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PubMed2005

Development of an oral biosensor for salivary amylase using a monodispersed silver for signal amplification.

An amperometric biosensor for monitoring the level of protein amylase in human saliva is described. A novel design and the preparation of amylase antibodies and antigens, essential for the development of the biosensor, are reported. The biosensor sensing elements comprise a layer of salivary antibody (or antigen) self-assembled onto Au-electrode via covalent attachment. Molecular recognition between the immobilized antibody and the salivary amylase proteins was monitored via an electroactive indicator (e.g., K(3)Fe(CN)(6)) or a monodispersed silver layer present in solution or electrochemically deposited onto the solid electrode. This electroactive indicator was oxidized or reduced and the resulting current change provided the analytical information about the concentration of the salivary proteins. The limit of detection of 1.57 pg/ml was obtained, in comparison to detection limits of 4.95 pg/ml obtained using potassium ferrocyanide as the redox probe and 10 ng/ml obtained using enzyme-linked immunosorbent assay. Cross-reactivity was tested against cystatin antibodies and was found to be less than 2.26%.

Amylases↗

PubMed2005

Amperometric biosensor for choline based on layer-by-layer assembled functionalized carbon nanotube and polyaniline multilayer film.

Conducting polymer film was prepared by electrochemical polymerization of aniline. Multiwalled carbon nanotubes (MWNTs) were treated with a mixture of concentrated sulfuric and nitric acid to introduce carboxylic acid groups to the nanotubes. By using the layer-by-layer method, homogeneous and stable MWNTs and polyaniline (PANI) multilayer films were alternately assembled on glassy carbon (GC) electrodes. Conducting polymer of PANI had three main functions: (i) excellent antiinterference ability, (ii) protection ability in favor of increasing the amount of the MWNTs immobilized on GC electrodes, and (iii) superior transducing ability. The protection effect of PANI film and the electrostatic interaction between positively charged PANI and negatively charged MWNTs both attributed to immobilizing abundant MWNTs stably, thereby enhancing the catalytic activity. The layer-by-layer assembled MWNTs and PANI-modified GC electrodes offered a significant decrease in the overvoltage for the H2O2 and were shown to be excellent amperometric sensors for H2O2 from +0.2V over a wide range of concentrations. As an application example, by linking choline oxidase (CHOD), an amplified biosensor toward choline was prepared. The choline biosensor exhibited a linear response range of 1x10(-6) to 2x10(-3) M with a correlation coefficient of 0.997, and the response time and detection limit (S/N=3) were determined to be 3 s and 0.3 microM, respectively. The antiinterference biosensor displays a rapid response and an expanded linear response range as well as excellent reproducibility and stability.

Alcohol Oxidoreductases↗

PubMed2005

Amperometric, screen-printed, glucose biosensor for analysis of human plasma samples using a biocomposite water-based carbon ink incorporating glucose oxidase.

This paper describes the optimisation of a screen-printing water-based carbon ink containing cobalt phthalocyanine (CoPC) and glucose oxidase (GOD) for the fabrication of a glucose biosensor. To optimise the performance of the biosensor, the loadings of the electrocatalyst (CoPC) and enzyme (GOD) were varied. It was found that the maximum linear range was achieved with a CoPC loading of 20% (m/m, relative to the mass of carbon) and a GOD loading of 628 U per gram of carbon. In our studies we chose to employ chronoamperometry, as this technique is commonly used for commercial devices. The optimum operating applied potential was found to be +0.5 V, following an incubation period of 60 s. The optimum supporting electrolyte was found to be 0.05 M phosphate buffer at pH 8.0, which resulted in a linear range of 0.2-5 mM, the former represents the detection limit. The sensitivity was 1.12 microA mM(-1). The effect of temperature was also investigated, and it was found that 40 degrees C gave optimal performance. The resulting amperometric biosensors were evaluated by measuring the glucose concentrations for 10 different human plasma samples containing endogenous glucose and also added glucose. The same samples were analysed by a standard spectrophotometric method, and the results obtained by the two different methods were compared. A good correlation coefficient (R(2) = 0.95) and slope (0.98) were calculated from the experimental data, indicating that the new devices hold promise for biomedical studies.

