An enhanced glucose biosensor using charge transfer techniques

An enhanced glucose biosensor based on a charge transfer technique glucose sensor (CTTGS) is described and demonstrated experimentally. In the proposed CTTGS, which is accumulation method (d-gluconate+H(+)) ion perception system, the quality of output signal with "signal integration cycles" is high. With the proposed CTTGS it is possible to amplify the sensing signals without an external amplifier by using an accumulation cycle. It can be supposed that measurements of small (d-gluconate+H(+)) ion fluctuation are difficult by ion-sensitive field effect transistor (ISFET) because the theoretical maximum sensitivity is only 59 mV/pH and the small output signals are buried in the 1/f noise component of the metal-insulator-semi-conductor field-effect transistor (MISFET). Therefore, the CTTGS has many advantages, such as high sensitivity, high accuracy, high signal-to-noise ratio (SNR), and has been successfully demonstrated using a charge transfer technique. The CTTGS exhibited excellent performance for glucose with a large span (1445 mV) and good reproducibility. Moreover, the CTTGS has good sensitivity in this range of 7.22mV/mM, a lower detection limit of about 0.01 mM/L and an upper detection limit of about 200 mM/L compared with amperometric glucose analysis which has been studied recently. Under optimum conditions, the proposed CTTGS exceeds the performance of the widely used ISFET glucose sensor. The sensitivity of the CTTGS (7.22 mV/mM) was seven times higher than that of the ISFET (1 mV/mM). Furthermore, the sensitivity obtained for human glucose levels was 29.06 mV/mM with a non-linear error of +/-0.27%; the linearity is y=0.0294x+1.8612 and R(2)=0.9999, which is acceptable for clinical application. Real sample analysis is investigated in blood glucose level by our developed CTTGS ISFET system.     

Internal supply of coenzyme to an amperometric glucose biosensor based on a chemically modified electrode

A biosensor for glucose using glucose dehydrogenase immobilized on a chemically modified graphite electrode was supplied with coenzyme, nicotinamide adenine dinucleotide (NAD+), through pores in the material. A graphite rod was hollowed out, leaving 0.3 mm at the end contacting the solution, filled with 10 mM NAD+ and pressurized. The response factor was 40% of that obtained when 2 mM NAD+ was mixed with the sample solution in a flow system. The coenzyme consumption was 11 microliters h-1 representing a 500-fold saving compared to supply through the bulk solution. The biosensor had a linear calibration curve from the detection limit, 1 microM, to 2 mM glucose and a repeatability of 0.3%. The graphite electrode was modified by adsorption of a bis-(benzophenoxazinyl)-terephthaloyl derivative in order to be able to oxidize NADH at 0 mV versus Ag/AgCl, 0.1 M KCl.     

Fabrication of a highly sensitive penicillin sensor based on charge transfer techniques

A highly sensitive penicillin biosensor based on a charge-transfer technique (CTTPS) has been fabricated and demonstrated in this paper. CTTPS comprised a charge accumulation technique for penicilloic acid and H(+) ions perception system. With the proposed CTTPS, it is possible to amplify the sensing signals without external amplifier by using the charge accumulation cycles. The fabricated CTTPS exhibits excellent performance for penicillin detection and exhibit a high-sensitivity (47.852 mV/mM), high signal-to-noise ratio (SNR), large span (1445 mV), wide linear range (0-25 mM), fast response time (<3s), and very good reproducibility. A very lower detection limit of about 0.01 mM was observed from the proposed sensor. Under optimum conditions, the proposed CTTPS outstripped the performance of the widely used ISFET penicillin sensor and exhibited almost eight times greater sensitivity as compared to ISFET (6.56 mV/mM). The sensor system is implemented for the measurement of the penicillin concentration in penicillin fermentation broth.     

