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.