A sensitive voltammetric sensor for specific recognition of vitamin C in human plasma based on MAPbI 3 perovskite nanorods

A novel and sensitive electrode was suggested for the rapid determination of ascorbic acid (AA) using a glassy carbon electrode (GCE) modified with synthesized MAPbI 3 and L-cys (L-cys/MAPbI 3 /GCE). Determination of ascorbic acid as an important component of the human diet due to help in decreasing blood pressure and improving endothelial function is crucial. The synthesized MAPbI 3 was characterized by different methods, including transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX)


Introduction
Ascorbic acid (AA), namely vitamin-C, is a six-carbon lactone produced by plants and some animal species but not by humans or other primates, and it must be obtained from food. Vitamin C's biological action is critical for the skin's proper functioning. L-ascorbic acid is the more physiologically active of the two forms of vitamin C found in nature in fruits and vegetables such as oranges, broccoli, leafy greens, grapefruit, and peppers, and is the more useful, but the "D" form can be made via chemical synthesis but has no significant biological role. L-ascorbic acid protects against hydroxyl radicals, superoxide, and singlet oxygen, among other things. Furthermore, it lowers the membrane-bound antioxidant -tocopherol. Diabetes, coronary artery disease, hypertension, and chronic heart failure benefit from L-ascorbic acid's endothelium-dependent vasodilation. Monitoring AA content should be regarded as an essential and relevant task for evaluating the quality of final food items, raw materials, and various other substances, considering the nutritional value and therapeutic AA characteristics.
Although a variety of analytical methods are utilized to determine AA, including spectroscopic [1], chromatographic [2,3], the electrochemical methods have been widely performed in recent years [4][5][6][7][8] because of their rapid response, high sensitivity, good stability, superior selectivity, and costeffectiveness [9][10][11]. However, enhancing electrochemical performance in real samples necessitates the development of well-defined chemical structures and porous nanomaterials [12][13][14]. Recently, there is a big jump in fine-tuning the properties of nanomaterials and utilizing them in various applications [15][16][17]. In terms of materials, methylammonium lead iodide (CH3NH3PbI3 or MAPbI3), a perovskite-like organic-inorganic metal halide, has emerged as a viable low-cost material for nextgeneration high-efficiency perovskite solar cells [18]. Furthermore, the sensitivity of perovskite materials allows this disadvantage to be turned into an advantage [19]. As far as we know, this is the first sensor system using MAPbI3 as a modifier that has appropriate results for measuring one of the main parts of the human diet. In biological systems, cysteine, a thiol-containing non-essential amino acid, plays a significant function. It's found in a lot of proteins and acts as a precursor for protein synthesis, and is also utilized as a modifier in the production of electrochemical sensors. In electrochemical investigations, cysteine and cysteine-containing materials adorned electrodes are particularly popular. In this study, L-cys were modified on MAPbI3, and the resulting nanomaterial was utilized to modify the GCE surface to fabricate the sensor. A voltammetric method was developed using L-cys@MAPbI3/GCE and utilized to determine AA in the human plasma samples. Furthermore, our study exhibits a sensitive and selective sensor for AA determination with a LOD value of 8.0 nM in a wider linear range of 0.02 to 11.4 µM. Finally, the sensor's applicability is demonstrated by its application to human plasma, which yielded satisfactory recovery results.

Materials:
Methylammonium iodide, lead iodide, oleic acid, oleylamine, L-cysteine were acquired from Sigma Aldrich Co. (Germany). A human plasma sample was also obtained from Sera-Flex Inc (Turkey). All chemicals were of analytical grade.

The synthesis procedure of MAPbI3
MAPbI3 nanorods were synthesized using a facile ligand-assisted deposition approach. To begin with, 16.0 mg methylammonium iodide and 40.0 mmol lead iodide were dissolved in 2.0 mL dimethylformamide (DMF) and stirred for 24 h. Following, 400.0 μL oleic acid and 50.0 μL oleylamine were introduced into the resultant dispersion and kept stirring for 4 h. Moreover, 200.0 μL of the mixture solution was poured into 5.0 mL chloroform solution, stirring at the dark conditions over 5.0 h to obtain a dark brownish solution as MAPbI3 nanorods were obtained [20].

Preparation of fabricated electrode
Before modification, the bare electrode was polished with aluminum slurries (1.0 and 0.05 μm) and then washed by using HNO3 (10 vol.%), ethanol, and distilled water, respectively. Afterward, 8.0 µL of MAPbI3 was dropped onto the GCE and allowed to dry at room temperature. In the following, 5.0 µL of L-cys solution (2.0 mg mL -1 ) was dropped on the MAPbI3 electrode surface and was left to dry at room temperature [21].

