Layer-layer assembly for extremely stable glucose sensors

A novel fabrication of an amperometric glucose sensor by layer after layer approach is described. The sensor electrode is fabricated by arranging a layer of Pt black, a layer of glucose oxidase (GOD) and a layer of stabilizer gelatin on a shapable electro-conductive (SEC) film surface. Finally, the dried layered-assembly is cross-linked by exposing to a diluted glutaraldehyde solution. The performance of the developed sensor is evaluated by a FIA system at 37°C and under a continuous polarization at 0.4 V (vs. Ag/AgCl). The sensitivity of the sensor was dependent on the amount of GOD loaded. The highest sensitivity (3.6 μA/mM cm⁻²) of the sensor was obtained at a GOD loading of 160 μg/cm², and the linear dynamic range was extended to 80 mM level when the sensor was covered with a polycarbonate membrane. The sensor shows an extremely stable response for several weeks and a storage stability of over 2 years.     

Minimally‐invasive Microneedle‐based Biosensor Array for Simultaneous Lactate and Glucose Monitoring in Artificial Interstitial Fluid

Here we report the first mediated pain free microneedle‐based biosensor array for the continuous and simultaneous monitoring of lactate and glucose in artificial interstitial fluid (ISF). The gold surface of the microneedles has been modified by electrodeposition of Au‐multiwalled carbon nanotubes (MWCNTs) and successively by electropolymerization of the redox mediator, methylene blue (MB). Functionalization of the Au‐MWCNTs/polyMB platform with the lactate oxidase (LOX) enzyme (working electrode 1) and with the FAD‐Glucose dehydrogenase (FADGDH) enzyme (working electrode 2) enabled the continuous monitoring of lactate and glucose in the artificial ISF. The lactate biosensor exhibited a high sensitivity (797.4±38.1 μA cm^?2 mM^?1), a good linear range (10–100 μM) with a detection limit of 3 μM. The performance of the glucose biosensor were also good with a sensitivity of 405.2±24.1 μA cm^?2 mM^?1, a linear range between 0.05 and 5 mM and a detection limit of 7 μM. The biosensor array was tested to detect the amount of lactate generated after 100 minutes of cycling exercise (12 mM) and of glucose after a normal meal for a healthy patient (10 mM). The results reveal that the new microneedles‐based biosensor array seems to be a promising tool for the development of real‐time wearable devices with a variety of sport medicine and clinical care applications.     

有序介孔碳/石墨烯/镍泡沫的制备及其对多巴胺的高灵敏度和高选择性检测

柔性生物传感器在可穿戴电子设备中有着广泛的应用前景. 为了获得柔性电化学多巴胺传感器,作者在本工作中首先在镍泡沫表面通过化学气相沉积生长石墨烯,随后通过高温碳化嵌段共聚物与酚醛树脂在石墨烯表面共组装形成的薄膜制备了有序介孔碳/石墨烯/镍泡沫（OMC/G/Ni）复合材料. 其中,镍泡沫可以为复合材料提供具有高导电性和良好柔韧性的金属骨架,而具有垂直排列介孔阵列的有序介孔碳层为复合材料提供了高的电活性表面积,且有利于活性位点的暴露. 值得注意的是,夹在有序介孔碳层和镍泡沫之间的石墨烯极大地增强了各组分之间的相容性,有利于进一步提升复合材料的电化学性能. 作为电化学传感器中的工作电极,OMC/G/Ni体现出优异的多巴胺检测能力. 不但具有宽的线性检测范围（0.05 ~ 58.75 μmol·L^-1）和低检测限（0.019 μmol·L^-1）,还具有良好的选择性、重现性和稳定性. 此外,OMC/G/Ni在弯曲状态下依旧能够保持对多巴胺的高检测能力,证明了其在柔性生物传感器中的应用潜力.     

基于表面活性剂单分子层修饰碳糊电极的一氧化氮电化学传感器及其应用

报道了一种表面活性剂单分子层修饰碳糊电极,并用于NO的高灵敏电化学检测。研究表明,表面活性剂通过烷基链在电极表面形成的疏水性单分子层微环境对NO的电化学响应具有较好的促进作用。其中,阳离子表面活性剂十六烷基三甲基溴化铵(CTAB)对NO的电化学氧化表现出最强的催化活性和增敏作用。在Nafion膜覆盖的CTAB修饰碳糊电极上,NO的安培响应与其浓度在3.6×10-8～1.8×10-5mol/L范围内呈良好的线性关系,检出限为1.8×10-8mol/L。该电极作为低成本、高灵敏的NO电化学传感器,被成功应用于大鼠肺组织细胞中NO释放的实时监测。     

