (~1350 cm −1 ), G band (~1580 cm −1 ), and 2D band (~2700 cm −1 ), correspond to random vibration of amorphous carbon (sp3 hybrid carbon) and in-plane vibration of graphitic carbon (sp2 hybrid carbon) [42]. The peak positions of Ti3C2Tx at 199 and 719 cm −1 are assigned to the out-of-plane vibrations of Ti and C atoms. The modes at 287, 369, and 624 cm −1 are the Eg group vibrations, including in-plane modes of Ti and C, and surface functional group atoms [43].  . SEM images of Ti 3 C 2 T x electrode (a) and Ti 3 C 2 T x /rGO electrode (c). (b,d) Regional enlarged view of (a,c) and the EDS mapping of element distribution of C, Ti, F, O, respectively. (e) Raman spectra of Ti 3 C 2 T x , rGO, and Ti 3 C 2 T x /rGO nanocomposite. (f) XPS survey spectra of Ti 3 C 2 T x /rGO, and Ti 2p spectra (g), C 1s spectra (h), O 1s spectra (i) spectra, respectively.
The Raman spectra of rGO, Ti 3 C 2 T x and Ti 3 C 2 T x /rGO are shown in Figure 2e. Three main peaks of rGO, namely the D band (~1350 cm −1 ), G band (~1580 cm −1 ), and 2D band (~2700 cm −1 ), correspond to random vibration of amorphous carbon (sp3 hybrid carbon) and in-plane vibration of graphitic carbon (sp2 hybrid carbon) [42]. The peak positions of Ti 3 C 2 T x at 199 and 719 cm −1 are assigned to the out-of-plane vibrations of Ti and C atoms. The modes at 287, 369, and 624 cm −1 are the E g group vibrations, including in-plane modes of Ti and C, and surface functional group atoms [43]. The peak positions of Ti 3 C 2 T x /rGO are located at 205, 287, 369, 624, 723, 1350, 1580, and 2700 cm −1 , verifying the existence of both Ti 3 C 2 T x and rGO in the composite. From the full XPS survey spectra of Ti 3 C 2 T x /rGO, F 1s, Ti 2s, O 1s and Ti 2p, C 1s appear at the binding energy of 684.8, 536.6, 496.9, 462.8 and 287.7 eV, respectively, as shown in Figure 2f, confirming the presence of four elements in the composite [44]. As depicted in Figure 2g, the Ti 2p narrow spectra of Ti 3 C 2 T x /rGO are divided into two parts: Ti 2p 3/2 and Ti 2p 1/2 . Ti 2p 3/2 spectra can be segmented into four components, which are located at 454.9 eV (Ti-C), 455.4 eV (Ti(II)), 456.3 eV (Ti(III)), and 458.8 eV (TiO 2 ). The Ti 2p 1/2 spectra can be fitted into three components, which are located at 461.1 eV (Ti-C), 462.1 eV (Ti(II)), and 462.6 eV (Ti(III)). Next, C 1s' XPS curve can be fitted into five components (Figure 2h), which correspond to 284.8 eV (C-C), 281.6 eV (C-Ti), 282.4 eV (C-Ti-O), 287.5 eV (C=O), and 288.6 eV (O-C=O) [45]. Notably, the peak of C=O and O-C=O are ascribed to the introduction of rGO and the closed interaction between Ti 3 C 2 T x and rGO [46]. The O 1s spectrum of Ti 3 C 2 T x /rGO is well fitted into two components (Figure 2i), which are centered at 531.8 eV (C-Ti-OH) and 529.6 eV (Ti-O-Ti). Therein, the oxygen-containing functional termination groups of Ti 3 C 2 T x /rGO were confirmed by the presence of C-Ti-OH bond [47]. The XPS consequence verifies the formation of Ti 3 C 2 T x /rGO heterostructure and is consistent with the previous results.

