Studies On Sensitization Of Luminol ECL And Its Application In Flow Injection Determination Of Reactive Oxygen Species

Electrochemiluminescence （ECL） has become an important and valuable detectionapproach in recent years due to its promising advantages such as high sensitivity, lowdetection limite and wide linear range. However, conducted as one of the most widelyused luminescent reagents, most of the ECL application of luminol has been limited inalkaline solution principally due to its less dissociation in neutral or acidic solution.How to fulfill the ECL of luminol in neutral even acidic medium has practicalsignificance since many biomolecules can only exist in. Up to now, there are only a fewreports about the ECL of luminol in neutral or acidic solution. There concentratedco-reactant such as NaBr or hydrogen peroxide is necessary to excite stronger ECLsignal of luminol. Despite the cost of reagent consumption, it will lead to thedescending reproducibility and/or introducing some interferents. In order to enhance theluminol ECL intensity in neutral or acidic solution and extend the pH range of luminolECL application, the following four aspects work has been done.A flow injection analysis （FIA） system with an ECL detector has been established，which include peristaltic pump, sample injector （six-way valve）, commixing cell andECL cell. A peristaltic pump and a sample injector with a5μl injection loop are appliedto construct the flow injection system. The system based on not only the layoutoptimization of sample injector, commixing cell and ECL cell but also a speciallydesigned flow-through ECL cell, which has some unique advantages such as simplestructure, almost zeroed dead-volume and easily to be assembled compared withconventional ECL flow cells. The ECL cell used ITO glass as both working electrodeand optical window. The developed ECL-FIA system has small injection volume andhigh sensitivity.A microemulsion enhanced ECL of luminol-H₂O₂was studied with the FIAtechnique. The results revealed that the microemulsion composed with cetyltrimethylammonium bromide （CTAB）, n-butanol, n-heptane and water greatlyenhanced the ECL especially in acidic medium. The ECL emission increased for20to2times in this microemulsion medium over the pH range of5.0to8.0compared to that inaqueous solution. The mechanism of enhancement of surfactant and microemulsion forluminol-H2O₂ECL was discussed. It is mainly based on the electrostatic interactionbetween luminol anion and the head group of surfactant, which causes the adsorptionand promotes the dissociation of luminol on the surfaces of the microemulsion droplets,favors the oxidation of luminol by the yielded reactive oxygen species （ROSs） duringelectrolysis. This research is very significant for ECL applications because of theextended practicable pH range which was suitable for environmental and biologicalsystems. As an example, this ECL-FIA technique can be applied for determination ofoligo proanthocyanidin （OPC） because of its antioxidant property and to evaluate thetotal antioxidant activity of the grape skin using OPC as an index.A nano-hybrid of nano-TiO₂supported AuAg alloy nanocluster was synthesizedand then modified onto the surface of indium tin oxide （ITO） to act as a workingelectrode for intensifying the ECL of luminol. The properties of the nano-hybrid and thefunctionalized electrode were characterized by TEM, AFM, electrochemistry andspectroscopy. Coupled with the use of microemulsion as carrier matrix, the ECLemission of luminol is greatly intensified in neutral even acidic phosphate buffersolution （PBS） without the requirement of any co-reactant. With the FIA approach, thisresearch very significantly extended the area of ECL applications because of theextended practicable pH range which was suitable for environmental or biologicaltargets etc. As an example, this ECL-FIA technique was successfully applied to evaluatethe total antioxidant activity of some fruits with satisfactory results.A copolymer of luminol with aniline （PLA） modified film was prepared onto theITO, nano-TiO₂functionalized ITO （TiO₂/ITO） and AuAg-TiO₂nano-hybridfunctionalized ITO （AuAg-TiO₂/ITO） electrode surface by cyclic voltammetry （CV） todevelop the reagentless ECL electrode for FIA. The properties of the prepared modifiedelectrodes were characterized by SEM, CV, electrochemical impedance spectroscopy（EIS） and ECL. The ECL emission of PLA/IT, PLA/TiO₂/ITO andPLA/AuAg-TiO₂/ITO modified electrodes were all greatly higher than the simplepolyluminol modified electrode （PL/ITO） especially the latter. ThePLA/AuAg-TiO₂/ITO modified electrode presented the most excellent quality of stability, reproducibility, higher background ECL emission and showed sensitiveresponse to ROSs thereafter to be applied for determination of hydrogen peroxide viaFIA mode. Under optimized conditions, an absolute detection limit of0.822pg ofhydrogen peroxide was obtained. Thus the hydrogen peroxide residue in disposablebamboo chopsticks was detected with satisfactory result. The research suggested a vastfuture of reagentless ECL-FIA technique.