Biosensing Techniques↗

PubMed2005

New immobilization method for immunoaffinity biosensors by using thiolated proteins.

A new immobilization method for immunoaffinity (IA) biosensors that ensures the high surface density and the stability of the IA layer was developed. For the immobilization of biomolecules, the molecular recognition protein was first thiolated by covalent conjugation of mercaptopropionic acid, and then the thiolated protein was attached on the gold surface of the transducer. In this work, horseradish peroxidase (HRP) and its antibody were used as a model antigen-antibody, and the following properties of the IA layer prepared by thiolated protein were estimated: (i) biological integrity of HRP after the immobilization process by using activity assay, (ii) charge transfer resistance by immobilization, (iii) mass loading by the surface plasmon resonance (SPR) biosensor, (iv) number of binding sites, and (v) feasibility test for the measurement of capacitive change by the antigen-antibody interaction. Based on these parameters, the immobilization method by using thiolated protein was determined to be feasible for application to IA biosensors.

Affinity Labels↗

PubMed2004

A stable three enzyme creatinine biosensor. 2. Analysis of the impact of silver ions on creatine amidinohydrolase.

The enzyme creatine amidinohydrolase is a clinically important enzyme used in the determination of creatinine in blood and urine. Continuous use biosensors are becoming more important in the clinical setting; however, long-use creatinine biosensors have not been commercialized due to the complexity of the three-enzyme creatinine biosensor and the lack of stability of its components. This paper, the second in a series of three, describes the immobilization and stabilization of creatine amidinohydrolase. Creatine amidinohydrolase modified with poly(ethylene glycol) activated with isocyanate retains significant activity after modification. The enzyme was successfully immobilized into hydrophilic polyurethanes using a reactive prepolymer strategy. The immobilized enzyme retained significant activity over a 30 day period at 37 degrees C and was irreversibly immobilized into the polymer. Despite being stabilized in the polymer, the enzyme remained highly sensitive to silver ions which were released from the amperometric electrodes. Computational analysis of the structure of the protein using the Gaussian network model suggests that the silver ions bind tightly to a cysteine residue preventing normal enzyme dynamics and catalysis.

Biosensing Techniques↗

PubMed2004

DNA biosensors based on self-assembled carbon nanotubes.

DNA biosensors based on self-assembled multi-walled carbon nanotubes (MWNTs) were described in this paper, in which the probe DNA oligonucleotides were immobilized by forming covalent amide bonds between carboxyl groups at the nanotubes and amino groups at the ends of the DNA oligonucleotides. Hybridization between the probe and target DNA oligonucleotides was confirmed by the changes in the voltammetric peak of the indicator of methylene blue. Our results demonstrate that the DNA biosensors based on self-assembled MWNTs had a higher hybridization efficiency compared to those based on random MWNTs. In addition, the developed DNA biosensors also had a high selectivity of hybridization detection.

Adsorption↗

PubMed2006

F0F1-ATPase as biosensor to detect single virus.

F(0)F(1)-ATPase within chromatophore was constructed as a biosensor (immuno-rotary biosensor) for the purpose of capturing single virus. Capture of virus was based on antibody-antigen reaction. The detection of virus based on proton flux change driven by ATP-synthesis of F(0)F(1)-ATPase, which was indicated by F1300, was directly observed by a fluorescence microscope. The results demonstrate that the biosensor loading of virus particles has remarkable signal-to-noise ratio (3.8:1) compared to its control at single molecular level, and will be convenient, quick, and even super-sensitive for detecting virus particles.

