Disposable electrodes modified with multi-wall carbon nanotubes for biosensor applications

This paper deals with the development of a disposable electrochemical sensor for the detection of hydrogen peroxide, using screen-printed carbon-based electrodes (SPCEs) modified with multi-wall carbon nanotubes (MWCNs) dispersed in a polyethylenimine (PEI) mixture. The modified sensors showed an excellent electrocatalytic activity towards the analyte, respect to the high overvoltage characterising unmodified screen-printed sensors. The composition of the PEI/MWCNT dispersion was optimised in order to improve the sensitivity and reproducibility. The optimised sensor showed good reproducibility (10% RSD calculated on three experiments repeated on the same electrode), whereas a reproducibility of 15% as RSD was calculated on electrodes from different preparations. Preliminary experiments carried out using glucose oxidase (GOD) as biorecognition element gave rise to promising results indicating that these new devices may represent interesting components for biosensor construction.     

Sensor and biosensor based on Prussian Blue modified gold and platinum screen printed electrodes

Gold (Au) and platinum (Pt) screen-printed electrodes were modified with Prussian Blue (PB) for the development of amperometric sensors selective for hydrogen peroxide detection. The sensors exhibited sensitivities towards H(2)O(2) equal to 2 A M(-1) cm(-2) for Au and 1 A M(-1) cm(-2) for Pt electrodes. The sensors were also employed as the basis for construction of glucose biosensors through further modification with crystallised glucose oxidase immobilised in a Nafion membrane. In order to improve the operational stability of the modified electrodes a buffer solution containing tetrabutylammonium toluene-4-sulfonate was used. The long-term performance of the sensors and biosensors were evaluated by continuous monitoring of hydrogen peroxide and glucose solutions (50 microM and 1 mM, respectively) in the flow-injection mode for 10 h.     

Glucose biosensor based on carbon black strips.

Amperometric biosensors for the determination of beta-D-glucose have been constructed. They were based on a porous matrix of carbon blacks--'Ketjenblack' (KB) and 'Shawinigan black' (SB) wet-proofed with polytetrafluorethylene. Glucose-sensitive elements were prepared by subsequent adsorptional immobilization of 1,1'-dimethylferrocene (DMFc) and nickel-ocene (Nc) on 'Shawinigan black' or tetracyanoquinodimethane (TCNQ) on 'Ketjenblack' together with Penicillium chrysogenum glucose oxidase. Maximum surface concentrations of DMFc, Nc and TCNQ on carbon black electrodes were 95, 116 and 151 nmol cm-2. The biosensor based on KB and TCNQ (KB-TCNQ biosensor) could be used at a potential of 0.5 V (vs. Ag/AgCl reference electrode) in the concentration range up to 7 mM. This biosensor possessed an approximately ten times higher sensitivity than the ones based on SB and DMFc (SB-DMFc biosensor) and on SB and Nc (SB-Nc biosensor) which acted at 0.3 V and 0.05 V, respectively. The biosensors were suitable for practical use longer than one week.     

Development of disposable amperometric sulfur dioxide biosensors based on screen printed electrodes

The possibility of developing amperometric biosensors for the measurement of SO(2) in flowing gas streams has been examined. Screen-printed carbon electrodes (SPCEs) were tailored with the enzyme sulfite oxidase and cytochrome c and the response is generated through the resulting enzymatic and electrocatalytic reactions involving SO(3)(2-), formed when SO(2) gas is dissolved in the supporting electrolyte. Two methods of integrating the enzyme and cytochrome c with the SPCE were investigated. In one design (b-type biosensor), the components were mixed thoroughly with the same ink used to produce the SPCEs, then the modified ink was spread over the working electrode. In the second approach the bio-components were dissolved in the supporting electrolyte and simply deposited on top of the transducer (s-type biosensor). Both devices gave linear responses over the range 4--50 ppm but the sensitivity of the s-type was approximately twice that of the b-type biosensor. In addition, the time taken to reach 90% of the maximum response (t(90%)) was 110 s for the s-type biosensor compared with 200 s for the b-type biosensor. These studies illustrate the successful use of biosensors for the detection of sulfur dioxide at the relatively low potential of +0.3 V versus Ag.AgCl and should provide useful alternatives for decentralised environmental studies.     

