1.Ab-25OHD/SPE/FMTAD

2.CYP27B1/GCE

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

3.SiO2/GO/Ni(OH)2/GCE

4.BSA/Ab-VD2/CD-CH/ITO

5.BSA/Anti-VD/Fe3O4 PANnFs/ITO

6.BSA/Ab-VD/Asp-Gd2O3NRs/ITO

7. 25OHD

8. 25OHD Antibody

9.IoT-Enabled Enzyme Embossed Biosensor

10.Au-Pt NPs/APTES/FTO

11.GCN-β-CD/Au nanocomposite

#### **3.1 Ab-25OHD/SPE/FMTAD**

The Ab-25OHD/SPE/FMTAD technology plays a crucial role in the determination of vitamin D levels in the body [26]. The Ab-25OHD/SPE/FMTAD technology uses an antibody-based assay to detect the presence of 25-hydroxyvitamin D (25OHD) in blood serum samples. 25OHD is the major circulating form of vitamin D and is used as a biomarker to determine vitamin D status. The assay is based on a competitive format, where a sample containing 25OHD competes with a labeled 25OHD conjugate for binding to a specific antibody. The amount of 25OHD in the sample is inversely proportional to the signal detected, and the assay provides a quantitative measurement of 25OHD levels in the sample [27, 28].

Compared to traditional methods for vitamin D measurement, such as liquid chromatography–tandem mass spectrometry (LC–MS/MS) and radioimmunoassay (RIA), the Ab-25OHD/SPE/FMTAD technology offers several advantages. It is highly specific, sensitive, and rapid, allowing for the measurement of 25OHD levels in small volumes of serum samples. The use of antibodies ensures the accuracy and precision of the assay, and the use of fluorescent detection provides a reliable and reproducible readout. The Ab-25OHD/SPE/FMTAD technology has been extensively validated and has demonstrated high concordance with LC–MS/MS, the gold standard for vitamin D measurement. Studies have shown that the technology offers improved accuracy and precision compared to other immunoassays, including RIA and chemiluminescence immunoassay (CLIA) methods [29].

In conclusion, the Ab-25OHD/SPE/FMTAD technology plays a critical role in determining vitamin D levels in the body. Its use can aid in the assessment of vitamin D status and inform clinical decisions regarding the prevention and management of vitamin D deficiency.

#### **3.2 CYP27B1/GCE**

An important super family of monooxygenases that can be found in various organisms is the cytochromes P450 (CYP450) [30, 31]. These enzymes are involved in the metabolism of a wide range of chemicals using different biotransformation

reactions and are associated with the synthesis of steroids, vitamins, lipids, and xenobiotic and drug metabolism [30–38]. These enzymes have therefore attracted enormous biotechnological attention, and they have been used in bioelectronic devices, biochips, bioreactors, and biosensor technologies [37–40]. The enzyme can be directly deposited on the electrode during direct electron transfer and it can be attached to the electrode using gold nanoparticles [41] or can be anchored to the electrode that is altered with polyelectrolyte multilayer films [42]. Within the cell, the cytochrome P450 27B1 (CYP27B1) enzyme, which belongs to CYP450, converts 25-hydroxyvitamin D (25(OH)D) into 1,25-dihydroxyvitamin D (1,25(OH)2D). 25(OH)D is considered the best marker of the body's vitamin D status [43, 44]. Vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol) are the two common forms of this nutrient. Detection of vitamin D deficiency is generally accomplished using commercial assays for 25(OH)D. Radioimmunoassays (RIA), high-pressure liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC–MS/MS) is used for determining 25(OH)D2 and 25(OH)D3 values in serum [45]. The human CYP27B1, a 55 kDa hemoprotein [45], is membrane-bound. Cobalt sepulchrate trichloride (Co(sep)3+), a non-native redox mediator, has been employed successfully with other CYP450s [45–47]. Synthetic mediators like Co(sep)3+ have many advantages such as facilitating quick, reversible electrochemistry, enhanced rates of reactions, and facile electrode design while maintaining flexibility in enzyme immobilization [48].

Ozbakir and Sambade [47] made an enzymatic electrode for 25(OH)D3 detection. First, the human CYP27B1 synthetic gene was produced by *E. coli*, purified, and its activity was determined by LC-MS/MS. Co(sep)3+, a redox mediator, was created and used to electrochemically monitor CYP27B1 activity. For this, a glassy carbon electrode was immobilized with a combination of pure CYP27B1, the redox mediator Co(sep)3+, and NafionR. The performance of the electrode was then assessed using cyclic and square wave voltammetry in the physiological range of 25(OH)D3 [49].

NADPH, 25(OH)D3Tris(ethylenediamine) colt(III) chloride trihydrate, 3-((3-cholamidopropyl) dimethylamino)-1-propanesulfonate (CHAPS), lithium carbonate, sodium diethyldithiocarbamate trihydrate, hexane, and dichloromethane were the materials employed together with FITC-conjugated secondary antibodies and anti-CYP27B1, anti-adrenodoxin, and adrenodoxin reductase primary antibodies. Reference electrodes used were Glassy carbon working electrode and Ag/AgCl [49, 50].

In the presence of NADPH as an electron donor, ADX, and ADR as electrontransfer proteins, CYP27B1 catalyzes the hydroxylation of 25(OH)D to 1,25(OH)2D. To calculate the quantity of 1,25(OH)2D and other potential byproducts that were created various 25(OH)D3 concentrations were used in the experiments, and they were compared to controls without CYP27B1. As the initial reactant concentration in the experiment climbed, the concentrations of the 1,25(OH)2D3 and (OH)2D3 isomers both increased linearly. The reaction that is most pertinent to the creation of a sensor for the detection of vitamin D involves the fact that consumption of the substrate rose when the quantity of CYP27B1 was raised in the assay [49, 51].

The GCE/NafionR/Co(sep)3+/CYP27B1 electrode has been demonstrated to be successful in detecting 25(OH)D3 in buffer within the physiological range (5–200 ng/ ml) using cyclic and square wave voltammetry. The sensor can detect 25(OH)D3 in the physiological range of concentrations from 5 to 200 ng/ml, but it will need to be optimized for the electrode assembly and method of detection to work in the complete range [24, 48, 50].