Development of a diamine biosensor

An amperometric diamine sensor is developed for clinical applications in diagnosis of bacterial vaginosis (BV). The sensor is based on crosslinked putrescine oxidase (PUO) which catalyzes the conversion of diamines (mainly putrescine and cadaverine) to products including hydrogen peroxide. The hydrogen peroxide is detected anodically at platinum electrode polarized at 0.5 V versus Ag/AgCl. Platinum-plated gold electrodes used as a substrate for the sensor construction, are batch-fabricated on a flexible polyimide foil (Kapton(R), DuPont). A three-electrode cell configuration is used in all amperometric measurements. The sensor construction is based on three layers: an inner layer to reject the interference effect of oxidizable molecules, an outer diffusion controlling layer, and in addition, an enzyme middle layer. The enzyme layer was immobilized by crosslinking PUO with bovine serum albumin (BSA) using glutaraldehyde (GA). An optimization study of the enzyme solution composition was carried out. With the optimized enzyme layer, the biosensor showed a very high sensitivity and fast response time of ca. 20 s. The sensor has a linear dynamic range from (0.5-300 muM) for putrescine that covers the expected biological levels of the analyte. Details on sensor fabrication and characterization are given in the present work.     

Covalent attachment of glucose oxidase to an Au electrode modified with gold nanoparticles for use as glucose biosensor

A feasible method to fabricate glucose biosensor was developed by covalent attachment of glucose oxidase (GOx) to a gold nanoparticle monolayer modified Au electrode. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) of ferrocyanide followed and confirmed the assemble process of biosensor, and indicated that the gold nanoparticles in the biosensing interface efficiently improved the electron transfer between analyte and electrode surface. CV performed in the presence of excess glucose and artificial redox mediator, ferrocenemethanol, allowed to quantify the surface concentration of electrically wired enzyme (Gamma(E)(0)) on the basis of kinetic models reported in literature. The Gamma(E)(0) on proposed electrode was high to 4.1 x 10(-12) mol.cm(-2), which was more than four times of that on electrode direct immobilization of enzyme by cystamine without intermediate layer of gold nanoparticles and 2.4 times of a saturated monolayer of GOx on electrode surface. The analytical performance of this biosensor was investigated by amperometry. The sensor provided a linear response to glucose over the concentration range of 2.0 x 10(-5)-5.7 x 10(-3) M with a sensitivity of 8.8 microA.mM(-1).cm(-2) and a detection limit of 8.2 microM. The apparent Michaelis-Menten constant (K(m)(app)) for the sensor was found to be 4.3 mM. In addition, the sensor has good reproducibility, and can remain stable over 30 days.     

PDMS-based gold electrode for sensing ascorbic acid

Electrode with optical shapes is appreciated in microfluidics. In this article, we reported a flexible poly(dimethylsiloxane) (PDMS)-based gold electrode for ascorbic acid detection. Gold nanoparticles were chemically deposited on PDMS and the composite film was applied as working electrode. The electrode could undergo deformation and display good response performance without damage. This biosensor could give quick response to ascorbic acid (AA) (<5s) and the currents were linear with concentrations of AA in range of 0.023-7.00 mM and 30-100 mM, respectively. Limit of detection was 0.008 mM (S/N=3). This biosensor has been applied to determine ascorbic acid content in vitamin C tablets and the results were consistent with traditional iodometric method.     

Sol-gel based amperometric biosensor incorporating an osmium redox polymer as mediator for detection of L-lactate

A novel amperometric biosensor for the determination of lactate was constructed by first immobilizing lactate oxidase and an osmium redox polymer ([Os(bpy)(2)(PVP)(10)Cl]Cl; abbreviated Os-polymer) on the surface of a glassy carbon electrode, followed by coating with a sol-gel film derived from methyltriethoxysilane (MTEOS). The electrooxidation current of this electrode was found to be diffusion controlled. In the presence of lactate, a clear electrocatalytic oxidation wave was observed, and lactate could be determined amperometrically at 400 mV versus Ag AgCl . The concentration range of linear response, slope of linear response and detection limit were 0.1-9 mM, 1.02 microA mM(-1), and 0.05 mM, respectively. Although L-ascorbate was electrooxidized at this potential, uric acid, paracetamol and glucose were found not to interfere.     

