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.     

An ISFET-based immunosensor for the detection of beta-Bungarotoxin

An ion-sensitive field effect transistor (ISFET)-based immunosensor was developed to detect/quantitate beta-Bungarotoxin (beta-BuTx), a potent presynaptic neurotoxin from the venom of Bungarus multicinctus. A murine monoclonal antibody (mAb 15) specific to beta-BuTx was immobilized onto silicon nitride wafers after silanization and activation with glutaraldehyde. A chip based enzyme linked-immunosorbantassay (ELISA) was performed to ascertain antigen binding to the immobilized antibody. To develop an electrochemical immunosensing system for the detection/quantitation of beta-BuTx, an ISFET was used as a solid phase detector. MAb 15 was immobilized on the gate region of the ISFET. The antigen antibody reaction was monitored by the addition of urease conjugated rabbit anti-beta-BuTx antibodies. The sensor can detect toxin level as low as 15.6 ng/ml. The efficacy of the sensor for the determination of beta-BuTx from B. multicinctus venom was demonstrated in mouse model. Toxin concentration was highest at the site of injection (748.0+/-26 ng/ml) and moderate amount was found in the plasma (158.5+/-13 ng/ml).     

Extended-gate FET-based enzyme sensor with ferrocenyl-alkanethiol modified gold sensing electrode

We developed a field-effect transistor (FET)-based enzyme sensor that detects an enzyme-catalyzed redox-reaction event as an interfacial potential change on an 11-ferrocenyl-1-undecanethiol (11-FUT) modified gold electrode. While the sensitivity of ion-sensitive FET (ISFET)-based enzyme sensors that detect an enzyme-catalyzed reaction as a local pH change are strongly affected by the buffer conditions such as pH and buffer capacity, the sensitivity of the proposed FET-based enzyme sensor is not affected by them in principle. The FET-based enzyme sensor consists of a detection part, which is an extended-gate FET sensor with an 11-FUT immobilized gold electrode, and an enzyme reaction part. The FET sensor detected the redox reaction of hexacyanoferrate ions, which are standard redox reagents of an enzymatic assay in blood tests, as a change in the interfacial potential of the 11-FUT modified gold electrode in accordance with the Nernstian response at a slope of 59 mV/decade at 25 degrees C. Also, the FET sensor had a dynamic range of more than five orders and showed no sensitivity to pH. A FET-based enzyme sensor for measuring cholesterol level was constructed by adding an enzyme reaction part, which contained cholesterol dehydrogenase and hexacyanoferrate (II)/(III) ions, on the 11-FUT modified gold electrode. Since the sensitivity of the FET sensor based on potentiometric detection was independent of the sample volume, the sample volume was easily reduced to 2.5 microL while maintaining the sensitivity. The FET-based enzyme sensor successfully detected a serum cholesterol level from 33 to 233 mg/dL at the Nernstian slope of 57 mV/decade.     

A novel glucose ENFET based on the special reactivity of MnO2 nanoparticles

Generally a glucose-sensitive enzyme field-effect transistor (ENFET) is based on local pH change in biomembranes resulted from the formation of gluconic acid. Here we proposed a glucose ENFET based on a new principle. The glucose ENFET was fabricated by coimmobilizing glucose oxidase (GOD) and MnO(2) nanoparticles on the gate of an ion-sensitive field-effect transistor (ISFET). The proposed glucose biosensor shows a significant local pH increase in the sensitive membrane with the increase of glucose concentration. The driving force of the pH change in our sensor is essentially different from all the other glucose ENFETs, including those prepared by bulk MnO(2). The special reaction ability of MnO(2) nanoparticles with hydrogen peroxide might be the main cause of the pH change. In addition, the influence of buffer concentration, pH and ionic strength on the glucose ENFET is investigated in detail. It is found that the ionic strength has little effect on the performance of the ENFET. Under optimal conditions, the proposed ENFET exhibits a linear response with glucose in the range of 0.025-1.90 mM, an extended dynamic upper limit of 3.5 mM glucose, and considerable good reproducibility and stability.     

