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1.
A feasible approach to construct multilayered enzyme film on the gold electrode surface for use as biosensing interface is described. The film was fabricated by alternate layer-by-layer deposition of periodate-oxidized glucose oxidase (GOx) and poly(allylamine) (PAA). The covalent attachment process was followed and confirmed by electrochemical impedance spectroscopy (EIS). X-ray diffraction (XRD) experiments revealed that the film was homogeneous and formed in an ordered manner with a thickness of 2.6 ± 0.1 nm per bilayer. The gold electrodes modified with the GOx/PAA multilayers showed excellent electrocatalytical response to the oxidation of glucose when ferrocenemethanol was used as an artificial redox mediator, which was studied by cyclic voltammetry (CV). From the analysis of voltammetric signals, the coverage of active enzyme on the electrode surface was estimated, which had a linear relationship with the number of GOx/PAA bilayers. This suggests that the analytical performance such as sensitivity, detection limit, and so on, is tunable by controlling the number of attached bilayers. The six GOx/PAA bilayer electrode exhibited a sensitivity of 15.1 μA mM−1 cm−2 with a detection limit of 3.8 × 10−6 M. In addition, the sensor exhibited good reproducibility and stability.  相似文献   

2.
We report a simple and rapid procedure that leads to incorporation of mediator and introduction of amine functionality onto the surface of screen-printed carbon electrodes (SPCE). The electrodes were doped with cobalt phthalocyanine (CoPc) by enhanced adsorption in a process that uses minimal amounts of this redox mediator as compared with CoPc loaded inks. The CoPc-doped SPCE showed a substantially increased sensitivity to hydrogen peroxide and thiocholine as compared to unmodified electrodes. This greatly facilitated their use as transducers for the construction of amperometric biosensors based on enzymes producing oxidizable products such as hydrogen peroxide or thiols. Immobilisation of enzymes including glucose oxidase, acetylcholinesterase and choline oxidase was achieved through their multi-contact electrostatic interaction with polyethyleneimine (PEI) which was electrodeposited on the surface of CoPc-doped electrodes in one step from ethanolic solution. The efficiency of enzyme immobilisation was shown to depend on the molecular weight of the PEI used, reaching a maximum for 25 kDa PEI. The biosensors shown sensitivity to glucose at 130 nA mM−1 (LOD 0.15 mM) and to acetylcholine at 70 nA mM−1 (LOD 0.10 mM) under +0.6 V. Detection of glucose has been demonstrated at +0.4 V with the sensitivity of 60 nA mM−1 and LOD of 0.33 mM. Possibility of the inhibition analysis of pesticides has been shown for acetylcholinesterase-based sensors.  相似文献   

3.
Creation and application of noble metal nanoclusters have received continuous attention. By integrating enzyme activity and fluorescence for potential applications, enzyme-capped metal clusters are more desirable. This work demonstrated a glucose oxidase (an enzyme for glucose)-functionalized gold cluster as probe for glucose. Under physiological conditions, such bioconjugate was successfully prepared by an etching reaction, where tetrakis (hydroxylmethyl) phosphonium-protected gold nanoparticle and thioctic acid-modified glucose oxidase were used as precursor and etchant, respectively. These bioconjugates showed unique fluorescence spectra (λem max = 650 nm, λex max = 507 nm) with an acceptable quantum yield (ca. 7%). Moreover, the conjugated glucose oxidase remained active and catalyzed reaction of glucose and dissolved O2 to produce H2O2, which quenched quantitatively the fluorescence of gold clusters and laid a foundation of glucose detection. A linear range of 2.0 × 10−6–140 × 10−6 M and a detection limit of 0.7 × 10−6 M (S/N = 3) were obtained. Also, another horseradish peroxidase/gold cluster bioconjugate was produced by such general synthesis method. Such enzyme/metal cluster bioconjugates represented a promising class of biosensors for biologically important targets in organelles or cells.  相似文献   