A novel, disposable, screen-printed amperometric biosensor for glucose in serum fabricated using a water-based carbon ink

Screen-printed amperometric glucose biosensors have been fabricated using a water-based carbon ink. The enzyme glucose oxidase (GOD) and the electro-catalyst cobalt phthalocyanine were mixed with the carbon ink prior to the screen-printing process; therefore, biosensors are prepared in a one-step fabrication procedure. Optimisation of the biosensor performance was achieved by studying the effects of pH, buffer strength, and applied potential on the analytical response. Calibration studies were performed under optimum conditions, using amperometry in stirred solution, with an operating potential of +500 mV versus SCE. The sensitivity was found to be 1170 nA mM(-1), with a linear range of 0.025-2 mM; the former represents the detection limit. The disposable amperometric biosensor was evaluated by carrying out replicate determinations on a sample of bovine serum. This was achieved by the method of multiple standard additions and included a correction for background currents arising from oxidizable serum components. The mean serum concentration was calculated to be 8.63 mM and compared well with the supplier's value of 8.3 mM; the coefficient of variation was calculated to be 3.3% (n=6).     

Layer-by-layer self-assembly of functionalized graphene nanoplates for glucose sensing in vivo integrated with on-line microdialysis system

In this work, a novel amperometric biosensor for hydrogen peroxide was fabricated through the layer-by-layer (LBL) self-assembling of amine-terminated ionic liquid (IL-NH(2)), and sulfonic acid (SO(3)(-)) functionalized graphene by covalent bonding. The modification of the two functionalities introduced positive and negative charge onto the surface of graphene respectively, thus facilitating the formation of a multilayer film denoted with {IL-RGO/S-RGO}(n) through electrostatic interaction and further immobilization of glucose oxidase (GOx). The resulting {IL-RGO/S-RGO}(n)/GOx/Nafion biosensor displayed an excellent response to glucose at a potential of -200 mV. Combined with on-line microdialysis system, the glucose biosensor in the on-line system showed good linear range from 10 μM to 500 μM with the detection limit of 3.33 μM (S/N=3). Consequently, the basal level of glucose in the striatum of anesthetic rats was calculated to be 0.376 ± 0.028 mM (mean ± s.d., n=3). The {IL-RGO/S-RGO}(n)/GOx/Nafion biosensor was further applied for in vivo sensing of the glucose level in the striatum when rats received intraperitoneal (i.p.) injection of 30 μL insulin, which resulted in an obvious decrease in the extracellular concentration of glucose within 30 min. The method was proved to be sensitive and reproducible, which enabled its promising application in physiology and pathology.     

An amperometric biosensor based on a composite of single-walled carbon nanotubes, plasma-polymerized thin film, and an enzyme

We report on an amperometric biosensor that is based on a nanocomposite of carbon nanotubes (CNT), a nano-thin plasma-polymerized film (PPF), and glucose oxidase (GOx) as an enzyme model. A mixture of the GOx and a CNT film is sandwiched with 10-nm-thick acetonitrile PPFs. Under PPF layer was deposited onto a sputtered gold electrode. To facilitate the electrochemical communication between the CNT layer and GOx, CNT was treated with nitrogen or oxygen plasma. The resulting device showed that the oxidizing current response due to enzymatic reaction was 4-16-fold larger than that with only CNT or PPF, showing that the PPF and/or plasma process is an enzyme-friendly platform for designing electrochemical communication from the reaction center of GOx to the electrode via CNTs. The optimized glucose biosensor showed high sensitivity (sensitivity of 42 microA mM(-1)cm(-2), correlation coefficient of 0.992, linear response range of 0.025-2.2 mM, and a detection limit of 6 microM at signal/noise ratio of 3, +0.8 V versus Ag/AgCl), high selectivity (almost no interference by 0.5 mM ascorbic acid) for glucose quantification, and rapid response (<4 s to reach 95% of maximum response). Additionally, the devices showed a small and stable background current (0.35+/-0.013 microA) compared with the glucose response (ca. 10 microA at 10mM glucose) and suitable reproducibility from sample-to-sample (<3%, n=4).     