Detection of DNA and proteins using amorphous silicon ion-sensitive thin-film field effect transistors

Amorphous silicon-based ion-sensitive field-effect transistors (a-Si:H ISFETs) are used for the label-free detection of biological molecules. The covalent immobilization of DNA, followed by DNA hybridization, and of the surface adsorption of oligonucleotides and proteins were detected electronically by the a-Si:H ISFET. The ISFET measurements are performed with an external Ag/AgCl microreference electrode immersed in 100mM phosphate buffer electrolyte with pH 7.0. Threshold voltage shifts in the transfer curve of the ISFETs are observed resulting from successive steps of surface chemical functionalization, covalent DNA attachment to the functionalized surface, surface blocking, and hybridization with a complementary target. The surface sensitivity achieved for DNA oligonucleotides is of the order of 1pmol/cm(2). Point-of-zero charge estimations were made for the functionalized surfaces and for the device surface after DNA immobilization and hybridization. The results show a correlation between the changes in the point-of-zero charge and the shift observed in the threshold voltage of the devices. Electronic detection of adsorbed proteins and DNA is also achieved by monitoring the shifts of the threshold voltage of the ISFETs, with a sensitivity of approximately 50nM.     

Non-Faradaic electrochemical detection of protein interactions by integrated neuromorphic CMOS sensors

Electronic detection of the binding event between biotinylated bovine serum albumen (BSA) and streptavidin is demonstrated with the chemoreceptive neuron MOS (CnuMOS) device. Differing from the ion-sensitive field-effect transistors (ISFET), CnuMOS, with the potential of the extended floating gate determined by both the sensing and control gates in a neuromorphic style, can provide protein detection without requiring analyte reference electrodes. In comparison with the microelectrode arrays, measurements are gathered through purely capacitive, non-Faradaic interactions across insulating interfaces. By using a (3-glycidoxypropyl)trimethoxysilane (3-GPS) self-assembled monolayer (SAM) as a simple covalent link for attaching proteins to a silicon dioxide sensing surface, a fully integrated, electrochemical detection platform is realized for protein interactions through monotone large-signal measurements or small-signal impedance spectroscopy. Calibration curves were created to coordinate the sensor response with ellipsometric measurements taken on witness samples. By monitoring the film thickness of streptavidin capture, a sensitivity of 25ng/cm2 or 2A of film thickness was demonstrated. With an improved noise floor the sensor can detect down to 2ng/(cm2mV) based on the calibration curve. AC measurements are shown to significantly reduce long-term sensor drift. Finally, a noise analysis of electrochemical data indicates 1/f(alpha) behavior with a noise floor beginning at approximately 1Hz.     

Electrochemical sensor based on photopolymeric membranes for determining urea

A new electrochemical enzymatic sensor based on the ion selective field effect transistors (ISFETs) and photocurable membrane was developed for the determination of urea. For the immobilization of urease on the gate surface of the ISFET a simple method, involving the use of liquid photocurable compositions on the basis of vinylpirollidone, oligouretanemetacrylate and oligocarbonatemetacrylate, was applied. The linearange of the response of the developed electrochemical sensor lies in the range 0.05-20 mM. The latter corresponds to the claims of the medical practice. The overall time of the analysis is 5-10 min. The effects of the buffer concentration and its pH as well as temperature and presence of ammonia ions in the measuring medium on the amplitude of the sensor response were estimated. The duration of sensor work is as shortest 40 days. The proposed sensor on the basis of the ISFET is promising for the express analysis of the level urea in blood, while the developed method of membrane preparation with entrapped enzyme can be combined with the integral technology of producing of the biosensors semiconductor transducers.     

