Amperometric biosensor for glutamate using prussian blue-based "artificial peroxidase" as a transducer for hydrogen peroxide

The specially deposited Prussian Blue denoted as "artificial peroxidase" was used as a transducer for hydrogen peroxide. The electrocatalyst was stable, highly active, and selective to hydrogen peroxide reduction in the presence of oxygen, which allowed sensing of H2O2 around 0.0 V (Ag/AgCl). Glutamate oxidase was immobilized on the surface of the Prussian Blue-modified electrode in a Nafion layer using a nonaqueous enzymology approach. The calibration range for glutamate in flow injection system was 1 x 10(-7)-1 x 10(-4) M. The lowest concentration of glutamate detected (1 x 10(-7) M) and the highest sensitivity in the linear range of 0.21 A M-1 cm-2 were achieved. The influence of reductants was practically avoided using the low potential of an indicator electrode (0.0 V Ag/AgCl). The attractive performance characteristics of the glutamate biosensor illustrate the advantages of Prussian Blue-based "artificial peroxidase" as transducer for hydrogen peroxide detection.     

Spongiform immobilization architecture of ionotropy polymer hydrogel coentrapping alcohol oxidase and horseradish peroxidase with octadecylsilica for optical biosensing alcohol in organic solvent

An organic-phase optical alcohol biosensor consisting of alcohol oxidase and horseradish peroxidase coimmobilized in a spongiform hydrogel matrix of hydroxethyl carboxymethyl cellulose, an adduct of 3-methoxy-4-ethoxy benzaldehyde, 4-tert-butylpyridinium acetohydrazone, silica gel particles, and octadecylsilica particles in conjunction with an optical oxygen transducer has been successfully fabricated. The novel enzyme entrapment structure was mainly characterized with desirable solvent permeability, high efficiency of mass transfer for reactants, and good accessibility and stability of the immobilized enzymes. The biosensor could work in water-miscible solvent such as a solvent mixture of acetonitrile and phosphate aqueous buffer, as well as hydrophobic organic solvent such as n-hexane. The biosensor had the highest sensitivity to methanol in both solvent systems. Under the stop-flow mode, the biosensor had the analytical working ranges from 80 microM to 90 mM methanol in n-hexane and 0.10 to 90 mM methanol in acetonitrile/buffer. When the biosensor functioned in n-hexane, it could take benzaldehyde as an alcohol substrate and was free from any pH disturbance. In the presence of coimmobilized horseradish peroxidase, the operational life of the biosensor was 60 assays and the shelf life was longer than two weeks. The biosensor has been satisfactorily applied to the determination of methanol in commercial gasoline-methanol blend samples.     

Scanning electrochemical microscopy. 44. Imaging of horseradish peroxidase immobilized on insulating substrates

Scanning electrochemical microscopy (SECM) was used to study horseradish peroxidase (HRP) immobilized with copolymer on insulating substrates (glass slide or polycarbonate membrane filter). Two methods were used to immobilize HRP: In the first, HRP was coimmobilized by cross-linking on a glass slide with a copolymer swelled in water to form a hydrogel; in the second, the same copolymer and avidin were coimmobilized on the glass slide and biotin-labeled HRP was conjugated to the avidin of the film. SECM was then used to detect the presence of the bound enzyme by observing the feedback current in a solution of benzoquinone and hydrogen peroxide, when hydroquinone was generated at the tip. A detection limit less than 7 x 10(5) HRP molecules within a approximately 7-microm-diameter area was demonstrated.     

Combined approach to the analysis of recombinant protein drugs using hollow-fiber flow field-flow fractionation, mass spectrometry, and chemiluminescence detection

The impurities present in recombinant protein drugs produced by large-scale refolding processes can not only affect the product safety but also interact with the expressed protein. To relate the impurity profile to conformation and functionality of the protein drug, analytical methods able not to degrade the sample components should be preferred. In this work, an urate oxidase (uricase) drug from Aspergillus flavus expressed in Saccharomyces cerevisiae, and a reagent-grade uricase from Candida sphaerica expressed in Escherichia coli, are analyzed by combining hollow-fiber flow field-flow fractionation with matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI/TOFMS) and with chemiluminescence enzyme activity assay. Preliminary detection and identification of sample impurities is performed by means of conventional methods such as RP HPLC with electrospray ionization quadrupole-TOF MS and MALDI/TOFMS with SDS PAGE and 2D SDS PAGE. Results show that the recombinant uricase samples obtained from different microorganisms have different impurities and different enzymatic activity and that different uricase oligomers are present in solution.     