Electrochemical Collaboration Behavior of Ti 3 C 2 T x /rGO towards DA
To investigate the electrochemcial response of Ti 3 C 2 T x /rGO towards DA and UA, CV scanning was performed on the Ti 3 C 2 T x /rGO electrode in PBS with 10 µM DA and 10 µM UA. As shown in Figure 3a, compared with the CV curve from blank PBS, there is an oxidation peak and a reduction peak in the CV curve of DA (Re 1 , Ox 1 ), and UA (Re 2 , Ox 2 ) [41]. Among them, Ox 1 and Ox 2 were specified as the characteristic peaks for qualitative and quantitative analysis of the electrochemical behavior of DA and UA, respectively. As shown in Figure 3b,c, DPV curves of electrochemical behaviors at a potential interval of 0.0-0.5 V were conducted in the presence of 10 µM DA on bare GCE, Ti 3 C 2 T x , rGO, and Ti 3 C 2 T x /rGO electrodes. The current intensity of the Ti 3 C 2 T x electrode exhibited higher than GCE, indicating that the porous Ti 3 C 2 T x electrode with good electrical conductivity promoted the electron transfer of DA oxidation. Compared to the Ti 3 C 2 T x electrode, the current intensity of the rGO electrode improved by nearly double, demonstrating the much better electrochemical performance of rGO than Ti 3 C 2 T x towards DA. Furthermore, the Ox 1 current intensity of Ti 3 C 2 T x /rGO electrode was much higher than the sum of rGO and Ti 3 C 2 T x , owing to the synergistic effect of the huge specific surface area of rGO and the porous Ti 3 C 2 T x electrode with good electrical conductivity. To assess the electrochemcial feasibility of various modified electrodes in 10 mM [Fe(CN) 6 ] 3−/4− , EIS was performed on bare GCE, Ti 3 C 2 T x , and Ti 3 C 2 T x /rGO electrodes with 10 mV amplitude of the AC voltage, as shown in Figure 3d. The semicircle diameter at higher frequencies in the Nyquist diagram indicates the interfacial electron transfer resistance (R ct ), which controls the electron transfer of [Fe(CN) 6 ] 3−/4− on the electrode surface [48]. The R ct values of GCE, Ti 3 C 2 T x , and Ti 3 C 2 T x /rGO electrodes were 1036.0, 628.8, and 369.6 Ω, respectively. The result reveals that the Ti 3 C 2 T x /rGO electrode greatly facilitates the electron transfer of the DA electrochemical reaction, which agrees with the former results. R s , R p , Q coat , and Q sub represent the solution resistance, pore resistance, coating constant phase, and double-layer constant phase, respectively, and the corresponding values are listed in Table S1.
To further investigate the synergistic effect of the Ti 3 C 2 T x /rGO nanocomposite, GO and Nafion were taken as the coating layer hybrid with Ti 3 C 2 T x and rGO as the coating layer hybridize with the Au electrode instead of the Ti 3 C 2 T x electrode, in comparison with the Ti 3 C 2 T x /rGO nanocomposite modified electrode. As shown in Figure 3e,f, the current intensity on the Ti 3 C 2 T x /Nafion electrode was lower than that of the Ti 3 C 2 T x electrode. This indicates that Nafion as an electric material is not suitable for hybridizing with Ti 3 C 2 T x to DA electrochemical reaction. The current intensity of the Ti 3 C 2 T x /rGO electrode was higher than of the Ti 3 C 2 T x /GO electrode, indicating that less oxygen-containing groups of rGO with better electrical conductivity exhibited greater facilitation of electron transfer reaction of DA. Interestingly, the current intensity of Au electrode/rGO electrode was lower than that of the Ti 3 C 2 T x /rGO electrode, demonstrating the advantage of the porous Ti 3 C 2 T x electrode to the smooth Au electrode towards DA electrochemical reaction, as displayed in Figure S2. To further reveal the superior electrochemical performance of rGO to Ti 3 C 2 T x , a comparison experiment of DA adsorption performance was conducted between Ti 3 C 2 T x and rGO water dispersions, as shown in Figure 3g. Ti 3 C 2 T x and rGO water dispersions containing 100 µM DA were prepared with sonification. After filtration by 0.22 µM membrane, the filtrates of Ti 3 C 2 T x and rGO dispersions both became much more transparent, and the colors of both film membranes were much darker. The result demonstrates that nanomaterials such as Ti 3 C 2 T x and rGO adsorption DA were mostly trapped on the membrane. Then, the DPV curves of electrochemical behaviors at a potential interval of 0.0-0.35 V were conducted on the Ti 3 C 2 T x /rGO electrode in electrolytes using original DA solutions, filtrates of the Ti 3 C 2 T x , and rGO dispersions containing 300 µM DA, respectively, as shown in Figure 3h. The adsorption consequence revealed that the DA adsorption performance of rGO was greater than that of Ti 3 C 2 T x . This may be due to the electrostatic interaction between positively charged DA (pKa = 8.87) and negatively charged rGO with oxygen-containing groups at pH 7.0, as well as the π-π interaction between the phenyl structure of DA and two-dimensional planar hexagonal carbon-carbon structure of graphene, rather than the electrostatic interaction between DA and negative Ti 3 C 2 T x only [49].  