**3.4 FMTAD and SiO2/GO/Ni(OH)2/GCE**

Ferrocene Methanol Triacid (FMTAD) is a redox-active compound that plays a key role in electrochemical sensing applications for the determination of vitamin D. In electrochemical sensing, FMTAD is used as an electron mediator to facilitate the transfer of electrons between the electrode and the target analyte, thereby improving the sensitivity, selectivity, stability, and reproducibility of the electrochemical sensing system [53–55].

SiO2/GO/Ni(OH)2/GCE is another type of electrochemical sensing platform that has been used for the determination of vitamin D. This platform consists of a GCE modified with silica nanoparticles (SiO2), graphene oxide (GO), and nickel hydroxide (Ni(OH)2). SiO2 acts as a support material, GO improves the conductivity of the electrode surface, and Ni(OH)2 enhances the electrocatalytic activity toward the oxidation of vitamin D [54].

The combination of FMTAD as an electron mediator and SiO2/GO/Ni(OH)2/GCE as an electrochemical sensing platform has been shown to improve the sensitivity and selectivity of the determination of vitamin D. For example, a recent study reported an ultrasensitive electrochemical immunosensor for 25-hydroxyvitamin D3 detection based on FMTAD and SiO2/GO/Ni(OH)2/GCE, with a detection limit of 0.06 pg./mL [55].

In summary, FMTAD and SiO2/GO/Ni(OH)2/GCE are two important components in the development of electrochemical sensing systems for the determination of vitamin D. FMTAD acts as an electron mediator to enhance the electrochemical response, while SiO2/GO/Ni(OH)2/GCE acts as an electrochemical sensing platform to improve the sensitivity and selectivity of the determination. The combination of these components has the potential to enable highly sensitive and specific detection of vitamin D levels in biological samples.

#### **3.5 BSA/ab-VD2/CD-CH/ITO**

BSA/Ab-VD2/CD-CH/ITO is a significant biosensor for the detection of vitamin D. Chitosan and carbon dots are used in its construction. Carbon dots (CD) have many useful properties, such as optical, biological, and electrical characteristics [56]. They are highly soluble [57, 58], have stable fluorescence, easy functionalization, and minimally toxic [59], high electrochemical response, [51, 60, 61], microwave pyrolysis produces a high yield of CD, and are cheaper [56]. The CDs are extremely water soluble and are generated in very small quantities, making it difficult to fabricate thin films or electrodes for their use in electrochemical biosensing. These CDs may be included into an appropriate matrix that can preserve their electrochemical application features.

A suitable matrix for the dispersion of nanomaterials, including CDs, is chitosan [61] for biosensor applications. Chitosan is nontoxic, biocompatible, and biodegradable, and because it is a biopolymer with high mechanical strength, it has great film-forming capabilities [61, 62]. In order to create a moderately conductive layer, conductive elements, including carbon nanomaterials, metal nanoparticles, redox mediators, and ionic solutions, can be added to chitosan to increase its conductivity [63, 64]. A biosensor to detect dopamine was created by Huang et al. [65] using a CDschitosan-modified GCE [66]. Vitamin D2 has been shown by Holick et al. to be just as important as vitamin D3 in sustaining human blood levels of 25-hydroxyvitamin D [42, 67]. Yeast ergosterol is exposed to ultraviolet light to produce vitamin D2. Using electrochemical and surface plasmon resonance (SPR), Carlucci et al. [68] created the first biological sensing platform based on gold nanoparticles (Au NPs) with 25OHD for vitamin D detection. According to their findings, the electrochemical transducer showed increased sensitivity at a lower LOD value (10 ng/mL) [67]. Both vitamin D2 and vitamin D3 are equally needed to maintain vitamin D levels, and both vitamins D2 and D3 must be identified to determine any type of vitamin D insufficiency [68, 69].