Biosensing Techniques↗

PubMed2005

The electrochemical behaviour of ferrocene in a photocurable poly(methyl methacrylate-co-2-hydroxylethyl methacrylate) film for a glucose biosensor.

A single-step fabrication of a glucose biosensor with simultaneous immobilization of both ferrocene mediator and glucose oxidase in a photocurable methacrylic film consisting of poly(methyl methacrylate-co-2-hydroxylethyl methacrylate) was reported. The entrapped ferrocene showed reversible redox behaviour in the photocured film and no significant leaching of both entrapped ferrocene and enzyme glucose oxidase was observed because of the low water absorption properties of the co-polymer films. From electrochemical studies, ferrocene entrapped in the co-polymer film demonstrated slow diffusion properties. A linear glucose response range of 2-11 mM was obtained at low applied potential of +0.25 V. The glucose biosensor fabricated by this photocuring method yielded sensor reproducibility and repeatability with relative standard deviation of <10% and long-term stability of up to 14 days. The main advantage of the use of photocurable procedure is that biosensor membrane fabrication can be performed in a single step without any lengthy chemical immobilization of enzyme.

Biosensing Techniques↗

PubMed2005

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.

Biosensing Techniques↗

PubMed2006

Parallel synthesis of libraries of anodic and cathodic functionalized electrodeposition paints as immobilization matrix for amperometric biosensors.

The integration of flexible anchoring groups bearing imidazolyl or pyridyl substituents into the structure of electrodeposition paints (EDP) is the basis for the parallel synthesis of a library containing 107 members of different cathodic and anodic EDPs with a high variation in polymer properties. The obtained EDPs were used as immobilization matrix for biosensor fabrication using glucose oxidase as a model enzyme. Amperometric glucose sensors based on the different EDPs showed a wide variation in their sensor characteristics with respect to the apparent Michaelis-Menten constant (KM(app)) representing the linear measuring range and the maximum current (Imax(app)). Based on these results first assumptions concerning the impact of different side chains in the EDP on the expected biosensor properties could be obtained allowing for an improved rational optimization of EDPs used as immobilization matrix in amperometric biosensors.

Biosensing Techniques↗

PubMed2006

Twenty years research in cholinesterase biosensors: from basic research to practical applications.

Over the last decades, cholinesterase (ChE) biosensors have emerged as an ultra sensitive and rapid technique for toxicity analysis in environmental monitoring, food and quality control. These systems have the potential to complement or replace the classical analytical methods by simplifying or eliminating sample preparation protocols and making field testing easier and faster with significant decrease in costs per analysis. Over the years, engineering of more sensitive ChE enzymes, development of more reliable immobilization protocols and progress in the area of microelectronics could allow ChE biosensors to be competitive for field analysis and extend their applications to multianalyte screening, development of small, portable instrumentations for rapid toxicity testing, and detectors in chromatographic systems. In this paper, we will review the research efforts over the last 20 years in fabricating AChE biosensors and the recent trends and challenges encounter once the sensor is used outside research laboratory for in situ real sample applications. The review will discuss the generations of cholinesterase sensors with their advantages and limitations, the existing electrode configurations and fabrication techniques and their applications for toxicity monitoring. We will focus on low-cost electrochemical sensors and the approaches used for enzyme immobilization. Recent works for achieving high sensitivity and selectivity are also discussed.

Biosensing Techniques↗

PubMed2006

Hydroxyl-containing antimony oxide bromide nanorods combined with chitosan for biosensors.