To further investigate the synergistic effect of the Ti3C2Tx/rGO nanocomposite, GO and Nafion were taken as the coating layer hybrid with Ti3C2Tx and rGO as the coating layer hybridize with the Au electrode instead of the Ti3C2Tx electrode, in comparison with the Ti3C2Tx/rGO nanocomposite modified electrode. As shown in Figure 3e,f, the current intensity on the Ti3C2Tx/Nafion electrode was lower than that of the Ti3C2Tx electrode. This indicates that Nafion as an electric material is not suitable for hybridizing with Ti3C2Tx to

Ti 3 C 2 T x /rGO Electrode Performance Optimization of DA Detection
To further improve the electrochemical performance of the proposed sensor, experimental parameters including the preparation of modified electrodes, electrolyte pH were optimized. To affirm the influence of the preparation of layer-by-layer structured Ti 3 C 2 T x /rGO electrode on the DPV response in PBS with 100 nM DA, various rGO masses including 0.03, 0.075, 0.15, 0.3, 0.75, and 1.5 µg were formed on the same Ti 3 C 2 T x electrode cast on 6.0 µg. As shown in Figure 4a, the background current of the DA detection peak in 0.124 V greatly increased with the rising mass of rGO, and the DA oxidation peak was obviously observed only when rGO mass adjusted to 0.075 µg in the fabrication of the Ti 3 C 2 T x /rGO electrode. The results reveal that the rising mass of rGO was not suitable for the nM concentration level of effective DA detection, and rGO mass was chosen as 0.075 µg. Similarly, to confirm the suitable mass of Ti 3 C 2 T x in the fabrication of Ti 3 C 2 T x /rGO electrode, various Ti 3 C 2 T x masses of 0.6, 1.5, 3, 6, 12, and 30 µg were firstly cast on GCE, and 0.075 µg GO was then dropped on and dried to perform the CV method of electrochemical reduction. The DPV response in PBS with 10 µM DA was then performed, as shown in Figure 4b,c. The Ti 3 C 2 T x mass was selected as 3 µg, and the corresponding optimal mass ratio of Ti 3 C 2 T x to rGO was 40:1.
To better determine if the mechanism of a lower rGO mass is suitable for trace level DA detection, CV and DPV curves at a potential interval of 0.0-0.5 V were performed in PBS on an rGO electrode cast with masses of 0.15 µg, 0.6 µg, and 3.0 µg respectively, as shown in Figure 4d,e. The capacitance can be calculated by CV methods with the following formula: , wherein, S refers to scan rate; ∆V refers to potential scan range; and i refers to current. The area A c of the CV curves determines the value of capacitance, when S and ∆V remain consistent. Obviously, the results reveal that the total capacitance formed on the rGO electrode and the background current both increased with the rising rGO mass. The structure of the EDL at the junction of a metal with an electrolyte solution conceives the layer to have two elements, known as "Helmholtz layer, and diffuse layer" [50]. The two elements interpret the existence of a capacitance C d of electrical double layer to be close to the solid/electrolyte interface, the Helmholtz capacitance C H , and diffuse layer capacitance C D , wherein C d −1 = C H −1 + C D −1 [51]. The thickness of the diffuse layer gives the distance from the solution up to the point where the electrostatic effect of the surface is felt by the ions [40]. According to the schematic diagram in Figure 4f,g, when the rGO mass modified on GCE increased, the total capacitance C d increased, and the diffuse layer capacitance C D increased. Thus, λ D decreased with respect to the rGO electrode modified with greater rGO mass, indicating that fewer DA biomolecules were capable of passing through EDL and reaching the surface of GO to achieve the voltammetric response of DA. The increasing mass of GO decreased λ D , suggesting that the detection limit of DA was raised to a higher level, and the result is consistent with Figure 4a.
