A disposable amperometric sensor screen printed on a nitrocellulose strip: a glucose biosensor employing lead oxide as an interference-removing agent

A new type of disposable amperometric sensor is devised by screen printing thick-film electrodes directly on a porous nitrocellulose (NC) strip. The chromatographic NC strip is then utilized to introduce various sample pretreatment layers. As a preliminary application, a glucose biosensor based on hydrogen peroxide detection is constructed by immobilizing glucose oxidase (GOx) on the NC electrode strip and by formulating a strong oxidation layer (i.e., PbO2) at the sample loading area, placed below the GOx reaction band. The screen-printed PbO2 paste serves as a sample pretreatment layer that removes interference by its strong oxidizing ability. Samples applied are carried chromatographically, via the PbO2 paste, to the GOx layer, and glucose is catalyzed to liberate hydrogen peroxide, which is then detected at the electrode surface. The proposed NC/PbO2 strip sensor is shown to be virtually insusceptible to interfering species such as acetaminophen and ascorbic and uric acids and to exhibit good performance, in terms of the sensor-to-sensor reproducibility (standard deviation, +/-0.026 - +/-0.086 microA), the sensitivity (slope, -0.183 microA/mM), and the linearity (correlation coefficient, 0.994 in the range of 0-10 mM).     

Glucose fast-response amperometric sensor based on glucose oxidase immobilized in an electropolymerized poly(o-phenylenediamine) film

o-Phenylenediamine has been used for glucose oxidase (GOx) immobilization on Pt electrodes by electrochemical polymerization at +0.65 V vs SCE. By this approach the enzyme is entrapped in a strongly adherent, highly reproducible thin membrane, whose thickness is around 10 nm. This one-step procedure produces a glucose sensor with a response time less than 1 s, an active enzyme loading higher than 3 units/cm2 of electrode surface, a high sensitivity, and a sufficiently wide linear range. The glucose response shows an apparent Michaelis-Menten constant, K'm = 14.2 mM, and a limiting current density, jmax of 181 microA/cm2. The product kD of partition and diffusion coefficients of glucose in the polymer film is on the order of 10(-13) cm2/s. Due to permselectivity characteristics of the membrane, the access of ascorbate, a common interfering species, to the electrode surface is blocked. To our knowledge, this represents the first report of a membrane capable, at the same time, of immobilizing GOx and rejecting ascorbate. The interesting electrode behavior can be rationalized by using an existing model predicting the amperometric response of an immobilized GOx system.     

ZnO nanonails: synthesis and their application as glucose biosensor

Well-crystallized zinc oxide nanonails were grown in a high density by thermal evaporation process and were used as supporting matrixes for glucose oxidase (GOx) immobilization to construct efficient glucose biosensor. The GOx attached to the surfaces of ZnO nanonails had more spatial freedom in its orientation, which facilitated the direct electron transfer between the active sites of immobilized GOx and electrode surface. The fabricated biosensor showed a high sensitivity of 24.613 microA cm(-2) mM(-1) with a response time less than 10 s. Moreover, it shows a linear range from 0.1 to 7.1 mM with a correlation coefficient of R = 0.9937 and detection limit of 5 microM.     

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.     

Multilayered assembly of dendrimers with enzymes on gold: thickness-controlled biosensing interface

A new approach to construct a multilayered enzyme film on the Au surface for use as a biosensing interface is described. The film was prepared by alternate layer-by-layer depositions of G4 poly(amidoamine) dendrimers and periodate-oxidized glucose oxidase (GOx). The cyclic voltammograms obtained from the Au electrodes modified with the GOx/dendrimer multilayers revealed that bioelectrocatalytic response is directly correlated to the number of deposited bilayers, that is, to the amount of active enzyme immobilized on the Au electrode surface. From the analysis of voltammetric signals, the coverage of active enzyme per GOx/dendrimer bilayer during the multilayer-forming steps was estimated, which demonstrates that the multilayer is constructed in a spatially ordered manner. Also, with the ellipsometric measurements, a linear increment of the film thickness was registered, supporting the formation of the proposed multilayered structure. The E5D5 electrode showed the sensitivity of 14.7 microA x mM(-1) glucose x cm(-2) and remained stable over 20 days under day-by-day calibrations. The proposed method is simple and would be applicable to the constructions of thickness- and sensitivity-controllable biosensing interfaces composed of multienzymes as well as a single enzyme.     

