title: Fluorescent Probe Encapsulated in SNAP-Tag Protein Cavity To Eliminate Nonspecific Fluorescence and Increase Detection Sensitivity.
abstract: Despite 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.
authors.affiliation: Department of Chemistry, and ‡Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University , 101 Sec. 2, Kuang Fu Road, Hsinchu 30013, Taiwan, ROC.
authors.forename: Yan-Syun
authors.initials: YS
authors.lastname: Zeng
authors.affiliation: Department of Chemistry, and ‡Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University , 101 Sec. 2, Kuang Fu Road, Hsinchu 30013, Taiwan, ROC.
authors.forename: Ruo-Cing
authors.initials: RC
authors.lastname: Gao
authors.affiliation: Department of Chemistry, and ‡Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University , 101 Sec. 2, Kuang Fu Road, Hsinchu 30013, Taiwan, ROC.
authors.forename: Ting-Wei
authors.initials: TW
authors.lastname: Wu
authors.affiliation: Department of Chemistry, and ‡Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University , 101 Sec. 2, Kuang Fu Road, Hsinchu 30013, Taiwan, ROC.
authors.forename: Chien
authors.initials: C
authors.lastname: Cho
authors.affiliation: Department of Chemistry, and ‡Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University , 101 Sec. 2, Kuang Fu Road, Hsinchu 30013, Taiwan, ROC.
authors.forename: Kui-Thong
authors.initials: KT
authors.lastname: Tan
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D002214:Capsules; D002934:Cinnamates / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D006862:Hydrogen Sulfide / Q000097:blood / Q000737:chemistry; D057230:Limit of Detection; D053644:Naphthalimides / Q000737:chemistry; D009601:Nitroreductases / Q000097:blood / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence
chemical_list: D002214:Capsules; D002934:Cinnamates; D005456:Fluorescent Dyes; D053644:Naphthalimides; D011506:Proteins; C018253:alpha-cyanocinnamate; D009601:Nitroreductases; D006862:Hydrogen Sulfide