Detection of Ascorbic Acid by Two-Dimensional Conductive Metal-Organic Framework-Based Electrochemical Sensors

The realization of efficient and accurate detection of biomolecules has become a key scientific issue in the field of life sciences. With the rapid development of nanotechnology, electrochemical sensors constructed from the superior physical and chemical properties of nanomaterials show faster and more accurate detection. Among nanomaterials, two-dimensional conductive MOF (2D cMOF) is considered to be a star material in electrochemical sensors due to its remarkable conductivity, high porosity, and stability. In this paper, a Cu3(HHTP)2/SPE electrochemical sensor for the detection of ascorbic acid (AA) was constructed by modifying 2D cMOF (Cu3(HHTP)2) on the surface of the screen-printed electrode (SPE). The sensor exhibited excellent catalytic activity in the detection of AA, with a lower detection limit of 2.4 μmol/L (S/N = 3) and a wide linear range of 25–1645 μmol/L. This high catalytic activity can be attributed to the abundant catalytic sites in Cu3(HHTP)2 and the rapid electron transfer between Cu+ and Cu2+, which accelerates the oxidation of AA. This work lays a foundation for the subsequent development of MOFs with special electrochemical catalytic properties and the integration of 2D cMOF into intelligent electrical analysis devices.


Introduction
Ascorbic acid (AA), also known as vitamin C, is an antioxidant that effectively scavenges free radicals in the body to reduce the incidence of cancer, and also plays a crucial role in biological metabolism [1][2][3][4].It is worth mentioning that both low-and high-AA levels are closely associated with certain diseases.A lack of AA in the body will lead to symptoms such as scurvy and bleeding gums, while excessive intake of AA causes stomach convulsion, diarrhea, and urinary stones [5,6].Therefore, the development of a simple, low-cost, and high-sensitivity AA detection pathway is essential for the early prevention of these diseases.Among the many detection methods of AA, electrochemical analysis has shown great application prospects due to its high sensitivity and fast response [7,8].However, it is difficult to determine AA using traditional electrodes such as glassy carbon and platinum as the redox reaction of AA on bare electrodes is irreversible and requires high overpotential [9].In addition, its high overpotential will cause electrode fouling, resulting in poor selectivity and reproducibility of AA detection [10].To break this deadlock, more and more studies have been carried out to modify electrodes with suitable electrochemically active materials, including metal nanoparticles, carbon materials, and polymers, for AA detection [11][12][13][14].Over the past few decades, the rapid development of nanomaterials has made a major breakthrough in terms of the activity of material-based electrochemical sensors [1,15,16], but their sensitivity and stability still need to be improved.Therefore, the further development of innovative electrode modifiers with high specific surface area, multiple active sites, and ultra-high stability is crucial for the detection of AA with high sensitivity.
Metal-organic frameworks (MOFs), as a novel porous material, have attracted extensive attention in the fields of catalysis [17], separation [18], energy storage [19], and drug delivery [20] due to their high porosity, large surface area, and structural diversity.Recently, MOFs have also been used for electrocatalytic sensing, but their inherent low conductivity severely affects the efficiency of charge transport, so they need to be combined with other conductive materials to improve their electrochemical performance [21,22].However, composite materials will reduce the specific surface area and stability of the MOF, and the composite interface is difficult to accurately control.Excitingly, the emergence of a two-dimensional conductive MOF (2D cMOF) has reversed this impasse.Two-dimensional cMOF not only inherits the inherent advantages of MOF, but is also endowed with high charge carrier mobility and excellent conductivity because of the unique π-π packing and π-d conjugation in their structure [23,24], which is a more ideal electrode modifier.Cu 3 (HHTP) 2 is a typical 2D cMOF with high electrical conductivity at room temperature and good water stability [25].Moreover, both the metal center and the ligand of Cu 3 (HHTP) 2 are redox sites [19], which is expected to further accelerate the electron transport of AA oxidation.All of these imply that Cu 3 (HHTP) 2 -modified electrodes have great potential to be excellent electrochemical sensor for the detection of AA.
In this work, Cu 3 (HHTP) 2 with high crystallization and specific surface area was synthesized through a simple solvothermal method.Subsequently, a Cu 3 (HHTP) 2 /SPE electrochemical sensor for the detection of AA was constructed by modifying 2D cMOF on the surface of SPE.As expected, the sensor detects AA with high sensitivity and wide linear range.The high catalytic activity of the Cu 3 (HHTP) 2 /SPE sensor can be ascribed to the abundant active sites and high conductivity of Cu 3 (HHTP) 2 , promoting the electron transfer in terms of AA oxidation and reducing reaction overpotential.This work provides a theoretical basis for the construction of 2D cMOF-based electrochemical sensors, and is expected to achieve rapid detection of AA in practical biological samples.