4.
In this work, we reported an efficient platinum nanoparticles functionalized nitrogen doped graphene (PtNPs@NG) nanocomposite for devising novel electrochemical glucose biosensor for the first time. The fabricated PtNPs@NG and biosensor were characterized using transmission electron microscopy, high-resolution transmission electron microscopy, X-ray photoelectron spectroscopy, static water contact angle, UV–vis spectroscopy, electrochemical impedance spectra and cyclic voltammetry, respectively. PtNPs@NG showed large surface area and excellent biocompatibility, and enhanced the direct electron transfer between enzyme molecules and electrode surface. The glucose oxidase (GOx) immobilized on PtNPs@NG nanocomposite retained its bioactivity, and exhibited a surface controlled, quasi-reversible and fast electron transfer process. The constructed glucose biosensor showed wide linear range from 0.005 to 1.1 mM with high sensitivity of 20.31 mA M−1 cm−2. The detection limit was calculated to be 0.002 mM at signal-to-noise of 3, which showed 20-fold decrease in comparison with single NG-based electrochemical biosensor for glucose. The proposed glucose biosensor also demonstrated excellent selectivity, good reproducibility, acceptable stability, and could be successfully applied in the detection of glucose in serum samples at the applied potential of −0.33 V. This research provided a promising biosensing platform for the development of excellent electrochemical biosensors.  相似文献   

5.
We are proposing for the first time the use of a Nafion/multi-walled carbon nanotubes dispersion deposited on glassy carbon electrodes (GCE) as a new platform for developing enzymatic biosensors based on the self-assembling of a chitosan derivative and different oxidases. The electrodes are obtained by deposition of a layer of Nafion/multi-wall carbon nanotubes dispersion on glassy carbon electrodes, followed by the adsorption of a chitosan derivative as polycation and glucose oxidase, l-aminoacid oxidase or polyphenol oxidase, as polyanions and biorecognition elements. The optimum configuration for glucose biosensors has allowed a highly sensitive (sensitivity = (0.28 ± 0.02) μA mM−1, r = 0.997), fast (4 s in reaching the maximum response), and highly selective (0% interference of ascorbic acid and uric acid at maximum physiological levels) glucose quantification at 0.700 V with detection and quantification limits of 0.035 and 0.107 mM, respectively. The repetitivity for 10 measurements was 5.5%, while the reproducibility was 8.4% for eight electrodes. The potentiality of the new platform was clearly demonstrated by using the carbon nanotubes/Nafion layer as a platform for the self-assembling of l-aminoacid oxidase and polyphenol oxidase. Therefore, the platform we are proposing here, that combines the advantages of nanostructured materials with those of the layer-by-layer self-assembling of polyelectrolytes, opens the doors to new and exciting possibilities for the development of enzymatic and affinity biosensors using different transdution modes.  相似文献   

6.
In this study, a multiplex fluorescence sensor for successive detection of Fe3+, Cu2+ and Hg2+ ions based on “on–off” of fluorescence of a single type of gold nanoclusters (Au NCs) is described. Any of the Fe3+, Cu2+ and Hg2+ ions can cause quenching fluorescence of Au NCs, which established a sensitive sensor for detection of these ions respectively. With the introduction of ethylene diamine tetraacetic acid (EDTA) to the system of Au NCs and metal ions, a restoration of fluorescence may be found with the exception of Hg2+. A highly selective detection of Hg2+ ion is, thus, achieved by masking Fe3+ and Cu2+. On the other hand, the masking of Fe3+ and Cu2+ leads to the enhancement of fluorescence of Au NCs, which in turn provides an approach for successive determination of Fe3+ and Cu2+ based on “on–off” of fluorescence of Au NCs. Moreover, this assay was applied to the successful detection of Fe3+, Cu2+ and Hg2+ in fish, a good linear relationship was found between these metal ions and the degree of quenched fluorescent intensity. The dynamic ranges of Hg2+, Fe3+ and Cu2+ were 1.96 × 10−10–1.01 × 10−9, 1.28 × 10−7–1.27 × 10−6 and 1.2 × 10−7–1.2 × 10−6 M with high sensitivity (the limit of detection of Fe3+ 2.0 × 10−8 M, Cu2+ 1.9 × 10−8 M and Hg2+ 2 × 10−10 M). These results indicate that the assay is suitable for sensitive detection of these metal ions even under the coexistence, which can not only determine all three kinds of metal ions successively but also of detecting any or several kinds of metal ions.  相似文献   