Microbial biosensor for detection of methyl parathion using screen printed carbon electrode and cyclic voltammetry

Whole cells of recombinant Escherichia coli were immobilized on the screen printed carbon electrode (SPCE) using glutaraldehyde. Recombinant E. coli was having high periplasmic expression of organophosphorus hydrolase enzyme, which hydrolyzes the methyl parathion into two products, p-nitrophenol and dimethyl thiophosphoric acid. Cells immobilized SPCE was studied under SEM. Cells immobilized SPCE was associated with cyclic voltammetry and cyclic voltammograms were recorded before and after hydrolysis of methyl parathion. Detection was calibrated based on the relationship between the changes in the current observed at +0.1 V potential, because of redox behavior of the hydrolyzed product p-nitrophenol. As concentration of methyl parathion was increased the oxidation current also increased. Only 20 μl volume of the sample was required for analysis. Detection range of biosensor was calibrated between 2 and 80 μM of methyl parathion from the linear range of calibration plot. A single immobilized SPCE was reused for 32 reactions with retention of 80% of its initial enzyme activity.     

DNA biosensor based on chitosan film doped with carbon nanotubes

A biosensor based on chitosan doped with carbon nanotube (CNT) was fabricated to detect salmon sperm DNA. Methylene blue (MB) was employed as a DNA indicator. It was found that CNTs can enhance the electroactive surface area threefold (0.28 +/- 0.03 and 0.093 +/- 0.06 cm(2) for chitosan-CNT- and chitosan-modified electrodes, respectively) and can accelerate the rate of electron transfer between the redox-active MB and the electrode. A low detection limit of 0.252 nM fish sperm DNA was achieved, and no interference was found in the presence of 5 microg/ml human serum albumin. The differential pulse voltammetry signal of MB was linear over the fish sperm DNA concentration range of 0.5-20 nM.     

Amperometric glucose biosensor based on multilayer films via layer-by-layer self-assembly of multi-wall carbon nanotubes, gold nanoparticles and glucose oxidase on the Pt electrode

A novel amperometric glucose biosensor based on the nine layers of multilayer films composed of multi-wall carbon nanotubes (MWCNTs), gold nanoparticles (GNp) and glucose oxidase (GOD) was developed for the specific detection of glucose. MWCNTs were chemically modified with the H₂SO₄–HNO₃ pretreatment to introduce carboxyl groups which were used to interact with the amino groups of poly(allylamine) (PAA) and cysteamine via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide cross-linking reaction, respectively. A cleaned Pt electrode was immersed in PAA, MWCNTs, cysteamine and GNp, respectively, followed by the adsorption of GOD, assembling the one layer of multilayer films on the surface of Pt electrode (GOD/GNp/MWCNTs/Pt electrode). Repeating the above process could assemble different layers of multilayer films on the Pt electrode. PBS washing was applied at the end of each assembly deposition for dissociating the weak adsorption. Film assembling and characterization were studied by transmission electron microscopy and quartz crystal microbalance, and properties of the resulting glucose biosensors were measured by electrochemical measurements. The marked electrocatalytic activity of Pt electrode based on multilayer films toward H₂O₂ produced during GOD enzymatic reactions with glucose permitted effective low-potential amperometric measurement of glucose. Taking the sensitivity and selectivity into consideration, the applied potential of 0.35 V versus Ag/AgCl was chosen for the oxidation detection of H₂O₂ in this work. Among the resulting glucose biosensors, the biosensor based on nine layers of multilayer films was best. It showed a wide linear range of 0.1–10 mM glucose, with a remarkable sensitivity of 2.527 μA/mM, a detection limit of 6.7 μM estimated at a signal-to-noise ratio of 3 and fast response time (within 7 s). Moreover, it exhibited good reproducibility, long-term stability and the negligible interferences of ascorbic acid, uric acid and acetaminophen. The study can provide a feasible approach on developing new kinds of oxidase-based amperometric biosensors, and can be used as an illustration for constructing various hybrid structures.     

