Nanobiocomposite Electrochemical Biosensor Utilizing Synergic Action of Neutral Red Functionalized Carbon Nanotubes

Nano-Micro Letters - An amperometric hydrogen peroxide biosensor using a nanobiocomposite based on neutral red modified carbon nanotubes and co-immobilized glucose oxidase and horseradish...     

Protein-modified nanocrystalline diamond thin films for biosensor applications

Diamond exhibits several special properties, for example good biocompatibility and a large electrochemical potential window, that make it particularly suitable for biofunctionalization and biosensing. Here we show that proteins can be attached covalently to nanocrystalline diamond thin films. Moreover, we show that, although the biomolecules are immobilized at the surface, they are still fully functional and active. Hydrogen-terminated nanocrystalline diamond films were modified by using a photochemical process to generate a surface layer of amino groups, to which proteins were covalently attached. We used green fluorescent protein to reveal the successful coupling directly. After functionalization of nanocrystalline diamond electrodes with the enzyme catalase, a direct electron transfer between the enzyme's redox centre and the diamond electrode was detected. Moreover, the modified electrode was found to be sensitive to hydrogen peroxide. Because of its dual role as a substrate for biofunctionalization and as an electrode, nanocrystalline diamond is a very promising candidate for future biosensor applications.     

Carbon nanotube-chitosan system for electrochemical sensing based on dehydrogenase enzymes

Multiwalled carbon nanotubes (CNT) were solubilized in aqueous solutions of a biopolymer chitosan (CHIT). The CHIT-induced solubilization of CNT facilitated their manipulations, including the modification of electrode surfaces for sensor and biosensor development. The colloidal solutions of CNT-CHIT were placed on the surface of glassy carbon (GC) electrodes to form robust CNT-CHIT films, which facilitated the electrooxidation of NADH. The GC/CNT-CHIT sensor for NADH required approximately 0.3 V less overpotential than the GC electrode. The susceptibility of CHIT to chemical modifications was explored in order to covalently immobilize glucose dehydrogenase (GDH) in the CNT-CHIT films using glutaric dialdehyde (GDI). The stability and sensitivity of the GC/CNT-CHIT-GDI-GDH biosensor allowed for the interference-free determination of glucose in the physiological matrix (urine). In pH 7.40 phosphate buffer solutions, linear least-squares calibration plots over the range 5-300 microM glucose (10 points) had slopes 80 mA M(-1) cm(-2) and a correlation coefficient 0.996. The detection limit was 3 microM glucose (S/N = 3). The CNT-CHIT system represents a simple and functional approach to the integration of dehydrogenases and electrodes, which can provide analytical access to a large group of enzymes for wide range of bioelectrochemical applications including biosensors and biofuel cells.     

Optimal environment for glucose oxidase in perfluorosulfonated ionomer membranes: improvement of first-generation biosensors

An optimal environment for glucose oxidase (GOx) in Nafion membranes is achieved using an advanced immobilization protocol based on a nonaqueous immobilization route. Exposure of glucose oxidase to water-organic mixtures with a high (85-95%) content of the organic solvent resulted in stabilization of the enzyme by a membrane-forming polyelectrolyte. Such an optimal environment leads to the highest enzyme specific activity in the resulting membrane, as desired for optimal use of the expensive oxidases. Casting solution containing glucose oxidase and Nafion is completely stable over 5 days in a refrigerator, providing almost absolute reproducibility of GOx-Nafion membranes. A glucose biosensor was prepared by casting the GOx-Nafion membranes over Prussian Blue-modified glassy carbon disk electrodes. The biosensor operated in the FIA mode allows the detection of glucose down to the 0.1 microM level, along with high sensitivity (0.05 A M(-1) cm(-2)), which is only 10 times lower than the sensitivity of the hydrogen peroxide transducer used. A comparison with the recently reported enzyme electrodes based on similar H2O2 transducers (transition metal hexacyanoferrates) shows that the proposed approach displays a dramatic (100-fold) improvement in sensitivity of the resulting biosensor. Combined with the attractive performance of a Prussian Blue-based hydrogen peroxide transducer, the proposed immobilization protocol provides a superior performance for first-generation glucose biosensors in term of sensitivity and detection limits.     