Inhibition and activation of glucose oxidase bioanodes for use in a self-powered EDTA sensor

Self-powered sensors are able to automatically signal the presence of a specific analyte without the aid of an external power source, making them useful as potential devices for batteryless sensing. Here, we present a self-powered enzymatic ethylenediaminetetraacetic acid (EDTA) sensor based on the inhibition and subsequent activation of glucose oxidase (GOx)-based bioelectrodes within the framework of a biofuel cell. Although EDTA is not redox-active, it is detected by the activation of a Cu(2+)-inhibited GOx bioanode in either a typical amperometric sensor (using a standard three-electrode setup) or in a self-powered sensor where the GOx bioanode is coupled to a platinum cathode. The sensors are able to detect concentrations of EDTA that correspond to the amount of Cu(2+) that is used to inhibit the enzymatic electrode. The self-powered sensor shows a greater than 10-fold increase in power output when it is activated by the presence of EDTA. This represents the first time that a non-redox-active analyte has been detected in a self-powered sensor that turns on in the presence of said analyte.     

Flexible carbon cloth electrode modified by hollow core-mesoporous shell carbon as a novel efficient bio-anode for biofuel cell

A new approach is described to produce an efficient electrode material for biofuel cells using flexible carbon cloth (FCC) and hollow core-mesoporous shell carbon (HCMSC) nanospheres as bio-anode materials. The bio-electrochemical activity of glucose oxidase (GOx) enzyme adsorbed on this bio-anode was evaluated, with the maximum anodic current density varying from 80 microA cm(-2) to 180 microA cm-2 for glucose concentrations up to 5.0 mmol L(-1) for the FCC modified electrode with HCMSCs. The open circuit cell voltage was E(0) = 380 mV, and the catalytic electro-oxidation current of glucose reached 0.1 mA cm(-2) at 0.0 V versus Ag/AgCl. This new system employing HCMSC-based FCC is promising toward novel bio-anodes for biofuel cells using glucose as a fuel.     

Fe3O4 magnetic nanoparticles as peroxidase mimetics and their applications in H2O2 and glucose detection

Artificial enzyme mimetics are a current research interest because natural enzymes bear some serious disadvantages, such as their catalytic activity can be easily inhibited and they can be digested by proteases. A very recently study reported by Yan et al. has proven that Fe(3)O(4) magnetic nanoparticles (MNPs) exhibit an intrinsic enzyme mimetic activity similar to that found in natural peroxidases, though MNPs are usually thought to be biological and chemical inert (Gao, L. Z.; Zhuang, J.; Nie, L.; Zhang, J. B.; Zhang, Y.; Gu, N.; Wang, T. H.; Feng, J.; Yang, D. L.; Perrett, S.; Yan, X. Y. Nat. Nanotechnol. 2007, 2, 577-583). In the present work, we just make use of the novel properties of Fe(3)O(4) MNPs as peroxidase mimetics reported by Yan et al. to detect H(2)O(2). The Fe(3)O(4) MNPs were prepared via a coprecipitation method. The as-prepared Fe(3)O(4) MNPs were then used to catalyze the oxidation of a peroxidase substrate 2,2'-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS) by H(2)O(2) to the oxidized colored product (see eq 1) which provides a colorimetric detection of H(2)O(2). As low as 3 x 10(-6) mol/L H(2)O(2) could be detected with a linear range from 5 x 10(-6) to 1 x 10(-4) mol/L via our method. More importantly, a sensitive and selective method for glucose detection was developed using glucose oxidase (GOx) and the as-prepared Fe(3)O(4) MNPs. The detection platforms for H(2)O(2) and glucose developed in the present work not only further confirmed that the Fe(3)O(4) MNPs possess intrinsic peroxidase-like activity but also showed great potential applications in varieties of simple, robust, and easy-to-make analytical approaches in the future.     