Effects of D-600 on intramembrane charge movement of polarized and depolarized frog muscle fibers

Intramembrane charge movement has been measured in frog cut skeletal muscle fibers using the triple vaseline gap voltage-clamp technique. Ionic currents were reduced using an external solution prepared with tetraethylammonium to block potassium currents, and O sodium + tetrodotoxin to abolish sodium currents. The internal solution contained 10 mM EGTA to prevent contractions. Both the internal and external solutions were prepared with impermeant anions. Linear capacitive currents were subtracted using the P-P/4 procedure, with the control pulses being subtracted either at very negative potentials, for the case of polarized fibers, or at positive potentials, for the case of depolarized fibers. In 63 polarized fibers dissected from Rana pipiens or Leptodactylus insularis frogs the following values were obtained for charge movement parameters: Qmax = 39 nC/microF, V = 36 mV, k = 18.5 mV. After depolarization we found that the total amount of movable charge was not appreciably reduced, while the voltage sensitivity was much changed. For 10 fibers, in which charge movement was measured at -100 and at 0 mV, Qmax changed from 46 to 41 nC/microF, while V changed from -41 to -103 mV and k changed from 20.5 to 30 mV. Thus membrane depolarization to 0 mV produces a shift of greater than 50 mV in the Q-V relationship and a decrease of the slope. Membrane depolarization to -20 and -30 mV, caused a smaller shift of the Q-V relationship. In normally polarized fibers addition of D-600 at concentrations of 50-100 microM, does not cause important changes in charge movement parameters. However, the drug appears to have a use-dependent effect after depolarization. Thus in depolarized fibers, total charge is reduced by approximately 20%. D-600 causes no further changes in the voltage sensitivity of charge movement in fibers depolarized to 0 mV, while in fibers depolarized to -20 and -30 mV it causes the same effects as that obtained with depolarization to 0 mV. These results are compatible with the idea that after depolarization charge 1 is transformed into charge 2. D-600 appears to favor the conversion of charge 1 into charge 2. Since D-600 also favors contractile inactivation, charge 2 could represent the state of the voltage sensor for excitation-contraction coupling in the inactivated state.     

An autonomous CMOS hysteretic sensor for the detection of desorption-free DNA hybridization

This paper describes a sensor for label-free, fully electrical detection of DNA hybridization based on capacitive changes in the electrode-electrolyte interface. The sensor measures capacitive changes in real time according to a charging-discharging principle that is limited by the hysteresis window. In addition, a novel autonomous searching technique, which exclusively monitors desorption-free hybridized electrodes among electrode arrays, enhances the performance of the sensor compared with conventional capacitive measurement. The sensor system achieves a detection range of 80 dB. The integrated circuit sensor is fabricated with a 0.35 μm CMOS process. The proposed sensor offers rapid, robust and inexpensive measurement of capacitance with highly integrated detection circuitry. It also facilitates quantitative evaluations of molecular densities on a chip with distinctive impedance variations by monitoring desorption-free hybridized electrodes. Our electrical biosensor has great potential for use with bio analytical tools and point-of-care diagnosis.     

Surface charge and lanthanum block of calcium current in bullfrog sympathetic neurons.

The density of surface charge associated with the calcium channel pore was estimated from the effect of extracellular ionic strength on block by La3+. Currents carried by 2 mM Ba2+ were recorded from isolated frog sympathetic neurons by the whole-cell patch-clamp technique. In normal ionic strength (120 mM N-methyl-D-glucamine, NMG), La3+ blocked the current with high affinity (IC50 = 22 nM at 0 mV). La3+ block was relieved by strong depolarization in a time- and voltage-dependent manner. After unblocking, open channels reblocked rapidly at 0 mV, allowing estimation of association and dissociation rates for La3+: k(on) = (7.2 +/- 0.7) x 10(8) M(-1) s(-1), k(off) = 10.0 +/- 0.5 s(-1). To assess surface charge effects, La3+ block was also measured in low ionic strength (12.5 mM NMG) and high ionic strength (250 mM NMG). La3+ block was higher affinity and faster by two- to threefold in 12.5 mM NMG, with little effect of 250 mM NMG. The data could be described by Gouy-Chapman theory with a surface charge density of approximately 1 e-/3000-4000 A2. These results indicate that there is a small but detectable surface charge associated with the pore of voltage-dependent calcium channels.     