Disposable potentiometric enzyme sensor for direct determination of organophosphorus insecticides

A potentiometric disposable enzyme sensor for the direct and fast determination of organophosphorus (OP) insecticides was developed by using an organophosphorus hydrolase (OPH) immobilized on an ion-selective electrode. The disposable screen-printed transducer was based on double matrix membrane technology which allows easy mass production. The potentiometric device consisted of a H(+)-sensitive electrode with integrated Ag/AgCl reference electrode. The electrodes were prepared with N,N-dioctadecylmethylamine as H(+)-sensitive ionophore and pH calibration resulted in slopes of 55 mV decade-1 over a pH range from 11 to 6. OPH was isolated from recombinant Escherichia coli DH5 alpha and immobilized within poly(carbamoyl sulfonate) prepolymer on the surface of the H(+)-sensitive electrode without any further fixation membrane. OPH catalyzes the hydrolytic cleavage of OP compounds which releases protons in a concentration proportional to hydrolyzed substrate. Sensor performance was investigated with regard to enzyme load, concentration, pH and temperature of the measuring buffer using paraoxon as analyte. Best sensitivity and response time were obtained with sensors prepared with 250 U of OPH and measuring at 37 degrees C in 1.0 mM HEPES buffer, pH 9.3, containing 100 mM NaCl. The enzyme sensor exhibited a linear calibration range of 0.01-0.15 mM chlorpyrifos, 0.05-0.35 mM diazinon, 0.05-0.4 mM paraoxon and 0.007-0.05 mM parathion, respectively. For all these analytes response times to reach 95% of maximum change in potential did not exceed 5 min. Sensors stored under dry conditions at 4 degrees C still showed 60% of initial hydrolytic rate after 70 d. The sensors even when stored dry were ready for measurements after 5 min incubation in measuring buffer. A range of putative interfering substances did not influence sensor response, and suitability of measuring OPs in soil extracts was ascertained.     

L-Malate-sensing electrode based on malate dehydrogenase and NADH oxidase

An L-malate-sensing electrode was constructed from an oxygen electrode and a layer containing immobilized malate dehydrogenase (MDH) and NADH oxidase. MDH catalyses the dehydrogenation of L-malate by NAD⁺ and NADH oxidase catalyses the regeneration of NAD⁺ with the use of oxygen. The regeneration enables the L-malate oxidation to proceed efficiently even in a medium of neutral pH. At pH 8.0, L-malate in the concentration range 5 μM–1.5 mM can be measured. The relative standard deviation for the measurement is 1.2% (L-malate concentration, 0.2 mM; n=10). The present L-malate-sensing electrode is stable for 8 weeks. A two-electrode sensor system consisting of the L-malate-sensing electrode and an L-lactate-sensing electrode based on lactate oxidase was prepared and applied to the simultaneous determination of the two components in wines.     

Application of screen-printed microband biosensors incorporated with cells to monitor metabolic effects of potential environmental toxins

Microband biosensors were fabricated from a screen-printed water-based carbon ink containing cobalt phthalocyanine redox mediator and glucose oxidase or lactate oxidase enzyme. The microbiosensors were characterised for their ability to monitor ferrocyanide and H₂O₂ in phosphate buffer solution: sigmoidal cyclic voltammograms, high current density values and steady-state amperometric responses confirmed the existence of radial-diffusion-limiting microelectrode behaviour. The lactate microband biosensors were then used, in conjunction with a screen-printed Ag/AgCl reference and platinum counter electrode, to monitor lactate levels in culture medium, with a linear range of 0.5–5 mM, sensitivity of 20 nA.mM^?1, and dynamic range up to >9 mM. The lactate microband biosensors could operate continuously in culture medium over extended times (up to 24 h) at 37 °C. These biosensors were then applied to detect changes in lactate release from cultured cells in response to toxic challenge: m-dinitrobenzene (500 μM) caused a reduction in lactate production by high-passage number HepG2 single cells; D-galactosamine (20 mM) induced release of lactate by HepG2 spheroid cultures. This novel use of microband biosensors in cell culture has the potential for further application in toxicity monitoring, in both environmental and pharmaceutical areas.     

Prussian Blue bulk modified screen-printed electrodes for H(2)O(2) detection and for biosensors

A sensor for H(2)O(2) amperometric detection based on a Prussian Blue (PB) bulk modified carbon screen-printed electrode was developed. It has been optimised with respect to the lowest limit of detection achieved. PB was made chemically by the reaction of FeCl(3) with K(4)[Fe(CN)(6)]. The resulting powder, obtained by forced crystallisation induced by acetone, was dried and activated at 150 degrees C for 10 h. PB microparticles (<38 mum) were prepared and mixed with carbon ink. The limit of detection achieved was 0.4 muM with the linear range up to 100 muM of H(2)O(2) with the sensitivity of 137 muA mM(-1) cm(-2), that was comparable with sensors based on electrodeposited PB film. The transducer was applied for a glucose biosensor, that exhibited LOD of 0.22 mM, linear range up to 3 mM, K(M)(app) of 4.6 mM, and the sensitivity of 3.21+/-0.16 muA mM(-1) cm(-2). The peroxide sensor, as well as the glucose biosensor, were totally insensitive to oxygen, ascorbate, urate, and paracetamol.     