7.
Miniaturized urea and glucose sensors prepared by immobilization of glucose oxidase or urease directly onto all solid-state contact PVC-NH2 membrane ammonium and hydrogen ion selective electrodes are described. The resulting biosensing membranes function equivalently to normal PVC membranes in terms of potentiometric response properties. The most important features of the glucose and urea sensors were high sensitivity, long life-time, easily built at a low cost, micro-construction and short response time. The characteristics of the glucose and urea sensors were examined in several buffer solutions at different concentrations and pH values. The influence of immobilization conditions on the dynamic response properties and life-time of the electrodes was studied. Under optimal conditions, the urea electrode showed a linear response between 5×10−2 and 5×10−4 M urea, while the glucose electrode showed a linear response between 5×10−2 and 1×10−4 M glucose.  相似文献   

8.
In this work, we reported a sandwiched luminol electrochemiluminescence (ECL) immunosensor using ZnO nanoparticles (ZnONPs) and glucose oxidase (GOD) decorated graphene as labels and in situ generated hydrogen peroxide as coreactant. In order to construct the base of the immunosensor, a hybrid architecture of Au nanoparticles and graphene by reduction of HAuCl4 and graphene oxide (GO) with ascorbic acid was prepared. The resulted hybrid architecture modified electrode provided an excellent platform for immobilization of antibody with good bioactivity and stability. Then, ZnONPs and GOD functionalized graphene labeled secondary antibody was designed for fabricating a novel sandwiched ECL immunosensor. Enhanced sensitivity was obtained by in situ generating hydrogen peroxide with glucose oxidase and the catalysis of ZnONPs to the ECL reaction of luminol–H2O2 system. The as-prepared ECL immunosensor exhibited excellent analytical property for the detection of carcinoembryonic antigen (CEA) in the range from 10 pg mL−1 to 80 ng mL−1 and with a detection limit of 3.3 pg mL−1 (S N−1 = 3). The amplification strategy performed good promise for clinical application of screening of cancer biomarkers.  相似文献   

9.
An amperometric nicotine inhibition biosensor has been substantially simplified and used for determination of nicotine in tobacco sample. Besides the use of single enzyme choline oxidase to replace bienzyme, the use of 1,4-benzoquinone as an electron mediator makes it possible to avoid the use of oxygen or hydrogen peroxide sensor as the internal transducer. Choline oxidase was immobilized on the carbon paste electrode through cross-linking with bovine serum albumin (BSA) by glutaraldehyde. In the presence of choline oxidase and its endogenous cofactor flavin-ademine dinneleotide (FAD), choline was oxidized into betaine while FAD was reduced to FADH2 which subsequently reduced 1,4-benzoquinone into hydroquinone. The later was finally oxidized at a relatively low potential of +450 mV versus saturated calomel electrode (SCE). Nicotine inhibits the activity of enzyme with an effect of decreasing of oxidation current. The experimental conditions were optimized. The electrode has a linear response to choline within 1.25×10−4 to 1.25×10−3 mol l−1. The nicotine measurements were carried out in 0.067 mol l−1phosphate buffer of pH 7.4 at an applied potential of 450 mV versus SCE. The electrode provided a linear response to nicotine over a concentration range of 2.0×10−5 to 9.2×10−4 mol l−1 with a detection limit of 1.0×10−5 mol l−1. The system was applied to the determination of nicotine in tobacco samples.  相似文献   

10.
Luque GL  Rodríguez MC  Rivas GA 《Talanta》2005,66(2):467-471
The performance of amperometric glucose biosensors based on the dispersion of glucose oxidase (GOx) and copper oxide within a classical carbon (graphite) paste composite is reported in this work. Copper oxide promotes an excellent electrocatalytic activity towards the oxidation and reduction of hydrogen peroxide, allowing a large decrease in the oxidation and reduction overpotentials, as well as an important enhancement of the corresponding currents. Therefore, it is possible to perform the glucose biosensing at low potentials where there is no interference even in large excess of ascorbic acid, uric acid or acetaminophen. The influence of the copper oxide and glucose oxidase content in the paste on the analytical performance of the bioelectrode is discussed. The resulting biosensor shows a fast response, a linear relationship between current and glucose concentration up to 1.35 × 10−2 M (2.43 g L−1) and a detection limit of 2.0 × 10−5 M. The effect of the presence of the enzyme in the composite material on the dispersion of the copper oxide particles is also discussed.  相似文献   