A fluorescence-based fiber optic biosensor for the flow-injection analysis of penicillin

A penicillin fiber optic sensor is described. The sensor is based on co-immobilization of a pH indicator, fluorescein isothiocyanate (FITC), and penicillinase on a preactivated biodyne B membrane attached to the end of a bifurcated optical fiber. The characteristics of the sensor are investigated in conjunction with a flow injection analysis system. The proposed sensor is reversible and responds to penicillin in the concentration range of 1 x 10(-4) to 5 x 10(-2) mol/L. The application of this sensor to penicillin analysis in some pharmaceutical samples is demonstrated.     

Development of a dimethyl ether (DME) sensor using platinum nanoparticles and thick-film printing

A portable and cost-effective technique to measure the dimethyl ether (DME) concentrations has been developed. It is based on an electrochemical principle measuring the oxidation current of DME at an applied potential of +0.2V versus a Ag/AgCl reference electrode. Thick-film printing technique is used for the fabrication of this DME sensor, and platinum nanoparticles in the crystallite size of 5.5 nm are used for the modification of the working electrode surface. This modification enhances the sensor performance significantly leading to a higher sensitivity of the sensor comparing to bare platinum electrode. Evaluation and characterization of this sensor are carried out over the DME concentration range of 0-7% (v/v), and a linear relationship between sensor outputs and the DME concentrations with an average R(2) of 0.996 exists. The reproducibility of the sensor is also very good. This electrochemically based DME sensor fabricated by thick-film screen printing technique and using the platinum nanoparticles to enhance its performance will be valuable and practical for the estimation of the airway mucosal blood flow.     

Surface charge and calcium channel saturation in bullfrog sympathetic neurons

Currents carried by Ba2+ through calcium channels were recorded in the whole-cell configuration in isolated frog sympathetic neurons. The effect of surface charge on the apparent saturation of the channel with Ba2+ was examined by varying [Ba2+]o and ionic strength. The current increased with [Ba2+]o, and the I-V relation and the activation curve shifted to more positive voltages. The shift of activation could be described by Gouy-Chapman theory, with a surface charge density of 1 e- /140 A2, calculated from the Grahame equation. Changes in ionic strength (replacing N-methyl-D-glucamine with sucrose) shifted the activation curve as expected for a surface charge density of 1 e-/85 A2, in reasonable agreement with the value from changing [Ba2+]o. The instantaneous I-V for fully activated channels also changed with ionic strength, which could be described either by a low surface charge density (less than 1 e-/1,500 A2), or by block by NMG with Kd approximately 300 mM (assuming no surface charge). We conclude that the channel permeation mechanism sees much less surface charge than the gating mechanism. The peak inward current saturated with an apparent Kd = 11.6 mM for Ba2+, while the instantaneous I-V saturated with an apparent Kd = 23.5 mM at 0 mV. This discrepancy can be explained by a lower surface charge near the pore, compared to the voltage sensor. After correction for a surface charge near the pore of 1 e-/1,500 A2, the instantaneous I-V saturated as a function of local [Ba2+]o, with Kd = 65 mM. These results suggest that the channel pore does bind Ba2+ in a saturable manner, but the current-[Ba2+]o relationship may be significantly affected by surface charge.     

