Voltammetric folic acid sensor based on nickel ferrite nanoparticles modified-screen printed graphite electrode

In this study, an electrochemical sensor for the quantification of folic acid with voltammetric detection in physiological conditions was constructed. For this purpose, nickel ferrite (NiFe 2 O 4 ) nanoparticles were used to modify the surface of a screen-printed graphite electrode (NiFe 2 O 4 /SPGE) and applied in the determination of folic acid. The modified electrode displays a strong electrochemical response to folic acid. Folic acid was determined electrochemically using the differential pulse voltammetry (DPV) technique with a detection limit of 0.09±0.001 µM in 0.2 – 147.0 µM linear range in phosphate buffer solution (PBS) at pH 7.0 with this NiFe 2 O 4 /SPGE sensor, which has the best electron transfer rate. Also, the sensitivity of the modified electrode was obtained as 0.1139 µA µM -1 . The NiFe 2 O 4 /SPGE sensor was successfully applied for the determination of folic acid in real samples.


Introduction
Vitamins are a group of organic compounds essential in very small amounts for the body's normal functioning [1]. Folic acid (N-[p-{[(2-amino-4-hydroxy-6-pteridinyl) methyl] amino}benzoyl]-L-glutamic acid), also called pteroylglutamic acid (PteGlu), is a water-soluble vitamin of B complex family. It is most commonly referred to as vitamin B9 [2,3]. Folic acid is an important substance for keeping the activity and health of critters and is essential for cell growth and division of the human body. It participates in lots of bodily reactions and mainly in the synthesis of nucleic acid and some important substances and promoting the synthesis of protein from amino acid [4][5][6]. Research over the past decades has shown that deficiency in folate concentration leads to neural tube defects in newborns and an increased risk of megaloblastic anemia, cancer, coronary heart disease, Alzheimer's disease, neurological disorders, and cardiovascular disease in children and adults. Furthermore, the requireement of folate increases during periods of rapid cell division and it is essential for pregnant women [7,8]. Hence, analytical methods for the determination of this important bioelement are needed. Several methods have been proposed for the determination of folic acid in real samples, including spectrophotometry [9], high-performance liquid chromatography [10], capillary electrophoresis [11], fluorimetric [12], colorimetry [13], and flow injection chemiluminescence method [14]. However, in most of the cases reported above, prior steps are required before the actual determination of folic acid. Also, these techniques consume a long time for analysis, are subject to interferences and require expensive reagents. These disadvantages do not make them applicable for rapid analytical determination.
The concept of modified electrodes is an exciting development in the field of electrochemistry. The electrocatalysis of slow electron transfer reactions is perhaps the most important feature of chemically modified electrodes. Such electrodes enhance the electron transfer rate by reducing the overpotential associated with a reaction. The importance of modified electrodes in electrochemical sensors is because of their high electron transfer rate, high sensitivity, selectivity and stability in analyzing the electrochemical behavior of the analyte. So it is very important to develop highly sensitive and precise analytical methods and material with good conductivity to modify electrode to detect the concentration of analyte effectively [30][31][32][33][34][35][36][37][38][39][40][41].
During the last years, the research outcomes related to nanomaterials have increased in different application fields due to the development in the preparation and application of these new materials [42][43][44][45][46]. Nanomaterials offer unique and specific electroanalysis properties only found in nanoscale materials. These properties derive from the enhanced diffusion of the target analyte based on convergent rather than linear diffusion, with a high surface area, enhanced selectivity, catalytic activity, and a high signal-to-noise ratio [47][48][49][50][51].
Magnetic nanoparticles provide significant levels of new functionality for electrochemistry due to their high surface area, effective mass transport, catalysis and control over the local microenvironment [52,53].
Magnetic nanoparticles (NPs) with the general formula MFe2O4 (M = Fe, Ni, Co, Cu, Mn, etc.) are the most popular materials in analytical biochemistry, medicine, removal of heavy metals and biotechnology and have been increasingly applied to immobilize proteins, enzymes, and other bioactive agents due to their unique advantages. Nickel-ferrite is one of the most malleable and important spinel compounds due to its typical ferromagnetic properties and high electrochemical stability. Moreover, NiFe2O4 NPs exhibit a high surface area and low mass transfer resistance. It is expected that NiFe2O4 could also be used as an electrocatalyst apart from its electronic and magnetic applications due to its conducting nature [54][55][56].
In this work, a screen-printed graphite electrode modified with the NiFe2O4 magnetic nanoparticles was used for sensitive voltammetric determination of folic acid and the modified electrode exhibited excellent electrocatalytic activity to folic acid.

Apparatus and chemicals
All the electrochemical measurements were carried out on a PGSTAT302N potentiostat/galvanostat Autolab. The measurement cell consisted of SPGE (DropSens; DRP-110: Spain) containing a graphite counter electrode, a graphite working electrode, and a silver pseudo-reference electrode.
Solution pH values were determined using a 713 pH meter combined with a glass electrode (Metrohm, Switzerland). All chemicals used were of analytical grade and were used as received without any further purification and were obtained from Merck. Orthophosphoric acid was utilized to prepare the phosphate buffer solutions (PBS), and sodium hydroxide was used to adjust the desired pH values (pH range between 2.0 and 9.0).

