Development of a microfluidic confocal fluorescence detection system for the hyphenation of nano-LC to on-line biochemical assays

One way to profile complex mixtures for receptor affinity is to couple liquid chromatography (LC) on-line to biochemical detection (BCD). A drawback of this hyphenated screening approach is the relatively high consumption of sample, receptor protein and (fluorescently labeled) tracer ligand. Here, we worked toward minimization of sample and reagent consumption, by coupling nano-LC on-line to a light-emitting diode (LED) based capillary confocal fluorescence detection system capable of on-line BCD with low-flow rates. In this fluorescence detection system, a capillary with an extended light path (bubble cell) was used as a detection cell in order to enhance sensitivity. The technology was applied to a fluorescent enhancement bioassay for the acetylcholine binding protein, a structural analog of the extracellular ligand-binding domain of neuronal nicotinic acetylcholine receptors. In the miniaturized setup, the sensitive and low void volume LED-induced confocal fluorescence detection system operated in flow injection analysis mode allowing the measurement of IC(50) values, which were comparable with those measured by a conventional plate reader bioassay. The current setup uses 50 nL as injection volume with a carrier flow rate of 400 nL/min. Finally, coupling of the detection system to gradient reversed-phase nano-LC allowed analysis of mixtures in order to identify the bioactive compounds present by injecting 10 nL of each mixture

CE of tricyclic antidepressant clomipramine and metabolites: electromigration and wall adsorption

CE of tricyclic antidepressants clomipramine and its metabolites demethylclomipramine, didemethylclomipramine and 8-hydroxyclomipramine resulted in partly extremely tailing peaks in bare fused-silica capillaries. Especially at high pH of the BGE this behavior was not unexpected as adsorption of the cationic analytes onto the negatively charged wall due to electrostatic attraction can be supposed. Less expected was the observation that peak tailing could not be overcome neither by using a capillary with dynamic coating with cationic CTAB added to the BGE, nor by the usage of a capillary permanently coated with polyvinyl alcohol (PVA), both operated at acidic pH. As this tailing was even more pronounced than with bare fused silica, and was suppressed upon addition ofMeCN to the BGE, nother source of adsorption than pure ion\u2013ion interaction seems plausible. In the bare silica apillary the mobility, m, of the analytes followed roughly the pHdependence of a monoacidic base, but two deviations from the sigmoid theoretical curve were evident: (i) even at low pH the mobilities were not constant; they decreased in contrary withpHover the entire range; (ii) the apparent pKa values of two analytes, derived at the pH with halve the mobility at low pH, are significantly smaller than the thermodynamic pKa. Upon modifying the expression for m = f(pH), and considering thepHdependence of the negative charge density at the wall by an additional term which takes chromatographic retention into account, an equation was derived which enables the description of the observed electromigration of the analytes as function of pH, pKa of analytes and surface silanol groups, actual mobility of analytes, distribution coefficient (or retention factor) due to adsorption including itspHdependence. The interplay of electrophoretic movement and residual adsorptive retention allowed to resolve the analytes finally in an uncoated capillary, namely at pH 7.65 (30 mM ionic strength), whereas at the cost of the robustness of the separation system

The effects of 4,7-di(pyrrolidin-1-yl) substituents on the reduction and oxidation mechanisms of 1,10-phenanthrolines: New perspectives in tailoring of phenantroline derivatives

The oxidation and reduction mechanisms of substituted 4,7-di(pyrrolidin-1-yl)-1,10-phenanthrolines were investigated in non-aqueous environment by means of cyclic voltammetry. Reduction of these derivatives leads to the formation of radical anion and subsequent reductive cleavage of pyrrolidine-1-yl moiety in overall ECE reduction processes. The regenerating formation of 1,10-phenanthroline was observed. IR spectroelectrochemistry and HPLC-MS/MS analysis were applied to support this result. The presence of pyrrolidine-1-yl moiety significantly thermodynamically facilitates the reduction of the 1,10-phenanthroline structure. Concerning oxidation, the primarily formed cation radical on nitrogen of the pyrrolidine moiety is further oxidized and undergoes a coupled nucleophilic addition of water and hydroxylated compound is formed as the oxidation product. Additionally, further opening of pyrrolidine ring and oxidative cleavage of alkyl are other proposed reactions. The results presented in this work pave the way for novel chelating agents with electrochemically controlled polarity. Furthermore, reductive regeneration of individual components of the molecular assemblies described in this work opens new directions in electrochemical technologies for the environmental protection