Multienzyme microbiosensor based on electropolymerized o-phenylenediamine for simultaneous in vitro determination of acetylcholine and choline

The electrochemical biosensors based on poly(o-phenylenediamine) (PoPD) and acetylcholinesterase (AChE) and choline oxidase (ChO) enzymes were fabricated on carbon fibre (CF) substrate. The electropolymerized PoPD was used to reduce the interfering substances. The electrode assembly was completed by depositing functionalized carbon nano tubes (FCNTs) and Nafion (Naf). Amperometric detection of acetylcholine (ACh) and choline (Ch) were realized at an applied potential of +750 mV vs Ag/AgCl (saturated KCl). At pH 7.4, the final assembly, Naf-FCNTs/AChE-ChO((10:1))/PoPD/CF(Elip), was observed to have high sensitivity towards Ch (6.3±0.3 μA mM(-1)) and ACh (5.8±0.3 μA mM(-1)), linear range for Ch (K(M)=0.52±0.03 mM) and ACh (K(M)=0.59±0.07 mM), and for Ch the highest ascorbic acid blocking capacity (97.2±2 1mM AA). It had a response time of <5s and with 0.045 μM limit of detection. Studies on different ratio (ACh/Ch) revealed that 10:1, gave best overall response.     

Iridium oxide pH sensor for biomedical applications. Case urea-urease in real urine samples

This work demonstrates the implementation of iridium oxide films (IROF) grown on silicon-based thin-film platinum microelectrodes, their utilization as a pH sensor, and their successful formatting into a urea pH sensor. In this context, Pt electrodes were fabricated on Silicon by using standard photolithography and lift-off procedures and IROF thin films were growth by a dynamic oxidation electrodeposition method (AEIROF). The AEIROF pH sensor reported showed a super-Nerstian (72.9±0.9mV/pH) response between pH 3 and 11, with residual standard deviation of both repeatability and reproducibility below 5%, and resolution of 0.03 pH units. For their application as urea pH sensors, AEIROF electrodes were reversibly modified with urease-coated magnetic microparticles (MP) using a magnet. The urea pH sensor provided fast detection of urea between 78μM and 20mM in saline solution, in sample volumes of just 50μL. The applicability to urea determination in real urine samples is discussed.     

A CMOS label-free DNA sensor using electrostatic induction of molecular charges

This paper reports a label-free biosensor for the detection of DNA hybridization. The proposed biosensor measures the surface potential on oligonucleotide modified electrodes using a direct charge accumulation method. The sensor directly and repeatedly measures the charges induced in the working electrode, which correspond to intrinsic negative charges in immobilized molecules. The sensor achieves an improved signal-to-noise ratio (SNR), through the oversampling effect of accumulation for charges and the differential architecture. The sensor also shows stable, robust, and reproducible measurement independent of slight changes in the reference voltage, unlike previous ion-sensitive field effect transistors (ISFETs), providing the benefits of choosing a wide variety of reference electrode materials. The proposed device is integrated with working electrodes, a reference electrode and readout circuits into one package via a 0.35 μm complementary metal-oxide-semiconductor (CMOS) process. The sensor achieves a detectable range of 88.3 dB and a detection limit of 36 μV for surface potential. It is demonstrated that the sensor successfully achieves specific detection of oligonucleotide sequences derived from the H5N1 avian influenza virus. The experiments show a limit of detection of 100 pM and include a single-base mismatch test in 18-mer oligonucleotides.     

An ISFET biosensor for the monitoring of maltose-induced conformational changes in MBP

Here we describe an ion sensitive field effect transistor (ISFET) biosensor, which was designed to monitor directly the surface charge of structurally altered maltose binding protein (MBP) upon stimulation with maltose. This study is the first report of the application of a FET biosensor to the monitoring of conformationally changed proteins. Consequently, a significant drop in current on the basis of the charge-dependent capacitance measurement has been clearly observed in response to maltose, but not for the glucose control, thereby indicating that the substrate-specific conformational properties of the target protein could be successfully monitored using the ISFET. Collectively, our results clearly suggest that ISFET provide a high fidelity system for the detection of maltose-induced structural alterations in MBP.     