11.
A long-life capillary enzyme bioreactor was developed that determines glucose concentrations with high sensitivity and better stability than previous systems. The bioreactor was constructed by immobilizing glucose oxidase (GOx) onto the inner surface of a 0.53 mm i.d. fused-silica capillary that was part of a continuous-flow system. In the presence of oxygen, GOx converts glucose to gluconic acid and hydrogen peroxide (H2O2). Hydrogen peroxide detection was accomplished using an amperometric electrochemical detector. The integration of this capillary reactor into a flow-injection (FIA) system offered a larger surface-to-volume ratio, reduced band-broadening effects, and reduced reagent consumption compared to packed column in FIA or other settings. To obtain operational (at ambient temp) and storage (at 4 °C) stability for 20 weeks, the glucose biosensing system was prepared using an optimal GOx concentration (200 mg/mL). This exhibited an FIA peak response of 7 min and a detection limit of 10 μM (S/N = 3) with excellent reproducibility (coefficient of variation, CV < 0.75%). It also had a linear working range from 101 to 104 μM. The enzyme activity in this proposed capillary enzyme reactor was well maintained for 20 weeks. Furthermore, 20 serum samples were analyzed using this system, and these correlated favorably (correlation coefficient, r2 = 0.935) with results for the same samples obtained using a routine clinical method. The resulting biosensing system exhibited characteristics that make it suitable for in vivo application.  相似文献   

12.
In the present study, a novel and ultrasensitive electrochemiluminescence (ECL) immunosensor based on luminol cathodic ECL was fabricated by using Au nanoparticles and Pt nanoparticles (nano-AuPt) electrodeposited on graphene–carbon nanotubes nanocomposite as platform for the detection of carcinoembryonic antigen (CEA). For this introduced immunosensor, graphene (GR) and single wall carbon nanotubes (CNTs) dispersed in chitosan (Chi-GR-CNTs) were firstly decorated on the bare gold electrode (GE) surface. Then nano-AuPt were electrodeposited (DpAu-Pt) on the Chi-GR-CNTs modified electrode. Subsequently, glucose oxidase (GOD) was employed to block the non-specific sites of electrode surface. When glucose was present in the working buffer solution, GOD immediately catalyzed the oxidation of glucose to in situ generate hydrogen peroxide (H2O2), which could subsequently promote the oxidation of luminol with an amplified cathodic ECL signal. The proposed immunosensor was performed at low potential (−0.1 to 0.4 V) and low concentration of luminol. The CEA was determined in the range of 0.1 pg mL−1 to 40 ng mL−1 with a limit of detection down to 0.03 pg mL−1 (S N−1 = 3). Moreover, with excellent sensitivity, selectivity, stability and simplicity, the as-proposed luminol-based ECL immunosensor provided great potential in clinical applications.  相似文献   

13.
You C  Yan X  Kong J  Zhao D  Liu B 《Talanta》2011,83(5):1507-1514
A strategy of protein-entrapment in bicontinuous gyroidal mesoporous carbon (BGMC) nanocomposite films is described. Herein, the quasi-reversible electron transfer of redox proteins (such as glucose oxidase and myoglobin) is probed and the associated biocatalytic activity is revealed. The apparent heterogeneous electron transfer rate constant of the immobilized glucose oxidase is up to 9.4 s−1, much larger than those in carbon nanotubes and some conventional mesoporous carbons. The BGMC based glucose biosensor enables the determination of glucose at a potential of 0.6 V (vs. SCE). Its detection limit is 1.0 × 10−5 M (signal-to-noise ratio, S/N = 3), the linear response is up to 7.49 mM and the detection sensitivity is 52.5 nA mM−1 Furthermore, a series of BGMCs with different pore sizes is designed and synthesized using sucrose or phenol formaldehyde resin to study the influences of pore sizes and carbon sources on the immobilization of redox proteins and on the heterogeneous electron transfer.  相似文献   