Elimination of electrooxidizable interferant-produced currents in amperometric biosensors.

Electrooxidizable ascorbate, urate, and acetaminophen that interfere with amperometric glucose assays are completely and rapidly oxidized by hydrogen peroxide in a multilayer electrode. The multilayer electrode is composed of an immobilized, but not electrically "wired", horseradish peroxidase (HRP) film coated onto a film of electrically "wired" glucose oxidase (GO). The "wired" enzyme is connected by a redox epoxy network to a vitreous carbon electrode. The current from the electrooxidizable interferants is decreased by their peroxidase-catalyzed preoxidation by a factor of 2500, and the glucose/interferant current ratio is increased 10(3)-fold. Undesired electroreduction of hydrogen peroxide can result when HRP is also "wired" to the electrode. Such unwanted "wiring" is prevented by incorporating an electrically insulating barrier layer between the wired GO film and the HRP film. The hydrogen peroxide necessary for elimination of interferants can be added externally, or when this is not possible, it can be generated in situ by means of a coupled enzyme reaction.     

Immobilized enzyme electrode for creatinine determination in serum

An immobilized enzyme electrode for continuous creatinine determination in blood serum is described. The enzymes creatinine amidohydrolase, creatine amidinohydrolase, and sarcosine oxidase are coimmobilized to the surface of the polypropylene membrane of a Clark-type electrode responsive to oxygen. The immobilized enzymes catalyze the decomposition of creatinine with the consumption of oxygen and thus permit the creatinine measurement. The whole assay takes less than 1 min. Effects of pH and temperature on electrode response are also described. The proposed technique offers a rapid, simple, and inexpensive means to determine creatinine in blood serum within the normal and abnormal ranges. The repeatability of the creatine determination in serum is 2.5% (relative standard deviation), and the detection limit is 3 x 10(-6) mol L-1. The results obtained by this method were compared to those obtained with the Technicon AutoAnalyzer SMAC system based on the Jaffé reaction; the correlation factor between the two methods was found to be r = 0.9997.     

A glucose sensor made of an enzymatic clay-modified electrode and methyl viologen mediator

A novel glucose sensor has been contrived by immobilizing glucose oxidase between two nontronite clay coatings on glassy carbon electrode with methyl viologen as mediator. The sandwich configuration proved to be very effective in the determination of glucose. The response of the glucose sensor was determined by measuring cyclic voltammetric peak current values under aerobic solution conditions. The effects of the amount of enzyme immobilized, the operating pH, and the common interferences on the response of the glucose sensor were studied. The detection limit was 5 μM, with a linear range extending to about 6 mM, giving a dynamic range of over 3 orders of magnitude for 0.1 mM methyl viologen. When stored in pH 7 phosphate buffer at 4 °C, the sensor shows almost no change in performance after operating for at least 2 months. A mechanism for the operation of the glucose sensor is also proposed.     

Nanoporous aluminum oxide as a novel support material for enzyme biosensors

To construct novel amperometric sensors for the detection of hydrogen peroxide and pyruvate, peroxidase and pyruvate oxidase were immobilized in self-supporting nanoporous alumina membranes those made by anodic oxidation. Pyruvate oxidase and other enzymes were enclosed in poly(carbamoylsulfonate) hydrogel and sucked into the nanoporous alumina structure before polymerization. The alumina membranes were investigated by scanning electron microscopy before and after the enzyme immobilization. In an amperometric flow detector cell, pyruvate and hydrogen peroxide were detected under flow injection analysis conditions in concentration ranges from 1 microM to 100 microM and 5 microM to 500 microM, respectively. The achieved operational stability showed that alumina membranes can be used to construct enzyme-modified electrodes.     