A hydroxyl-containing antimony oxide bromide (AOB) nanorods was synthesized by a hydrothermal method. TEM and SEM images showed that the as-prepared AOB nanorods were very copious with diameters of about 50 nm. The AOB nanorods could be easily combined with biopolymer chitosan (Chi) to form an organic-inorganic hybrid material, and a biocompatible, crack-free and porous Chi-AOB composite film could be readily obtained. Horseradish peroxidase (HRP) was chosen as a model protein to construct a reagentless mediator-free third-generation HRP biosensor. UV-visible and FTIR spectroscopy revealed that HRP entrapped in the composite film could retain its native secondary structure. A pair of stable and well-defined redox peaks of HRP with a formal potential of about -0.24 V (vs. Ag/AgCl) in a pH 7.0 phosphate-buffered solution (PBS) were obtained at the HRP-Chi-AOB composite film modified glassy carbon (GC) electrode. With advantages of organic-inorganic hybrid materials, dramatically facilitated direct electron transfer of HRP and excellent bioelectrocatalytic activity towards H(2)O(2) were demonstrated. The apparent Michaelis-Menten constant K(M)(app) was calculated to be 7.5mum, indicating that HRP entrapped in the composite film possessed high affinity to H(2)O(2) and exhibited high enzymatic activity. The prepared biosensor displayed good sensitivity and reproducibility, wide linear range, low detection limit, fast response and excellent long-term stability. The Chi-AOB composite film could be used efficiently for the entrapment of other redox-active proteins and may find wide potential applications in biosensors, biocatalysis, biomedical devices and bioelectronics.

Antimony↗

PubMed2004

A biosensor for the analysis of acetonitrile.

A biosensor for monitoring acetonitrile was constructed. A mixed culture was taken from a degradation reactor and mounted on top of a Clark electrode. The amperometric biosensor was placed in a flow-through cell and integrated into a flow injection system. The metabolic response in terms of oxygen consumption was well correlated to the concentration of acetonitrile in standard solutions. However, when the reaction products, acetic acid and ammonia, were also present, the response was erratic, due to the additional metabolic reaction on acetate. By introducing a hydrophobic barrier it was possible to eliminate the negative influence of these charged products and thus to improve the operational selectivity of the sensor. The biosensor showed good stability for analysis during at least 6 days and future work will focus on using it for monitoring and control of degradation processes.

Acetonitriles↗

PubMed2004

A novel method to construct a third-generation biosensor: self-assembling gold nanoparticles on thiol-functionalized poly(styrene-co-acrylic acid) nanospheres.

A novel method for fabrication of horseradish peroxidase (HRP) biosensor has been developed by self-assembling gold nanoparticles on thiol-functionalized poly(styrene-co-acrylic acid) (St-co-AA) nanospheres. At first, a cleaned gold electrode was immersed in thiol-functionalized poly(St-co-AA) nanosphere latex prepared by emulsifier-free emulsion polymerization of St with AA and function with dithioglycol to assemble the nanospheres, then gold nanoparticles were chemisorbed onto the thiol groups. Finally, horseradish peroxidase was immobilized on the surface of the gold nanoparticles. The sensor displayed an excellent electrocatalytical response to reduction of H2O2 without the aid of an electron mediator. The sensor was highly sensitive to hydrogen peroxide with a detection limit of 4.0 micromoll(-1), and the linear range was from 10.0 micromoll(-1) to 7.0 mmoll(-1). The biosensor retained more than 97.8% of its original activity after 60 days of use. Moreover, the studied biosensor exhibited good current repeatability and good fabrication reproducibility.

Acrylic Resins↗

PubMed2004

Determination of beta-D-glucose using flow injection analysis and composite-type amperometric tubular biosensors.

An amperometric tubular cell involving composite biosensors for the determination of beta-d-glucose in a flow injection analysis (FIA) system is proposed. Diverse configurations and parameters are evaluated to improve the system's response. The configuration producing less noise resulted when the biosensor was located closer to the auxiliary electrode, which also required coupling both electrodes within the system under a continuous flow regime. Further, we report on the influence of the active area of the biosensor and of the flow rate used. Statistical analyses of the data revealed two regions with a linear response range for the determination of beta-d-glucose, with a detection limit of 4.7 x 10(-4) M and in the low concentration region a sensitivity of 17.46 +/- 0.12 microAM(-1). At the beta-d-glucose concentrations studied there was no evidence of enzymatic saturation. An increment on the ionic strength of the sample and carrier passing through the analysis system decreases its sensitivity. The reproducibility of the analytical system in terms of its standard deviation was 2.9% with a 95% confidence level, having a lifetime that lasted at least 100 days. beta-d-Glucose was determined in different commercial medical glucose-containing solutions, the experimental results are in good agreement with those reported by the manufacturer.