14.
Dai Z  Fang M  Bao J  Wang H  Lu T 《Analytica chimica acta》2007,591(2):195-199
We have constructed a glucose biosensor by immobilizing glucose oxidase (GOD) on titanium-containing MCM-41 (Ti-MCM-41) modified screen-printed electrodes. The strategy of the sensing method is to monitor the extent of the decrease of the reduction current of O2 upon adding glucose at a selected potential. The detection can be done at the applied potential of −0.50 V and can efficiently exclude the interference from commonly coexisted substances. The constructed sensor has a high sensitivity to glucose (5.4 mAM−1 cm−2) and a linear response range of 0.10-10.0 mM. The detection limit is 0.04 mM at a signal-to-noise ratio of 3. The sensor also shows high stability and remains its catalytic activity up to 60 °C. The biocompatibility of Ti-MCM-41 means that this immobilization matrix not only can be used for immobilizing GOD but also can be extended to other enzymes and bioactive molecules, thus providing a promising platform for the development of biosensors.  相似文献   

15.
Chang G  Tatsu Y  Goto T  Imaishi H  Morigaki K 《Talanta》2010,83(1):61-65
Optical biosensor arrays for rapidly determining the glucose concentrations in a large number of beverage and blood samples were developed by immobilizing glucose oxidase (GOD) on oxygen sensor layer. Glucose oxidase was first encapsulated in silica based gels through sol-gel approach and then immobilized on 96-well microarrays integrated with oxygen sensing film at the bottom. The oxygen sensing film was made of an organically modified silica film (ORMOSIL) doped with tris(4,7-diphenyl-1,10-phenanthroline) ruthenium dichloride (Ru(dpp)3Cl2). The oxidation reaction of glucose by glucose oxidase could be monitored through fluorescence intensity enhancement due to the oxygen consumption in the reaction. The luminescence changing rate evaluated by the dynamic transient method (DTM) was correlated with the glucose concentration with the wide linear range from 0.1 to 5.0 mM (Y = 13.28X − 0.128, R = 0.9968) and low detection limit (0.06 mM). The effects of pH and coexisting ions were systemically studied. The results showed that the optical biosensor arrays worked under a wide range of pH value, and normal interfering species such as Na+, K+, Cl, PO43−, and ascorbic acid did not cause apparent interference on the measurement. The activity of glucose oxidase was mostly retained even after 2-month storage, indicating their long-term stability.  相似文献   

16.
In this work a method is presented for the enzymatic determination of glucose using fluorescence anisotropy. During the enzymatic reaction a change in the fluorescence anisotropy of the glucose oxidase (GOx) is produced; the reaction time at which this change appears (tm) depends on the glucose concentration. A theoretical study has been developed which enables: (a) the correlation of this change in anisotropy with changes in the intensity and the lifetime of the enzyme fluorescence; from this a model which could be generalized to other flavo-enzymes is proposed; (b) the linking of tm with glucose concentration.After optimisation, the proposed method allows the determination of glucose over the range 100-1000 mg l−1. The detection limit is 90 mg l−1and the reproducibility is better than 4% (n = 6, [glucose] = 250 mg l−1). Anisotropy is more selective than conventional fluorescence intensity, and this method has therefore been applied to direct glucose determination in fruit juices without the interference caused by the inner filter effect.  相似文献   

