A convenient and economical strategy for multiple-target electrochemiluminescence detection using peroxydisulfate solution.

As various aptasensors are adopted in clinical diagnosis, the development of convenient multiple-target determination is a field of ever-increasing interests. Herein, a label-free and amplified electrochemiluminescence (ECL) sensing platform was constructed to detect multiple targets of hemin, glucose and thrombin (TB) using peroxydisulfate (S2O82-) solution, which was one of the most convenient and economical ECL systems. It was worth mentioning that the target-induced bi-enzyme cascade catalysis reaction was developed to increase the ECL response strongly of S2O82- solution due to the production of (1O2)2* from the inter-reaction between reactive oxygen species (ROS) and sulfate radical (SO4•-). Specifically, with the layer-by-layer assembly of multi-walled carbon nanotubes (MWCNTs), glucose oxidase (GOx) and gold nanoparticles (AuNPs) as the interface, the guanine-rich (G-rich) thrombin aptamer (TBA) was anchored for hemin (target 1) detection, due to the electrocatalysis of hemin/G-quadruplex as a horseradish peroxidase mimicking DNAzyme (HRP-DNAzyme) towards dissolved oxygen for ROS generation. Second, in the presence of glucose (target 2), the ECL intensity was improved because glucose was the substrate of the bi-enzyme cascade catalysis reaction. Third, when TB (target 3) was sequentially incubated based on the above-mentioned aptasensor, the bi-enzyme catalysis was inhibited to decrease the ECL signal, due to the steric hindrance effect of the TB protein. As a result, the aptasensor achieved the nanomolar detection for hemin (3.33 nM), the micromolar detection for glucose (0.33 µM) and the femtomolar detection for TB (3.33 fM), respectively.

Ultra-Sensitive Nano Optical Sensor Samarium-Doxycycline Doped in Sol Gel Matrix for Assessment of Glucose Oxidase Activity in Diabetics Disease.

A low cost and very sensitive method for the determination of the activity of glucose oxidase enzyme in different diabetics serum samples was developed. The method based on the assessment of the H2O2 concentration produced from the reaction of the glucose oxidase (GOx) enzyme with glucose as substrate in the serum of diabetics patients by nano optical sensor Sm-doxycycline doped in sol gel matrix. H2O2 enhances the luminescence intensity of all bands of the nano Sm-doxycycline complex [Sm-(DC)2]+ doped in sol-gel matrix, especially the 645 nm band at λex = 400 nm and pH 7.0 in water. The influence of the different analytical parameters that affect the luminescence intensity of the nano optical sensor, e.g. pH, H2O2 concentration and foreign ions concentrations were studied. The remarkable enhancement of the luminescence intensity of nano optical sensor [Sm-(DC)2]+ complex in water at 645 nm by the addition of various concentrations of H2O2 was successfully used as an optical sensor for the assessment of the activity of the glucose oxidase enzyme in different diabetics serum samples. The calibration plot was achieved over the activity range 0.1-240 U/L with a correlation coefficient of 0.999 and a detection limit of 0.05 U/L.

Biosensing of glucose in flow injection analysis system based on glucose oxidase-quantum dot modified pencil graphite electrode.

A novel amperometric glucose biosensor was proposed in flow injection analysis (FIA) system using glucose oxidase (GOD) and Quantum dot (ZnS-CdS) modified Pencil Graphite Electrode (PGE). After ZnS-CdS film was electrochemically deposited onto PGE surface, GOD was immobilized on the surface of ZnS-CdS/PGE through crosslinking with chitosan (CT). A pair of well-defined reversible redox peak of GOD was observed at GOD/CT/ZnS-CdS/PGE based on enzyme electrode by direct electron transfer between the protein and electrode. Further, obtained GOD/CT/ZnS-CdS/PGE offers a disposable, low cost, selective and sensitive electrochemical biosensing of glucose in FIA system based on the decrease of the electrocatalytic response of the reduced form of GOD to dissolved oxygen. Under optimum conditions (flow rate, 1.3mL min(-1); transmission tubing length, 10cm; injection volume, 100µL; and constant applied potential, -500mV vs. Ag/AgCl), the proposed method displayed a linear response to glucose in the range of 0.01-1.0mM with detection limit of 3.0µM. The results obtained from this study would provide the basis for further development of the biosensing using PGE based FIA systems.

