Highly sensitive fluorescent detection of 25-HydroxyvitaminD3 using
truncated affinity-improved DNA aptamers
Abstract
Vitamin D insufficiency is closely related to various kinds of metabolic
diseases. Acted as a marker of vitamin D status, 25-HydroxyvitaminD3
detection possesses important practical significance. In this study,
highly sensitive fluorescent detection of 25-HydroxyvitaminD3 using
truncated affinity-improved aptamers were developed, based on
fluorescence intensity changes of PicoGreen (PG) generated upon binding
to double-stranded DNA (dsDNA) formed through hybridization of aptamer
and corresponding complementary strand. Four truncated aptamers were
obtained by intercepting the small hairpin loop as the functional domain
and retaining double helix structural domains of different lengths that
exist in the selected original 25-HydroxyvitaminD3 aptamer. Under the
optimized PG concentration, we conducted comparison experiments for
affinity and specificity of these four truncated aptamers. Among them,
the shortest aptamer with only 21 bp, D3-4, was found to show the
highest affinity and specificity to 25-HydroxyvitaminD3, with the limit
of detection of 0.04μg/mL, which solved the problem that original long
aptamer could not applied for this fluorescent detection of
25-HydroxyvitaminD3. The truncated 25-HydroxyvitaminD3-specific aptamer
with highly enhanced affinity performs promising application in
sensitive detection of 25-HydroxyvitaminD3.