Flow-through Micro Sensor Using Immobilized Peroxidase with Chemiluminometric FIA System for Determining Hydrogen Peroxide

Abstract

The development of a rapid assay for hydrogen peroxide has great practical potential because H2O2 is an important analyte in the fields of environmental chemistry, 1-3 clinical chemistry 4 and food chemistry. 5 Many chemiluminometric flow injection (FI) systems 1,[6][7][8] have been developed for the rapid detection of H2O2, because chemiluminescence (CL) reaction kinetics are sufficiently fast and the combination of CL with FI methods makes it possible to mix samples and reagents rapidly with a high degree of reproducibility. Further, the characteristics of the FI systems are better sensitivity, larger linear calibration range, and simpler optical system. Among them, FI systems based on an H2O2-dependent peroxidase (POD) catalyzed oxidation of luminol are suited for the rapid and sensitive detection of H2O2 because of the low background level with the CL reaction.9-11 Flow-through sensors packed with immobilized POD have been developed, 12-16 since the instantaneous CL that is emitted immediately after mixing of the reagents is monitored by the integration of the reaction and detection in a flow cell. Horseradish POD (HPOD) and microbial POD were immobilized on a glass-disk, 12 glass beads, 13 immuno-affinity membranes, 14 activated nylon membranes, 15 or vinyl polymer beads. 16 In the sensor systems, though the maximum sample throughput went up to 200 h -1 , 16 it was required to prepare a large quantity of reagent solution and to continuously deliver it into the flow cell. From the position of green chemistry, this is undesirable based on environmental and resource considerations.As an alternative to full-size FI systems, miniaturized systems that offer the promise of increased throughput, as well as lower sample and reagent consumption, have been developed. [17][18][19] Using micromachining techniques, flow cells having channels and hollows have been fabricated based on the silicon wafer. 20,21 An enhancement of the analytical performance can be achieved by an overall reduction in the dimensions of the flow channel.In the present work, a single-line chemiluminometric FI system using a luminol solution as a carrier solution was designed for the fast determination of H2O2, and a miniaturized flow-through sensor based on immobilized POD was fabricated by micromachining techniques.To obtain a higher performance, flow rate, sample and detection volumes and connecting capillaries were reduced. The sensor consisted of an anisotropically etched silicon chip and a glass plate. The chip had a spiral channel and two penetrated holes. To form the capillary, the chip was anodically attached to a glass plate. Microbial POD was covalently immobilized with 3-(trimethoxysilyl)propyldietylenetriamine (TSDT) and glutaraldehyde (GA) on the inner surface of the capillary; the microbial one was selected because of a higher maximum rate for the CL reaction than that of HPOD. A spiral capillary was placed in front of a window of a photomultiplier tube and used as a flow cell. This method was applied to the determination of H2O2 in ...

In vivo and in vitro sensitization of leukemic cells to adriamycin-induced apoptosis by pentoxifyllineInvolvement of caspase cascades and IκBα phosphorylation

“…We also used an affinity-purified polyclonal antibody directed against phospho(Ser32) IB␣ (Calbiochem, Nottingham, England, 400002) that was raised with a synthetic phosphopeptide corresponding to amino acid residues surrounding the phosphorylated Ser32 of human IB␣ [35][36][37][38]. Secondary antibody-horseradish peroxidase-luminol (mouse/rabbit) was used to detect specific antigen-antibody interactions with enhanced chemiluminescence (ECL kit; Roche Molecular 1520709) [39,40].…”

Direct electrocatalytic reduction of hydrogen peroxide based on Nafion and copper oxide nanoparticles modified Pt electrode

“…Accurate and reliable determination of hydrogen peroxide (H 2 O 2 ) has been widely investigated using chromatography [31], chemiluminscence [32] and electrochemistry [33,34] technologies, because of its increasing application in the areas of environment, biology, food, industry and clinical laboratory [35][36][37]. Among these methods, electrochemical technique, especially enzyme/protein-modified electrode has been frequently applied [38][39][40], owing to the advantages of simple equipment, low operating expense and fast analytical time.…”

Effects of ferrocene derivative on the physico-chemical and electrocatalytic properties of ordered mesoporous carbon

“…Therefore the detection of hydrogen peroxide is very important in biomedical and environmental applications [9,10]. For this aim, various methods have been developed, such as chromatography [11], chemiluminescence [12], and electrochemical technologies [13,14]. Electrochemical sensors, such as amperometric biosensors, have been appeared as the most convenient tools for hydrogen peroxide determination [15,16], especially enzyme/protein modified electrode has been usually used [17,18].…”