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1.
A biosensor design involving coimmobilization of fructose dehydrogenase (FDH) and inulinase (INU) on a gold nanoparticle-cysteamine (Cyst) self-assembled monolayer (SAM)-modified gold electrode (Au(coll)-Cyst-AuE), for the determination of the carbohydrate inulin in foodstuffs, is reported. Tetrathiafulvalene (TTF), used as the mediator, was also coimmobilized by crosslinking with glutaraldehyde. INU catalyzes the hydrolysis of inulin, forming fructose that is detected through the fructose dehydrogenase system by the electrochemical oxidation of TTF at the bioelectrode. The variables involved in the preparation and performance of both the single enzyme FDH biosensor and the bienzyme inulin biosensor were optimized. The FDH-Au(coll)-Cyst-AuE biosensor exhibited rapid and sensitive response to fructose, allowing the obtention of improved analytical characteristics for the determination of fructose with respect to other FDH electrochemical biosensors. Moreover, the lifetime of this biosensor was 35 days. The bienzyme INU/FDH-Au(coll)-Cyst-AuE biosensor provided a calibration plot for inulin in the (5-100)x10(-6) M linear range, with a detection limit of 6.6 x 10(-7) mol L(-1). One single bienzyme biosensor responded within the control limits, set at +/-3x the standard deviation of the currents measured on the first day of use, for more than 5 months. Furthermore, the biosensor exhibited high selectivity with respect to other carbohydrates. The usefulness of the biosensor was evaluated by the rapid determination of inulin in food products involving minimization of the fructose interference.  相似文献   

2.
Glutathione (L-gamma-glutamyl-L-cysteinyl-L-glycine; GSH) forms a surface monolayer on gold nanoparticles by tethering via sulfur bonds (Au:GSH). In the present study, glucose oxidase (GOx; EC 1.1.3.4) was immobilized by covalent chemical coupling reactions on to Au:GSH nanoparticles and the enzyme coupled nanoparticles formed a stable colloid (stable for several weeks) in water. The immobilized enzyme was investigated for electrochemical characteristics to monitor the FAD (prosthetic group of the GOx) redox potentials. Various concentrations of substrate (glucose) were added to check the oxidation characteristics. It was observed that with increase in substrate concentrations, the oxidation rate increased proportionally with the current. The present study demonstrated that GOx was effectively coupled to the gold nanoparticle (Au:GSH). The coupled nanoparticle system could be used in a potential biosensor application. Similarly, other enzymes (e.g., horseradish peroxidase) could be immobilized to the Au:GSH nanoparticles via the peptide arm (GSH) to achieve the desired characteristics needed for a specific application in biosensor.  相似文献   

3.
For the first time glucose oxidase (GOx) was successfully co-deposited on nickel-oxide (NiO) nanoparticles at a glassy carbon electrode. In this paper we present a simple fabrication method of biosensor which can be easily operated without using any specific reagents. Cyclic voltammetry was used for electrodeposition of NiO nanoparticle and GOx immobilization. The direct electron transfer of immobilized GOx displays a pair of well defined and nearly reversible redox peaks with a formal potential (E(0')) of -0.420 V in pH 7 phosphate buffer solution and the response shows a surface controlled electrode process. The surface coverage and heterogeneous electron transfer rate constant (k(s)) of GOx immobilized on NiO film glassy carbon electrode are 9.45 x 10(-13)mol cm(-2) and 25.2+/-0.5s(-1), indicating the high enzyme loading ability of the NiO nanoparticles and great facilitation of the electron transfer between GOx and NiO nanoparticles. The biosensor shows excellent electrocatalytical response to the oxidation of glucose when ferrocenmethanol was used as an artificial redox mediator. Furthermore, the apparent Michaelis-Menten constant 2.7 mM, of GOx on the nickel oxide nanoparticles exhibits excellent bioelectrocatalytic activity of immobilized enzyme toward glucose oxidation. In addition, this glucose biosensor shows fast amperometric response (3s) with the sensitivity of 446.2nA/mM, detection limit of 24 microM and wide concentration range of 30 microM to 5mM. This biosensor also exhibits good stability, reproducibility and long life time.  相似文献   