A flow injection system, comprising a biosensor based on a screen-printed carbon electrode containing Meldola’s Blue-Reinecke salt coated with glucose dehydrogenase, for the measurement of glucose

A biosensor for the measurement of glucose in serum has been developed, based on a screen-printed carbon electrode modified with Meldola’s Blue-Reinecke salt, coated with the enzyme glucose dehydrogenase (from Bacillus sp.), and nicotinamide adenine dinucleotide coenzyme (NAD⁺). A cellulose acetate layer was deposited on top of the device to act as a permselective membrane. The biosensor was incorporated into a commercially available, thin-layer, amperometric flow cell operated at a potential of only +0.05 V versus Ag/AgCl. The mobile phase consisted of 0.2 M phosphate buffer (pH 7.0) containing 0.1 M potassium chloride solution, and a flow rate of 0.8 ml min⁻¹ was used throughout the investigation. The biosensor response was linear over the range of 0.075-30 mM glucose, with the former representing the detection limit. The precision of the system was determined by carrying out 20 repeat injections of a 5-mM glucose standard, and the calculated coefficient of variation was 3.9%. It was demonstrated that this biosensor system could be applied to the direct measurement of glucose in serum without pretreatment. Therefore, this would allow high-throughput analysis, at low cost, for this clinically important analyte.     

An enzyme-chromogenic surface plasmon resonance biosensor probe for hydrogen peroxide determination using a modified Trinder's reagent

An absorption-based surface plasmon resonance (SPR(Abs)) biosensor probe has been developed for simple and reproducible measurements of hydrogen peroxide using a modified Trinder's reagent (a chromogenic reagent). The reagent enabled the determination of the hydrogen peroxide concentration by the development of deep color dyes (lambda(max)=630nm) through the oxidative coupling reaction with N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline sodium salt monohydrate (MAOS; C(13)H(20)NNaO(4)S.H(2)O) and 4-aminoantipyrine (4-AA) in the presence of hydrogen peroxide and horseradish peroxidase (HRP). In the present study, urea as an adduct of hydrogen peroxide for color development could be omitted from the measurement solution. The measurement solution containing 5mM hydrogen peroxide was deeply colored at a high absorbance value calculated as 46.7cm(-1) and was directly applied to the SPR(Abs) biosensing without dilution. The measurement was simply performed by dropping the measurement solution onto the surface of the SPR sensor probe, and the SPR(Abs) biosensor response to hydrogen peroxide was obtained as a reflectivity change in the SPR spectrum. After investigation of the pH profiles in the SPR(Abs) biosensor probe, a linear calibration curve was obtained between 1.0 and 50mM hydrogen peroxide (r=0.991, six points, average of relative standard deviation; 0.152%, n=3) with a detection limit of 0.5mM. To examine the applicability of this SPR(Abs) biosensor probe, 20mM glucose detection using glucose oxidase was also confirmed without influence of the refractive index in the measurement solution. Thus, the SPR(Abs) biosensor probe employing the modified Trinder's reagent demonstrated applicability to other analyte biosensing tools.     

Construction of an amperometric glucose biosensor based on the immobilization of glucose oxidase onto electrodeposited Pt nanoparticles-chitosan composite film

One-step construction of Pt nanoparticles-chitosan composite film (PtNPs-CS) was firstly proposed as a novel immobilization matrix for the enzymes to fabricate glucose biosensor. This novel interface embedded in situ PtNPs in CS hydrogel was developed by one-step electrochemical deposition in solution containing CS and chloroplatinic acid (H(2)PtCl(6)). Several techniques, including scanning electron microscopy (SEM), cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and chronoamperometry were employed to characterize the assembly process and performance of the biosensor. Under the optimized experimental conditions, the resulting biosensor exhibited excellent linear behavior in the concentration range from 1.2 μM to 4.0 mM for the quantitative analysis of glucose with a limit of detection of 0.4 μM at a signal-to-noise ratio of 3. The apparent Michaelis-Menten constant (K(M)(app)) was evaluated to be 2.4 mM, showing good affinity. The proposed biosensor offered good amperometric responses to glucose due to the nanostructured sensing film provided plenty of active sites for the immobilization of glucose oxidase (GOD).     