Results and Discussion
The structure and morphology of as-synthesized Cu 3 (HHTP) 2 was characterized by X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectrometry and field emission scanning electron microscopy (FE-SEM).As shown in Figure 1a, the XRD pattern of Cu 3 (HHTP) 2 exhibits three prominent peaks at 4.7 • , 9.5 • , and 12.8 • , corresponding to the (100), (200), and (210) reflection planes.The minor and broad peak at 28 • can be attributed to the (004) crystal plane of Cu 3 (HHTP) 2 [26].All the peaks of Cu 3 (HHTP) 2 display good agreement with the reported characterization [27] and simulated data, demonstrating the formation of Cu 3 (HHTP) 2 .In the FT-IR spectrum (Figure 1b), the characteristic bands at 1457 cm −1 , 1303 cm −1 , and 575 cm −1 belong to the C=C, C-O, and Cu-O vibration in Cu 3 (HHTP) 2 , respectively [28], and the formation of coordination between Cu and O atoms further proves the successful synthesis of Cu 3 (HHTP) 2 .Additionally, the vibration bands located at 1648 cm −1 and 1374 cm −1 are assigned to the C=O and O-H bonds, where the existence of C=O bonds indicates the partial oxidization of organic ligands (HHTP) in the hydrothermal reaction, while the O-H bonds suggest that a small number of hydroxyl groups are not involved in the coordination reaction [1,19].The SEM image (Figure 1c) of the obtained Cu 3 (HHTP) 2 reveals particle-shaped morphology with an average diameter of 40 nm, also matching well with previous reports [26].
It is well known that porous material is conducive to catalytic performance, so a N 2 adsorption and desorption experiment was performed to explore the pore structure of Cu 3 (HHTP) 2 .As can be seen from Figure 1d, the gas adsorption increases rapidly at low relative pressure, presenting the characteristic Type I isotherm (IUPAC classification); meanwhile, it indicates the microporosity of Cu 3 (HHTP) 2 .In addition, the Brunauer-Emmett-Teller (BET) specific surface area of the obtained Cu 3 (HHTP) 2 is calculated to be 236 m 2 g −1 and the average pore diameter is about 1.4 nm (Figure S1).The above results confirm the prepared Cu 3 (HHTP) 2 has a large number of internal porous channels that increase the contact area and efficient electron transport between the catalyst and AA.m 2 g −1 and the average pore diameter is about 1.4 nm (Figure S1).The above results confirm the prepared Cu3(HHTP)2 has a large number of internal porous channels that increase the contact area and efficient electron transport between the catalyst and AA.Next, X-ray photoelectron spectroscopy (XPS) was used to verify the element composition and electronic structure of Cu3(HHTP)2.The results of the XPS survey spectrum (Figure 2a) reveal the existence of C, O, and Cu elements in the obtained Cu3(HHTP)2.Moreover, the XPS spectrum of C 1s (Figure 2b) displays three peaks at the binding energy of 288.49, 286.60, and 284.79 eV, which can be attributed to C=O, C-O, and C-C bonds, respectively [29].As shown in Figure 2c (the XPS spectrum of O 1s), the coexistence of C-O (532.76 eV) and C=O (531.22 eV) [30,31] indicates the formation of semiquinone due to the in situ partial oxidation, as mentioned above (FT-IR results).Furthermore, the peaks at 934.34 and 932.46 eV in the XPS spectrum of Cu 2p (Figure 2d) belong to Cu 2+ and Cu + , respectively [32], and the existence of Cu + indicates unsaturated coordination in Cu3(HHTP)2 [27,33].It should be noted that the electronic conductivity of Cu3(HHTP)2 characterized by the four-point method (Figure S2) is 2.4 S m −1 .The excellent conductivity of Cu3(HHTP)2 can be attributed to the fact that the coexistence of Cu + and Cu 2+ facilitates the delocalization of electrons.The unique structure of 2D cMOF, with high conductivity, is expected to accelerate charge transfer during the electrocatalytic oxidation of AA.Next, X-ray photoelectron spectroscopy (XPS) was used to verify the element composition and electronic structure of Cu 3 (HHTP) 2 .The results of the XPS survey spectrum (Figure 2a) reveal the existence of C, O, and Cu elements in the obtained Cu 3 (HHTP) 2 .Moreover, the XPS spectrum of C 1s (Figure 2b) displays three peaks at the binding energy of 288.49, 286.60, and 284.79 eV, which can be attributed to C=O, C-O, and C-C bonds, respectively [29].As shown in Figure 2c (the XPS spectrum of O 1s), the coexistence of C-O (532.76 eV) and C=O (531.22 eV) [30,31] indicates the formation of semiquinone due to the in situ partial oxidation, as mentioned above (FT-IR results).Furthermore, the peaks at 934.34 and 932.46 eV in the XPS spectrum of Cu 2p (Figure 2d) belong to Cu 2+ and Cu + , respectively [32], and the existence of Cu + indicates unsaturated coordination in Cu 3 (HHTP) 2 [27,33].It should be noted that the electronic conductivity of Cu 3 (HHTP) 2 characterized by the four-point method (Figure S2) is 2.4 S m −1 .The excellent conductivity of Cu 3 (HHTP) 2 can be attributed to the fact that the coexistence of Cu + and Cu 2+ facilitates the delocalization of electrons.The unique structure of 2D cMOF, with high conductivity, is expected to accelerate charge transfer during the electrocatalytic oxidation of AA.