4.
A new amperometric biosensor for the detection of sugars was prepared. A glassy carbon electrode was modified with Prussian blue (PB) nanoparticles protected by chitosan (CS) and poly(diallyldimethylammonium chloride) (PDDA), and then gold nanoparticles were assembled onto the electrode followed by the assembly of 4-mercaptophenylboronic acid (MPBA) onto the surface of gold nanoparticles through a sulfur–Au bond to fabricate a self-assembled biosensor. The PB nanoparticles protected by CS and PDDA were characterized using transmission electron microscopy and UV–vis absorption spectroscopy. The characterization of the self-assembled electrode was investigated by cyclic voltammetry and electrochemical impedance spectroscopy. The pK a values of the MPBA monolayer before and after combining with sugars were determined. The fabricated electrode exhibited excellent performances for determining d(+)-glucose, d(+)-mannose, and d(−)-fructose on the basis of the change in i p of the Fe(CN)63−/4− ion in the presence of sugars.  相似文献   

5.
A highly stable functional composite film was prepared using polyazetidine prepolymer (PAP) with peroxidase from horseradish (HRP) and/or glucose oxidase (GOx). The good permeability of the PAP layer to classical electrochemical mediators, as evaluated by the determination of the diffusion coefficient of different redox molecules, is of great importance in view of the use of PAP as an immobilizing agent in second-generation biosensor development. Cyclic voltammetry of the HRP-PAP layer on a glassy carbon electrode (GCE) showed a pair of stable and quasi-reversible peaks for the HRP-Fe((III))/Fe((II)) redox couple at about -370 mV vs. Ag/AgCl electrode in pH 6.5 phosphate buffer. The electrochemical reaction of HRP entrapped in the PAP film exhibited a surface-controlled electrode process. This film and the successive modifications (HRP-PAP self-assembled monolayer (SAM) modified Au electrode) were used as a biological catalyst (hydrogen peroxide transducers) for glucose biosensors, after coupling to GOx. Both HRP/GOx-PAP and HRP/GOx-PAP SAM third generation biosensors were prepared and characterized. The use of PAP as immobilizing agent offers a biocompatible micro-environment for confining the enzyme and foreshadows the great potentiality of this immobilizing agent not only in theoretical studies on protein direct electron transfer but also from an applications point of view in the development of second- and third-generation biosensors.  相似文献   

6.
One of the challenges in electrochemical biosensor design is gaining a fundamental knowledge of the processes underlying immobilisation of the molecules onto the electrode surface. This is of particular importance in biocomposite sensors where concerns have arisen as to the nature of the interaction between the biological and synthetic molecules immobilised. We examined the use of the Quartz Crystal Microbalance with Dissipation (QCM-D) as a tool for fundamental analyses of a model sensor constructed by the immobilisation of cobalt(II) phthalocyanine (TCACoPc) and glucose oxidase (GOx) onto a gold-quartz electrode (electrode surface) for the enhanced detection of glucose. The model sensor was constructed in aqueous phase and covalently linked the gold surface to the TCACoPc, and the TCACoPc to the GOx, using the QCM-D. The aqueous metallophthalocyanine (MPc) formed a multi-layer over the surface of the electrode, which could be removed to leave a monolayer with a mass loading that compared favourably to the theoretical value expected. Analysis of frequency and dissipation plots indicated covalent attachment of glucose oxidase onto the metallophthalocyanine layer. The amount of GOx bound using the model system compared favourably to calculations derived from the maximal amperometric functioning of the electrochemical sensor (examined in previously-published literature, Mashazi, P.N., Ozoemena, K.I., Nyokong, T., 2006. Electrochim. Acta 52, 177-186), but not to theoretical values derived from dimensions of GOx as established by crystallography. The strength of the binding of the GOx film with the TCACoPc layer was tested by using 2% SDS as a denaturant/surfactant, and the GOx film was not found to be significantly affected by exposure to this. This paper thus showed that QCM-D can be used in order to model essential processes and interactions that dictate the functional parameters of a biosensor.  相似文献   

7.
An amperometric glucose biosensor based on an n-alkylamine-stabilized palladium nanoparticles (PdNPs)-glucose oxidase (GOx) modified glassy carbon (GC) electrode has been successfully fabricated. PdNPs were initially synthesized by a biphase mixture of water and toluene method using n-alkylamines (dodecylamine, C??-NH? and octadecylamine, C??-NH?) as stabilizing ligands. The performance of the PdNPs-GOx/GC biosensor was studied by cyclic voltammetry. The optimum working potential for amperometric measurement of glucose in pH 7.0 phosphate buffer solution is -0.02 V (vs. Ag/AgCl). The analytical performance of the biosensor prepared from C??-PdNPs-GOx is better than that of C??-PdNPs-GOx. The C??-PdNPs-GOx/GC biosensor exhibits a fast response time of ca. 3s, a detection limit of 3.0 μM (S/N=3) and a linear range of 3.0 μM-8.0 mM. The linear dependence of current density with glucose concentration is 70.8 μA cm?2 mM?1. The biosensor shows good stability, repeatability and reproducibility. It has been successfully applied to determine the glucose content in human blood serum samples.  相似文献   