Rapid detection of avian influenza H5N1 virus using impedance measurement of immuno-reaction coupled with RBC amplification

Avian influenza virus (AIV) subtype H5N1 was first discovered in the 1990 s and since then its emergence has become a likely source of a global pandemic and economic loss. Currently accepted gold standard methods of influenza detection, viral culture and rRT-PCR, are time consuming, expensive and require special training and laboratory facilities. A rapid, sensitive, and specific screening method is needed for in-field or bedside testing of AI virus to effectively implement quarantines and medications. Therefore, the objective of this study was to improve the specificity and sensitivity of an impedance biosensor that has been developed for the screening of AIV H5. Three major components of the developed biosensor are immunomagnetic nanoparticles for the separation of AI virus, a microfluidic chip for sample control and an interdigitated microelectrode for impedance measurement. In this study polyclonal antibody against N1 subtype was immobilized on the surface of the microelectrode to specifically bind AIV H5N1 to generate more specific impedance signal and chicken red blood cells (RBC) were used as biolabels to attach to AIV H5N1 captured on the microelectrode to amplify impedance signal. RBC amplification was shown to increase the impedance signal change by more than 100% compared to the protocol without RBC biolabels, and was necessary for forming a linear calibration curve for the biosensor. The use of a second antibody against N1 offered much greater specificity and reliability than the previous biosensor protocol. The biosensor was able to detect AIV H5N1 at concentrations down to 10(3) EID(50)ml(-1) in less than 2h.     

A screen-printed microband glucose biosensor system for real-time monitoring of toxicity in cell culture

Microband biosensors, screen-printed from a water-based carbon ink containing cobalt phthalocyanine redox mediator and glucose oxidase (GOD) enzyme, were used to monitor glucose levels continuously in buffer and culture medium. Five biosensors were operated amperometrically (E(app) of +0.4V), in a 12-well tissue culture plate system at 37°C, using a multipotentiostat. After 24 h, a linear calibration plot was obtained from steady-state current responses for glucose concentrations up to 10 mM (dynamic range 30 mM). Within the linear region, a correlation coefficient (R(2)) of 0.981 was obtained between biosensor and spectrophotometric assays. Over 24 h, an estimated 0.15% (89 nmol) of the starting glucose concentration (24 mM) was consumed by the microbiosensor. The sensitivity of the biosensor response in full culture medium was stable between pHs 7.3 and 8.4. Amperometric responses for HepG2 monolayer cultures decreased with time in inverse proportionality to cell number (for 0 to 10(6) cell/ml), as glucose was being metabolised. HepG2 3D cultures (spheroids) were also shown to metabolise glucose, at a rate which was independent of spheroid age (between 6 and 15 days). Spheroids were used to assay the effect of a typical hepatotoxin, paracetamol. At 1 mM paracetamol, glucose uptake was inhibited by 95% after 6 h in culture; at 500 μM, around 15% inhibition was observed after 16 h. This microband biosensor culture system could form the basis for an in vitro toxicity testing system.     

A high-performance glucose biosensor based on monomolecular layer of glucose oxidase covalently immobilised on indium-tin oxide surface

In this paper, a mediatorless amperometric glucose biosensor based on direct covalent immobilisation of monomolecular layer of glucose oxidase (GOx) on a semiconducting indium-tin oxide (ITO) is demonstrated. The abundance of surface hydroxyl functional group of ITO allows it to be used as a suitable platform for direct covalent immobilisation of the enzyme for sensor architecture. The anodic current corresponding to electrochemical oxidation of the enzymatic product, hydrogen peroxide, at a sputtered Pt electrode at 0.500 V (vs. SCE) was obtained as the sensor signal. It was found that the biosensor based on the direct immobilisation scheme shows a fast biosensor response, minimum interference from other common metabolic species and ease of biosensor miniaturisation. A linear range of 0-10 mM of glucose was demonstrated, which exhibits a high sensitivity as far as performance per immobilised GOx molecule is concerned. A detection limit as low as 0.05 mM and long-term stability were observed. Even more important, the biosensor design allows fabrication through a dry process. These characteristics make it possible to achieve mass production of biosensor compatible with the current electronic integrated circuit manufacturing technologies.     

Amperometric glucose biosensor based on single-walled carbon nanohorns

The biosensing application of single-walled carbon nanohorns (SWCNHs) was demonstrated through fabrication of an amperometric glucose biosensor. The biosensor was constructed by encapsulating glucose oxidase in the Nafion-SWCNHs composite film. The cyclic voltammograms for glucose oxidase immobilized on the composite film displayed a pair of well-defined and nearly symmetric redox peaks with a formal potential of -0.453 V. The biosensor had good electrocatalytic activity toward oxidation of glucose. To decrease detection potential, ferrocene monocarboxylic acid was used as a redox mediator. The mediated glucose biosensor shows a linear range from 0 to 6.0 mM. The biosensor shows high sensitivity (1.06 microA/mM) and stability, and can avoid the commonly coexisted interference. Because of impressive properties of SWCNHs, such as high purity and high surface area, SWCNHs and their composites are expected to be promising material for biomolecular immobilization and biosensing applications.     

