Figure captions
Figure 1. Schematic illustration of PG-based fluorescent detection for 25-HydroxyvitaminD3. The PicoGreen dye inserted into the minor groove of dsDNA duplex when forming dsDNA through hybridization of 25-HydroxyvitaminD3-specific aptamer and corresponding complementary strand, that generating significant fluorescence enhancement. In the test group, specific binding of 25-HydroxyvitaminD3 and aptamers occurred prior to dsDNA formation, and small amount of remaining free aptamers reacted with complementary strands.
Figure 2. The secondary structures of original aptamer and truncated aptamers. The secondary structures of original VDBA14 aptamer (A and B) and truncated aptamers including D3-1 (C), D3-2 (D), D3-3 (E) and D3-4 (F) were obtained from mfold software program. While maintaining higher affinity to 25-HydroxyvitaminD3, the VDBA14 were heuristically truncated to four aptamers with different sequences by intercepting the small hairpin loop and retaining double helix regions of different lengths that existed in VDBA14 aptamer.
Figure 3. The fluorescence intensities change depending on PG concentrations. The concentration of PG fluorescent dye is an important parameter influencing the detection performance. For VDBA 14, D3-3 and D3-4 aptamers, the fluorescence intensities reached basically stable peak and did not change obviously when 10×PG were added, indicating that 10×PG were enough to mark dsDNA. For D3-1 and D3-2 aptamers, increasing PG concentration did not lead to obvious intensity increase when adding 20×PG.
Figure 4. Affinity and specificity evaluation of aptamers. A) Affinity evaluation of aptamers with different sequences to 25-HydroxyvitaminD3. Through comparison, D3-3 and D3-4 gave rise to significant fluorescence intensity decrease, indicating these two truncated aptamers possessed higher affinity than original VDBA14 aptamer. B) Specificity evaluation of D3-3 and D3-4 for 25-HydroxyvitaminD3 compared to VC, VB12 and FC. By comparison, D3-3 had poorer specificity than D3-4 to 25-HydroxyvitaminD3.
Figure 5. Calibration plot with fluorescence intensity against different concentrations of 25-HydroxyvitaminD3 . The calibration plot with fluorescence intensity of PG/aptamer duplex mixture against different concentrations of 25-HydroxyvitaminD3 from 0.04μg/mL to 0.8μg/mL were displayed. Linear decreases in fluorescence intensity (F) were appeared with increasing 25-HydroxyvitaminD3concentrations, for the reason that increased concentrations of 25-HydroxyvitaminD3 caused binding of targets to more aptamers and less remaining aptamer/complementary strand DNA duplexes were formed.