To explore the performance of Cu 3 (HHTP) 2 in the electrocatalytic oxidation of AA, the 2D cMOF was coated on the SPE working electrode to prepare the Cu 3 (HHTP) 2 /SPE sensor, and evaluated by cyclic voltammetry (CV) at the sweep speed of 100 mV/s with or without 1 mM AA.First of all, the condition optimization tests were carried out.The result (Figure S3) exhibits that, within a certain range, the current of the AA oxidation peak increases with the concentration of Cu 3 (HHTP) 2 (1 to 4 mg/mL).However, with the further increase in concentration, the current of the AA oxidation peak is reversed.The results show that a proper concentration of Cu 3 (HHTP) 2 effectively enhances the catalytic efficiency of the SPE working electrode, whereas too high concentration will increase the resistivity and affect the performance of the sensor.Furthermore, it can be seen from Figure S4 that the peak current of the AA reaches its maximum at pH = 6 and then gradually decreases with the increase in pH.This can be attributed to the fact that the destruction of the AA intramolecular hydrogen bond under alkaline conditions will reduce its stability and the current of the oxidation peak.Based on this, followup electrochemical tests were performed at pH = 6 and a Cu 3 (HHTP) 2 concentration of 4 mg/mL.As shown in Figure 3a, the CV curve of Cu 3 (HHTP) 2 /SPE exhibits two strong oxidation peaks at 0.28 V and −0.21 V in 0.1 M PBS, which can be assigned to the oxidation potentials of Cu + to Cu 2+ and C-O to C=O bonds (ligand), respectively, while the peak of bare SPE at 0.48 V is the oxidation of AA.The abundant redox active centers of Cu 3 (HHTP) 2 and the close oxidation potential between AA and Cu + /Cu 2+ imply Cu 3 (HHTP) 2 with the ability of accelerating the oxidation of AA.As expected, the oxidation current of AA on Cu 3 (HHTP) 2 /SPE (1.98 × 10 −5 A) is nearly four times higher than that on bare SPE (5.01 × 10 −6 A).In addition, in comparison with the initial potential on bare SPE (0.48 V), the lower oxidation potential of AA on Cu 3 (HHTP) 2 /SPE (0.27 V) confirms that the Cu 3 (HHTP) 2 with outstanding electrocatalytic efficiency promotes the oxidation of AA.Although the above BET results find that Cu 3 (HHTP) 2 has a high specific surface area, the electroactive surface area (ESA) more directly reflects the effective area of the electrode surface involved in the reaction.Therefore, the ESA of Cu 3 (HHTP) 2 /SPE was tested and calculated using the K 4 [Fe(CN) 6 ] system and Randles-Sevcik equation, respectively.The results show that the ESA of Cu 3 (HHTP) 2 /SPE is 0.72 cm 2 (detailed calculations are provided in the Section 3.2.5),which is significantly larger than that of the traditional inorganic material-modified electrodes [34,35].This further indicates that Cu 3 (HHTP) 2 -modified SPE possesses more active sites, and as a consequence, it could effectively promote AA oxidation.Based on those and the above structure analysis, the excellent electrochemical performance of Cu 3 (HHTP) 2 can be ascribed to the following three points: To explore the performance of Cu3(HHTP)2 in the electrocatalytic oxidation of A the 2D cMOF was coated on the SPE working electrode to prepare the Cu3(HHTP)2/S sensor, and evaluated by cyclic voltammetry (CV) at the sweep speed of 100 mV/s with without 1 mM AA.First of all, the condition optimization tests were carried out.The res (Figure S3) exhibits that, within a certain range, the current of the AA oxidation peak creases with the concentration of Cu3(HHTP)2 (1 to 4 mg/mL).However, with the furth above structure analysis, the excellent electrochemical performance of Cu3(HHTP)2 can be ascribed to the following three points: (1) Cu3(HHTP)2 contains active sites (the coexistence of Cu + /Cu 2+ ) for AA oxidation; (2) the high conductivity of Cu3(HHTP)2 accelerates the electron transfer of AA oxidation; (3) different from traditional carbon materials, the unique pore structure of Cu3(HHTP)2-MOF endows it with a high specific surface area, which provides more active sites for the electrocatalytic oxidation of AA.To verify the kinetics of the electrocatalytic oxidation, CV tests with different AA concentrations and sweep speeds were carried out.The CV curves of Cu3(HHTP)2/SPE at different AA concentrations in Figure 3b show that the current increases along with the AA concentration and the oxidation peak moves towards a higher potential with the increase in AA concentration.Moreover, in Figure 3c, with the increase in scan rates, the current gradually increases and the AA oxidation peak shifts in the positive direction.Also, Figure 3d shows that the linear correlation of peak current with v 1/2 can be described as y = −0.342x− 2.984 (R 2 = 0.99).These results manifest that the oxidation rate of AA on the Cu3(HHTP)2/SPE electrode is limited by diffusion-controlled kinetics [36].
Then, the detection range and limit of the Cu3(HHTP)2/SPE sensor were also studied.As exhibited in Figure 4a,b  To verify the kinetics of the electrocatalytic oxidation, CV tests with different AA concentrations and sweep speeds were carried out.The CV curves of Cu 3 (HHTP) 2 /SPE at different AA concentrations in Figure 3b show that the current increases along with the AA concentration and the oxidation peak moves towards a higher potential with the increase in AA concentration.Moreover, in Figure 3c, with the increase in scan rates, the current gradually increases and the AA oxidation peak shifts in the positive direction.Also, Figure 3d shows that the linear correlation of peak current with v 1/2 can be described as y = −0.342x− 2.984 (R 2 = 0.99).These results manifest that the oxidation rate of AA on the Cu 3 (HHTP) 2 /SPE electrode is limited by diffusion-controlled kinetics [36].