Biosensor based on polyaniline-Prussian Blue/multi-walled carbon nanotubes hybrid composites

In this work, a novel route for fabrication polyaniline (PANI)-Prussian Blue (PB) hybrid composites is proposed by the spontaneous redox reaction in the FeCl(3)-K(3)[Fe(CN)(6)] and the aniline solution. With the introduction of multi-walled carbon nanotubes (MWNTs), the PANI-PB/MWNTs system shows synergy between the PANI-PB and MWNTs which amplified the H(2)O(2) sensitivity greatly. A linear range from 8 x1 0(-8) to 1 x 10(-5)M and a high sensitivity 508.1 8 microA microM cm(2) for H(2)O(2) detection are obtained. The composites also show good stability in neutral solution. A glucose biosensor was further constructed by immobilizing glucose oxidase (GOD) with Nafion and glutaraldehyde on the electrode surface. The performance factors influencing the resulted biosensor were studied in detail. The biosensor exhibits excellent response performance to glucose with the linear range from 1 to 11 mM and a detection limit of 0.01 mM. Furthermore, the biosensor shows rapid response, high sensitivity, good reproducibility, long-term stability and freedom of interference from other co-existing electroactive species.     

Enzymatically induced formation of neodymium hexacyanoferrate nanoparticles on the glucose oxidase/chitosan modified glass carbon electrode for the detection of glucose

The formation of neodymium hexacyanoferrate (NdHCF) nanoparticles (NPs) on the surface of glucose oxidase/chitosan (GOx/CHIT) modified glass carbon electrode induced by enzymatic reaction was described and characterized. CHIT can be used not only as enzyme immobilizer, but also to provide active sites for NPs growth. Results showed that the optimized conditions of the GOx/CHIT film induced NdHCF NPs for the biosensing of glucose were 1.0mM Nd(3+) and 20.0mM Fe(CN)(6)(3-). The biocatalyzed generation of NdHCF NPs enabled the development of an electrochemical biosensor for glucose. The calculated apparent Michaelis-Menten constant was 7.5mM. The linear range for glucose detection was 0.01-10.0mM with the correlation coefficient of 0.9946, and the detection limit was 5muM (S/N=3). Furthermore, this system avoids the interferences of other species during the biosensing process and can be used for the determination of glucose in human plasma samples.     

Enzymatic glucose biosensor based on CeO2 nanorods synthesized by non-isothermal precipitation

Cerium oxide nanorods (CeO(2) NRs) were synthesized without templates through a low cost and simple non-isothermal precipitation method. The structure and morphology of CeO(2) NRs were characterized by X-ray diffraction and transmission electron microscopy. The CeO(2) NRs films, deposited on indium tin oxide (ITO)-coated glass substrates through electrophoretic deposition, were used for the immobilization of glucose oxidase (GOx). Field emission scanning electron microscopy, Fourier transform infrared spectroscopy, cyclic voltammetry, and electrochemical impedance spectroscopy were used to characterize the CeO(2) NRs/ITO and GOx/CeO(2) NRs/ITO electrodes. The GOx/CeO(2) NRs/ITO electrode exhibits a linear range for the detection of glucose from 2 to 26 mM (correlation coefficient: 0.99) at 1-2s response time. Biosensor sensitivity is 0.165 μA mM(-1) cm(-2) with 100 μM detection limit. The anti-interference ability of the biosensor was also examined. The mediator-less application of CeO(2) NRs for glucose sensing was demonstrated.     