Development of a flow amperometric enzymatic method for the determination of total glucosinolates in real samples

The first amperometric flow analyzer, based on the biosensor concept, capable of determining total glucosinolates in real samples, is described. Myrosinase was immobilized on aminopropyl-modified controlled pore glass, which was then used for the construction of a packed-bed reactor. Myrosinase catalyzes the hydrolysis of glucosinolates (sinigrin) to glucose (among the other products), which is then oxidized by the action of glucose oxidase to produce hydrogen peroxide. The glucose enzyme electrode is based on a multimembrane architecture and was mounted on an amperometric flow cell (hydrogen peroxide detection at a platinum anode poised at +0.65 V vs Ag/AgCl/3M KCl). Different membrane types and different activation procedures were tested. The system was optimized to various working parameters, either as a glucose electrode or as a glucosinolate analyzer. The interference effect of various compounds was also investigated. Application of the method to real samples was carried out using glucose/glucose, hydrolyzed sinigrin and glucose/sinigrin solution as calibrators of the glucose electrode and the glucosinolate analyzer. Deviations due to the enantioselectivity of glucose oxidase to the beta-glucose anomer were observed, and a data elaboration protocol is proposed. The possibility of the simultaneous determination of glucose and glucosinolates is also demonstrated.     

A biomimetic phospholipid/alkanethiolate bilayer immobilizing uricase and an electron mediator on an Au electrode for amperometric determination of uric acid.

A biomimetic bilayer membrane immobilizing uricase (urate oxidase; EC 1.7.3.3) (UOx) and a redox agent of 1-methoxy-5-methylphenazinium (MMP) was fabricated on an Au electrode substrate with use of the Au substrate coated with a self-assembled monolayer of n-octanethiolate (OT/Au) and L-alpha-phosphatidylcholine beta-oleoyl-gamma-palmitoyl (PCOP). The preparation was carried out by successively immersing an Au electrode substrate in an ethanol solution of OT, an MMP aqueous solution, and a suspension of proteoliposome formed by PCOP containing UOx and MMP. The prepared electrode exhibited such fast steady amperometric responses to uric acid as to allow its determination within 20 s after injecting uric acid, indicating that UOx-catalyzed electrochemical oxidation of uric acid was accomplished with assistance of electron mediation by MMP between UOx and the Au substrate. An increase in the response currents with increasing concentration of uric acid was obtained in a concentration range of uric acid found in healthy human blood. Any interference in the current response that is caused by direct anodic oxidation of uric acid or ascorbic acid was not observed at the prepared sensor electrode because the densely packed bilayer effectively blocked the diffusion of these substrates toward the Au surface, making it possible to determine amperometrically uric acid at the electrode with high precision.     

Paper bioassay based on ceria nanoparticles as colorimetric probes

We report the first use of redox nanoparticles of cerium oxide as colorimetric probes in bioanalysis. The method is based on changes in the physicochemical properties of ceria nanoparticles, used here as chromogenic indicators, in response to the analyte. We show that these particles can be fully integrated in a paper-based bioassay. To construct the sensor, ceria nanoparticles and glucose oxidase were coimmobilized onto filter paper using a silanization procedure. In the presence of glucose, the enzymatically generated hydrogen peroxide induces a visual color change of the ceria nanoparticles immobilized onto the bioactive sensing paper, from white-yellowish to dark orange, in a concentration-dependent manner. A detection limit of 0.5 mM glucose with a linear range up to 100 mM and a reproducibility of 4.3% for n = 11 ceria paper strips were obtained. The assay is fully reversible and can be reused for at least 10 consecutive measurement cycles, without significant loss of activity. Another unique feature is that it does not require external reagents, as all the sensing components are fixed onto the paper platform. The bioassay can be stored for at least 79 days at room temperature while maintaining the same analytical performance. An example of analytical application was demonstrated for the detection of glucose in human serum. The results demonstrate the potential of this type of nanoparticles as novel components in the development of robust colorimetric bioassays.     

A hydrogen peroxide biosensor based on peroxidase activity of hemoglobin in polymeric film

A Hydrogen peroxide (H2O2) biosensor, based on hemoglobin (Hb) and ortho-phenylenediamine (o-PD) gold electrode, was fabricated. Hb was immobilized onto the electrode surface by electrochemical polymerize method with o-PD. The designed biosensor showed a well defined redox peak which was attributed to the direct electrochemical response of Hb. The immobilized Hb exhibited an excellent electrocatalytical response to the reduction of hydrogen peroxide, enabling the sensitivity determination of H2O2. Factors and performances such as pH, potential, influencing the designed biosensor, were studied carefully. The amperometric detection of H2O2 was carried out at -300 mV in phosphate buffer solution (PBS) (0.1 M) with pH 6.0. This biosensor showed a fast amperometric response (less then 5 s) to H2O2. The levels of the (Relative standard deviation) RSDs (< 3.5%) for the entire analyses reflected a highly reproducible sensor performance. Using the optimized conditions, the detection limit of the biosensor was 1 x 10(-7) M and linear range was from 5 x 10(-6) to 1.25 x 10(-4) M. In addition, this sensor showed long-term stability and good sensitivity.     