Toward the development of smart and low cost point-of-care biosensors based on screen printed electrodes

Screen printing technology provides a cheap and easy means to fabricate disposable electrochemical devices in bulk quantities which are used for rapid, low-cost, on-site, real-time and recurrent industrial, pharmaceutical or environmental analyses. Recent developments in micro-fabrication and nano-characterization made it possible to screen print reproducible feature on materials including plastics, ceramics and metals. The processed features forms screen-printed disposable biochip (SPDB) upon the application of suitable bio-chemical recognition receptors following appropriate methods. Adequacy of biological and non-biological materials is the key to successful biochip development. We can further improve recognition ability of SPDBs by adopting new screen printed electrode (SPE) configurations. This review covers screen-printing theory with special emphasis on the technical impacts of SPE architectures, surface treatments, operational stability and signal sensitivity. The application of SPE in different areas has also been summarized. The article aims to highlight the state-of-the-art of SPDB at the laboratory scale to enable us in envisaging the deployment of emerging SPDB technology on the commercial scale.     

Glucose biosensor based on glucose oxidase immobilized at gold nanoparticles decorated graphene-carbon nanotubes

Biopolymer pectin stabilized gold nanoparticles were prepared at graphene and multiwalled carbon nanotubes (GR-MWNTs/AuNPs) and employed for the determination of glucose. The formation of GR-MWNTs/AuNPs was confirmed by scanning electron microscopy, X-ray diffraction, UV–vis and FTIR spectroscopy methods. Glucose oxidase (GOx) was successfully immobilized on GR-MWNTs/AuNPs film and direct electron transfer of GOx was investigated. GOx exhibits highly enhanced redox peaks with formal potential of −0.40 V (vs. Ag/AgCl). The amount of electroactive GOx and electron transfer rate constant were found to be 10.5 × 10⁻¹⁰ mol cm⁻² and 3.36 s⁻¹, respectively, which were significantly larger than the previous reports. The fabricated amperometric glucose biosensor sensitively detects glucose and showed two linear ranges: (1) 10 μM – 2 mM with LOD of 4.1 μM, (2) 2 mM – 5.2 mM with LOD of 0.95 mM. The comparison of the biosensor performance with reported sensors reveals the significant improvement in overall sensor performance. Moreover, the biosensor exhibited appreciable stability, repeatability, reproducibility and practicality. The other advantages of the fabricated biosensor are simple and green fabrication approach, roughed and stable electrode surface, fast in sensing and highly reproducible.     

Glucose biosensor based on electrodeposition of platinum nanoparticles onto carbon nanotubes and immobilizing enzyme with chitosan-SiO(2) sol-gel

A novel amperometric biosensor, based on electrodeposition of platinum nanoparticles onto multi-walled carbon nanotube (MWNTs) and immobilizing enzyme with chitosan-SiO(2) sol-gel, is presented in this article. MWNTs were cast on the glass carbon (GC) substrate directly. An extra Nafion coating was used to eliminate common interferents such as acetaminophen and ascorbic acids. The morphologies and electrochemical performance of the modified electrodes have been investigated by scanning electron microscopy (SEM) and amperometric methods, respectively. The synergistic action of Pt and MWNTs and the biocompatibility of chitosan-SiO(2) sol-gel made the biosensor have excellent electrocatalytic activity and high stability. The resulting biosensor exhibits good response performance to glucose with a wide linear range from 1 microM to 23 mM and a low detection limit 1 microM. The biosensor also shows a short response time (within 5s), and a high sensitivity (58.9 microAm M(-1)cm(-2)). In addition, effects of pH value, applied potential, rotating rate, electrode construction and electroactive interferents on the amperometric response of the sensor were investigated and discussed in detail.     

