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Fluorescent Probe Encapsulated in SNAP-Tag Protein Cavity To Eliminate Nonspecific Fluorescence and Increase Detection Sensitivity
journal contribution
posted on 2016-07-27, 14:48 authored by Yan-Syun Zeng, Ruo-Cing Gao, Ting-Wei Wu, Chien Cho, Kui-Thong TanDespite the promising improvements
made recently on fluorescence
probes for the detection of enzymes and reactive small molecules,
two fundamental problems remain: weaker fluorescence of many dyes
in aqueous buffers and strong nonspecific signals in samples containing
high protein levels. In this paper, we introduce a novel fluorescent
probe encapsulated in protein cavity (FPEPC) concept as demonstrated
by SNAP-tag protein and three environment-sensitive fluorescence probes
to overcome these two problems. The probes were constructed by following
the current probe design for enzymes and reactive small molecules
but with an additional benzylguanine moiety for selective SNAP-tag
conjugation. The SNAP-tag conjugated probes achieved quantitative
nitroreductase and hydrogen sulfide detection in blood plasma, whereas
analyte concentrations were overestimated up to 700-fold when bare
fluorescent probes were employed for detection. Furthermore, detection
sensitivity was increased dramatically, as our probes displayed 390-fold
fluorescence enhancement upon SNAP-tag conjugation, in stark contrast
to the weak fluorescence of the free probes in aqueous solutions.
Compared with the conventional approaches where fluorescent probes
are encapsulated into polymers and nanoparticles, our simple and general
approach successfully overcame many key issues such as dye leakage,
long preparation steps, inconsistent dye–host ratios, difficulty
in constructing in situ in a complex medium, and limited application
to detect only small metabolites.
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Keywords
detection sensitivityprobe encapsulatedhydrogen sulfide detectionfluorescence probesblood plasmaanalyte concentrationsprotein levelsprobe designDetection SensitivityNonspecific Fluorescencebenzylguanine moietydye leakageFPEPCpreparation stepsfluorescence enhancementFluorescent Probe Encapsulatedprotein cavity
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