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Liquid Chromatography
Published in Ernő Pungor, A Practical Guide to Instrumental Analysis, 2020
Amino acid analysis is widely used in biochemistry. Determination of primary structure of proteins and peptide sequencing all require a sensitive and selective method for amino acid analysis. For the analysis of protein structure possessing only small amounts of sample are available. Amino acids processing no significant chromophores or fluorophores in addition to this in a complex mixture large number of substance may be interfere with amino acids. Amino acid analyzers were developed based on ion exchange and operated in post-column derivatization mode utilized either ninhidrin or other fluorogenic reagents such as o-phthalaldehyde. These instruments have been used in different fields, but they have some shortcomings: high cost of instrumentation, long analysis time, and inadequate separation and detection limit for some solutes such as proline, hydroxyproline, cysteine, and cystine. Using HPLC most of the shortcomings can be overcome. It has been demonstrated that o-phthalaldehyde in the presence of either ethanethiol or mercaptoethanol can react with amino acids rapidly.
Molecular Imaging of Viable Cancer Cells
Published in Shoogo Ueno, Bioimaging, 2020
Aminopeptidases are also good targets for fluorescence imaging of tumors, since they play essential roles in many diseases and some of them exhibit altered expression levels in the pathological context. In order to develop activatable probes for aminopeptidases, we synthesized and evaluated a series of hydroxymethyl rhodamine derivatives and found an intriguing difference of intramolecular spirocyclization behavior: hydroxymethyl rhodamine green (HMRG) takes an open xanthene form, whereas the acetylated derivative, Ac-HMRG, exists as a closed spirocyclic structure in aqueous solution at physiological pH (Figure 2.10).31 Based on this distinctive property, we designed and developed highly sensitive fluorogenic probes for aminopeptidases by incorporating substrate peptide moieties into the HMRG scaffold. These probes exist in the colorless and non-fluorescent spirocyclic form at the physiological pH of 7.4, but are converted to HMRG, which exists in the fluorescent xanthene form, by one-step hydrolysis upon reaction with the target aminopeptidase, resulting in a rapid and significant fluorescence activation.
Monitoring Apoptosis and Anticancer Drug Activity in Single Cells Using Nanosensors
Published in Tuan Vo-Dinh, Nanotechnology in Biology and Medicine, 2017
The enzyme substrate–based sensing format utilizes an enzyme substrate that is chemically immobilized onto an optical transducer element. Cleavage of the substrate results in the release of a fluorophore or chromophore, which can be detected in the evanescent field of the nanobiosensor. Thus, caspase activity in response to apoptosis stimuli can be assayed in caspase activation pathways by using either a fluorophore or a chromophore attached to a suitable peptide substrate. Fluorogenic substrates tend to offer better sensitivity than their colorimetric counterparts. Fluorogenic substrates consisting of fluorophores, for example, methyl coumarin or rhodamine, are covalently linked to peptides via amino terminal groups, thereby suppressing both the visible absorption and fluorescence of the fluorophore. Upon enzymatic cleavage of the nonfluorescent substrate, the fluorophore is released and it can be excited and detected at the appropriate excitation and emission wavelength. It is important to use fluorescence emission filters to produce excellent signal-to-background ratios [27–30].
Self-assembly as a key player for materials nanoarchitectonics
Published in Science and Technology of Advanced Materials, 2019
Katsuhiko Ariga, Michihiro Nishikawa, Taizo Mori, Jun Takeya, Lok Kumar Shrestha, Jonathan P. Hill
Application of self-assembly concepts is not limited to organic substances. Nanoarchitectonics strategy upon self-assembly is applicable to systems with metallic and inorganic substances in which self-assembly of inorganic materials themselves and hybrid assemblies between organic and inorganic substances can be considered [169–177]. As self-assembly of functional parts on gold surfaces for sensing applications, Prins and co-workers demonstrated fluorescence sensing systems through nanoarchitectonics on Au nanoparticles [178]. On the surface of Au nanoparticles, functional moieties such as 1,4,7-triazacyclononane Zn(II) complex and a 1,4,7,10-tetraazacyclododecane Zn(II) complex were immobilized together with fluorogenic probe molecules. Upon selective binding of guest molecules to the complex part, release of the fluorogenic probe molecules from the surface of nanoparticles to solution resulted in fluorescent signals. This strategy can be applied in versatile combination of recognition and fluorescent functional units, which possibly enables to nanoarchitect various sensing systems for many kinds of chemical inputs. Harlé, Fujiwara, and co-workers immobilized malachite green derivatives as a self-assembled monolayer (SAM) on TiO2 [179]. The malachite green derivatives were characterized at negative excited state redox levels for more appropriate in use of dye-sensitized solar cell applications.