17.
This work reports a sequential-injection analysis (SIA) method for the enzymatic assay of glucose with soluble glucose oxidase (GOD) and on-line sample dilution with chemiluminescence (CL) detection. A zone of sample was aspirated in the holding coil of the SIA manifold and, if necessary, was diluted on-line by means of an auxiliary dilution conduit. Then, a zone of GOD was aspirated adjacent to the sample zone and a stopped-flow period was applied to allow the enzymatic reaction to proceed with production of hydrogen peroxide. Then, zones of a catalyst (Co(II) solution) and alkaline luminol were aspirated into the holding coil. Finally, the flow was reversed and the stacked zones were sent to a flow-cell located in front of a photomultiplier tube (PMT) that monitored the CL intensity. The linear dynamic range was 1 × 10−5-1 × 10−3 mol L−1 glucose, the coefficient of variation at 8 × 10−5 mol L−1 of glucose was sr = 3.1% (n = 8), the limit of detection at the 3σ level was cL = 1 × 10−6 mol L−1 and the sampling frequency was 28 h−1. With on-line dilution by a factor of 1/200, the linear range could be extended up to 0.2 mol L−1 glucose. The advantages of the proposed method are the simple manifold and instrumentation used, the scope for automated on-line dilution, the low consumption of sample and reagents and the elimination of enzyme immobilisation procedures. The method was applied to the analysis of commercial drinks and honey with percent relative errors in glucose determination in the range 100 ± 6.1%.  相似文献   

18.
In the present work, we have included for the first time diamond nanoparticles (DNPs) in a sol–gel matrix derived from (3-mercaptopropyl)-trimethoxysilane (MPTS) in order to improve electron transfer in a lactate oxidase (LOx) based electrochemical biosensing platform. Firstly, an exhaustive AFM study, including topographical, surface potential (KFM) and capacitance gradient (CG) measurements, of each step involved in the biosensing platform development was performed. The platform is based on gold electrodes (Au) modified with the sol–gel matrix (Au/MPTS) in which diamond nanoparticles (Au/MPTS/DNPs) and lactate oxidase (Au/MPTS/DNPs/LOx) have been included. For the sake of comparison, we have also characterized a gold electrode directly modified with DNPs (Au/DNPs). Secondly, the electrochemical behavior of a redox mediator (hydroxymethyl-ferrocene, HMF) was evaluated at the platforms mentioned above. The response of Au/MPTS/DNPs/LOx towards lactate was obtained. A linear concentration range from 0.053 mM to 1.6 mM, a sensitivity of 2.6 μA mM−1 and a detection limit of 16 μM were obtained. These analytical properties are comparable to other biosensors, presenting also as advantages that DNPs are inexpensive, environment-friendly and easy-handled nanomaterials. Finally, the developed biosensor was applied for lactate determination in wine samples.  相似文献   

19.
A novel enzyme reactor with co-immobilization of β-galactosidase and glucose oxidase in calcium alginate fiber (CAF) and amine modified nanosized mesoporous silica (AMNMS) was prepared which incorporate the adsorption and catalysis of AMNMS with the cage effect of the polymer to increase catalytic activity and stability of immobilized enzyme. The enzyme reactor was applied to prepare a chemiluminescence (CL) flow-through biosensor for determination of lactose combined with a novel luminol-diperiodatonickelate (DPN) CL system we reported. It shows that the CL flow-through biosensor possesses long lifetime, high stability, high catalytic activity and sensitivity. The relative CL intensity was linear with the lactose concentration in the range of 8 × 10−8-4 × 10−6 g mL−1 with the detection limit of 2.7 × 10−8 g mL−1 (3σ). It has been successfully applied to the determination of lactose in milk.  相似文献   

20.
Chunhui Xiao 《Talanta》2010,80(5):1719-9434
Polymerized ionic liquid-wrapped carbon nanotubes (PIL-CNTs) were firstly designed for direct electrochemistry and biosensing of redox proteins. The CNTs were coated successfully with polymerized ionic liquid (PIL) layer, as verified by transmission electron microscopy (TEM), thermogravimetric analysis (TGA) and Fourier transform infrared (FT-IR) spectroscopy. The PIL-CNTs were dispersed better in water and showed superior electrocatalysis toward O2 and H2O2 comparing to pristine CNTs and the mixture of IL monomer and CNTs. With glucose oxidase (GOD) as a protein model, the direct electrochemistry of the redox protein was investigated on the PIL-CNTs modified glassy carbon (GC) electrode and excellent direct electrochemical performance of GOD molecules was observed. The proposed biosensor (GOD/PIL-CNTs/GC electrode) displayed good analytical performance for glucose with linear response up to 6 mM, response sensitivity of 0.853 μA mM−1, good stability and selectivity.  相似文献   

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