A glucose biosensor employing a stable artificial peroxidase based on ruthenium purple anchored cinder

Iron-enriched industrial waste cinder (CFe*) has been recycled for efficient and stable anchoring of Ru(CN)6(4-) to the formation of a hybrid ruthenium purple complex. The cinder/ruthenium purple hybrid-modified carbon paste electrode (designated as CPE/CFe*-RP) was worked out for hydrodynamic analysis of H2O2 at a low detecting potential of 0.0 V versus Ag/AgCl in pH 7 ammonium buffer solution. The highly active, selective, and stable electrocatalytic system with a function similar to peroxidase enzyme shows a linear calibration curve up to 0.8 mM H2O2 at a rotation rate of 3600 rpm with slope and detection limit (S/N = 3) of 0.11 microA/microM and 33 nM, respectively. Interference by direct electrochemical oxidation of easily oxidizable substances can be prevented as a result of the low detecting potential of the working system. A glucose biosensor was further constructed by coating with glucose oxidase and Tosflex on the CPE/CFe*-RP (denoted as CPE/CFe*-RP/GOx/Ts). The proposed CPE/CFe*-RP/GOx/Ts with a two-layer configuration, that is, enzyme and protecting layers, exhibits good operational performance in terms of response time, linearity, detection limit, and lifetime.     

Investigation of carbon nanofibers as support for bioactive substances

In this paper we have studied the adsorption properties of various bio-active systems onto the surface of carbon nanofibers (CNF) synthesized by chemical vapor deposition (CVD). Amino acids (alanine, aspartic acid, glutamic acid) and glucose oxidase (GOx) were adsorbed on CNF and the results were compared with those obtained when activated carbon (AC) was used as support. CNF and AC properties (hydrophilic or hydrophobic properties) were characterized by the pH value, the concentration of acidic/basic sites and by naphthalene adsorption. CNF with immobilized GOx was additionally investigated as a highly sensitive glucose biosensor. An amperometric method was used in an original manner to detect the changes in the specific activity of GOx, immobilized longer time on CNF. The method demonstrates that not the whole enzyme adsorbed onto CNF can catalyze the oxidation of glucose from the solution.     

Magnetic nanoparticle-enhanced biosensor based on grating-coupled surface plasmon resonance

A highly sensitive surface plasmon resonance (SPR) biosensor employing magnetic nanoparticle (MNP) assays is presented. In the reported approach, MNPs simultaneously served as "vehicles" for rapid delivery of target analyte from a sample to the sensor surface and as labels increasing the measured refractive index changes that are associated with the binding of target analyte. An optical setup based on grating-coupled surface plasmon resonance (GC-SPR) was used with a magnetic field gradient applied through the sensor chip for manipulating with MNPs on its surface. Iron oxide MNPs and a sensor surface with metallic diffraction grating were modified with antibodies that specifically recognize different epitopes of the analyte of interest. The sensitivity of the biosensor was investigated as a function of mass transport of the analyte to the sensor surface driven by diffusion (free analyte) or by the magnetic field gradient (analyte bound to MNPs). Immunoassay-based detection of β human chorionic gonadotropin (βhCG) was implemented to evaluate the sensitivity of the MNP-enhanced GC-SPR biosensor scheme. The results reveal that the sensitivity of βhCG detection was improved by 4 orders of magnitude compared with the regular SPR sensor with direct detection format, and a limit of detection below pM was achieved.     