Nano-moles detection of tumor specific biomarker DNA for colorectal cancer detection using vertically aligned multi-wall carbon nanotubes based flexible electrodes

A genosensor, flexible and low cost as well, has been fabricated to efficiently detect colorectal cancer cell by targeting CEACAM5, a tumor biomarker. Sensing electrode was fabricated with efficient transfer of a pattern of vertically aligned multi-wall carbon nanotubes on flexible polyethylene terephthalate (PET) substrate by hot press technique, a technologically advanced one. The purpose was to make the sensor device stretchable, bendable, transparent, disposable and heat resistant; without losing the pristine character of the electrode material before and after transfer process. Fabricated sensor was characterized by cyclic voltammetry (CV) and electrochemical impedance spectra (EIS). The sensor response was checked by adding target DNA within a range of 50–250 μM and sensor showed a detection limit of 0.92μM; exhibited good linearity with a regression coefficient of 0.96 obtained through calibration plot. The sensor was found to be highly sensitive, stable and good specificity as binding occurs only with the complementary DNA. Besides, the genosensor showed relative standard deviation of 6.5 % and 6.7 % at respective 100 μM and 150 μM complementary DNA concentrations. Reproducibility and stability are two crucial parameters where the developed sensor have scored extremely well.     

Glucose biosensor based on titanium dioxide-multiwall carbon nanotubes-chitosan composite and functionalized gold nanoparticles

In this paper, a new glucose biosensor was prepared. At first, Prussian blue (PB) was electrodeposited on a glassy carbon electrode (GCE) modified by titanium dioxide-multiwall carbon nanotubes-chitosan (TiO₂-MWNTs-CS) composite, and then gold nanoparticles functionalized by poly(diallyldimethylammonium chloride) (PDDA-Au) were adsorbed on the PB film. Finally, the negatively charged glucose oxidase (GOD) was self-assembled on to the positively charged PDDA-Au. The electrochemical performances of the modified electrodes had been studied by cyclic voltammetry (CV) and amperometric methods, respectively. In addition, the stepwise fabrication process of the as-prepared biosensor was characterized by scanning electron microscopy. PDDA-Au nanoparticles were characterized by ultraviolet–vis absorption spectroscopy and transmission electron microscopy. Under the optimal conditions, the as-prepared biosensor exhibited a good response performance to glucose with a linear range from 6 μM to 1.2 mM with a detection limit of 0.1 μM glucose (S/N = 3). In addition, this work indicated that TiO₂-MWNTs-CS composite and PDDA-Au nanoparticles held great potential for constructing biosensors.     

Adsorption and adhesion of blood proteins and fibroblasts on multi-wall carbon nanotubes

This article concerns the investigation of blood protein adsorption on carbon paper and multi-wall carbon nanotubes (MWCNTs). Mouse fibroblast cell adhesion and growth on MWCNTs was also studied. The results showed that fibrinogen adsorption on carbon paper was much lower than that on MWCNTs, which means that platelets readily aggregate on the surface of MWCNTs. Mouse fibroblast cells implanted on MWCNTs tended to grow more prolifically than those implanted on carbon paper. The cell concentration observed on MWCNTs increased from 1.2×10⁵/mL for a single day culture to 2×10⁵/mL for a 7-day culture. No toxicity reaction was observed during the culturing period. These results indicated that MWCNTs possessed excellent tissue compatibility. Supported by open-program of State Key Laboratory of Bioelectronics, Southeast University.     