Iodophenol blue-enhanced luminol chemiluminescence and its application to hydrogen peroxide and glucose detection.

In this study, we found that iodophenol blue can enhance the weak chemiluminescence (CL) of luminol-H2O2 system. With the aid of CL spectral, electron spin resonance (ESR) spectral measurements and studies on the effects of various free radical scavengers on the iodophenol blue-enhanced luminol-H2O2 system, we speculated that iodophenol blue may react with H2O2 and oxygen to produce oxidizing radical species such as OH(•) and O2(•-) resulting the formation of (1)O2. The generated (1)O2 may react with luminol anion generating an unstable endoperoxide and subsequent 3-aminophthalate* (3-APA*). When the excited-state 3-APA returned to the ground-state, an enhanced CL was observed. Based on the H2O2 concentration dependence of the catalytic activity of iodophenol blue, a cheap, simple, sensitive CL assay for the determination of H2O2 was established. Under the optimum experimental conditions, a linear relationship between the relative CL intensity and H2O2 concentration in the range of 0.025-10 µM was obtained. As low as 14 nM H2O2 can be sensitively detected by using the proposed method. The relative standard deviation for 5, 1 and 0.25 µM H2O2 was 2.58%, 5.16% and 4.66%, respectively. By combining the glucose oxidase (GOx)-catalyzed oxidation reaction, CL detection of glucose was realized. The linear range of glucose detection was 0.1-30 µM with a detection limit of 0.06 µM. The proposed method has been applied to the detection of glucose in diluted serum.

Chip-based amperometric enzyme sensor system for monitoring of bioprocesses by flow-injection analysis.

A microfluidic chip integrating amperometric enzyme sensors for the detection of glucose, glutamate and glutamine in cell-culture fermentation processes has been developed. The enzymes glucose oxidase, glutamate oxidase and glutaminase were immobilized by means of cross-linking with glutaraldehyde on platinum thin-film electrodes integrated within a microfluidic channel. The biosensor chip was coupled to a flow-injection analysis system for electrochemical characterization of the sensors. The sensors have been characterized in terms of sensitivity, linear working range and detection limit. The sensitivity evaluated from the respective peak areas was 1.47, 3.68 and 0.28 µAs/mM for the glucose, glutamate and glutamine sensor, respectively. The calibration curves were linear up to a concentration of 20 mM glucose and glutamine and up to 10 mM for glutamate. The lower detection limit amounted to be 0.05 mM for the glucose and glutamate sensor, respectively, and 0.1 mM for the glutamine sensor. Experiments in cell-culture medium have demonstrated a good correlation between the glutamate, glutamine and glucose concentrations measured with the chip-based biosensors in a differential-mode and the commercially available instrumentation. The obtained results demonstrate the feasibility of the realized microfluidic biosensor chip for monitoring of bioprocesses.

A high-sensitive and fast-fabricated glucose biosensor based on Prussian blue/topological insulator Bi2Se3 hybrid film.

A novel and fast-fabricated Prussian blue (PB)/topological insulator Bi(2)Se(3) hybrid film has been prepared by coelectrodeposition technique. Taking advantages of topological insulator in possessing exotic metallic surface states with bulk insulating gap, Prussian blue nanoparticles in the hybrid film have smaller size as well as more compact structure, showing excellent pH stability even in the alkalescent solution of pH 8.0. Based on the Laviron theory, the electron transfer rate constant of PB/Bi(2)Se(3) hybrid film modified electrode was calculated to be 4.05 ± 0.49 s(-1), a relatively big value which may be in favor of establishing a high-sensitive biosensor. An amperometric glucose biosensor was then fabricated by immobilizing glucose oxidase (GOD) on the hybrid film. Under the optimal conditions, a wide linear range extending over 3 orders of magnitude of glucose concentrations (1.0 × 10(-5)-1.1 × 10(-2)M) was obtained with a high sensitivity of 24.55 µA mM(-1) cm(-2). The detection limit was estimated for 3.8 µM defined from a signal/noise of 3. Furthermore, the resulting biosensor was applied to detect the blood sugar in human serum samples without any pretreatment, and the results were comparatively in agreement with the clinical assay.

