**4. BSA/anti-VD/Fe3O4 PANnFs/ITO**

Vitamin-D (Vit-D) is crucial for the human body due to its role in calcium and bone metabolism. Previously utilized biosensors included CYP27B1/GCE and Ab-25OHD/SPE/FMTAD, which required complicated manufacturing processes and took more time [71, 72]. The potential uses of one-dimensional nanoscale materials, such as nanofibers, nanorods, and nanowires, in a variety of industries, biomedical engineering, and other sectors have received significant study attention [73, 74]. One-dimensional nanoscale systems have an advantage in the field of biosensors because of their surface morphology and distinctive one-dimensional arrangement,

### *Vitamin D Detection Using Electrochemical Biosensors: A Comprehensive Overview DOI: http://dx.doi.org/10.5772/intechopen.112212*

which allows for quick charge transfer in the axial direction [75, 76]. Several methods have been devised to create well-ordered nanofibers [77]. Electrospinning is the most prospective of these methods and has garnered a lot of interest since it has a number of noteworthy benefits, including being straightforward, quick, easy to use, and economical for mass production [78]. Biocompatible nanostructures containing polymer nanofibers have been utilized in the past to enhance the biosensor's features, such as sensitivity, response time, and stability. The surface area, flexibility, and electrochemical characteristics of nanofibers can be enhanced by the interaction of polymers with nanostructured materials (metal or metal oxide NPs) [77, 78].

In the most recent work by Chauhan, Gupta, and Solanki [79], magnetite (Fe3O4) NPs with polyacrylonitrile nanofibers (Fe3O4-PANnFs) were made utilizing the electrospinning process. Due to its low cost, high carbon content, ease of electrospinning, great solubility, improved mechanical qualities, and higher thermal stability, polyacrylonitrile (PAN) was chosen [18, 79]. Additionally, PAN is easily dissolved in solvents, including dimethylformamide (DMF), dimethylacetamide, and dimethyl sulfoxide [80, 81], but is only fully soluble in DMF at higher amounts [81]. Due to its magnetic characteristics and possible uses in the sectors of biology, pharmacy, diagnostics, and sensors, Fe3O4 NPs' non-toxicity and biocompatibility played a significant role [82, 83]. In addition, Fe3O4 NPs can be linked to proteins, enzymes, drugs, nucleotides, or proteins [84, 85]. Biomolecules attach to the surface of Fe3O4 NPs by physical and chemical forces [86, 87]. Additionally, the Fe3O4-PANnFs' nitrile group is readily convertible to a carboxyl group, aiding in the binding of biomolecules by creating a web of nanofibers for bio-sensing [88].

Polyacrylonitrile (PAN, MW = 150,000) was employed as a raw material in research [89]. The reagents used were dimethylformamide (DMF) and sodium hydroxide (NaOH) phosphate buffer solution (PBS) of pH 7.0. N-hydroxysuccinimide (NHS), N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), bovine serum albumin (BSA), ferric chloride hexahydrate (FeCl3.6H2O) (97%), and ferrous chloride tetrahydrate (FeCl2.4H2O, 97%), a monoclonal vitamin D3 antibody (Anti-VD) and (Antigen) Vitamin D3. Glass plates with an indium tin oxide (ITO) coating with specifications of 1.1 mm in thickness, 25 sq-1 in sheet resistance, and 90% transmittance.

Through the use of co-precipitation, Fe3O4NPs were created. To discover the ideal concentration of PAN solution for nanofiber manufacturing, several concentrations of PAN (8–10%) in DMF were created. At 9 and 10% PAN solutions, the diameter of the fibers was measured in the microscale, whereas at 8% PAN solution, the fibers were measured in the nanoscale range. Additionally, varied amounts of Fe3O4 NPs were distributed in 3 mL of DMF in order to create PANnFs with Fe3O4 NPs integrated. The Fe3O4 NPs were then equally distributed by adding this solution dropwise into 7 mL of DMF that had PAN and ultrasonically dispersing it for 60 minutes. Before being employed for electrospinning, this solution was swirled at 300 rpm overnight at ambient temperature (25 C). Fe3O4NPs in PAN solution at a concentration of 0.02 g resulted in consistent, bead-free nanofibers. As a result, the optimized condition for all studies was 0.02 g of Fe3O4NPs in PAN (8%) solution. Fe3O4-PANnFs was electrospun and straight away collected on ITO electrodes (0.5 cm<sup>2</sup> ) for 15 minutes at a flow rate of 0.2 mL/hr. and a voltage of 16 kV. The needle tip to collector distance was maintained at 18 cm. The Fe3O4-PANnFs/ITO electrode was dried overnight in a vacuum at 25°C. An aliquot of 5 L of nafion solution (0.5 wt.% in isopropanol) was cast on the layer of Fe3O4-PANnFs/ITO to improve the adhesion of Fe3O4-PANnFs to the ITO surface. At 25°C, room temperature, all electrodes were created. The

Fe3O4-PANnFs/ITO electrode was functionalized by hydrolyzing it in a NaOH (2 M) solution at 50°C for one hour. The PAN's nitrile (C N) ≡ group was largely changed during this hydrolysis process to carboxyl [34] and amine functional groups, which were then further modified with desirable biomolecules (Anti-VD). In phosphate buffer (pH 7.4), a new stock solution of Anti-VD (50 g mL−1) was made. The previous carboxyl functionalized Fe3O4-PANnFs/ITO electrode surface was then drop casted with 10 L of Anti-VD stock solution, which was then left in a humid chamber for 6 hours at room temperature (25°C). In order to prevent the unspecific binding sites on the immunoelectrode surface, 10 L of BSA (100 g mL−1) was evenly placed on the electrode surface and left for 4 hours. When not in use, these BSA/Anti-VD/Fe3O4- PANnFs/ITO immunoelectrode were kept at 4°C.

