Studies On Hyphenated Technique Of Microchip Electrophoresis With Electrochemiluminescent Detection

Including in the fast progress in development of microchip analytic technique in the past decade, studies on modification of microchennal and miniaturization of detector appear to be the hot research topics. Electrochemiluminescence (ECL), also known as electrogenerated chemiluminescence, is an excellent analytic method that possessed the advantages of both electrochemical and chemiluminescent methods. It has the benefits such as simplicity, inexpensive instrumentation, low background noise, high sensitivity, good controllability and wide dynamic range. The present paper focuses on the modification of microchannel, fabrication of ECL detection cell and their hyphenated technique.A poly(dimethylsiloxane) (PDMS) microchip with an amperometric detector was developed for the electrophoretic separation and determination of neurotransmitters. Although the polystyrene (PS) microsphere is widely used as the modifier for chromatography, there is no report on electrophoretic separation on self-assembled nano-PS modified PDMS microchannel. To enhance the separation efficiency, the positive charged PS nano-sphere (PSNS) has been solvothermal synthesized and was self-assembly modified onto a PDMS microchannel to obtain a quasi-ordered PSNS monolayer. Followed by driving through the PSS solution, the final PSNS/PSS modified layer was built on the channel surface. Thus a stable electroosmotic ?ow (EOF) and high separation efficiency are obtained in resulted modified microchannel. Under optimized conditions, dopamine, epinephrine, catechol, and serotonin are acceptably baseline separated in this 3.5 cm length separation channel with the theoretical plate number from 4.6×104 to 2.1×105 per meter and resolution from 1.29 to 12.5, is obviously higher than some reported papers. The practicability of this microchip is validated by the recovery test with cerebrospinal ?uid as real sample which resulted from 91.7% to 106.5%.ECL is a simple and sensitive method for analytical detection. It provides a real-time analytical approach. An ECL choline biosensor is developed by drop- coating of choline oxidase (ChOx) onto a carbon nanotubes (CNTs) / potassium ferricyanide modified platinum electrode with ECL of luminol as readout signal. Due to the improvement of biocompatibility and electron transfer of electrode surface from CNTs, meanwhile the activation for enzyme and the ECL emission from K3Fe(CN)6, the developed biosensor possesses excellent analytical properties. It gives optimal results while the Pt basal electrode was modified with 4μL of 0.33g/L CNTs dispersoid, 2μL of 0.1mol/L K3Fe(CN)6 and 1.5U of ChOx. In the PBS buffer (pH 7.4) containing 8×10-6mol/L luminol, the ECL signal linearly responded the concentration of choline from 1×10-7mol/L to 4×10-3mol/L (r=0.994) with detection limit of 1.21×10-8mol/L under 30°C of detection temperature. The developed biosensor was applied to assay the concentration of choline in rat blood sample. The result of 0.268 mg/100mL was obtained with average recovery of 101.1%. It shows a fast response to choline with good reproducibility.Indium tin oxide (ITO) glass is generally used as the substrate of modified electrode, but the authors have once found a very significant phenomenon when using the indium tin oxide (ITO) glass as the anode in electrochemical studies. There were notable luminescent signals had been observed when a pulse potential was applied on the ITO electrode in alkaline solution without any luminescent reagent.The mechanism of this luminescence was discussed in details. After all of the studies, it was revealed that the yielded reactive oxygen species (ROSs) as O2??, OH˙ and H2O2 during the electrolysis and whereafter the produced singlet oxygen (1O2) acted as the indispensable right-emitting entity and the ITO was a critical determinant intensifier.Since the first work on the ECL of silicon quantum dots (QDs) was reported, the ECL phenomena of CdS, CdSe and CdTe in the presence of co-reactants have been studied, and further applied for the development of ECL sensors. There the TGA-capped CdTe QDs were synthesized in our lab by an improved method, which had a high quantum yield of over 50%, and an ECL electrode has been developed which was constructed based on the immobilization of TGA-capped CdTe QDs by nanocomposite of CNTs and chitosan (Chit) on ITO glass. The developed ECL electrode displayed high ECL intensity, excellent stability and good biocompatibility. It provides an excellent platform for further applications. To miniaturize the analytical detector is one of the trends of instrumental analysis. A nano-liter sized ?ow-cell is developed for constructing a ?ow injection analytic (FIA) system with ECL detection. The CdTe QDs composite modified ECL electrode is applied as the working electrode in this ?ow-cell. It has been demonstrated to have the efficient anodic ECL performance with the triethylamine (TEA) as the co-reactant. The ?ow-cell gives the stable ECL background under optimized conditions for parameters such as electrolytic pulse, concentration of TEA and ?ow rate, etc. The sensitive ECL quenching response of dopamine (DA) is realized on this FIA system within the linear range from 10 pM to 4 nM and a detection limit as low as 3.6 pM. It is practically used to determine the neurotransmitters in cerebro-spinal ?uid (CSF) with DA as the index and with an average recovery of 94%.And then the nano-liter sized ECL detection cell was hyphenated with PDMS microchip to construct a microchip-ECL electrophoretic system. The preliminary study of this microchip-ECL system reveals the practicability after the investigation on pivotal factors such as the effect of high voltage on luminescence detection and the distance between microchannel and detector. There is a confirmable electrophoretic peak of dopamine to be recorded in this microchip-ECL electrophoretic system. The study provides the foundation of experimental technique, and suggests the direction for the further researches. 1, Apply the channel modification technique with PSNS to improve the separation efficiency, outline of electrophoretic peak and therefore the detection sensitivity; 2, Optimize the matching degree between separation channel and the detection cell to improve the detection performance; 3, Adopt the technique of sampling-enrichment to conquer the limitation of little sampling quantity to enhance the detection sensitivity.