Trueness assessment for serum glucose measurement using commercial systems through the preparation of commutable reference materials.

BACKGROUND: Commutable reference materials (RMs) are suitable for end-users for evaluating the metrological traceability of values obtained using routine measurement systems. We assessed the performance of 6 routine measurement systems with validated secondary RMs. METHODS: We tested the homogeneity, stability, and commutability of 5 minimally processed human serum pools according to the standard guidelines. The serum pools were assigned values as per the reference procedure of the United States Centers for Disease Control and were used to evaluate the trueness of results from 6 commercial measurement systems based on enzymatic methods: 3 glucose oxidase (GOD) and 3 hexokinase (HK) methods. RESULTS: The prepared RMs were validated to be sufficiently homogenous, stable, and commutable with the patient samples. Method bias varied for different systems: GOD01, -0.17 to 2.88%; GOD02, 1.66 to 4.58%; GOD03, -0.17 to 3.14%; HK01, -3.48 to -0.85%; HK02, -3.83 to -0.11%, and HK03, -1.82 to -0.27%. CONCLUSIONS: We observed that the prepared serum glucose RMs were qualified for trueness assessment. Most of the measurement systems met the minimal quality specifications.

Colorimetric detection of urine glucose based ZnFe2O4 magnetic nanoparticles.

In this paper, we discovered that ZnFe(2)O(4) magnetic nanoparticles (MNPs) possess intrinsic peroxidase-like activity. ZnFe(2)O(4) MNPs exhibit several advantages such as high catalytic efficiency, good stability, monodispersion, and rapid separation over other peroxidase nanomimetics and horseradish peroxidase (HRP). ZnFe(2)O(4) MNPs were used as a colorimetric biosensor for the detection of urine glucose. This method is simple, inexpensive, highly sensitive, and selective for glucose detection using glucose oxidase (GOx) and ZnFe(2)O(4) MNPs with a linear range from 1.25 × 10(-6) to 1.875 × 10(-5) mol L(-1) with a detection limit of 3.0 × 10(-7) mol L(-1). The color change observable by the naked eyes based on the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) is the principle for the sensing of urine glucose level.

A low-cost, simple, and rapid fabrication method for paper-based microfluidics using wax screen-printing.

Wax screen-printing as a low-cost, simple, and rapid method for fabricating paper-based microfluidic devices (µPADs) is reported here. Solid wax was rubbed through a screen onto paper filters. The printed wax was then melted into the paper to form hydrophobic barriers using only a hot plate. We first studied the relationship between the width of a hydrophobic barrier and the width of the original design line. We also optimized the heating temperature and time and determined the resolution of structures fabricated using this technique. The minimum width of hydrophilic channel and hydrophobic barrier is 650 and 1300 µm, respectively. Next, our fabrication method was compared to a photolithographic method using the reaction between bicinchoninic acid (BCA) and Cu(1+) to demonstrate differences in background reactivity. Photolithographically defined channels exhibited a high background while wax printed channels showed a very low background. Finally, the utility of wax screen-printing was demonstrated for the simultaneous determination of glucose and total iron in control human serum samples using an electrochemical method with glucose oxidase and a colorimetric method with 1,10-phenanthroline. This study demonstrates that wax screen-printing is an easy-to-use and inexpensive alternative fabrication method for µPAD, which will be especially useful in developing countries.

Glucose concentration determination based on silica sol-gel encapsulated glucose oxidase optical biosensor arrays.