Reagentless glucose biosensor based on direct electron transfer of glucose oxidase immobilized on colloidal gold modified carbon paste electrode

The direct electrochemistry of glucose oxidase (GOD) adsorbed on a colloidal gold modified carbon paste electrode was investigated. The adsorbed GOD displayed a pair of redox peaks with a formal potential of -(449+/-1) mV in 0.1 M pH 5.0 phosphate buffer solution. The response showed a surface-controlled electrode process with an electron transfer rate constant of (38.9+/-5.3)/s determined in the scan rate range from 10 to 100 mV/s. GOD adsorbed on gold colloid nanoparticles maintained its bioactivity and stability. The immobilized GOD could electrocatalyze the reduction of dissolved oxygen and resulted in a great increase of the reduction peak current. Upon the addition of glucose, the reduction peak current decreased, which could be used for glucose detection with a high sensitivity (8.4 microA/mM), a linear range from 0.04 to 0.28 mM and a detection limit of 0.01 mM at a signal-to-noise ratio of 3sigma. The sensor could exclude the interference of commonly coexisted uric and ascorbic acid.     

Multilayer assembly of positively charged polyelectrolyte and negatively charged glucose oxidase on a 3D Nafion network for detecting glucose

In this paper, a novel amperometric glucose biosensor was constructed by alternative self-assembly of positively charged poly(diallydimethylammonium chloride) (PDDA) and negatively charged glucose oxidase (GOx) onto a 3D Nafion network via electrostatic adsorption. The amount of Nafion in the electrode and the number of the (PDDA/GOx)_n multilayers were optimized to develop a sensitive and selective glucose biosensor. Under optimal conditions, the glucose biosensor with (PDDA/GOx)₅ multilayers exhibited remarkable electrocatalytic activity, capable of detecting glucose with enhanced sensitivity of 9.55 μA/mM cm² and a commendably low detection limit of 20 μM (S/N = 3). A linear response range of 0.05–7 mM (a linear correlation coefficient of 0.9984, n = 20) was achieved. In addition, the glucose biosensor demonstrated superior selectivity towards glucose over some interferents, such as ascorbic acid (AA) and uric acid (UA), at an optimized detection potential of 0.6 V versus Ag/AgCl reference.     

Construction of a glucose sensor based on a screen-printed electrode and a novel mediator pyocyanin from Pseudomonas aeruginosa

Pyocyanin is the blue phenazine pigment produced by Pseudomonas aeruginosa. Pyocyanin production using immobilized cells was investigated. The maximum production of pyocyanin was obtained using cells immobilized in kappa-carrageenan. Moreover, 0.01% PO4(3-), 0.2% Mg(2+), 0.001% Fe(2+), 1% glycerine, 0.8% leucine and 0.8% dl-alanine were also essential for pyocyanin production. Pyocyanin was purified by chloroform extraction and silica gel column chromatography. An amperometric biosensor system using a screen-printed electrode and pyocyanin as mediator were also developed for a more accurate determination of glucose concentration. Pyocyanin, which exists in the oxidated form, was reduced by the reaction between glucose oxidase and glucose. The reduced form was then converted back to the oxidized form by an oxidative reaction on the electrode. There was a linear relation ship between sensor output currents and glucose concentrations ranging from 1 to 20mM under the following conditions: -200 mV of the applied potential, pH 5.0, and 10 U of the immobilized enzyme. The coefficient of variation was below 3% (n = 5) for the glucose sensor.     

Covalent conjugation of avidin with dye-doped silica nanopaticles and preparation of high density avidin nanoparticles as photostable bioprobes

A sensitive, selective and stable amperometric glucose biosensor employing novel PtPd bimetallic nanoparticles decorated on multi-walled carbon nanotubes (PtPd-MWCNTs) was investigated. PtPd-MWCNTs were prepared by a modified Watanabe method, and characterized by XRD and TEM. The biosensor was constructed by immobilizing the PtPd-MWCNTs catalysts in a Nafion film on a glassy carbon electrode. An inner Na?on film coating was used to eliminate common interferents such as uric acid, ascorbic acid and fructose. Finally, a highly porous surface with an orderly three-dimensional network enzyme layer (CS-GA-GOx) was fabricated by electrodeposition. The resulting biosensor exhibited a good response to glucose with a wide linear range (0.062-14.07 mM) and a low detection limit 0.031 mM. The biosensor also showed a short response time (within 5 s), and a high sensitivity (112 μA mM(-1)cm(-2)). The Michaelis-Menten constant (K(m)) was determined as 3.3 mM. In addition, the biosensor exhibited high reproducibility, good storage stability and satisfactory anti-interference ability. The applicability of the biosensor to actual serum sample analysis was also evaluated.