Amperometric sulfide detection using Coprinus cinereus peroxidase immobilized on screen printed electrode in an enzyme inhibition based biosensor

In the present work, an amperometric inhibition biosensor for the determination of sulfide has been fabricated by immobilizing Coprinus cinereus peroxidase (CIP) on the surface of screen printed electrode (SPE). Chitosan/acrylamide was applied for immobilization of peroxidase on the working electrode. The amperometric measurement was performed at an applied potential of -150 mV versus Ag/AgCl with a scan rate of 100 mV in the presence of hydroquinone as electron mediator and 0.1M phosphate buffer solution of pH 6.5. The variables influencing the performance of sensor including the amount of substrate, mediator concentration and electrolyte pH were optimized. The determination of sulfide can be achieved in a linear range of 1.09-16.3 μM with a detection limit of 0.3 μM. Developed sensor showed quicker response to sulfide compared to the previous developed sulfide biosensors. Common anions and cations in environmental water did not interfere with sulfide detection by the developed biosensor. Cyanide interference on the enzyme inhibition caused 43.25% error in the calibration assay which is less than the amounts reported by previous studies. Because of high sensitivity and the low-cost of SPE, this inhibition biosensor can be successfully used for analysis of environmental water samples.     

Amperometric choline biosensor based on multiwalled carbon nanotubes/zirconium oxide nanoparticles electrodeposited on glassy carbon electrode

Perturbation of the tubulin/microtubule dynamic in cells is perhaps the single most important mode of action of anticancer drugs. Standard methods for identifying and evaluating compounds for their ability to alter tubulin polymerization are low throughput, labor intensive, expensive, or make their assessment in vitro. Here we report a method to rapidly quantify the extent of tubulin polymerization in whole cells using flow cytometry, and we use this technique to evaluate compounds that stabilize and destabilize microtubule formation. This facile method is useful for conveniently, quantitatively, and cost-effectively comparing small molecules that perturb tubulin polymerization.     

A novel nanobiocomposite based glucose biosensor using neutral red functionalized carbon nanotubes

The performance of a new glucose biosensor based on the combination of biocatalytic activity of glucose oxidase (GOx) with the electrocatalytic properties of CNTs and neutral red (NR) for the determination of glucose is described. This sensor is comprised of a multiwalled carbon nanotubes (MWNTs) conduit functionalized with NR and Nafion (Nf) as a binder and glucose oxidase as a biocatalyst. Neutral red was covalently immobilized on carboxylic acid groups of the CNTs via carbodiimide reaction. The functionalized MWNTs were characterized by microscopic, spectroscopic and thermal methods. The MWNT-NR-GOx-Nf nanobiocomposite was prepared by mixing the GOx solution with NR functionalized CNTs followed by mixing homogeneously with Nafion. The performance of the MWNT-NR-GOx-Nf nanobiocomposite modified electrode was examined by electrochemical impedance spectroscopy and cyclic voltammetry. The catalytic reduction of hydrogen peroxide liberated from the enzymatic reaction of glucose oxidase upon glucose with NR functionalized CNTs leads to the selective detection of glucose. The excellent electrocatalytic activity and the influence of nanobiocomposite film result in good characteristics such as low potential detection of glucose with a large determination range from 1 x 10(-8) to 1 x 10(-3)M with a detection limit of 3 x 10(-9)M glucose, a short response time (with 4s), good stability and anti-interferent ability. The improved electrocatalytic activity and stability made the MWNT-NR-GOx-Nf nanobiocomposite biosensor system a potential platform to immobilize different enzymes for other bioelectrochemical applications.     

