title: In-capillary probing of quantum dots and fluorescent protein self-assembly and displacement using Förster resonance energy transfer.
abstract: Herein, a Förster resonance energy transfer system was designed, which consisted of CdSe/ZnS quantum dots donor and mCherry fluorescent protein acceptor. The quantum dots and the mCherry proteins were conjugated to permit Förster resonance energy transfer. Capillary electrophoresis with fluorescence detection was used for the analyses for the described system. The quantum dots and mCherry were sequentially injected into the capillary, while the real-time fluorescence signal of donor and acceptor was simultaneously monitored by two channels with fixed wavelength detectors. An effective separation of complexes from free donor and acceptor was achieved. Results showed quantum dots and hexahistidine tagged mCherry had high affinity and the assembly was affected by His6-mCherry/quantum dot molar ratio. The kinetics of the self-assembly was calculated using the Hill equation. The microscopic dissociation constant values for out of- and in-capillary assays were 10.49 and 23.39 μM, respectively. The capillary electrophoresis with fluorescence detection that monitored ligands competition assay further delineated the different binding capacities of histidine containing peptide ligands for binding sites on quantum dots. This work demonstrated a novel approach for the improvement of Förster resonance energy transfer for higher efficiency, increased sensitivity, intuitionistic observation, and low sample requirements of the in-capillary probing system.
authors.affiliation: School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, People's Republic of China.
authors.forename: Jianhao
authors.initials: J
authors.lastname: Wang
authors.affiliation: School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, People's Republic of China.
authors.forename: Jie
authors.initials: J
authors.lastname: Fan
authors.affiliation: School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, People's Republic of China.
authors.forename: Jinchen
authors.initials: J
authors.lastname: Li
authors.affiliation: School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, People's Republic of China.
authors.forename: Li
authors.initials: L
authors.lastname: Liu
authors.affiliation: School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, People's Republic of China.
authors.forename: Jianpeng
authors.initials: J
authors.lastname: Wang
authors.affiliation: School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, People's Republic of China.|Key Laboratory of Synthetic Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, People's Republic of China.
authors.forename: Pengju
authors.initials: P
authors.lastname: Jiang
authors.affiliation: School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, People's Republic of China.
authors.forename: Xiaoqian
authors.initials: X
authors.lastname: Liu
authors.affiliation: School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, People's Republic of China.|State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing, Jiangsu, People's Republic of China.
authors.forename: Lin
authors.initials: L
authors.lastname: Qiu
mesh_terms: D002623:Chemistry Techniques, Analytical / Q000295:instrumentation / Q000379:methods*; D019075:Electrophoresis, Capillary; D031541:Fluorescence Resonance Energy Transfer; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism*; D010455:Peptides; D011485:Protein Binding; D045663:Quantum Dots / Q000378:metabolism*; D000097573:Red Fluorescent Protein
chemical_list: D008024:Ligands; D008164:Luminescent Proteins; D010455:Peptides; D000097573:Red Fluorescent Protein
keywords: Förster resonance energy transfer; assembly; capillary electrophoresis; fluorescent protein; quantum dots