Nucleic acid-modulated silver nanoparticles: a new electrochemical platform for sensing chloride ion

Inorganic nanomaterials have generated considerable interest in connection to the design of biosensors. Here we exploit the DNA-induced generation of silver nanoparticles for developing an electrical biosensing protocol for chloride ions. Conjugated with thiol modified oligonucleotide, silver nanoparticles were template-synthesized and immobilized on gold electrode. During cyclic voltammogram (CV) scans, the silver nanoparticles were oxidized at high potential to form a layer of Ag/AgCl complex in the presence of Cl(-), giving off sharp solid state redox signals. Under the optimum condition, the electrode responded to Cl(-) over a dynamic range of 2.0 × 10(-5)-0.01 M, with a detection limit of 5.0 × 10(-6) M. Moreover, the specific solubility product constant-based anion recognition made the electrode applicable at a wide pH range and in complex biological systems. To demonstrate the analytical applications of this sensor in real samples, the Cl(-) concentrations in human urine were measured without any sample pretreatment. Urinary Cl(-) detected by the proposed sensor ranged from 110 to 200 mM, which was comparable to the results obtained by standard silver titration.     

Voltammetric response of phenylboronic acid monolayer-modified gold electrode to sugars.

The surface of a gold (Au) disk electrode was modified with a self-assembled monolayer consisting of phenylboronic acid moiety to fabricate a voltammetric sensor sensitive to sugars. The modified Au electrode exhibited a voltammetric response to sugars in the presence of Fe(CN)6(3-) ion in the sample solution at neutral pH. The peak current of the cyclic voltammograms decreased depending on the type and concentration of sugars. The dynamic range of the electrode is 3 - 100 mM for glucose and mannose and 1 - 30 mM for fructose. The sugar sensor can be used repeatedly after rinsing in 10 mM acetate buffer (pH 4.5).     

A polypyrrole-based amperometric ammonia sensor

An ammonia sensor is described in this work. The sensing membrane is a thin layer of oxidized polypyrrole (PPy) on a platinum substrate. This sensor is used as the working electrode in a conventional three-electrode system for amperometric measurement of ammonia in aqueous solutions in the potential range of + 0.2 to + 0.4 V (vs. Ag/AgCl). Contact with ammonia causes a current to flow through the electrode. This current is proportional to the concentration of free ammonia in the solution and ammonium ions do not contribute to the measured signal. The signal is due to reduction of PPy by ammonia with subsequent oxidation of PPy by the external voltage source. The sensor is able to detect ammonia reproducibly at the muM level. The main interference is the doping effect of small anions such as Cl(-) and NO(3)(-), also giving a response on PPy at the mM level. This anionic response can, to a certain degree, be reduced by covering the polymer surface with dodecyl sulfate. The sensor gradually loses its activity when exposed to ammonia concentrations greater than 1 mM. The sensor has been tested by the flow injection analysis technique.     

Gamma-irradiation immobilization of lactate oxidase in poly(vinyl alcohol) on platinized graphite electrodes

A novel method for enzyme immobilization in a polymer matrix was examined with lactate oxidase (LOD) to make a sensor for lactate. Poly(vinyl alcohol) (PVAL) and LOD were applied in layers on platinized graphite electrodes and cross-linked by exposure to a ⁶⁰Co gamma radiation source. When the sensor is dipped in lactate solution, the product of the enzymatic reaction, hydrogen peroxide, is detected at +300 mV vs. Ag/AgCl. The LOD-PVAL lactate sensor exhibits a fast response (10–50 s), a linear range between 26 μM and 1.7 mM, a detection limit of 13 μM and a sensitivity of 2.94 μA mmol^?1. The sensitivity and the linearity of the electrode were improved considerably by bubbling oxygen continuously through the lactate solution. Optimum response to lactate was obtained with a radiation dose of 3–10 Mrad. LOD was found to be active in the presence of the polymer under radiation doses as high as 40 Mrad. Repeated use of the sensors under various conditions showed a stable and reproducible response to lactate for over 80 days.     