Monitoring of dihydroxyacetone production during oxidation of glycerol by immobilized Gluconobacter oxydans cells with an enzyme biosensor

A bi-enzymatic biosensor for monitoring of dihydroxyacetone production during oxidation of glycerol by bacterial cells of Gluconobacter oxydans is presented. Galactose oxidase oxidizes dihydroxyacetone efficiently producing hydrogen peroxide, which reacts with co-immobilized peroxidase and ferrocene pre-adsorbed on graphite electrode. This mediator-based bi-enzymatic biosensor possesses very high sensitivity (4.7 μA/mM in phosphate buffer), low detection limit (0.8 μM, signal/noise = 3), short response time (22 s, 95% of steady-state) and broad linear range (0.002-0.55 mM in phosphate buffer). The effect of pH, temperature, type of buffer, as well as different stabilizers (combinations of a polyelectrolyte and a polyol) on the sensor performance were carefully optimized and discussed. Dihydroxyacetone produced during a batch conversion of glycerol by the pectate-immobilized bacteria in an air-lift reactor was determined by the biosensor and by reference spectrophotometric method. Both methods were compared and were in a very good correlation. The main advantage of the biosensor is a very short time needed for sample analysis (less than 1 min).     

A novel amperometric bienzymatic biosensor based on alcohol oxidase coupled PVC reaction cell and nanomaterials modified working electrode for rapid quantification of alcohol

Abstract

A new amperometric sensor has been fabricated for sensitive and rapid quantification of ethanol. The biosensor assembly was prepared by covalently immobilizing alcohol oxidase (AOX) from Pichia pastoris onto chemically modified surface of polyvinylchloride (PVC) beaker with glutaraldehyde as a coupling agent followed by immobilization of horseradish peroxidase (HRP), silver nanoparticles (AgNPs), chitosan (CHIT), carboxylated multi-walled carbon nanotubes (c-MWCNTs) and nafion (Nf) nanocomposite onto the surface of Au electrode (working electrode). Owing to properties such as chemical inertness, light weight, weather resistance, corrosion resistance, toughness and cost-effectiveness, PVC membrane has attracted a growing interest as a support for enzyme immobilization in the development of biosensors. The amperometric biosensor displayed optimum response within 8?s at pH 7.5 and 35°C temperature. A linear response to alcohol in the range of 0.01mM–50?mM and 0.0001?µM as a minimum limit of detection was displayed by the proposed biosensor with excellent storage stability (190?days) at 4°C. The sensitivity of the sensor was found to be 155?µA mM^?1?cm^?2. A good correlation (R²?=?0.99) was found between alcohol level in commercial samples as evaluated by standard ethanol assay kit and the current biosensor which validates its performance.     

Glucose Biosensor Based on a Glassy Carbon Electrode Modified with Polythionine and Multiwalled Carbon Nanotubes

A novel glucose biosensor was fabricated. The first layer of the biosensor was polythionine, which was formed by the electrochemical polymerisation of the thionine monomer on a glassy carbon electrode. The remaining layers were coated with chitosan-MWCNTs, GOx, and the chitosan-PTFE film in sequence. The MWCNTs embedded in FAD were like “conductive wires” connecting FAD with electrode, reduced the distance between them and were propitious to fast direct electron transfer. Combining with good electrical conductivity of PTH and MWCNTs, the current response was enlarged. The sensor was a parallel multi-component reaction system (PMRS) and excellent electrocatalytic performance for glucose could be obtained without a mediator. The glucose sensor had a working voltage of −0.42 V, an optimum working temperature of 25°C, an optimum working pH of 7.0, and the best percentage of polytetrafluoroethylene emulsion (PTFE) in the outer composite film was 2%. Under the optimised conditions, the biosensor displayed a high sensitivity of 2.80 µA mM⁻¹ cm⁻² and a low detection limit of 5 µM (S/N = 3), with a response time of less than 15 s and a linear range of 0.04 mM to 2.5 mM. Furthermore, the fabricated biosensor had a good selectivity, reproducibility, and long-term stability, indicating that the novel CTS+PTFE/GOx/MWCNTs/PTH composite is a promising material for immobilization of biomolecules and fabrication of third generation biosensors.