Then, the detection range and limit of the Cu 3 (HHTP) 2 /SPE sensor were also studied.As exhibited in Figure 4a,b, AA concentration in the range of 25 µmol/L-1645 µmol/L has a certain linear correlation, and the linear equation is as follows: y = −0.0345x− 2 × 10 −6 (R 2 = 0.99).Additionally, the detection limit was 2.44 µmol at a signal-to-noise ratio (S/N) of 3. According to the aforementioned findings, Cu 3 (HHTP) 2 /SPE shows great potential as an excellent AA sensor.Meanwhile, the selectivity and stability of the sensor are also crucial in electrochemical detection, so differential pressure pulse voltammetry (DPV) technologies and current responses at different times are used to evaluate the anti-interference ability and stability of the electrode.In Figure S5a,b, it can be seen that other common interferences (Glu, KCl, Gly, and CA, 10-times interference) also show relatively weak peaks near the oxidation potential of AA.To further explore the selectivity of the sensor to AA, these interferences (10-times interference) were added to AA to verify the effect on current signals of AA.As shown in Figure 4c,d, there is almost no change in the DPV current response of Cu 3 (HHTP) 2 /SPE when AA coexists with other interferences, such as Glu, KCl, and Gly.Although the DPV current signal of AA and CA is 1.2 times higher than that of pure AA, the addition of CA is as high as 2 mM (note that the concentration of CA in the human body is only 0.092 mM [37]), indicating that the interference of CA is negligible.These Molecules 2024, 29, 2413 6 of 11 results show that the sensor possesses high selectivity for AA.Furthermore, the Ip value of Cu 3 (HHTP) 2 /SPE is still as high as 86% (relative to the initial current) after 5 days (Figure S6a,b); as a result, the sensor has high stability.Moreover, the feasibility of the Cu 3 (HHTP) 2 /SPE sensor for the detection of AA in actual serum samples (mouse serum) was verified by standard addition experiments.As shown in Table 1, the relative standard deviations and recovery measured at AA concentrations of 25, 55, and 285 µmol/L are 4.87%/98.6%,4.08%/98.0%,and 0.67%/99.0%,respectively, indicating that the constructed Cu 3 (HHTP) 2 /SPE sensor can be extended to practical applications and a general adaptative platform can be built for later disease diagnosis.Furthermore, to further verify the stability and reproducibility of the Cu 3 (HHTP) 2 /SPE sensor, electrode uncertainty experiments were also carried out.The results further suggest that the sensor has good stability as a consequence of the low uncertainty of 0.171 (Table S1).
peaks near the oxidation potential of AA.To further explore the selectivity of the sensor to AA, these interferences (10-times interference) were added to AA to verify the effect on current signals of AA.As shown in Figure 4c,d, there is almost no change in the DPV current response of Cu3(HHTP)2/SPE when AA coexists with other interferences, such as Glu, KCl, and Gly.Although the DPV current signal of AA and CA is 1.2 times higher than that of pure AA, the addition of CA is as high as 2 mM (note that the concentration of CA in the human body is only 0.092 mM [37]), indicating that the interference of CA is negligible.These results show that the sensor possesses high selectivity for AA.Furthermore, the Ip value of Cu3(HHTP)2/SPE is still as high as 86% (relative to the initial current) after 5 days (Figure S6a,b); as a result, the sensor has high stability.Moreover, the feasibility of the Cu3(HHTP)2/SPE sensor for the detection of AA in actual serum samples (mouse serum) was verified by standard addition experiments.As shown in Table 1, the relative standard deviations and recovery measured at AA concentrations of 25, 55, and 285 µmol/L are 4.87%/98.6%,4.08%/98.0%,and 0.67%/99.0%,respectively, indicating that the constructed Cu3(HHTP)2/SPE sensor can be extended to practical applications and a general adaptative platform can be built for later disease diagnosis.Furthermore, to further verify the stability and reproducibility of the Cu3(HHTP)2/SPE sensor, electrode uncertainty experiments were also carried out.The results further suggest that the sensor has good stability as a consequence of the low uncertainty of 0.171 (Table S1).Finally, according to the above XPS and CV results, a possible catalytic mechanism is proposed (Scheme 1).The Cu + /Cu 2+ in Cu 3 (HHTP) 2 are the activity sites of the electrochemical sensor.During the oxidation of AA, Cu + loses an electron to become Cu 2+ , and then Cu 2+ gains an electron from AA to promote the oxidation.The electrode material with high conductivity accelerates the electron transfer of the AA oxidation and endows the sensor with a better electrochemical signal.Finally, according to the above XPS and CV results, a possible catalytic mechanism is proposed (Scheme 1).The Cu + /Cu 2+ in Cu3(HHTP)2 are the activity sites of the electrochemical sensor.During the oxidation of AA, Cu + loses an electron to become Cu 2+ , and then Cu 2+ gains an electron from AA to promote the oxidation.The electrode material with high conductivity accelerates the electron transfer of the AA oxidation and endows the sensor with a better electrochemical signal.Scheme 1. Proposed mechanism of a Cu3(HHTP)2/SPE sensor for AA detection.
The materials were as follows: copper acetate monohydrate (Cu(OAc

