Abstract
For maintaining the healthy metabolic status, vitamin D is a beneficial metabolite stored majorly in its pre-activated form, 25-hydroxyvitamin D3 (25(OH)D3). Due to its important role in bone strengthening, the study was planned to quantify 25(OH)D3 levels in our blood. Quantification techniques for 25(OH)D3 are costly thus requiring a need for a low cost, and sensitive detection methods. In this work, an economic, and sensitive sensor for the detection of 25(OH)D3 was developed using aptamer and graphene oxide (GO). Aptamer is an oligonucleotide, sensitive towards its target, whereas, GO with 2D nanosheets provides excellent quenching surface. Aptamer labeled with fluorescein (5’, 6-FAM) is adsorbed by π–π interaction on the GO sheets leading to quenching of the fluorescence due to Förster resonance energy transfer (FRET). However, in the presence of 25(OH)D3, a major portion of aptamer fluorescence remains unaltered, due to its association with 25(OH)D3. However, in the absence, aptamer fluorescence gets fully quenched. Fluorescence intensity quenching was monitored using fluorescence spectrophotometer and agarose gel based system. The limit of detection of 25(OH)D3 by this method was found to be 0.15 µg/mL whereas when GO-COOH was used, limit of detection was improved to 0.075 µg/mL. Therefore, this method could come up as a new sensing method in the field of vitamin D detection.
Highlights
Vitamin D (Vit D) is a major regulatory fat soluble secosteroid moiety governing the skeletal growth and calcium homeostasis
In order to be used as sensors, aptamers can be labeled with fluorophores, gold nanoparticles and quantum dots that can be visualized on their respective binding[20,21,22]
FRET (Förster resonance energy transfer) is the phenomenon of energy exchange between molecules associated in < 10 nm of range[26]
Summary
Vitamin D (Vit D) is a major regulatory fat soluble secosteroid moiety governing the skeletal growth and calcium homeostasis. Immunoassays having poor antibody specificity with issues of cross-reactivity while chromatography techniques involving costly instruments that require trained staff for o peration[6,7] Due to these cost limitations and high maintenance issues, status monitoring of 25(OH)D3 is hampered. Chauhan et al, have been working on different procedures for Vit-D detection and have come up with electrochemical sensors using polyacrylonitrile nanofibers with incorporated magnetic nanoparticles and gadolinium oxide nanorods They were able to obtain enhanced sensitivity of metal oxide nanorods as compared to n anofibers[11]. Aptamers have successfully come up as a detection tool that is stable, cheap and reproducible[13] They are oligonucleotide equivalent of antibodies having specificity towards particular metabolites[14]. Considering the stability, specificity and low cost of aptamers we planned to prepare a FRET based assay for Vit-D detection with the help of above explained VDBA-14 aptamer
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