Optical biosensor arrays for rapidly determining the glucose concentrations in a large number of beverage and blood samples were developed by immobilizing glucose oxidase (GOD) on oxygen sensor layer. Glucose oxidase was first encapsulated in silica based gels through sol-gel approach and then immobilized on 96-well microarrays integrated with oxygen sensing film at the bottom. The oxygen sensing film was made of an organically modified silica film (ORMOSIL) doped with tris(4,7-diphenyl-1,10-phenanthroline) ruthenium dichloride (Ru(dpp)(3)Cl(2)). The oxidation reaction of glucose by glucose oxidase could be monitored through fluorescence intensity enhancement due to the oxygen consumption in the reaction. The luminescence changing rate evaluated by the dynamic transient method (DTM) was correlated with the glucose concentration with the wide linear range from 0.1 to 5.0mM (Y=13.28X-0.128, R=0.9968) and low detection limit (0.06 mM). The effects of pH and coexisting ions were systemically studied. The results showed that the optical biosensor arrays worked under a wide range of pH value, and normal interfering species such as Na(+), K(+), Cl(-), PO(4)(3-), and ascorbic acid did not cause apparent interference on the measurement. The activity of glucose oxidase was mostly retained even after 2-month storage, indicating their long-term stability.

Fabrication of an implantable fine needle-type glucose sensor using gamma-polyglutamic acid.

Implantable fine needle-type glucose sensors with an outer diameter of less that 0.2 mm were fabricated using a low-cost and non-animal origin polyamide, gamma-polyglutamic acid (PGA) as a glucose oxidase (GOx) immobilizing material. Two types of PGA, gamma-polyglutamic acid (PGAH) and gamma-polyglutamic acid sodium salt (PGANa), were employed to prepare GOx immobilized film by the covalent attachment of GOx using water-soluble carbodiimide (EDC). Nafion/cellulose acetate composite film and polyurethane/polydimethylsiloxane composite film were employed as a permselective inner film and a biocompatible outer film, respectively. The procedure of enzyme-immobilized film fabrication affected the stability of the sensor; that is, GOx immobilized film prepared by pouring a mixture solution of GOx and EDC on a PGA precoated surface showed higher sensor stability than that prepared by pouring a mixture solution of GOx, PGA and EDC. Although, obvious differences in the sensor properties were not observed between the use of PGANa and PGAH, the electrode prepared with PGAH had a lower swelling degree. The glucose sensors prepared with both PGANa and PGAH were practically not affected by the existence of electroactive compounds, such as uric acid, and provided long-term stability for approximately 5 weeks. These sensors also showed good performance in horse serum.

Zinc oxide nanoparticles/glucose oxidase photoelectrochemical system for the fabrication of biosensor.

Nanosized semiconductor crystals can increase efficiency of photochemical reactions and greatly improve the catalytic activity of enzymes to generate novel photoelectrochemical systems. In this work, glucose oxidase (GOx)/zinc oxide (ZnO) is selected as a model system to assess the photovoltaic effect of semiconductor nanoparticles on the enzyme electrode. UV-spectrum and circular dichroism (CD) results show that the structure of GOx is preserved after conjugation with ZnO nanoparticles. The current response of the enzyme electrode containing ZnO nanoparticles increases from 0.82 to 21 microA cm(-2) in the solution of 10 mM beta-D-glucose. Furthermore, after irradiating the enzyme electrode with UV light for 2 h, the current response can be increased nearly 30% and the detection limit can be lowered about two orders compared with the catalytic reactions in the dark, which indicate that a technique to fabricate a novel photocontrolled enzyme-based biosensor may be developed.

Electrochemically deposited nanocomposite film of CS-Fc/Au NPs/GOx for glucose biosensor application.

A simple and controllable electrodeposition method is described to fabricate a homogeneous chitosan-ferrocene/Au nanoparticles/glucose oxidase (CS-Fc/Au NPs/GOx) nanocomposite film. The morphologies and electrochemistry of the nanocomposite film were investigated by using scanning electron microscopy (SEM) and electrochemical techniques including electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV), respectively. The ferrocene group (Fc) covalently bounded chitosan hybrid (CS-Fc) can effectively avoid the leakage of Fc and retain its electrochemical activity. Further immobilization of Au NPs into the CS-Fc matrix enhances both the charge-transport properties of the composite and bioaffinity to enzyme. Biosensor based on this CS-Fc/Au NPs/GOx film has advantages of fast response, excellent reproducibility and high stability. This biosensor shows a linear response to glucose in the concentration range from 0.02 to 8.66 mM with a detection limit of 5.6 microM at a signal-to-noise ratio of 3. The present method offers a facile way to fabricate biosensors and bioelectronic devices.