A bio-electrode was created by Sarkar, Bohidar, and Solanki for the detection of vitamin D2 [70]. In this case, a glass substrate coated with indium tin oxide (ITO) was used to drop cast a thin layer of CD-CH in order to detect vitamin D2 using differential pulse voltammetry (DPV). Using EDC-NHS chemistry, a particular antibody against vitamin D2 (Ab-VD2) and bovine serum albumin (BSA) was immobilized on CD-CH/ITO film. This immunoelectrode was BSA/Ab-VD2/CD-CH/ITO, and it had a linear detection range of 1–50 ng mL−1 for Ag VD2. Citric acid (CA), ethylenediamine (EDA), chitosan (CH) and 1-ethyl-3-(3-dimethylaminopropyl)- carbodiimide (EDC), polyclonal antibody against to vitamin D2 (Ab-VD2), antigen as vitamin D2 (Ag-VD2), bovine serum albumin (BSA), hydroxysuccinimide (NHS), potassium ferrocyanide (K4[Fe(CN)6]), potassium ferricyanide (K3[Fe(CN)6]), sodium chloride salt, and ethanol are the materials used.

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

Following the method described by Zhai et al. [59] a bottom-up technique using microwave irradiation was used to synthesize CDs [59]. 1 gm of CA was dissolved in 10 mL of distilled water for the creation of CDs. After that, 50 μL of EDA was added to promote carbonization. This transparent solution was cooked. The final volume created with deionized water was crimson, brown thick solid frothy stuff. The resultant dark solution was dialyzed against water and contained CDs and CA residue. Chitosan (1%) was made in 10 mL of (1%) acetic acid. 20 μL of CDs were combined with 10 mL of chitosan solution (1%), creating a CD-CH solution. We took all of the identically sized ITO-coated glass substrates (1.5 × 0.5 cm) and hydrolyzed them. A newly made CD-CH solution was cast onto an ITO glass substrate that had been hydrolyzed, dried in a 60°C oven, and then presented as a CD-CH/ITO electrode. Onto the ITO surface, a drop of CH solution (without CDs) was cast. Ab-VD2 was produced as a fresh solution in phosphate buffer saline (PBS, pH -7.4) containing sodium azide at a concentration of 25 g/L. In PB (pH 7), a solution of NHS (0.1 M) and EDC (0.4 M) was produced. EDC (an activator) and NHS (a coupling agent) were combined with Ab-VD2. The CD-CH/ITO electrode was coated with 20 μL of Ab-VD2 solution and stored at room temperature for 4 hours in a humid environment. PBS was used to rinse the Ab-VD2/CD-CH/ITO bioelectrode to get rid of any excess antibodies. Finally, BSA (10 mL of a concentration of 1 mg/mL) was placed onto the bioelectrode to inhibit any nonspecific active sites. When not in use, the BSA/Ab-VD2/CD-CH/ITO bioelectrode was stored at 4°C.

High-resolution transmission electron microscopy (HR-TEM) was used to determine the shape and size of the CDs. A cyclic voltammogram was used to track the oxidation and reduction of this BSA/Ab-VD2/CDCH/ITO bioelectrode. The FRA method was used to measure the impedance at the bioelectrode-electrolyte interface. The CDs exhibited a uniformly distributed, spherical structure without any obvious agglomeration. The hydrophilic nature of the electrode after modification with CDs increased significantly, indicating that the supports are appropriate for interacting with antibodies made in PBS.

By using the cyclic voltammetry (CV) technique at a scan rate of 50 mVs-1 in PBS containing [Fe(CN)6]3−/4, the electrochemical behavior of the BSA/Ab-VD2/ CD-CH/ITO electrode was investigated. The *Ipa* (anodic peak current) rose around three times (376 A) when CH was modified with CDs. The interaction of macromolecule-sized Ab-VD2 and BSA with freely accessible functional groups on the electrode surface caused the electrochemical current to decrease after the immobilization of Ab-VD2 and BSA onto CD-CH/ITO electrode surface.

Ag-VD2 was detected using the differential pulse voltammetry (DPV) method within the potential range of −0.2 to 0.5 V, with a potential step of 5 mV, pulse amplitude of 25 mV, and pulse period of 50 ms. In PBS containing [Fe(CN)6]3−/4-, the response of BSA/Ab-VD2/CD-CH/ITO was measured in the range of 1–50 ng mL−1 of Ag-VD2. The response of the bioelectrode was first measured in the absence of any Ag-VD2 and then with various concentrations of Ag-VD2 (1, 10, 20, 30, 40, and 50 ng mL−1). The peak current value rose as Ag-VD2 concentration increased. The interaction between Ag-VD2 and Ab-VD2 increased the peak current's magnitude [70].
