Materials
25-hydroxyvitaminD3 (25(OH)D3), 25-hydroxyvitaminD2 (25(OH)D2), Chenodeoxycholic acid (CDA), Tris, Sodium chloride, Graphite flakes from Sigma (www.sigmaaldrich.com), Agarose from Lonza (www.lonza.com). Aptamer specific to vitamin D was taken from literature36 and synthesized with 6-carboxyfluorescein (FAM) modifications at its 5’end by IDT. 96 well black round bottom plate from Corning, Thermoscientific (www.thermoscientific.com). Mice serum was taken from animal house facility at National Agri-Food Biotechnology Institute, Mohali, India under the ethical no. NABI/2039/CPCSEA/IAEC/2019.
Synthesis and characterizations of Graphene Oxide (GO)
GO was prepared by modified Hummers’ method 37. Briefly, a 9:1 mixture of concentrated H2SO4/H3PO4 (360:40 mL) was added to a mixture of graphite flakes (3.0 g, 1 wt. equiv.) and KMnO4 (18.0 g, 6 wt. equiv.). The reaction was then heated to 50°C and stirred for 12 h. The reaction was cooled to RT and poured onto ice (400 mL) with 30% H2O2 (3 mL). Then filtered through muslin cloth. The filtrate was centrifuged (5380g for 30mins), and supernatant decanted. The remaining solid material was then washed two times in succession with 200 mL of water, 200 mL, 30% HCl, and 200 mL, ethanol and after each wash, the mixture was filtered through muslin cloth with the filtrate being centrifuged (5380g for 30mins) and the supernatant decanted away. The remaining material after washing was coagulated with 200 mL of ether, and centrifuged at 5380g for 30mins to remove ether. The pellet obtained was dissolved in water and 0.1M NaOH was added to make homogenous suspension followed by dialysis using deionized water and lastly lyophilized overnight at room temperature to find the concentration of the obtained GO. Prepared graphene oxide was characterized by UV-Vis spectroscopy, TEM imaging and AFM analysis.
Optimization of Graphene Oxide concentration, temperature and pH for efficient quenching and regaining assays
Aptamer (stock 1µM) was prepared in 20 mM phosphate buffer, 25 mM NaCl, pH 7.4. To estimate the optimum concentration of GO, its varying concentrations (0, 12.5, 25, 50, 100 µg/ml) were incubated with aptamer, 100 nM (working concentration) for 10 minutes in 250 µl reaction mixture at room temperature. Centrifugation was followed at 18800g for 20mins. Supernatant obtained was separated, used for fluorescence estimations and for visualized confirmation on agarose gel (3%).
To study the effects of pH on the binding, 5 µg/mL of 25(OH)D3 was incubated with aptamer in varying reaction buffers, 20 mM sodium acetate buffer (pH-3.5, 5.5), 20 mM phosphate buffer (pH-7.4), 20 mM tris buffer (pH-8.3, 9.2). In order to find the optimum temperature, reaction mixture containing aptamer and 25(OH)D3, was incubated at 4⁰C, 16⁰C, 25⁰C, 37⁰C and 50⁰C. After the appropriate condition for standardization was completed over 45 minutes, GO (optimized as above, 50 µg/ml) was added to the reaction mixture followed by centrifugation and later estimations.
Fluorescence detection of Vitamin D3
In a 250µL reaction mixture, aptamer (100 nM) and 25(OH)D3 (0.15, 0.3, 0.6, 1.25, 2.5, 5, 10 and 20 µg/mL) were incubated in 20 mM phosphate buffer (25 mM NaCl, pH7.4) for 45 minutes with gentle mixing at 450 rpm in thermomixer (Eppendorf). GO (50 µg/mL) was added and allowed to mix for 10 mins at room temperature. In control experiment, only aptamer and GO were taken without 25(OH)D3. Centrifugation was followed with supernatant removal without disturbing the pellet and analysis by fluorescence estimation and agarose gel based visualization.
Selectivity and real-time sample analysis
Closely resembling analytes, 25(OH)D3, 25(OH)D2 and CDA was used for the specificity check of the assay. 5 µg/mL of each biomolecule was diluted in 20 mM phosphate buffer, pH 7.4. Optimized concentrations of aptamer and GO was added and analyzed using the same procedure as given above for the detection of 25(OH)D3.
In order to determine practical applications in human serum sample, mice serum was taken as a real-time sample. Serum was diluted to 50% with phosphate buffer, spiked up with 25(OH)D3 in dilutions ranging from 0.15, 0.3, 0.6, 1.25, 2.5, 5,10 µg/mL were used for the estimations. For the control experiment, non-spiked sample of 50% mice serum was used. Rest of the assay procedure was followed as described above.