Development of a respirometric biochip for embryo assessment.

A prototype respirometric biochip dedicated to monitoring oxygen consumption of preimplantation embryos has been developed. The biochip comprises a linear array of eight flow-through microchambers profiled on silicon substrate, and functions together with a phosphorescent oxygen sensitive probe and fluorescence plate reader detection. A high level of sensitivity to changes in dissolved oxygen was achieved through miniaturisation and optimization of biochip geometry, and incorporation of appropriate sealing and humidification systems. The biochips have allowed characterisation of oxygen consumption, by 2 cell and blastocyst stage preimplantation mouse embryos, through monitoring as few as ten preimplantation embryos over a one-hour time period. They provide a non-invasive, simple and convenient means for assessing preimplantation embryo metabolism.

Detection of outliers and establishment of targets in external quality assessment programs.

BACKGROUND: The establishment of target values is important in external quality assessment (EQA) programs since most of the programs use the target as the assessment criterion. Results submitted to EQA programs usually are not Gaussian-distributed due to the contamination with outliers. The traditional or robust statistical method can be chosen for the truncation of outliers. We investigated the two methods when setting targets for glucose in an EQA program. METHODS: The results of glucose were analyzed as an all-methods group and divided into four subgroups according to the analytical methods prior to testing of each data distribution. Based on the distribution, the traditional or robust statistical method was used to detect outliers. After removal of outliers, the data distributions were retested and if Gaussian distributions were obtained, the mean values were used as the target. RESULTS: Original data sets were not Gaussian-distributed for all tested groups. Therefore, the robust statistical method was employed to detect outliers. After truncation of outliers, the data sets showed Gaussian distributions and the means were used to set target values. CONCLUSIONS: The targets of glucose were determined for all-methods and individual methods from the mean values following removal of outliers using the robust statistical method. This led to comparable targets among the tested groups.

Immobilization and stabilization of invertase on Cajanus cajan lectin support.

Use of lectins as ligands for the immobilization and stabilization of glycoenzymes has immense application in enzyme research and industry. But their widespread use could be limited by the high cost of their production. In the present study preparation of a novel and inexpensive lectin support for use in the immobilization of glycoenzymes containing mannose or glucose residues in their carbohydrate moiety has been described. Cajanus cajan lectin (CCL) coupled covalently to cyanogen bromide activated Seralose 4B could readily bind enzymes such as invertase, glucoamylase and glucose oxidase. The immobilized and glutaraldehyde crosslinked preparations of invertase exhibited high resistance to inactivation upon exposure to enhanced temperature, pH, denaturants and proteolysis. Binding of invertase to CCL-Seralose was however found to be readily reversible in the presence of 1.0 M methyl alpha-D mannopyranoside. In a laboratory scale column reactor the CCL-Seralose bound invertase was stable for a month and retained more than 80% of its initial activity even after 60 days of storage at 4 degrees C. CCL-Seralose bound invertase exhibited marked stability towards temperature, pH changes and denaturants suggesting its potential to be used as an excellent support for the immobilization of other glycoenzymes as well.

Evaluation of rapid methods for the determination of okadaic acid in mussels.

AIMS: Two different screening methods, a Buffalo Green Monkey cytotoxicity test and a biosensor test, have been considered to replace the official mouse bioassay in monitoring for okadaic acid (OA) levels in mussels. METHODS AND RESULTS: Diarrhoetic shellfish poison-contaminated mussels from the Adriatic Sea were assayed in parallel by means of the mouse bioassay and both alternative methods. Both the cytotoxicity test and the biosensor test showed high sensitivity (OA 0.01 mg g-1 hepatopancreas and 0.002 mg g-1 hepatopancreas, respectively) and a high correlation with the mouse bioassay (r=0.932, P < 0.001 and r=- 0.850, P < 0.001, respectively). CONCLUSION: Both methods are efficacious, quick, inexpensive and provide data on the amount of toxin present in mussels. SIGNIFICANCE AND IMPACT OF THE STUDY: Both methods, besides allowing the simultaneous assay of a great number of samples, comply with the ethical need to reduce the use of animals in the laboratory.