Preparation of real sample
For the preparation procedure for the human plasma as the real sample, firstly, 1.0 mL of human plasma was treated with 1.0 mL of acetonitrile to eliminate the proteins. After that, the solution was centrifuged over 20 minutes at 10 °C at 6000 rpm. Before being put into the electrochemical cell, the samples were diluted to a certain concentration using B-R buffer at pH 7.0. [14].

Figure 1. XRD pattern of MAPbI3
Intensity, a.u. SEM and TEM images of pristine MAPbI3 are displayed in Figure 2. As shown in Figures 2A and 2B, the pristine MAPbI3 nanorods have a size of 50.0-200.0 nm, relatively smooth surfaces, and high crystallinity. SEM image ( Figure 2C) indicates the formation of highly crystalline, dense, and pinholefree MAPbI3. Elemental composition and percentage of as-synthesized MAPbI3 were validated through EDX analysis( Figure 2D). It was clearly observed sharp peaks relative to Pb, N, and I. Electrochemical characterization of the L-cys@MAPbI3/GCE: The effect of the modification of electrode surface was studied via DPV techniques. Figure 3 showed the DPV response of the bare electrode, MAPbI3/GCE, and L-cys@MAPbI3/GCE to a fixed concentration of AA (0.5 µM). Obviously, it can be seen that the presence of the MAPbI3 and L-cys increased the signal magnitude of the glassy carbon-based electrode to determine AA. In other words, such a described behavior can be related to the electrocatalytic activity of L-cys@MAPbI3/GCE for electro-oxidation of AA.   Figure 4B. The charge transfer resistance magnitude at the respective electrodes was ascribed to the diameters of the depressed semicircles. As shown, the charge transfer resistance at L-cys@MAPbI3/GCE surface is decreased about 1.7-fold compared to the bare electrode (23.57 kΩ), indicating fast electron transfer on the electrode surface [27]. Results confirmed that the presence of L-cys and MAPbI3 increases the electrode surface's conductivity [28].

pH and CV studies
The effect of pH on the oxidation of 1.0 μM AA was depicted in Figure 5A. The pH value of the B-R buffer was adjusted using 0.1 M HCl and NaOH. The oxidation currents of AA were observed in the pH range between 2.0 to 8.0 in 0.1 M B-R buffer. The peak current enhanced when the pH increased until pH 7.0, and after pH 7.0, the peak current decreased with a further increase of pH. Finally, pH 7 was selected for further experiments.
The diffusion-controlled mechanism at L-cys@MAPbI3/GCE was investigated using the linear relationship between the square root of the scan rates and current peak at different scan rates (10.0 to 400.0 mV s -1 ). The oxidation peak changed to positive potentials with increasing scan rates, as seen in Figure 5B. As shown in Figure 5C, the anodic peak currents enhanced linearly with the square root of scan rate, indicating that the redox reaction of the electrodes was a diffusion-controlled process [29]. Therefore, the electrocatalytic behavior of the electrode was improved. As for an irreversible electrochemical reaction, the Epa is determined by the following (Eq. 1): According to the linear relationship of Epa against ln  (Figure 5D), the value of n was observed to be approximately 1.0. By plotting I versus t -1/2 and Cottrell's equation (Figure 6), the diffusion coefficient (D) was estimated as 8.99×10 -7 cm 2 s -1 .  Figure 7B). The limit of detection (LOD) was 8.0 nM AA, according to the definition of LOD = 3sb/m [30]. cys@MAPbI3/GCE has a lower detection limit for AA compared with other modified electrodes (Table 1).

Reproducibility, repeatability
The reproducibility, repeatability, and selectivity of L-cys@MAPbI3/GCE were investigated by recording DPV of 1.0 µM AA at pH 7.0. The relative standard deviations (RSD) of 2.3 and 1.2 % for five successive recorded signals (repeatability) and five independents (reproducibility) of L-cys@MAPbI3/GCE, respectively that confirmed outstanding repeatability and reproducibility for L-cys@MAPbI3/GCE as an electroanalytical sensor toward AA.

Determination of ascorbic acid in the human plasma sample
The utilization of the developed sensor L-cys@MAPbI3/GCE in the real sample was also observed using the standard addition method in human plasma samples. The recovery of the spiked samples was obtained from 97.5 to 102.8 %. Results show that the developed electrochemical sensor will be useful for diagnosing biological samples.

Conclusions
In summary, a sensor based on L-cys@MAPbI3 was fabricated on a glassy carbon electrode to determine ascorbic acid. The results offered a well-defined oxidation peak for oxidation of ascorbic at L-cys@MAPbI3/GCE, which were large enough to determine AA. Moreover, the fabricated L-cys@MAPbI3/GCE exhibited acceptable results as a working electrode with a low detection limit, appropriate reproducibility, and repeatability. The fabricated sensor was successfully utilized to analyze AA in the real sample. The as-fabricated electrode could be an outstanding candidate as an alternative analytical approach for determining the trace amount of AA in clinical samples.