A potentiometric polypyrrole-based glucose biosensor

A new concept is described for monitoring a biomolecule with a sensor having an enzyme entrapped in a conducting polymer. This is based on the sensitivity of the electroactive polymer itself to changes of pH in solution. The concept has been investigated for a glucose sensor with glucose oxidase (GOD) immobilized in a polypyrrole (PPy) layer on an inert platinum electrode. Measurements with a Pt/PPy/GOD electrode for glucose concentrations in the physiological range gave a linear correlation with logarithm of concentration over one decade with a satisfactory dynamic response. There was practically no change of slope or range of linear response to glucose after several days of use; this was in contrast to the amperometric response of the detector when there was about a 50% loss of sensitivity.     

Using Transparent Adhesive Tape as New Substrate for Integrated Flexible Enzymatic Sensor: Good Adhesion and Better Printability

Substrate plays an essential role in the construction of flexible electrode and related wearable sensors. Compared with conventional flexible substrates such as Polyethylene terephthalate (PET), the common transparent adhesive tape exhibits the unique advantages in the non-adhesive surface with good printability, allowing the conductive layer to be deposited directly on its surface, and in another adhesive surface with good fastening, thus facilitating the fabrication of as-prepared electrode in subsequent wearable sensors. Herein, we constructed a new type of flexible sensor to detect ascorbic acid which is closely related to human health in sweat by integrating flexible electrode based on transparent adhesive tape with potentiostat that incorporate the critical signal conditioning, processing, and transmission functions. Experiment results show that resulting electrode still has the good electrochemistry performance even after 1000 bending cycles (20 % bending degrees). By connecting as-prepared flexible electrode to the potentiostat to carry out real time analysis, the resulting sensor exhibits excellent sensitivity, detection limit and repeatability (0.15 V detection potential vs printed Ag/AgCl reference electrode, 3.8 μM detection limit, 25 μM-1 mM linearity, and good selectivity).     

In situ synthesis of thulium(III) hexacyanoferrate(II) nanoparticles and its application for glucose detection

Thulium hexacyanoferrate (TmHCF) nanoparticles (NPs) were in situ synthesized within the chitosan film on the electrode surface by a biocatalyzed reaction. The properties of the obtained nanoparticles are characterized with scanning electron microscope (SEM) and energy-dispersive X-ray (EDX). The optimized conditions for the formation of TmHCF NPs were 16 mM Fe(CN)₆³⁻ and 1.5 mM Tm³⁺ with an accumulation time of 20 min. Based on process of in situ synthesis of TmHCF NPs, a novel biosensor for glucose was designed, and there is a linear relationship between the current response of TmHCF NPs and glucose concentration. The linear range for glucose detection was 0.02–0.4 mM (r = 0.9975, n = 5) and 0.4–13.6 mM (r = 0.9935, n = 10) and the detection limit was 6 μM at a signal-to-noise ratio of 3.     

DNA/Poly(p-aminobenzensulfonic acid) composite bi-layer modified glassy carbon electrode for determination of dopamine and uric acid under coexistence of ascorbic acid

A nano-composite of DNA/poly(p-aminobenzensulfonic acid) bi-layer modified glassy carbon electrode as a biosensor was fabricated by electro-deposition method. The DNA layer was electrochemically deposited on the top of electropolymerized layer of poly(p-aminobenzensulfonic acid) (Pp-ABSA). Scanning electron microscopy, X-ray photoelectron spectroscopy and electrochemical impedance spectrum were used for characterization. It demonstrated that the deposited Pp-ABSA formed a 2-D fractal patterned nano-structure on the electrode surface, and which was further covered by a uniform thin DNA layer. Cyclic voltammetry and electrochemical impedance spectrum were used to characterize the deposition, and demonstrated the conductivity of the Pp-ABSA layer. The biosensor was applied to the detection of dopamine (DA) and uric acid (UA) in the presence of ascorbic acid (AA). In comparison with DNA and Pp-ABSA single layer modified electrodes, the composite bi-layer modification provided superior electrocatalytic actively towards the oxidation of DA, UA and AA, and separated the originally overlapped differential pulse voltammetric signals of UA, DA and AA oxidation at the bare electrode into three well-defined peaks at pH 7 solution. The peak separation between AA and DA, AA and UA was 176 mV and 312 mV, respectively. In the presence of 1.0 mM AA, the anodic peak current was a linear function of the concentration of DA in the range 0.19-13 microM. The detection limit was 88 nM DA (s/n=3). The anodic peak current of UA was also a linear function of concentration in the range 0.4-23 microM with a detection limit of 0.19 microM in the presence of 0.5 mM AA. The superior sensing ability was attributed to the composite nano-structure. An interaction mechanism was proposed.