Specific oligonucleotides such as telomere DNA and aptamer often undergo conformational changes upon ligand binding. Composite reagent composed of o-phthalaldehyde and β-mercaptoethanol（OPAME） has been extensively ...Specific oligonucleotides such as telomere DNA and aptamer often undergo conformational changes upon ligand binding. Composite reagent composed of o-phthalaldehyde and β-mercaptoethanol（OPAME） has been extensively applied to fluorescent detection of amino compounds based on the reaction of primary amino-group, herein we proposed a general spectrofluorometry for ions and small molecules due to conformational changes upon ligand binding taking K^＋ and ATP as examples. In a borate controlled buffer medium, telomere DNA could react with OPAME, giving a thio-subtituted isoindole compound with strong fluorescence emission at 455 nm when excited at 340 nm. It was found that however, the fluorescence emission was greatly reduced in the presence of K^＋ since the formation of the quadruplex structure inhibits the reaction activity of amino-groups of telomere DNA. In order to testify the general application of OPAME reagent based on the conformational change of oligonucleotides, we further proposed a sensitive method of ATP based on its highly selective interaction with ATP-aptamer. The above mentioned applications show that the spectrofluorometry with the aid of OPAME reagent is simple, label free that is expected to be potentially general for DNA conformational change-based target detection.展开更多
A rapid method has been developed based on the sample preparation procedure named as QuEChERS （Quick, Easy, Cheap, Effective, Rugged and Safe）, combined with reversed-phase high performance liquid chromatography wit...A rapid method has been developed based on the sample preparation procedure named as QuEChERS （Quick, Easy, Cheap, Effective, Rugged and Safe）, combined with reversed-phase high performance liquid chromatography with fluorescence detector and C18 column after precolumn derivatization using o-phthalaldehyde and 2-mercaptoethanol to determine dopamine in porcine muscle. Methanol and deionized water （0.1% acetic acid, v/v） with a ratio of 60：40 was used as mobile phase. The flow rate was 0.8 mL/min and dopamine was eluted within 15 min. The linearity range was 0.003-8 μg/mL with r=0.9992. The detection limit for dopamine was 4 μg/kg and the quantification limit was 9 μg/kg. Recovery studies were carried out at 0.1, 0.5 and 1.0 mg/kg fortification levels and the average recoveries obtained ranged from 90.4% to 98.2% with relative standard deviations between 3.5% and 8.1%. The method was found to be suitable for detection of dopamine in animal product tissues at the maximum residue level.展开更多
The response of extracellular matrix(ECM) to dynamic cell signals is of great significance for the regulation of cell behavior. In the present study, we prepared a type of matrix metalloproteinase(MMP)-sensitive degra...The response of extracellular matrix(ECM) to dynamic cell signals is of great significance for the regulation of cell behavior. In the present study, we prepared a type of matrix metalloproteinase(MMP)-sensitive degradable hydrogels(MSDHs) via the catalyst-free o-phthalaldehyde(OPA)/amine cross-linking reaction between o-phthalaldehyde-grafted four-arm poly(ethylene glycol)(4aPEG-OPA) and an MMP-sensitive degradable peptide. The gelation rates and storage moduli of MSDHs and the MMP-insensitive hydrogels(MIHs) based on an MMP-insensitive scramble peptide were comparable and dependent on the concentrations of precursor polymers. MSDHs were degradable while MIHs were stable in the presence of proteinase in vitro.The degradation of MSDHs was obviously faster than that of MIHs after subcutaneous injection into rats. In addition, both types of poly(ethylene glycol)/peptide hydrogels displayed excellent cytocompatibility in vitro, and showed good histocompatibility in vivo in the subcutaneous layer of rats. Furthermore, the proliferation of several MMP-expressing cell lines including MDA-MB-231 cells within MSDHs was obviously faster than that in MIHs, indicating the influence of metabolism-mediated scaffold degradation on the cell proliferation. This study provides a new biocompatible and biodegradable 3 D cell culture interactive platform for regulation of cell behavior.展开更多
基金Supported by the National Natural Science Foundation of China(No.20775061)
文摘Specific oligonucleotides such as telomere DNA and aptamer often undergo conformational changes upon ligand binding. Composite reagent composed of o-phthalaldehyde and β-mercaptoethanol（OPAME） has been extensively applied to fluorescent detection of amino compounds based on the reaction of primary amino-group, herein we proposed a general spectrofluorometry for ions and small molecules due to conformational changes upon ligand binding taking K^＋ and ATP as examples. In a borate controlled buffer medium, telomere DNA could react with OPAME, giving a thio-subtituted isoindole compound with strong fluorescence emission at 455 nm when excited at 340 nm. It was found that however, the fluorescence emission was greatly reduced in the presence of K^＋ since the formation of the quadruplex structure inhibits the reaction activity of amino-groups of telomere DNA. In order to testify the general application of OPAME reagent based on the conformational change of oligonucleotides, we further proposed a sensitive method of ATP based on its highly selective interaction with ATP-aptamer. The above mentioned applications show that the spectrofluorometry with the aid of OPAME reagent is simple, label free that is expected to be potentially general for DNA conformational change-based target detection.
基金supported by the Natural Science Foundation of Shaanxi Province (no.2009jm4002-1)
文摘A rapid method has been developed based on the sample preparation procedure named as QuEChERS （Quick, Easy, Cheap, Effective, Rugged and Safe）, combined with reversed-phase high performance liquid chromatography with fluorescence detector and C18 column after precolumn derivatization using o-phthalaldehyde and 2-mercaptoethanol to determine dopamine in porcine muscle. Methanol and deionized water （0.1% acetic acid, v/v） with a ratio of 60：40 was used as mobile phase. The flow rate was 0.8 mL/min and dopamine was eluted within 15 min. The linearity range was 0.003-8 μg/mL with r=0.9992. The detection limit for dopamine was 4 μg/kg and the quantification limit was 9 μg/kg. Recovery studies were carried out at 0.1, 0.5 and 1.0 mg/kg fortification levels and the average recoveries obtained ranged from 90.4% to 98.2% with relative standard deviations between 3.5% and 8.1%. The method was found to be suitable for detection of dopamine in animal product tissues at the maximum residue level.
基金the National Natural Science Foundation of China(Grant Nos.51973218,51622307,21574127,51520105004)the Youth Innovation Promotion Association CAS。
文摘The response of extracellular matrix(ECM) to dynamic cell signals is of great significance for the regulation of cell behavior. In the present study, we prepared a type of matrix metalloproteinase(MMP)-sensitive degradable hydrogels(MSDHs) via the catalyst-free o-phthalaldehyde(OPA)/amine cross-linking reaction between o-phthalaldehyde-grafted four-arm poly(ethylene glycol)(4aPEG-OPA) and an MMP-sensitive degradable peptide. The gelation rates and storage moduli of MSDHs and the MMP-insensitive hydrogels(MIHs) based on an MMP-insensitive scramble peptide were comparable and dependent on the concentrations of precursor polymers. MSDHs were degradable while MIHs were stable in the presence of proteinase in vitro.The degradation of MSDHs was obviously faster than that of MIHs after subcutaneous injection into rats. In addition, both types of poly(ethylene glycol)/peptide hydrogels displayed excellent cytocompatibility in vitro, and showed good histocompatibility in vivo in the subcutaneous layer of rats. Furthermore, the proliferation of several MMP-expressing cell lines including MDA-MB-231 cells within MSDHs was obviously faster than that in MIHs, indicating the influence of metabolism-mediated scaffold degradation on the cell proliferation. This study provides a new biocompatible and biodegradable 3 D cell culture interactive platform for regulation of cell behavior.