The effect of pH on the electrochemical response of the Ti 3 C 2 T x /rGO electrode was conducted in the range from 3.0 to 11.0, as shown in Figure 4h. The oxidation peak potentials of DA shifted negatively with the increased electrolyte pH, ascribing to an improvement in the reversibility of the investigated faradic process that involves the deprotonation of DA, followed by the protonation of the amine group in DA to form a cation [52,53]. The value of the peak current reached the maximum at pH 7.0 and was selected as the optimal pH value. The electrochemical behavior of various electrodes was performed by CV in 10 mM [Fe(CN) 6 ] 3−/4− containing 0.1 M KCl electrolyte solution at scan rates ranging from 20 to 260 mV s −1 (Figure 4i). The observed peak currents (I pa and I pc ) both increased linearly, with the square root of scan rates as shown in Figure 4j, indicating that the Ti 3 C 2 T x /rGO electrodes were controlled by diffusion [54]. The effect of pH on the electrochemical response of the Ti3C2Tx/rGO electrode was conducted in the range from 3.0 to 11.0, as shown in Figure 4h. The oxidation peak potentials of DA shifted negatively with the increased electrolyte pH, ascribing to an improvement in the reversibility of the investigated faradic process that involves the deprotonation of DA, followed by the protonation of the amine group in DA to form a cation [52,53]. The value of the peak current reached the maximum at pH 7.0 and was selected as the optimal pH value. The electrochemical behavior of various electrodes was performed by CV in 10 mM [Fe(CN)6] 3−/4− containing 0.1 M KCl electrolyte solution at scan rates ranging from 20 to 260 mV s −1 (Figure 4i). The observed peak currents (Ipa and Ipc) both increased linearly, with the square root of scan rates as shown in Figure 4j, indicating that the Ti3C2Tx/rGO electrodes were controlled by diffusion [54].

Electrochemical Determination of DA and UA with Different Concentrations
The quantitative electrochemical detection of DA and UA on the Ti3C2Tx/rGO electrode was conducted via DPV measurements, as shown in Figure 5a,b. An increase in peak current value was recorded with the increasing concentration of DA in a range from 9.5

Electrochemical Determination of DA and UA with Different Concentrations
The quantitative electrochemical detection of DA and UA on the Ti 3 C 2 T x /rGO electrode was conducted via DPV measurements, as shown in Figure 5a,b. An increase in peak current value was recorded with the increasing concentration of DA in a range from 9.5 nM to 100 µM, and the increasing concentration of UA in a range from 300 nM to 1000 µM, respectively. Correspondingly, the inset graphic of Figure 5a,b depicts the enlarged view in the potential range from 0.0-0.3 V and 0.1-0.4 V to clearly show the variations of DPV curves ranging from 0.0-100 nM DA and 0.0-1.0 µM DA, respectively. As presented in Figure 5c, the calibration curve of DA and UA was obtained from the average of peak current data. According to the calibration curve, the linear range of DA detection was in a range from 0.1 to 100 µM, and UA detection was in a range from 1 to 1000 µM, respectively. The linear regression equation of DA was I pc (µA) = 0.413 lg DA (µM) − 5.780 (R 2 = 0.993), and the linear regression equation of UA was I pc (µA) = 0.529 lg UA (µM) − 0.209 (R 2 = 0.994). The limit of detection (LOD) of DA and UA on the Ti 3 C 2 T x /rGO electrode was determined as 9.5 nM and 300 nM, respectively. current data. According to the calibration curve, the linear range of DA detection was in a range from 0.1 to 100 μM, and UA detection was in a range from 1 to 1000 μM, respectively. The linear regression equation of DA was Ipc (μA) = 0.413 lg DA (μM) − 5.780 (R 2 = 0.993), and the linear regression equation of UA was Ipc (μA) = 0.529 lg UA (μM) − 0.209 (R 2 = 0.994). The limit of detection (LOD) of DA and UA on the Ti3C2Tx/rGO electrode was determined as 9.5 nM and 300 nM, respectively. Specifically, the quantitative electrochemical detection of DA and UA were conducted by DPV measurements on six individual electrodes. Compared with graphenebased modified electrodes prepared using various methods for simultaneous detection of DA and UA, as shown in Figure 5d, our Ti3C2Tx/rGO electrode achieved a relatively low simultaneous detection LOD of DA and UA and a four-order-magnitude linear range with convenience and efficiency. The corresponding literatures are listed in Table 1. Specifically, the quantitative electrochemical detection of DA and UA were conducted by DPV measurements on six individual electrodes. Compared with graphene-based modified electrodes prepared using various methods for simultaneous detection of DA and UA, as shown in Figure 5d, our Ti 3 C 2 T x /rGO electrode achieved a relatively low simultaneous detection LOD of DA and UA and a four-order-magnitude linear range with convenience and efficiency. The corresponding literatures are listed in Table 1.