Microfabricated electrophoresis chip for bioassay of renal markers

A novel capillary electrophoresis chip-based detection system for simultaneous measurements of the renal markers creatine, creatinine, p-aminohippuric acid, and uric acid is described. Fluid control is used for mixing the sample with the enzymes creatininase (CA), creatinase (CI), and sarcosine oxidase (SOx) and for separating the neutral hydrogen peroxide end product from the anionic p-aminohippuric and urate species. The 'total' (creatinine and creatine) signal was measured with the running buffer containing all three enzymes, while the creatine signal alone was recorded by mixing the sample with the CI-SOx solution. Creatinine concentrations are measured by comparing the response in the presence and absence of CA. The peroxide product and the oxidizable p-aminohippuric and uric acids are detected electrochemically at a downstream gold-coated thick-film amperometric detector. The four renal markers are readily measured within 5 min, while creatinine/creatine within less than 2 min. Factors influencing the performance, including the level of three enzymes, separation voltage, and detection potential, are optimized. Applicability to urine samples is demonstrated. Such a multianalyte microchip detection device would allow renal function testing to be performed more rapidly, easily, and economically in the point-of-care setting.     

Nanophase-separated amphiphilic conetworks as versatile matrixes for optical chemical and biochemical sensors

As a novel class of sensor matrixes, nanophase-separated amphiphilic polymeric conetworks (APCNs) open a new dimension for optical chemical and biochemical sensing. These conetworks consist of a hydrophilic phase-we used poly(2-hydroxyethyl acrylate), poly(2-(dimethylamino)ethyl acrylate), or polycationic poly(2-(trimethylammonium)ethyl acrylate)-and of a hydrophobic phase-poly(dimethylsiloxane). Sensors can be prepared by simple impregnation of the matrix. Due to nanophase separation, there is a spatial separation between areas in which the indicator reagents are well immobilized and areas that advantageously take care of the diffusive transport of the analyte, whereby these functionalities of the contrary phases can be exchanged. Thanks to the huge interface between the contrary phases, the accessibility of the indicator reagents is good, which makes it possible to design sensors with high sensitivity. To demonstrate the advantages of APCNs as matrixes, different prototypes of sensors were prepared, e.g., one to determine gaseous chlorine based on its reaction with immobilized o-tolidine and another to determine vaporous acids based on immobilized bromophenol blue dianions. As a breakthrough in biochemical sensing, we are also able to present an easily producible, optically transparent biochemical sensor to determine peroxides in nonpolar organic media-based on coimmobilized horseradish peroxidase and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate).     

FIA型微生物探针动态响应过程的研究Ⅰ──谷氨酸的测定

将固定化细胞柱和简单的流动注射系统相结合，以大肠杆菌（Ｅ．Ｃｏｌｉ）为工作菌株配合ＣＯ＿２选择性电极，制成谷氨酸微生物传感器。它具有以下特性：传感器对谷氨酸溶液的适用测量浓度范围为４００～２０００ｍｇ／Ｌ谷氨酸；一个样品的检测在２０ｍｉｎ内完成；固定化细胞柱的使用寿命在４周以上；除Ｌ－精氨酸和Ｌ－谷氨酰胺外，其余氨基酸均不干扰传感器对谷氨酸的测定。     

Disposable biosensor based on a hemoglobin colloidal gold-modified screen-printed electrode for determination of hydrogen peroxide

A disposable reagentless hydrogen peroxide biosensor based on the direct electrochemistry of hemoglobin immobilized on a colloidal gold-modified screen-printed carbon electrode (Hb-Au-SPCE) was proposed. The electrochemical behavior of immobilized Hb at a SPCE was studied for the first time. The electrode reaction of immobilized Hb showed a surface-controlled process with an electron transfer rate constant of (0.40 /spl plusmn/ 0.02) s/sup -1/ determined in the scan rate range from 25 to 200 mV s/sup -1/. The Hb-Au-SPCE exhibited an electrocatalytic activity toward the reduction of hydrogen peroxide with a K/sub M//sup app/ value of 1.8 mM, which was allowed to be used as a disposable sensor for determination of hydrogen peroxide with a linear range from 1.0 /spl times/ 10/sup -5/ M to 3.2 /spl times/ 10/sup -4/ M, a detection limit of 5.5 /spl times/ 10/sup -6/ M at 3/spl sigma/, a high sensitivity, fast response, and good selectivity, accuracy, and reproducibility. The disposable reagentless sensor was stable, low cost, and simple to use for detection of hydrogen peroxide in real samples.     

