Electrochemical, detection processes

Post-column on-line derivatisation is carried out in a special reactor situated between the column and detector. A feature of this technique is that the derivatisation reaction need not go to completion provided it can be made reproducible. The reaction, however, needs to be fairly rapid at moderate temperatures and there should be no detector response to any excess reagent present. Clearly an advantage of post-column derivatisation is that ideally the separation and detection processes can be optimised separately. A problem which may arise, however, is that the most suitable eluant for the chromatographic separation rarely provides an ideal reaction medium for derivatisation this is particularly true for electrochemical detectors which operate correctly only within a limited range of pH, ionic strength and aqueous solvent composition. 

There are mainly three types of transducers used in immunosensors electrochemical, optical, and microgravimetric transducers. The immunosensors may operate either as direct immunosensors or as indirect ones. For direct immunosensors, the transducers directly detect the physical or chemical effects resulting from the immunocomplex formation at the interfaces, with no additional labels used. The direct immunosensors detect the analytes in real time. For indirect immunosensors, one or multiple labeled bio-reagents are commonly used during the detection processes, and the transducers should detect the signals from the labels. These indirect detections used to need several washing and separation steps and are sometimes called immunoassays. Compared with the direct immunosensors, the indirect immunosensors may have higher sensitivity and better ability to defend interference from non-specific adsorption. 

Electrochemical detection is based on an electrochemical reaction of the analyte in the mobile phase and is therefore more sensitive to HPLC condition changes than detection based on the relatively stable process of light absorption (uv) or emission (fluorescence). 

Anions play key roles in chemical and biological processes. Many anions act as nucleophiles, bases, redox agents or phase transfer catalysts. Most enzymes bind anions as either substrates or cofactors. The chloride ion is of special interest because it is crucial in several phases of human biology and in disease regulation. Moreover, it is of great interest to detect anionic pollutants such as nitrates and phosphates in ground water. Design of selective anion molecular sensors with optical or electrochemical detection is thus of major interest, however it has received much less attention than molecular sensors for cations. 

In this light we will now consider some examples of electrochemically detectable molecular reorganisations induced by electron transfer processes, which can be chemically more appealing than the simple variations of bond distances/angles discussed up to now. 

The ability to modulate electrochemical reactivity and effectively switch OFF the reaction was extended further by Wang and coworkers [174] to control, on-demand, the separation and detection processes in microfiuidic devices. In this work, the catalytic nickel nanowires were placed, reoriented and removed on-demand at the exit of the separation channel of the microfiuidic chip, offering unique possibilities for controlling externally, events inside and outside a microchannel. 

Research has been done showing that rapid pressnre-driven LC analysis can be done with little solvent consumption, demonstrating this as a viable process analytical tool. Using electrokinetic nanoflow pumps LC can be miniaturized to the point of being a sensor system. Developments in terms of sampling to enable sampling directly from a process stream, to the separation channel on a chip are critical for the application of miniaturized process LC. The components (valves and pumps) required for hydrodynamic flow systems appear to be a current limitation to the fnll miniatnrization of LC separations. Detection systems have also evolved with electrochemical detection and refractive index detection systems providing increased sensitivity in miniaturized systems when compared to standard UV-vis detection or fluorescence, which may require precolumn derivatization. 

Liquid chromatographic methods based on ultraviolet and/or electrochemical detection have also been developed (622-625). In the earliest of these methods (623), tissues were extracted with mcthanol/water (1 1), and the evaporated residue was taken up in dichloromethanc. The extract was then injected on a Kieselgel Merckosorb SI-60 liquid chromatographic column, and eluted with dichloromethane/ethanol/water. Monitoring at 280 nm allowed 5 ppb thiouracils to be readily detected in the tissue samples. In the latest method (625), cattle plasma samples were extracted with etliyl acetate in presence of ethylenediamine-tetraacetate (EDTA). The addition of ED I A could significantly improve the efficiency of the extraction process. Remarkable improvement of the ethyl acetate... 

Immusoft is a software that has been developed to perform computer-driven assays in our microchips. This software has a user-friendly graphical user interface, and it enables control of the pump, the valves and the electrochemical detection system, as well as the development of specific assay protocols, the running of simultaneous or sequential experiments in eight parallel microchannels, the automatic read-out of the results and the processing of the obtained data. These different functions are managed by way of three main menus, named Method, Analysis and Results, and the software also comprises two additional items dedicated to the setting of the computing parameters and to the maintenance of the instrumentation. 

Fig. 32.1. PCR reactor for the real-time electrochemical detection of Salmonella enterica serovar Typhimurium ATCC 14028 based on the doubly labeled PCR amplification performed with the magnetic bead primer. White dots show the non-specific electrochemical signal processing the negative PCR control, while the black dots show the increasing signal of DNA IS200 doubly labeled amplicon onto magnetic beads. In all cases, 60 pg AntiDig-HRP were used. Other experimental details are medium, phosphate buffer 0.1 mol L-1, KC1 0.1 mol L-1, pH 7.0 mediator, hydroquinone 1.81 mmol L 1 substrate, H202 4.90 mmol L 1 applied potential = -0.1 V (vs. Ag/AgCl).

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