Repeatability, Reproducibility, Interference, and Real Sample Analysis
In order to study the repeatability of the Ti 3 C 2 T x /rGO electrode for DA detection (10 µM), the Ox 1 peak currents in DPV curves were repeatedly measured 11 times on the same electrode at a potential interval of 0.0-0.5 V. As shown in Figure 5e, the reduction peak potentials of DPV curves were consistent at 0.129 V, and these curves overlapped well. The relative standard deviation (RSD) of peak currents was 3.57%. The reproducibility of the Ti 3 C 2 T x /rGO electrode was performed in the presence of 10 µM DA by using six individual electrodes in DPV curves, as shown in Figure 5f, and the RSD was 3.92%. The results indicate that the Ti 3 C 2 T x /rGO electrode has good repeatability and reproducibility. The anti-interference of the Ti 3 C 2 T x /rGO electrode was investigated via DPV curves in PBS containing various concentrations of DA ranging from 0.1 µM to 10 µM in the presence of 30 µM UA as interfering substances, as shown in Figure 5g. Similarly, the anti-interference was performed in PBS containing UA ranging from 1 µM to 100 µM in the presence of 10 µM DA, as presented in Figure 5h. When compared to the calibration curve of DA and UA detection individually in Figure 5a,b, the anti-interference results indicate that DA and UA did not induce obvious interference in the DPV determination of each other. The anti-interference of Ti 3 C 2 T x /rGO electrode in the presence of other potential interfering substances as 100 µM glucose, 100 µM ascorbic acid (AA), 100 µM H 2 O 2 , and 10 µM isoniazid in PBS containing 3 µM DA and 3 µM UA was investigated via DPV curves, as shown in Figure S3. The results indicate that our constructed sensor will not be affected by these molecules during testing. To evaluate the practical application performance of Ti 3 C 2 T x /rGO electrodes for simultaneous detection of DA and UA, human serum was selected as real samples for analysis using the standard addition technique. The serum samples were centrifuged at 6000 rpm for 5 min, and the supernatants were collected and diluted 100 times with PBS. Then KCl was added to 0.1 M of the serum samples, and the pH was adjusted to 7.0 to perform appropriate electrochemical detection of DA and UA [56]. Serum samples were then spiked with 0.1, 0.3 µM DA, and 1, 3 µM UA, respectively, and the DPV curves of Ti 3 C 2 T x /rGO electrode were extracted, as shown in Figure 5i. The results demonstrate the accuracy and reliability of the fabricated sensor, indicating that the proposed Ti 3 C 2 T x /rGO electrode exhibited good potential for simultaneously detecting DA and UA practically.
Thus, our fabricated Ti 3 C 2 T x /rGO electrode with a double-deck layer was applied as the biosensor for the simultaneous detection of DA and UA successfully. The detection sensitivity of the Ti 3 C 2 T x /rGO electrode was greatly improved with the adjustment to Debye length. Our proposed Ti 3 C 2 T x /rGO electrode displayed good repeatability, selectivity, and proved suitable for real sample analysis.

Conclusions
In summary, a Ti 3 C 2 T x /rGO heterostructure with a double-deck layer was fabricated through electrochemical reduction. The rGO was modified on the porous Ti 3 C 2 T x electrode as the biosensor for the simultaneous detection of DA and UA. The Debye length λ D is regulated by the alteration of rGO on the surface of the Ti 3 C 2 T x electrode. λ D decreased with respect to the rGO electrode modified with a greater rGO mass, indicating that fewer DA biomolecules were capable of passing through EDL and reaching the surface of GO to achieve the voltammetric response of DA. Thus, the proposed Ti 3 C 2 T x /rGO sensor had an excellent performance in the detection of DA and UA, with a wide linear range from 0.1-100 µM to 1-1000 µM and a low detection limit from 0.0095 to 0.3 µM, respectively. Additionally, the proposed Ti 3 C 2 T x /rGO electrode displayed good repeatability, selectivity, and proved suitable for real sample analysis.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/bios11110462/s1. Figure S1: CV curves of Ti 3 C 2 T x /rGO electrode on electrochemical reduction of GO to rGO. Figure S2: Performance comparison of DPV curves on Ti 3 C 2 T x /rGO and Au/rGO electrode with 10 µM DA in PBS and the corresponding current values. Figure S3: The anti-interference of our electrode in the presence of 100 µM glucose, 100 µM ascorbic acid, 100 µM H 2 O 2 and 10 µM isoniazid with PBS containing 3 µM DA and 3 µM UA. Table S1: The fitting parameters of EIS for GCE, Ti 3 C 2 T x and Ti 3 C 2 T x /rGO electrode.