Preparation of modified electrode
NiFe2O4/SPGEs were prepared by modifying the bare working electrode of an SPGE using the dropcasting method. Briefly, 4 µL of the solution of NiFe2O4 NPs (1 mg/mL) were dropped onto the working electrode surface and dried at room temperature. The obtained electrode was noted as NiFe2O4/SPGE.
The surface area of NiFe2O4/SPGE and the bare SPGE were obtained by CV using 1 mM K3Fe(CN)6 at different scan rates. Using Randles-Ševčik formula for NiFe2O4/SPGE, the electrode surface was found to be 0.109 cm 2 which was about 3.5 times greater than bare SPGE.

Electrochemical behavior of folic acid at the surface of various electrodes
The effect of the electrolyte pH on the oxidation of 100.0 μM folic acid was investigated at NiFe2O4/SPGE using DPV measurements in the PBS in the pH range from 2.0 to 9.0. According to the results, the oxidation peak current of folic acid depends on the pH value and increases with increasing pH until it reaches the maximum at pH 7.0 and then decreases at higher pH values. The optimized pH corresponding to the higher peak current was 7.0, indicating that protons are involved in the reaction of folic acid oxidation. Scheme 1 demonstrates the electrooxidation process of folic acid.   The results showed that the oxidation of folic acid is very weak on the surface of the bare SPGE, but the presence of NiFe2O4 NPs in SPGE could enhance the peak current and decrease the oxidation potential (decreasing the overpotential). A substantial negative shift of the currents starting from oxidation potential for folic acid and a dramatic increase of the current indicates the catalytic ability of NiFe2O4/SPGE to folic acid oxidation. The results showed that the use of NiFe2O4 nanoparticle (curve b) definitely improved folic acid oxidation characteristics, partly due to the excellent characteristics of NiFe2O4 NPs, such as good electrical conductivity and high chemical stability.

Effect of scan rate on the determination of folic acid at NiFe2O4/SPGE
The influence of potential scan rate (ν) on Ip of 70.0 μM folic acid at the NiFe2O4/SPGE was studied by linear sweep voltammetry (LSV) at various sweep rates ( Figure 2). As shown in Figure 2, the peak currents of folic acid grow with the increasing scan rates and there are good linear relationships between the peak currents and ν 1/2 (square root of scan rate) (Figure 2   To obtain further information on the rate-determining step, the Tafel plot for oxidation of 70.0 µM folic acid at the surface of NiFe2O4/SPGE using the data derived from the raising part of the current-voltage curve has been recorded in Figure 3. Using the slope of Tafel at a scan rate of 5 mV/s, the value of the electron transfers coefficient (α) was determined as 0.68.

Chronoamperometric studies
The electrochemical oxidation of folic acid by a NiFe2O4/SPGE was also studied by chronoamperometry. Chronoamperometric measurements of different concentrations of folic acid at NiFe2O4/SPGE were done by setting the working electrode potential at 650 mV ( Figure 4). In chronoamperometric studies, we have determined folic acid's diffusion coefficient, D. The experimental plots of I versus t −1/2 with the best fits for different concentrations of folic acid were employed (Figure 4 A). The slopes of the resulting straight lines were then plotted versus the folic acid concentrations (Figure 4 B), from whose slope and using the Cottrell equation (1): We calculated a diffusion coefficient of 6.610 -5 cm 2 s -1 for folic acid.

Calibration curve and limit of detection
Since DPV has a much higher current sensitivity than cyclic voltammetry, we used the DPV method for the determination of folic acid. Figure 5 (inset) shows DPVs of different concentrations of folic acid and the obtained calibration curves (step potential = 0.01 V and pulse amplitude = 0.025 V). The results showed a linear segment for folic acid concentration from 0.2 to 147.0 μM folic acid ( Figure 5), with a regression equation of Ip = 0.1139Cfolic acid+ 1.1599 (R 2 = 0.9992, n = 8). The detection limit, LOD, was obtained by using the equation (2): where Sb is the standard deviation of the blank response (n = 15) and m is the slope of the calibration plot. The limit of detection was determined to be 0.09±0.001 μM for folic acid.

Real sample analysis
To investigate the applicability of the proposed sensor for the voltammetric determination of folic acid in real samples, we selected urine and folic acid tablet samples for the analysis of folic acid contents. The folic acid contents were measured after sample preparation using the standard addition method. The results are given in Table 2. According to the table, the recovery values within 97.5 to 103.3 % confirm the powerful ability of NiFe2O4/SPGE for the determination of folic acid in real samples.

Conclusion
In this work, a simple, rapid and sensitive electrochemical detection method has been developed for the determination of folic acid. NiFe2O4 nanoparticles modified SPGE as a voltammetric sensor to improve the detection sensitivity. The sensitivity (0.1139 µA µM -1 ), detection limit (0.09 ± 0.001 µM) and linear response range (0.2 to 147.0 µM) for the NiFe2O4/SPGE modified electrode make it an efficient way for determination of folic acid. Real sample applications were carried out to prove the applicability and precision of the novelty-produced electrode. The amount of folic acid in real samples was obtained satisfactorily with high recovery values by the standard addition method.