Electrode Pretreatment
The screen-printed electrode (SPE) was connected to the electrochemical workstation.Cyclic voltammetric tests were performed in 0.5 mol/L H 2 SO 4 solution with a scanning voltage range of −1.0~1.0V and a scanning rate of 100 mV/s until the baseline stabilized, and then the test ended.The electrode surface was rinsed with deionized water and blow-dried.

The Synthesis of Cu 3 (HHTP) 2
A solvothermal method was used to synthesize Cu 3 (HHTP) 2 .First, 0.320 g of Cu(OAc) 2 -H 2 O and 0.260 g of HHTP were weighed and dissolved in 100 mL and 125 mL of CH 4 O, respectively.Then, the above two solutions were mixed and stirred for 10 min at room temperature, transferred to a sealed volumetric flask, and placed in a 65 • C blast drying oven for 24 h to obtain blue-black solid.The solid was washed by methanol and acetone.Finally, it was dried in a vacuum at 80 • C for 12 h to obtain the Cu 3 (HHTP) 2 .
3.2.3.The Preparation of Cu 3 (HHTP) 2 /SPE 4 mg of Cu 3 (HHTP) 2 was placed into 1 mL of CH 4 O and ultrasonicated for 30 min for uniform dispersion.Subsequently, 2 µL of the dispersion was applied dropwise on the surface of the working electrode of the SPE.Then, it was dried at room temperature to obtain the Cu 3 (HHTP) 2 /SPE, which was stored at room temperature for spare use.

