Calcein Chemiluminescence Reagent Chemiluminescence Reaction And Its Analytical Applications Research

It is well known that chemiluminescence(CL) method is an important detection method,with the advantages of low detection limit,wide linear dynamic range and inexpensive instruments.The chemiluminescent reagent is the basis of CL analysis.A chemiluminescent reagent with steady application performance,high CL ratio and wide application range is very important to improve the analytical sensitivity and widen the application range of the CL analysis.To our preliminary investments,some fluorescent reagents could also be used as CL reagent. They could absorb the energy of certain reaction producing CL and related substances can be determined.This thesis includes a review and a research section.The review summarized the status of the CL study,includes the application of CL,such as the combination of CL with other techniques,CL imaging technique,nanometer technique,the integration of the experiment apparatus and the CL immunoassay,and the development of each CL systems.In the research section,the CL characteristics of calcein,a fluorescent reagent,was studied. The CL system of potassium ferricyanide-calcein,potassium permanganate-calcein, cerium(â…£)-calcein,N-bromosuccinimide(NBS)-calcein and N-chlorosuccinimide(NCS)-calcein was developed.The CL behaviors of some metal ions and organic substances were studied and the mechanisms of these CL reactions were discussed.The CL methods of some substances were established.The research section includes seven parts.1.Novel chemiluminescence system with calcein as chemiluminescent reagentChemiluminescence was observed when calcein reacted with potassium ferricyanide.Several metal ions and organic compounds could enhance this CL signal.The possible mechanisms for the CL reaction and the enhance effect were discussed via the studies of the CL kinetic characteristics, the CL spectra of some related reactions,the UV absorption spectra and the fluorescence spectra of some substances.The merits of figures for some substances with CL activity were obtained.A new CL system with calcein as CL reagent was established preliminarily.2.Study on other calcein chemiluminescence systemsThe CL behaviors of some substances includes metal ions and some pharmaceutical preparations were investgated in potassium permanganate-calcein CL system,NCS-calcein CL system and NBS-calcein CL system.A lot of substances were found had CL activities.The CL methods of some substamces were established under the optimized conditions.3.Determination ofketotifen with potassium ferricyanide-calcein chemiluminescence systemIt was found that calcein could absorb the chemical energy of the oxidizing reaction between potassium ferricyanide and ketotifen accompanied with CL emission.For the first time,CL system with calcein as CL reagent was established.Employing this CL system,a CL method to determine ketotifen was founded.The linear range of the method was 2.0×10^-8～6.0×10^-6 g·mL^-1 with a detection limit of 8×10^-9 g·mL^-1.The relative standard deviation was 2.1%for 5.0×10^-7 g·mL^-1 ketotifen(n=11).Furthermore,this method was applied to the determination of ketotifen in the tablets successfully.The mechanism of the CL reaction was discussed simply.4.Determination of ketotifen by using calcein as chemiluminescence reagentChemiluminescence was observed when potassium ferricyanide reacted with the mixture of calcein and ketotifen.Interestedly,the CL intensity would be enhanced by trace amounts of Mg²⁺ and the CL intensity was strongly dependent on ketotifen concentration.Based on this phenomenon, a flow injection CL method was established for the determination of ketotifen.The possible CL mechanism is proposed based on the kinetic characteristic of the CL reaction,CL spectrum, ultraviolet(UV) spectra and fluorescent spectra.The CL intensity was correlated linearly with concentration of ketotifen over the range of 6.0×10^-9 to 2.0×10^-7 g·mL^-1 and the detection limit was 3×10^-9 g·mL^-1.The relative standard deviation was 1.8%for 2.0×10^-8 g·mL^-1 ketotifen(n=11). This method was applied to the determination of ketotifen in the tablets successfully.5.Determination of fenfluramine based on potassium permanganate-calcein chemiluminescence systemPost-chemiluminescence was observed when fenfluramine was injected into the mixture after the CL reaction of calcein-potassium permanganate.Based on this phenomenon,a flow injection CL method was established for the determination of fenfluramme.The possible CL mechanism was proposed.The CL intensity was correlated linearly with the concentration of fenflummine over the range of 1.0×10^-7 to 6.0×10^-6 g·mL^-1 and the detection limit was 6×10^-8 g·mL^-1.The relative standard deviation was 2.2%for 5.0×10^-7 g·mL^-1 fenfluramine(n=11).This method was applied to the determination of fenfluramine in weight-reducing tonic successfully.6.Determination of trace aluminum(â…¢) using a novel cerium(â…£)-calcein chemiluminescence detectionA simple,sensitive and selective CL method was developed for the direct determination of aluminum(Al).This method is based on that the weak CL of cerium(â…£)-calcein can be greatly enhanced by Al(â…¢).The calibration curve was linear over the range 2.0×10^-10～4.0×10^-8 g·mL^-1 with a detection limit of 8×10^-11 g·mL^-1(3σ).The RSD was 2.5%by 11 replicated determinations of 1.0×10^-9 g·mL^-1 Al(â…¢).The proposed method has been used to determine the concentration of Al (â…¢) in real water samples with satisfactory results.The mechanism of the CL reaction was also discussed.7.Determination ofthioridazine with potassium permanganate-calcein chemiluminescence systemPotassium permanganate-calcein CL system was established with calcein as CL reagent. Thioridazine was found to be able to produce CL in this system and formaldehyde could enhance this CL signal.Based on this,a CL method to determine thioridazine was established.The linear range of the method was 2.0×10^-8～6.0×10^-6 g·mL^-1 with a detection limit of 8×10^-9 g·mL^-1.The relative standard deviation was 2.1%for 1.0×10^-7 g·mL^-1 thioridazine(n=11).Furthermore,this method was applied to the determination of thioridazine in the tablets and plasma.The results were satisfactory after the control experiment with the standard method and the recovery test.The mechanism of the CL reaction was discussed simply.