Wet-chemical green synthesis of L-lysine amino acid stabilized biocompatible iron-oxide magnetic nanoparticles

In this paper, we report a novel method for the synthesis of L-Lysine (lys) amino acid coated maghemite (gamma-Fe2O3) magnetic nanoparticles (MNPs). The facile and cost effective method permitted preparation of the high-quality superparamagnetic gamma-Fe2O3 MNPs with hydrophilic and biocompatible nature. For this work, first we synthesized magnetite phase Fe3O4/lys by wet chemical method and oxidized to y-Fe2O3 in controlled oxidizing environment, as evidenced by XRD and VSM magnetometry. The crystallite size and magnetization of gamma-Fe2O3/lys MNPs was found to be 14.5 nm, 40.6 emu/gm respectively. The surface functionalization by L-lysine amino acid and metal-ligand bonding was also confirmed by FTIR spectroscopy. The hydrodynamic diameter, colloidal stability and surface charge on MNPs were characterized by DLS and zeta potential analyser.     

Positive potential operation of a cathodic electrogenerated chemiluminescence immunosensor based on luminol and graphene for cancer biomarker detection

In this work, we report a cathodic electrogenerated chemiluminescence (ECL) of luminol at a positive potential (ca. 0.05 V vs Ag/AgCl) with a strong light emission on the graphene-modified glass carbon electrode. The resulted graphene-modified electrode offers an excellent platform for high-performance biosensing applications. On the basis of the cathodic ECL signal of luminol on the graphene-modified electrode, an ECL sandwich immunosensor for sensitive detection of cancer biomarkers at low potential was developed with a multiple signal amplification strategy from functionalized graphene and gold nanorods multilabeled with glucose oxidase (GOx) and secondary antibody (Ab(2)). The functionalized graphene improved the electron transfer on the electrode interface and was employed to attach the primary antibody (Ab(1)) due to it large surface area. The gold nanorods were not only used as carriers of secondary antibody (Ab(2)) and GOx but also catalyzed the ECL reaction of luminol, which further amplified the ECL signal of luminol in the presence of glucose and oxygen. The as-proposed low-potential ECL immunosensor exhibited high sensitivity and specificity on the detection of prostate protein antigen (PSA), a biomarker of prostate cancer that was used as a model. A linear relationship between ECL signals and the concentrations of PSA was obtained in the range from 10 pg mL(-1) to 8 ng mL(-1). The detection limit of PSA was 8 pg mL(-1) (signal-to-noise ratio of 3). Moreover, the as-proposed low-potential ECL immunosensor exhibited excellent stability and reproducibility. The graphene-based ECL immunosensor accurately detected PSA concentration in 10 human serum samples from patients demonstrated by excellent correlations with standard chemiluminescence immunoassay. The results suggest that the as-proposed graphene ECL immunosensor will be promising in the point-of-care diagnostics application of clinical screening of cancer biomarkers.     

Multiresponsive Supramolecular Theranostic Nanoplatform Based on Pillar[5]arene and Diphenylboronic Acid Derivatives for Integrated Glucose Sensing and Insulin Delivery

A closed‐loop “smart” insulin delivery system with the capability to mimic pancreatic cells will be highly desirable for diabetes treatment. This study reports a multiple stimuli‐responsive insulin delivery platform based on an explicit supramolecular strategy. Self‐assembled from a well‐designed amphiphilic host–guest complex formed by pillar[5]arene and a diphenylboronic acid derivative and loaded with insulin and glucose oxidase, the obtained insulin‐GOx‐loaded supramolecular vesicles can selectively recognize glucose, accompanied by the structure disruption and efficient release of the entrapped insulin triggered by the high glucose concentration as well as the in situ generated H₂O₂ and acid microenvironment during the GOx‐promoted specific oxidation of glucose into gluconic acid. Moreover, such a “smart” supramolecular theranostic nanoplatform is able to function as both a glucose sensor and a controlled insulin delivery actuator. In vivo experiments further demonstrate that this smart supramolecular nanocarrier shows fast response to hyperglycemic circumstances and can effectively regulate the glucose levels in a mouse model of type I diabetes.     