Inmovilización covalente de glucosa oxidasa y peroxidasa / Covalent immobilization of oxidase glucose and peroxidase

Las enzimas por su función catalítica tienen amplia aplicación en infinidad de procesos tecnológicos y en los últimos 15 a han marcado avances significativos en la industria. Dentro de la Industria Farmacéutica y Biológica, la dedicada a los medios diagnósticos ha recibido también el impacto de la introducción de este tipo de productos, soportando en la actualidad tecnologías tan importantes como el inmunoensayo enzimático, el diagnóstico en química clínica y la química seca, donde las técnicas de inmovilización alcanzan un desarrollo cada vez mayor por el incremento de la estabilidad que se logra con estos sistemas. Se presentan ensayos de inmovilización covalente de las enzimas glucosa oxidasa y peroxidasa sobre papel de filtro Whatman No. 2. Fueron ensayadas 2 variantes: la inmovilización covalente de un polímero soluble de las enzimas y la inmovilización covalente de una solución de las enzimas libres. Los resultados del proceso se evaluaron frente a soluciones de referencia de glucosa en concentraciones entre 2,0 y 55,0 mmol/L. Las mejores respuestas se encontraron con el más bajo porcentaje de inmovilización en el caso del enlazamiento del polímero, y para la solución de las enzimas libres correspondió al más alto grado de inmovilización logrado

Enzyme immobilization on a low-cost magnetic support: kinetic studies on immobilized and coimmobilized glucose oxidase and glucoamylase.

Glucose oxidase (GOx) and glucoamylase (GA) were immobilized and coimmobilized through their carbohydrate moieties onto polyethyleneimine-coated magnetite crosslinked with glutaraldehyde and derivatized with adipic dihydrazide. The carbohydrates were oxidized with sodium periodate, and at optimal concentration, their Vm increased up to 18% for GOx and up to 16% for GA. After immobilization, a remaining activity as high as 88% and 70% for GA with maltose and maltodextrin respectively as substrates was obtained, independently of the particle loading. On the contrary, the remaining activity of GOx strongly decreased at high particle loading. Nevertheless, half of its initial activity was recovered at low loading and was not significantly affected when GA was coimmobilized by saturating the reactive groups left on the particle. The Vm of both immobilized enzymes was improved by crosslinking their carbohydrates with adipic dihydrazide, a treatment which allows further coimmobilization of the other enzyme on a second layer.

[Biosensor development in clinical analysis]. / Evolution des capteurs biologiques en analyse clinique.

The use of enzymes immobilized or as markers formed the subject of more than thousand publications in the field of industry or biomedical applications, during the last five years. Recently, some authors published works concerning immobilization of total microorganisms for catalytic purposes, others use the enzymatic activity for marking molecules involved in immunological analysis processes. Together industrial biotechnology and medical analysis laboratory are interested with the evolution of these procedures involving the activity of immobilized enzymes. Enzyme immobilization allowed the lowering of analysis costs for, in this case, the enzyme can be used several times. We take account of the two main cases which are encountered during utilization of immobilized enzymes of analytical purposes. The enzyme is used directly for the catalysed reaction or it is used as enzymatic marker. These both aspects are developed mainly for the elaboration of enzymatic and immunoenzymatic electrodes and the realization of automatic computerized devices allowing continuous estimation of numerous biological blood parameters. From these two precise examples, glucose and antigen determination, the authors show the evolution of these technologies in the field of immobilized enzymes or captors and the analysis of signals given by these electrodes requiring a computerized treatment. This new technology opens to important potentialities in the analytical field. The automatization of these devices allowing the control in real time, will probably make easier the optimization steps of procedures actually used in the biomedical sphere.