RESUMO
The development of a separation-based sensor for catecholamines based on microdialysis (MD) coupled to microchip electrophoresis (ME) with electrochemical (EC) detection is described. The device consists of a pyrolyzed photoresist film working electrode and a poly(dimethylsiloxane) microchip with a flow-gated sample injection interface. The chip was partially reversibly sealed to the glass substrate by selectively exposing only the top section of the chip to plasma. This partially reversible chip/electrode integration process not only allows the reuse of the working electrode but also greatly enhanced the reproducibility of electrode alignment with the separation channel. The developed MD-ME-EC system was then tested using l-DOPA, 3-O-MD, HVA, DOPAC, and dopamine standards, which were separated in less than 100 seconds using a background electrolyte consisting of 15 mM sodium phosphate (pH 7.4), 15 mM sodium dodecyl sulfate, and 2.5 mM boric acid. A potential of +1.0 V vs. Ag/AgCl was used for amperometric detection of the analytes. The device was evaluated for on-line monitoring of the conversion of l-DOPA to dopamine in vitro and for monitoring dopamine release in an anesthetized rat in vivo following high K+ stimulation. The system was able to detect stimulated dopamine release in vivo but not endogenous levels of dopamine.
Assuntos
Encéfalo/metabolismo , Catecolaminas/análise , Dopamina/análise , Eletroquímica/métodos , Eletrodos , Eletroforese em Microchip/métodos , Levodopa/análise , Animais , Catecolaminas/isolamento & purificação , Dopamina/isolamento & purificação , Levodopa/isolamento & purificação , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Microchip electrophoresis with amperometric detection (ME-EC) is a useful tool for the determination of redox active compounds in complex biological samples. In this review, a brief background on the principles of ME-EC is provided, including substrate types, electrode materials, and electrode configurations. Several different detection approaches are described, including dual-channel systems for dual-electrode detection and electrochemistry coupled with fluorescence and chemiluminescence. The application of ME-EC to the determination of catecholamines, adenosine and its metabolites, and reactive nitrogen and oxygen species in microdialysis samples and cell lysates is also detailed. Lastly, approaches for coupling of ME-EC with microdialysis sampling to create separation-based sensors that can be used for near real-time monitoring of drug metabolism and neurotransmitters in freely roaming animals are provided. Graphical abstract.
Assuntos
Técnicas Eletroquímicas/métodos , Eletroforese em Microchip/métodos , Animais , Eletrodos , Desenho de Equipamento , Camundongos , Microdiálise , OvinosRESUMO
It is well documented that the structure, and thus function, of nucleic acids depends on the chemical environment surrounding them, which often includes potential proteinaceous binding partners. The nonpolar amino acid side chains of these proteins will invariably alter the polarity of the local chemical environment around the nucleic acid. However, we are only beginning to understand how environmental polarity generally influences the structural and energetic properties of RNA folding. Here, we use a series of aqueous-organic cosolvent mixtures to systematically modulate the solvent polarity around two different RNA folding constructs that can form either secondary or tertiary structural elements. Using single-molecule Förster resonance energy transfer spectroscopy to simultaneously monitor the structural and energetic properties of these RNAs, we show that the unfolded conformations of both model RNAs become more compact in apolar environments characterized by dielectric constants less than that of pure water. In the case of tertiary structure formation, this compaction also gives rise to more energetically favorable folding. We propose that these physical changes arise from an enhanced accumulation of counterions in the low dielectric environment surrounding the unfolded RNA.
RESUMO
Rapid monitoring of concentration changes of neurotransmitters and energy metabolites is important for understanding the biochemistry of neurological disease as well as for developing therapeutic options. This paper describes the development of a separation-based sensor using microchip electrophoresis (ME) with electrochemical (EC) detection coupled to microdialysis (MD) sampling for continuous on-line monitoring of adenosine and its downstream metabolites. The device was fabricated completely in PDMS. End-channel electrochemical detection was accomplished using a carbon fiber working electrode embedded in the PDMS. The separation conditions for adenosine, inosine, hypoxanthine, and guanosine were investigated using a ME-EC chip with a 5-cm long separation channel. The best resolution was achieved using a background electrolyte consisting of 35 mM sodium borate at pH 10, 15% dimethyl sulfoxide (DMSO), and 2 mM sodium dodecyl sulphate (SDS), and a field strength of 222 V/cm. Under these conditions, all four purines were separated in less than 85 s. Using a working electrode detection potential of 1.4 vs Ag/AgCl, the limits of detection were 25, 33, 10, and 25 µM for adenosine, inosine, hypoxanthine, and guanosine, respectively. The ME-EC chip was then coupled to microdialysis sampling using a novel all-PDMS microdialysis-microchip interface that was reversibly sealed. This made alignment of the working electrode with the end of the separation channel much easier and more reproducible than could be obtained with previous MD-ME-EC systems. The integrated device was then used to monitor the enzymatic conversion of adenosine to inosine in vitro.