Glucose oxidase-modified carbon-felt-reactor coupled with peroxidase-modified carbon-felt-detector for amperometric flow determination of glucose

Glucose oxidase (GOx) and horseradish peroxidase (HRP) were covalently immobilized on a porous carbon-felt (CF) by using cyanuric chloride (CC) as a linking reagent. The resulting GOx-modified-CF (GOx-ccCF) was used as column-type enzyme reactor and placed on upstream of the HRP-ccCF-based H₂O₂ flow-detector to fabricate amperometric flow-biosensor for glucose. Sensor setting conditions and the operational conditions were optimized, and the analytical performance characteristics of the resulting flow-biosensor were evaluated. The chemical modification of the GOx via CC was found to be effective to obtain larger catalytic activity as compared with the physical adsorption. Under the optimized conditions (i.e., volume ratio of the GOx-ccCF-reactor to the HRP-ccCF-detector is 1.0; applied potential is − 0.12 V vs. Ag/AgCl; carrier pH is 6.5; and carrier flow rate is 4.3 ml/min), highly selective and quite reproducible peak current responses toward glucose were obtained: the RSD for 30 consecutive injections of 3 mM glucose was 1.04%, and no serious interferences were observed for fructose, ethanol, uric acid, urea and tartaric acid for the amperometric measurements of glucose. The magnitude of the cathodic peak currents for glucose was linear up to 5 mM (sensitivity, 6.38 ± 0.32 μA/μM) with the limit detection of 9.4 μM (S/N = 3, noise level, 20 nA). The present GOx-ccCF-reactor and HRP-ccCF-detector-coupled flow-glucose biosensor was utilized for the determination of glucose in beverages and liquors, and the analytical results by the sensor were in fairly good agreement with those by the conventional spectrophotometry.     

Polymerase chain reaction coupling with magnetic nanoparticles-based biotin-avidin system for amplification of chemiluminescent detection signals of nucleic acid

A novel method was established through the detection of chemiluminescent signals of nucleic acid hybridization based on magnetic nanoparticles (MNPs) and PCR. 5' amino- modified specific probes were immobilized on the surface of silanized MNPs by Schiff reaction between amino and aldehyde group. The probes were used to capture the synthetic biotin-dUTP-labeled DNA fragments which were obtained by polymerase chain reaction (PCR). Then these complexes were bonded with streptavidin-modified alkaline phosphatase (SA-AP). Finally the chemiluminescent signals were detected by adding 3-(2'-spiroadamantane)- 4-methoxy -4-(3"-phosphoryloxy) phenyl-1, 2-dioxetane (AMPPD) which was the substrate reagent of AP. The concentration of probes which were immobilized on the surface of MNPs was studied, how to reduce the adsorption of SA-AP on the surface of MNPs was also researched. It was shown that 12.5 pmol of probes were immobilized on 1 mg of MNPs. Aldehyde-MNPs modified with probes could adsorb SA-AP, affecting the sensitivity of chemiluminescene consequently. Reduction of aldehyde group by sodium borohydride and blocking the bare position of MNPs with bovine serum albumin (BSA) could decrease the background of chemiluminescence, and this method has good specificity in detection of chloramphenicol acetyltransferase (CAT) gene.     

Biocatalytically induced growth of gold nanoshells: using enzyme reaction for the controllable fabrication of nanomaterials

In the present work, the enzymatically controlled growth process of gold nanoshells (GNSs) in the presence of O2/glucose/glucose oxidase (GOx) and its chloroaurate ion electron acceptor is described. The biocatalytically stimulated growth process is one of the bio-inspired synthetic procedures directed by biological molecules which occur under ambient conditions. It is found that hydrogen peroxide (H2O2) could enlarge the gold nanoparticles (GNPs) on the surface of GNSs precursor composites, of which the preadsorbed GNPs serve as nucleation sites for further gold deposition. Here, GOx is harnessed for its unparalled level of catalytic activity and substrate specificity while H2O2 is produced as a by-product during the oxidation of D-glucose to gluconic acid by GOx. Then the bio-generated H2O2 is used as the reducing agent in the catalytic deposition process of GNSs formation. During the procedure, the localized surface plasmon resonance peaks range across hundreds of nanometers from visible to near infrared region accompanying by the resultant formation of uniform and continuous core-shell nanostructures. The corresponding optical, morphological and enzyme kinetic properties are all well investigated. The novel protocol offers a new perspective for the bio-directed synthesis method in nanotechnology.     