Biosensing Techniques↗

PubMed2004

Novel biosensors for quantitative phytic acid and phytase measurement.

Phytase (EC 3.1.3.26) and phytic acid (myo-inositol hexaphosphate) play an important environmental role in poultry industry and have a health aspect in food industry. Novel biosensors have been developed for simple, one step quantitative phytic acid and phytase detection. A system based on the sequentially acting enzyme phytase and pyruvate oxidase (POD) was employed for the development of phytase and phytic acid biosensors. Poly(carbamoylsulphonate) (PCS) hydrogel immobilized POD electrode was applied for the detection of phytase. It was based on the indication of phosphate ions produced by the hydrolysis of phytic acid. The phytase biosensor showed a linear response ranging from 0.5 to 6.0 units/ml. A bi-enzyme sensor based on co-immobilization of phytase and POD was developed for the detection of phytic acid on the basis of amperometric detection of the enzymatically-generated hydrogen peroxide at 0.6 V versus Ag/AgCl. It showed a linear response ranging from 0.2 to 2.0 mM with a detection limit of 0.002 mM.

6-Phytase↗

PubMed2004

A novel approach to improve specificity of algal biosensors using wild-type and resistant mutants: an application to detect TNT.

A new genetic approach was developed for increasing specificity of microalgal biosensors. This method is based on the use of two different genotypes jointly to detect a given pollutant: (i) a sensitive genotype to obtain sensitivity; and (ii) a resistant mutant to obtain specificity. The method was tested by the development of a microalgal biosensor for the detection of the explosive 2,4,6-trinitrotoluene (TNT) using a wild-type strain (DcG1wt) of Dictyosphaerium chlorelloides (Chlorophyceae) as the sensitive organism, and a TNT-resistant mutant, obtained from DcG1wt strain by a modified Luria-Delbrück fluctuation analysis. The inhibition of chlorophyll a fluorescence of PSII by TNT was used as the biological signal. Significant differences in maximal fluorescence of light-adapted algae (F'(m)) between wild-type DcG1wt cells and TNT-resistant mutants, were observed in all the TNT concentrations tested (from 0.5 to 31.3 mg l(-1)) after only 3 min of exposure. Resistant mutants always exhibited significant higher F'(m) values in the presence of TNT than wild-type cells. These results suggest that the use of two different genotypes (sensitive and resistant to a given pollutant) jointly is a useful method to improve microalgal biosensors specificity.

Biosensing Techniques↗

PubMed2004

Amperometric ATP biosensor based on polymer entrapped enzymes.

A dual enzyme electrode for the detection of adenosine-5'-triphosphate (ATP) at physiologically relevant pH levels was developed by co-immobilization of the enzymes glucose oxidase (GOD) and hexokinase (HEX) using pH-shift induced deposition of enzyme containing polymer films. Application of a simple electrochemical procedure for the co-immobilization of the enzymes at electrode surfaces exhibits a major improvement of sensitivity, response time, reproducibility, and ease of fabrication of ATP biosensors. Competition between glucose oxidase and hexokinase for the substrate glucose involving ATP as a co-substrate allows the determination of ATP concentrations. Notable control on the immobilization process enables fabrication of micro biosensors with a diameter of 25 microm. The presented concept provides the technological basis for a new generation of fast responding, sensitive, and robust biosensors for the detection of ATP at physiological pH values with a detection limit of 10 nmol l(-1).

Adenosine Triphosphate↗