8.
A novel cholesterol biosensor was prepared based on gold nanoparticles-catalyzed luminol electrogenerated chemiluminescence (ECL). Firstly, l-cysteine-reduced graphene oxide composites were modified on the surface of a glassy carbon electrode. Then, gold nanoparticles (AuNPs) were self-assembled on it. Subsequently, cholesterol oxidase (ChOx) was adsorbed on the surface of AuNPs to construct a cholesterol biosensor. The stepwise fabrication processes were characterized with cyclic voltammetry and atomic force microscopy. The ECL behaviors of the biosensor were also investigated. It was found that AuNPs not only provided larger surface area for higher ChOx loading but also formed the nano-structured interface on the electrode surface to improve the analytical performance of the ECL biosensor for cholesterol. Besides, based on the efficient catalytic ability of AuNPs to luminol ECL, the response of the biosensor to cholesterol was linear range from 3.3 μM to 1.0 mM with a detection limit of 1.1 μM (S/N=3). In addition, the prepared ECL biosensor exhibited satisfying reproducibility, stability and selectivity. Taking into account the advantages of ECL, we confidently expect that ECL would have potential applications in biotechnology and clinical diagnosis.  相似文献   

9.
Xian Y  Hu Y  Liu F  Xian Y  Wang H  Jin L 《Biosensors & bioelectronics》2006,21(10):1996-2000
In this paper, we report a novel glucose biosensor based on composite of Au nanoparticles (NPs)-conductive polyaniline (PANI) nanofibers. Immobilized with glucose oxidase (GOx) and Nafion on the surface of nanocomposite, a sensitive and selective biosensor for glucose was successfully developed by electrochemical oxidation of H2O2. The glucose biosensor shows a linear calibration curve over the range from 1.0x10(-6) to 8.0x10(-4) mol/L, with a slope and detection limit (S/N=3) of 2.3 mA/M and 5.0x10(-7) M, respectively. In addition, the glucose biosensor system indicates excellent reproducibility (less than 5% R.S.D.) and good operational stability (over 2 weeks).  相似文献   

10.
A new procedure for fabricating deoxyribonucleic acid (DNA) electrochemical biosensor was developed based on covalent immobilization of target single-stranded DNA (ssDNA) on Au electrode that had been functionalized by direct coupling of sol-gel and self-assembled technologies. Two siloxanes, 3-mercaptopropyltrimethoxysiloxane (MPTMS) and 3-glycidoxypropyltrimethoxysiloxane (GPTMS) were used as precursors to prepare functionally self-assembly sol-gel film on Au electrode. The thiol group of MPTMS allowed assembly of MPTMS sol-gel on gold electrode surface. Through co-condensation between silanols, GPTMS sol-gel with epoxide groups interconnected into MPTMS sol-gel and enabled covalent immobilization of target NH(2)-ssDNA through epoxide/amine coupling reaction. The concentration of MPTMS and GPTMS influenced the performance of the resulting biosensor due to competitive sol-gel process. The linear range of the developed biosensor for determination of complementary ssDNA was from 2.51 x 10(-9) to 5.02 x 10(-7)M with a detection limit of 8.57 x 10(-10)M. The fabricated biosensor possessed good selectivity and could be regenerated. The covalent immobilization of target ssDNA on self-assembled sol-gel matrix could serve as a versatile platform for DNA immobilization and fabrication of biosensors.  相似文献   