Different doses of Vit-D3 (antigen) from 1 to 100 ng mL−1 were generated by serially diluting a stock solution (1 mg mL−1) in 100% ethanol in order to test the sensitivity of constructed immunoelectrode for Vit-D3. Electrospinning tools were used to create Fe3O4-PANnFs/ITO electrodes. X-ray diffractometer was used to analyze Fe3O4- PANnFs' crystal structure. SEM method was used to examine the surface morphology of the Fe3O4-PANnFs/ITO and Anti-VD modified (Anti-VD/Fe3O4-PANnFs/ITO) immunoelectrode. Because of the hydrophilic nature of Fe3O4-PANnFs and the availability of more polar groups (COO- and NH3+) of Anti-VD on the electrode surface, nanofibers came closer after the adsorption of Anti-VD, resulting in the formation of a honeycomb-like structure after Anti-VD immobilization onto Fe3O4-PANnFs/ ITO electrode surface. As a result, Fe3O4-PANnFs promoted Anti-VD's adsorption to the electrode surface. Results show that Anti-VD was successfully immobilized on the Fe3O4-PANnFs/ITO electrode surface. Few sites of Anti-VD were therefore accessible for interaction with redox species in the case of the Anti-VD/Fe3O4-PANnFs/ITO electrode due to covalent contact between the functionalized surface of Fe3O4-PANnFs and Anti-VD.

The BSA/Anti-VD/Fe3O4-PANnFs/ITO immunoelectrode diffusion coefficient was determined to be 1.66 10–12 cm<sup>2</sup> s−1. BSA/Anti-VD/Fe3O4-PANnFs/ITO immunoelectrode surface concentration was measured as 6.56 × 10−9 mol cm−2. For the BSA/ Anti-VD/Fe3O4-PANnFs/ITO immunoelectrode, the electron transfer rate constant (Ks) was determined to be 0.56 s−1.

At a scan rate of 50 mV/s, the electrochemical response of the BSA/Anti-VD/ Fe3O4-PANnFs/ITO immunoelectrode was investigated as a function of Vit-D3 concentrations (10–100 ng mL−1). When the Vit-D3 concentration was between 10 and 90 ng mL−1, a linear increase in the Differential Pulse Voltammetry (DPV) peak current was seen; however, after that point, it became saturated. It was claimed that an immunochemical reaction occurred when antigen (Vit-D3) interacted with the surface of the immunosensor electrode, and as a result, a change in isoelectric point (IEP) was seen. On a single BSA/Anti-VD/Fe3O4-PANnFs/ITO immunoelectrode, DPV responses were observed for each concentration (10–100 ng mL−1) of vitamin D3. When Vit-D3 concentration and peak current value were plotted, the linear equation was discovered (**Figure 1**).

### **4.1 Selectivity**

When the BSA/Anti-VD/Fe3O4-PANnFs/ITO immunoelectrode was in contact with interfering substances like oxalic acid (1 mM), glucose (4 mM), cholesterol (4 mM), uric acid (0.5 mM), ascorbic acid (0.1 mM), and urea (2 mM), there was little change in the magnitude of the current, indicating that it is fairly selective [87, 88].

*Vitamin D Detection Using Electrochemical Biosensors: A Comprehensive Overview DOI: http://dx.doi.org/10.5772/intechopen.112212*

#### **Figure 1.**

*(a) DPV response of immunoelectrode (BSA/anti-VD/Fe3O4-PANnFs/ITO) as a function of antigen concentrations; (b) calibration graph between current peak and antigen concentration and (c) Hanes-wolf plot between (antigen concentrations) and (antigen concentration/current) [79].*

#### **4.2 Comparison with other biosensors**

For the detection of 25-OH vitamin D (25-OHD), Carlucci et al. [68] employed two techniques: surface plasmon resonance (SPR) and electrochemical affinity biosensors. By using the SPR approach, they were able to achieve a linearity of


#### **Table 2.**

*Biosensing parameters of BSA/anti-VD/Fe3O4-PANnFs/ITO immunosensor compared with other biosensors for Vit-D3.*

5–50 g mL−1, a sensitivity of 4.8 g mL−1, and a limit of detection (LOD) of 1 g mL−1. The electrochemical method, on the other hand, demonstrated linearity from 20 to 200 ng mL−1, sensitivity of 0.020 A ng−1 mL cm−2, and LOD of 10 ng mL−1. Using an enzyme-modified electrode, Ozbakir and Sambade [47] reported the detection of 25-OH Vitamin-D3 [25(OH)D3]. They created the enzyme (CYP27B1) using a synthetic human cytochrome P45027B1 gene, and they tested the enzyme's activity using LC–MS. This enzyme was fixed using pH-adjusted nafion and cobalt sepulchrate trichloride [Co(sep)3+] as a redox mediator on GCE. Cyclic and square wave voltammetry techniques were used to evaluate the activity of the bioelectrodes. The artificial biosensor displayed the data in a buffer with a concentration range that is physiological (5–200 ng mL−1) (**Table 2**).