A wireless, remote query glucose biosensor based on a pH-sensitive polymer

This paper describes a wireless, remote query glucose biosensor using a ribbonlike, mass-sensitive magnetoelastic sensor as the transducer. The glucose biosensor is fabricated by first coating the magnetoelastic sensor with a pH-sensitive polymer and upon it a layer of glucose oxidase (GOx). The pH-responsive polymer swells or shrinks, thereby changing mass, respectively, in response to increasing or decreasing pH values. The GOx-catalyzed oxidation of glucose produces gluconic acid, inducing the pH-responsive polymer to shrink, which in turn decreases the polymer mass. In response to a time-varying magnetic field, a magnetoelastic sensor mechanically vibrates at a characteristic resonance frequency, the value of which inversely depends on sensor mass loading. As the magnetoelastic films are magnetostrictive, the vibrations launch magnetic flux that can be remotely detected using a pickup coil. Hence, changes in the resonance frequency of a passive magnetoelastic transducer are detected on a remote query basis, without the use of physical connections to the sensors.The sensitivity of the glucose biosensors decreases with increasing ionic strength; at physiological salt concentrations, 0.6 mmol/L of glucose can be measured. At glucose concentrations of 1-10 mmol/L, the biosensor response is reversible and linear, with the detection limit of 0.6 mmol/L corresponding to an error in resonance frequency determination of 20 Hz. Since no physical connections between the sensor and the monitoring instrument are required, this sensor can potentially be applied to in vivo and in situ measurement of glucose concentrations.     

Electrical communication between glucose oxidase and electrodes mediated by phenothiazine-labeled poly(ethylene oxide) bonded to lysine residues on the enzyme surface

A series of glucose oxidase (GOx) hybrids (GOx-phe-nothiazine-labeled poly(ethylene oxide) (PT-PEO)) capable of direct electrical communication with electrodes is synthesized by covalently modifying PT-PEO to lysine residues on the enzyme surface. The length of the PEO chain and the number of PT groups are systematically altered. After the PT-PEO modification, all the hybrids maintain more than 50% of enzyme activity relative to that of native GOx, although loss of the activity becomes greater with increasing PEO chain length. The catalytic current, i(cat), is observed at a potential more positive than 0.55 V after the addition of glucose, due to the intramolecular electron transfer (El) from reduced forms of flavin adenine dinucletide (FADH2/FADH) to PT+ that are electrogenerated at the electrode. The i(cat) value increases with the number of PT groups, indicating that most of the modified PT groups act as mediators. The magnitude of the i(cat) increase depends on the PEO chain length and reveals a maximum for PT-PEO with the molecular weight of 3,000. In contrast, the i(cat) is almost constant for GOx-2-(10-phenothiazyl)propionic acid (PT-PA) hybrids with more than two PT groups synthesized by covalently modifying PT-PA to surface lysines, indicating that only a few key PT groups function as mediators. The maximum rate constant (130 s(-1)) for the ET from FADH2/FADH to PT+ is obtained for the GOx hybrid modified with five PT-PEO groups with the molecular weight of 3,000.     

Enzymatic Growth of Quantum Dots: Applications to Probe Glucose Oxidase and Horseradish Peroxidase and Sense Glucose

Three innovative assays are developed for the detection of enzymatic activities of glucose oxidase (GOx) and horseradish peroxidase (HRP) by the generation of CdS quantum dots (QDs) in situ using non‐conventional enzymatic reactions. In the first assay, GOx catalyzes the oxidation of 1‐thio‐β‐D ‐glucose to give 1‐thio‐β‐D ‐gluconic acid. The latter is spontaneously hydrolyzed to β‐D‐gluconic acid and H₂S, which in the presence of cadmium nitrate yields fluorescent CdS nanoparticles. In the second assay HRP catalyzes the oxidation of sodium thiosulfate with hydrogen peroxide generating H₂S and consequently CdS QDs. The combination of GOx with HRP, allowed quantification of glucose in plasma by following growth of fluorescent QDs.