Enzyme-immobilized Langmuir-Blodgett film for a biosensor

A lipid-protein monolayer for a biosensor was prepared utilizing a Langmuir- Blodgett technique. The enzyme glucose oxidase was used as the protein. Three types of lipid were chosen to change the surface charge of the polar group. The enzyme was immobilized on the lipid monolayer by adsorption from the subphase solution onto the lipid monolayer on the air/water interface. It was found that the lipid-enzyme interaction was dominated by electrostatic forces, and the characteristics of the film can be controlled by expansion and recompression of the adsorbed monolayer. Finally, a glucose sensor was fabricated by depositing the film onto a hydrogen peroxide electrode.     

High-performance immunoaffinity chromatography and chemiluminescent detection in the automation of a parathyroid hormone sandwich immunoassay

An automated sandwich immunoassay was developed based on high-performance immunoaffinity chromatography and chemiluminescent detection, using the determination of parathyroid hormone (PTH) in plasma as a model system. In this method, injections of plasma and acridinium ester-labeled anti-(1-34 PTH) antibodies were made onto a column containing immobilized anti-(44-68 PTH) antibodies. Upon elution, PTH and its associated labeled antibody were combined with an alkaline peroxide postcolumn reagent, and the resulting light production was measured. Factors considered in optimizing this system included the column's dissociation properties, the rate of light production in the postcolumn reactor, and the use of sequential vs simultaneous injection of sample and labeled antibody. The final system developed required 6 min per plasma injection, following a 1-h incubation of sample with labeled antibody. The response was linear over 2-3 orders of magnitude and the lower limit of detection for a 66-microL plasma sample was only 16 amol, or 2.4 x 10(-13) M. Overall, this method had a precision and response similar to those of manual PTH methods but required 24-fold less time to perform. By using different immobilized and labeled antibodies, this method could easily be adapted for use with other analytes.     

Peroxidase model electrodes: heme peptide modified electrodes as reagentless sensors for hydrogen peroxide

A peroxidase model electrode was devised for reagentless sensing of hydrogen peroxide (H2O2). A small model molecule, which mimics the vicinity of the reaction center of a redox enzyme, can communicate electrochemically with an electrode. Heme nonapeptide (MW congruent to 1600) having peroxidase activity was adopted as a peroxidase model compound and was covalently immobilized on a tin oxide (SnO2) electrode as a roughly monomolecular layer. The modified electrode thus obtained responded to H2O2 at concentrations down to 10(-6) M without electron mediator or promoter, at a mild potential of +150 or +300 mV vs Ag/AgCl. In a batch system, the response reached a steady state in a few seconds. Measurements were possible also in a flow system with an assay time of 0.5-1.0 min/sample. The steady-state response of the electrode was kinetically analyzed.     

Cyanide detection using a substrate-regenerating, peroxidase-based biosensor

An enzyme-based, dual working electrode system is described for the sensing of cyanide. Horseradish peroxidase (HRP) is incorporated as the sensing element. A continuous monitoring of oxidative activity by the enzyme results through the generation and regeneration of substrates at the electrode surfaces. Thus, HRP is oxidized by hydrogen peroxide generated from dissolved oxygen, at the primary electrode, and then reduced through the secondary electrode by mediated electron transfer using ferrocene as a carrier. Ferrocene regeneration at this electrode is proportional to the intrinsic activity of HRP. The dynamics of the system are investigated by using a rotating ring-disk electrode. The enzyme is immobilized to provide better control over its catalytic activity and to increase the lifetime of the biosensor. Cyanide inhibition of current can be modeled by reversible binding kinetics. Detection of cyanide is possible in submicromolar (ppb) concentrations, with a half maximal response at 2 microM. The response time for detection of introduced cyanide is within 1 s. The sensor can be operated between 5 and 40 degrees C, and cyanide inhibition is unaffected by pH changes between 5 and 8. The sensor is reproducible for cyanide determination and is stable for over 6 months.