11.
A novel electrochemical biosensor was developed for detecting short DNA oligonucleotide of Bacillus thuringiensis (Bt) transgenic sequence based on Ag nanoparticle aggregates. To fabricate this DNA biosensor, the thiol-modified capture DNA (cDNA) was first anchored on gold (Au) electrode, and then the target DNA (tDNA) was hybridized with the immobilized cDNA. Subsequently, the probe DNA (pDNA) functionalized by biotinylated Ag nanoparticle was associated with the fixed tDNA, and the single Ag nanoparticle label was obtained (cited as SAg label). Finally, dissociative biotinylated Ag nanoparticle was bound to the resultant biotinylated SAg label assembled on Au electrode by virtue of bridge molecule streptavidin (SA) through biotin-SA specific interaction, which could lead to in situ aggregate of Ag nanoparticles on Au electrode and induce a novel tag including multiple Ag nanoparticles (cited as MAg tag). The novel tag exhibited excellent electroactive property in the solid-state Ag/AgCl process and was successfully applied to Bt transgenic sequence assay. A detection limit of 10 fM was achieved, which was improved by three orders of magnitude as compared to the SAg label. Furthermore, this novel DNA biosensor demonstrated a good selectivity towards tDNA.  相似文献   

12.
Zhao H  Ju H 《Analytical biochemistry》2006,350(1):138-144
A bilayer of the polyelectrolytes poly(dimethyldiallylammonium chloride) (PDDA) and poly(sodium 4-styrenesulfonate) (PSS) was formed on a 3-mercapto-1-propanesulfonic-acid-modified Au electrode. Subsequently, multiwall carbon nanotubes (MWCNTs) wrapped by positively charged PDDA were assembled layer-by-layer with negatively charged glucose oxidase (GOx) onto the PSS-terminated bilayer. Electrochemical impedance spectroscopy and atomic force microscopy were adopted to monitor the regular growth of the PDDA-MWCNTs/GOx bilayers. Using GOx as a model enzyme, the assembled multilayer membranes showed some striking features such as the adsorbed form of GOx on individual MWCNT, uniformity, good stability, and electrocatalytic activity toward oxygen reduction. Based on the consumption of dissolved oxygen during the oxidation process of glucose catalyzed by the immobilized GOx, a sensitive amperometric biosensor was developed for the detection of glucose up to 5.0 mM with a detection limit of 58 microM. The sensitivity increased with increasing sensing layers up to five bilayers. Ascorbic acid and uric acid did not cause any interference due to the use of a low operating potential. The present method showed high reproducibility for the fabrication of carbon-nanotubes-based amperometric biosensors.  相似文献   

13.
Polyaniline protected gold nanoparticles (PPAuNPs) were electrophoretically deposited onto a gold electrode, and utilized to fabricate an electrochemical cortisol biosensor. Cortisol specific monoclonal antibody (C-Mab) was covalently immobilized onto the surface of a PPAuNP/Au electrode using N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) chemistry. BSA was employed for blocking nonspecific adsorption on the electrode surface. PPAuNP formation and BSA/C-Mab/PPAuNP/Au electrode fabrication were characterized using transmission electron microscopy, atomic force microscopy and electrochemical impedance techniques, respectively. Cyclic voltammetry and differential pulse voltammetric techniques were used to determine the cortisol concentration in a phosphate buffer saline (PBS) solution. Results confirmed that the PPAuNP based electrode was stable during repeated scans and exhibited repeatable redox peaks. Further, the BSA/C-Mab/PPAuNP/Au electrode in the PBS buffer accurately detected cortisol in the range of 1 pM-100 nM with a sensitivity of 1.63 μAM(-1). The biosensor was found to be selective against BSA and 17-α-hydroxy progesterone. This research establishes the feasibility of using a PPAuNP based matrix for a label and mediator free electrochemical biosensor for cortisol, a stress biomarker.  相似文献   

14.
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).  相似文献   

15.
A nanobiocomposite film consisted of polypyrrole (PPy), functionalized multiwalled carbon nanotubes (cMWNTs), and glucose oxidase (GOx) were electrochemically synthesized by electrooxidation of 0.1M pyrrole in aqueous solution containing appropriate amounts of cMWNTs and GOx. Potentiostatic growth profiles indicate that the anionic cMWNTs is incorporated within the growing PPy-cMWNTs nanocomposite for maintaining its electrical neutrality. The morphology of the PPy-cMWNTs nanocomposite was characterized by scanning electron microscopy (SEM). The PPy-cMWNTs nanocomposite was deposited homogeneously onto glassy carbon electrode. The amperometric responses vary proportionately to the concentration of hydrogen peroxide at the PPy-cMWNTs nanocomposite modified electrode at an operating potential of 0.7V versus Ag/AgCl (3M). The results indicate that the electroanalytical PPy-cMWNTs-GOx nanobiocomposite film was highly sensitive and suitable for glucose biosensor based on GOx function. The GOx concentration within the PPy-cMWNTs-GOx nanobiocomposite and the film thickness are crucial for the performance of the glucose biosensor. The amperometric responses of the optimized PPy-cMWNTs-GOx glucose biosensor (1.5 mgmL(-1) GOx, 141 mCcm(-2) total charge) displayed a sensitivity of 95 nAmM(-1), a linear range up to 4mM, and a response time of about 8s.  相似文献   