Spectroelectrochemical Properties of 1,10-Phenanthroline Substituted by Phenothiazine and Carbazole Redox-active Units

Complexes of 1,10-phenanthrolines with cations of transition metals have broad range of applications. This work aims at designing and investigating phenothiazine and carbazole substituted 1,10-phenanthrolines as ligands for future complexes with transient metal cations. The combined electrochemical, spectroelectrochemical and DFT studies were employed to demonstrate the effect of broken symmetry in substituted 4,7-di(phenothiazine)-1,10-phenanthrolines on their spectroelectrochemical properties. A reversible color change (new absorption band around 500 nm) due to phenothiazine radical cation was observed in the first oxidation step. Results further indicate that phenothiazine substituents behave as two equivalent but almost electronically isolated redox centres. The work additionally presents a comprehensive reaction mechanistic study of oxidation and reduction processes complemented by HPLC-MS/MS identification

Electric conductivity measurements employing 3D printed electrodes and cells

The electric conductivity is one the most routinely inspected characteristics of aqueous media, being employed in monitoring drinking water quality and determining thermodynamic properties of electrolytes. In this work, we utilize fused deposition modelling 3D printing (3DP) to manufacture a platform composed of supporting cells and sensing electrodes for electric conductivity measurements of aqueous solutions. For the first time, the electric conductivity of liquid electrolytes is sensed by 3DP electrodes, employing a direct electrode/electrolyte contact. Conductivity measurements performed in the presented 3DP platform are controlled by an electronic circuitry developed employing a programmable system on chip prototyping kit. The entire conductometric setup was validated employing commercial conductance standards as well as in-lab prepared aqueous solutions of potassium chloride and formic and acetic acid as representatives of strong and weak electrolytes. Conductivity measurements enabled the correct determination of limiting molar conductivity (for potassium chloride) and dissociation constants (for the two weak acids). Finally, the functionality of the presented platform was confirmed by measuring conductivity of various bottled water samples. Results obtained in this work pave the wave for further development and applications of conductometers based on 3DP electrodes and cells

THE INFLUENCE OF THE HOST-GUEST INTERACTION ON THE OXIDATION OF NATURAL FLAVONOID DYES

The influence of the molecular cavity protection on degradation processes of bioorganic compounds quercetin and luteolin used as the original dyes in old tapestries was studied. The degradation processes were studied by electrochemical methods in aqueous media. The products of the exhaustive electrolysis were separated and identified by GC-MS analysis. Cyclic voltammetry characteristics indicate that the inclusion complex is formed. The inclusion affects the redox potentials of both oxidation waves related to the different dissociation forms of the flavonoid molecule. It was shown that decomposition products formed by the oxidation of quercetin are stabilized in the cavity of β-cyclodextrin, including the main oxidation product 2(3′,4′-dihydroxybenzoyl)-2,4,6-trihydroxybenzofuran-3(2H)-one. The formation of the 1:1 inclusion complex of luteolin with β-cyclodextrin is supported by the enhancement of fluorescence intensity. In the case of quercetin, a decrease of fluorescence intensity occurs when 1:1 inclusion complex with β-cyclodextrin is formed

Electrochemical current-sensing atomic force microscopy in conductive solutions

Insulated atomic force microscopy probes carrying gold conductive tips were fabricated and employed as bifunctional force and current sensors in electrolyte solutions under electrochemical potential control. The application of the probes for current-sensing imaging, force and current-distance spectroscopy as well as scanning electrochemical microscopy experiments was demonstrated