Electrochemical Test
Cyclic voltammetry (CV) and differential pressure pulse voltammetry (DPV) were carried out using an electrochemical workstation CHI660E with a solution of 0.1 mol/L PBS.The CV had a range of −1 to 1 V and a scanning rate of 100 mV/s.The DPV scanning range was from −1 to 1 V, with a potential increment of 0.005 V, pulse amplitude of 0.05 V, and pulse width of 0.3 s.

Calculation of Electrochemically Surface Area (ESA)
The ESA of Cu 3 (HHTP) 2 /SPE was determined and calculated according to the literature [38]

Conclusions
In this experiment, Cu 3 (HHTP) 2 was synthesized and then deposited on the surface of SPE to prepare a Cu 3 (HHTP) 2 /SPE sensor for the detection of AA.The sensor's linear range of AA detection was 25-1645 µmol/L, with the linear equation of y = −0.0345x− 2 × 10 −6 (R 2 = 0.99) and the detection limit of 2.44 µmol/L (S/N = 3).The sensor also showed good anti-interference, reproducibility, and stability.In addition, the sensor was used to detect AA in actual mouse serum samples, which presented good recoveries, confirming the feasibility of the sensor for AA detection in real biological samples.
, AA concentration in the range of 25 µmol/L-1645 µmol/L has a certain linear correlation, and the linear equation is as follows: y = −0.0345x− 2 × 10 −6 (R 2

Figure 4 .
Figure 4. (a) The DPV of Cu3(HHTP)2/SPE for detection of AA with different concentrations.(b) The relationship between the current and AA concentration.(c) The DPV of Cu3(HHTP)2/SPE in 200 µM

Figure 4 .
Figure 4. (a) The DPV of Cu 3 (HHTP) 2 /SPE for detection of AA with different concentrations.(b) The relationship between the current and AA concentration.(c) The DPV of Cu 3 (HHTP) 2 /SPE in 200 µM AA with 2 mM KCl, Glu, Gly, and CA, respectively.(d) Bar chart of DPV response current of Cu 3 (HHTP) 2 /SPE to AA and AA with interferences.

Table 1 .
Detection of AA in actual serum samples.