16.
Huang Y  Qin X  Li Z  Fu Y  Qin C  Wu F  Su Z  Ma M  Xie Q  Yao S  Hu J 《Biosensors & bioelectronics》2012,31(1):357-362
Enzyme immobilization is one of the key factors in constructing high-performance enzyme biosensors and biofuel cells (BFCs). Herein, we propose a new protocol for efficient immobilization of a glycoprotein enzyme based on the interaction of the 1, 2- or 1, 3-diols in the glycoprotein with a boronic acid functionalized monomer. Briefly, casting a mixture of glucose oxidase (GOx) and anilineboronic acid (ABA) followed by a NaAuCl(4) solution to an Au-plated Au electrode surface yielded a GOx-poly(ABA) (PABA)-gold nanoparticle (Au(nano)) bionanocomposite, and chitosan (CS) was then cast and air-dried. In the present protocol, the small-sized Au(nano) or Au subnanostructures can form near/on the enzyme molecule, which greatly promotes the electron transfer of enzymatic reaction and enhances the amperometric responses. The thus-prepared CS/GOx-PABA-Au(nano)/Au-plated Au electrode worked well in the first-/second generation biosensing modes and as a bioanode in a monopolar biofuel cell, with analytical or cell-power performance superior to those of most analogues hitherto reported.  相似文献   

17.
A simple and relatively cheap glucose biosensor based on a combination of gold nanoparticles (Au NPs) and glucose oxidase (GO(x) ) immobilized on a bioplatform eggshell membrane was established. Scanning electron microscopy showed successful immobilization of Au NPs/GO(x) on the eggshell membrane. The effects of pH, phosphate buffer concentration, and temperature on the glucose biosensor were studied in detail. The biosensor shows a linear response at a glucose concentration range of 5-525 μM. The detection limit of the biosensor is 2.5 μM (S/N = 3). The biosensor exhibits good repeatability with RSD = 3.6% (n = 6), good operational stability with over 300 measurements and long-term storage stability with a shelf life of at least 6 months. The response time is less than 60 s. The glucose level in commercial food samples has been successfully determined. The proposed work shows potential to develop cost-effective biosensors for biotechnological, biomedical and industrial use.  相似文献   

18.
Development of reagentless biosensors implies the tight and functional immobilisation of biological recognition elements on transducer surfaces. Specifically, in the case of amperometric enzyme electrodes, electron-transfer pathways between the immobilised redox protein and the electrode surface have to be established allowing a fast electron transfer concomitantly avoiding free-diffusing redox species. Based on the specific nature of different redox proteins and non-manual immobilisation procedures possible biosensor designs are discussed, namely biosensors based on (i) direct electron transfer between redox proteins and electrodes modified with self-assembled monolayers; (ii) anisotropic orientation of redox proteins at monolayer-modified electrodes; (iii) electron-transfer cascades via redox hydrogels; and (iv) electron-transfer via conducting polymers.  相似文献   

19.
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.  相似文献   

20.
A surface modification procedure for the creation of self-assembled monolayers (SAMs) that can be used as a scaffold for double-stranded DNA (dsDNA) incorporation onto the gold surfaces is described. The SAMs of an azidohexane thiol derivative were prepared on the Au electrode and then used for the immobilization of dsDNA. The electrochemical characteristics of dsDNA onto the SAM-modified gold electrode were investigated by cyclic voltammetry and electrochemical impedance spectroscopy, and the surface concentration of dsDNA onto the SAMs surface was estimated. The interaction of dsDNA with the anticancer drug, taxol (paclitaxel), was also studied on the surface of DNA/SAM/Au electrode. The observed decrease in the guanine oxidation peak current was used to monitor the interaction of taxol with DNA. The resulting Langmuir isotherm for taxol binding to DNA at the modified electrode was used to evaluate the binding constant of taxol-DNA. The results obtained supported the groove binding interaction of taxol with DNA. The modified electrode was used as a sensitive sensor for quantification of taxol in human serum sample.  相似文献   

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