Bienzymatic glucose biosensor based on co-immobilization of peroxidase and glucose oxidase on a carbon nanotubes electrode

A bienzymatic glucose biosensor was proposed for selective and sensitive detection of glucose. This mediatorless biosensor was made by simultaneous immobilization of glucose oxidase (GOD) and horseradish peroxidase (HRP) in an electropolymerized pyrrole (PPy) film on a single-wall carbon nanotubes (SWNT) coated electrode. The amperometric detection of glucose was assayed by potentiostating the bienzymatic electrode at -0.1 versus Ag/AgCl to reduce the enzymatically produced H(2)O(2) with minimal interference from the coexisting electroactive compounds. The single-wall carbon nanotubes, sandwiched between the enzyme loading polypyrrole (PPy) layer and the conducting substrate (gold electrode), could efficiently promote the direct electron transfer of HRP. Operational characteristics of the bienzymatic sensor, in terms of linear range, detection limit, sensitivity, selectivity and stability, were presented in detail.     

Design and development of a highly stable hydrogen peroxide biosensor on screen printed carbon electrode based on horseradish peroxidase bound with gold nanoparticles in the matrix of chitosan

The design and development of a screen printed carbon electrode (SPCE) on a polyvinyl chloride substrate as a disposable sensor is described. Six configurations were designed on silk screen frames. The SPCEs were printed with four inks: silver ink as the conducting track, carbon ink as the working and counter electrodes, silver/silver chloride ink as the reference electrode and insulating ink as the insulator layer. Selection of the best configuration was done by comparing slopes from the calibration plots generated by the cyclic voltammograms at 10, 20 and 30 mM K(3)Fe(CN)(6) for each configuration. The electrodes with similar configurations gave similar slopes. The 5th configuration was the best electrode that gave the highest slope. Modifying the best SPCE configuration for use as a biosensor, horseradish peroxidase (HRP) was selected as a biomaterial bound with gold nanoparticles (AuNP) in the matrix of chitosan (HRP/AuNP/CHIT). Biosensors of HRP/SPCE, HRP/CHIT/SPCE and HRP/AuNP/CHIT/SPCE were used in the amperometric detection of H(2)O(2) in a solution of 0.1M citrate buffer, pH 6.5, by applying a potential of -0.4V at the working electrode. All the biosensors showed an immediate response to H(2)O(2). The effect of HRP/AuNP incorporated with CHIT (HRP/AuNP/CHIT/SPCE) yielded the highest performance. The amperometric response of HRP/AuNP/CHIT/SPCE retained over 95% of the initial current of the 1st day up to 30 days of storage at 4 degrees C. The biosensor showed a linear range of 0.01-11.3mM H(2)O(2), with a detection limit of 0.65 microM H(2)O(2) (S/N=3). The low detection limit, long storage life and wide linear range of this biosensor make it advantageous in many applications, including bioreactors and biosensors.     

Glucose biosensor based on Au nanoparticles-conductive polyaniline nanocomposite

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).     

Highly sensitive biosensor based on bionanomultilayer with water-soluble multiwall carbon nanotubes for determination of phenolics

A highly sensitive biosensor was developed based on bionanomultilyer with water-soluble carbon nanotubes (CNTs). The water-soluble poly(allylamine hydrochloride)-wrapped multiwall carbon nanotubes (PAH-MWNTs) can be obtained for the first time relying on the function of barbiturates, which provides a useful avenue for CNT application in material science and biosensor technology. Based on this, the PAH-MWNTs/horseradish peroxidase (HRP) bionanomultilayer was prepared via layer-by-layer (LBL) assembly. Electrochemical impedance spectroscopy, atomic force microscopy and UV-vis spectra were adopted to monitor the uniform LBL assembly of the homogeneous bionanomultilayer. The bionanomultilayer was used to construct a phenolic biosensor. Under the optimal conditions, the biosensor presented a linear response for catechol from 0.1 to 20.4muM, with a detection limit of 0.06muM. A series of phenolics were detected by the bionanomultilayer biosensor. The introduced MWNTs in the biosensor provided a suitable microenvironment to retain the HRP activity and acted as a transducer for amplifying the electrochemical signal of the product of the enzymatic reaction. So the developed bionanomultilayer biosensor exhibited a fast, sensitive and stable detection.