基于二氧化硅球腔阵列的葡萄糖传感器研究

在二氧化硅球腔阵列电极上,通过电沉积普鲁士蓝和直接吸附葡萄糖氧化酶,制备了一种新型葡萄糖生物传感器。该传感器对酶催化反应产物过氧化氢的选择性催化还原特性可实现对葡萄糖的检测,实验结果表明,传感器的最佳工作电位是－0．3V,测试溶液的最佳pH值为6．0。在选定的工作条件下,传感器的线性范围为2．49×10-5－2．42×10-3mol／L,检测极限值为7．2×10-6mol／L（S／N=3）,米氏常数为1．136mmol／L。该方法制备的生物传感器能有效降低干扰,具有潜在的应用价值。     

Spatial organization of peptide nanotubes for electrochemical devices

A novel biosensor for hydrogen peroxide was developed by combining the known properties of microperoxidase-11 (MP11) as an oxidation catalyst, and the interesting properties of diphenylalanine peptide nanotubes (PNTs) as a supporting matrix to allow a good bioelectrochemical interface. In this case, the synthesized MP11/PNTs were immobilized onto the ITO electrode surface via layer-by-layer (LBL) deposition, using poly(allylamine hydrochloride) (PAH) as positively charged polyelectrolyte layers. The PNTs provide a favorable microenvironment for MP11 to perform direct electron transfer to the electrode surface. The resulting electrodes showed a pair of well-defined redox peaks with formal potential at about −343 mV (versus SCE) in phosphate buffer solution (pH 7). The experimental results also demonstrated that the resulting biosensor exhibited good electrocatalytic activity to the reduction of H₂O₂ with a sensitivity of 9.43 μA cm⁻² mmol⁻¹ L, and a detection limit of 6 μmol L⁻¹ at the signal-to-noise ratio of 3. Moreover, we also observed that the peptides self-assembly can be influenced upon changing the pH of the solution. Alkaline solution appears to favor the packing of diphenylalanine nanotubes being closer than acidic or neutral conditions. The study proved that the combination of PNTs with MP11 is able to open new opportunities for the design of enzymatic biosensors with potential applications in practice.     

Electrochemical biosensing platforms using platinum nanoparticles and carbon nanotubes

Platinum nanoparticles with a diameter of 2-3 nm were prepared and used in combination with single-wall carbon nanotubes (SWCNTs) for fabricating electrochemical sensors with remarkably improved sensitivity toward hydrogen peroxide. Nafion, a perfluorosulfonated polymer, was used to solubilize SWCNTs and also displayed strong interactions with Pt nanoparticles to form a network that connected Pt nanoparticles to the electrode surface. TEM and AFM micrographs illustrated the deposition of Pt nanoparticles on carbon nanotubes whereas cyclic voltammetry confirmed an electrical contact through SWCNTs between Pt nanoparticles and the glassy carbon (GC) or carbon fiber backing. With glucose oxidase (GOx) as an enzyme model, we constructed a GC or carbon fiber microelectrode-based biosensor that responds even more sensitively to glucose than the GC/GOx electrode modified by Pt nanoparticles or CNTs alone. The response time and detection limit (S/N = 3) of this biosensor was determined to be 3 s and 0.5 microM, respectively.     

Electrocatalytic reduction of H2O2 on thiolate graphene oxide covalently to bonded palladium nanoparticles

The electrocatalytic reduction of hydrogen peroxide on thioalted graphene oxide (t-GO) covalent bonded to palladium nanoparticles was used as the basis of an H2O2 biosensor. Poly (diallydimethylammonium chloride)-coated t-GO-Pd on glassy carbon electrodes was easily and quickly prepared and gave sensitive measurements of H2O2 concentration. The Pd nanoparticles covalently bonded to the thiolated graphene oxide were characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, and energy dispersive X-ray spectroscopy. Comparable results for H2O2 determination were obtained from cyclic voltammetric and amperometric measurements. The proposed H2O2 biosensor exhibited a wide linear range of 10 microM to 10 mM, and a low detection limit of 0.22 microM (S/N = 3), at an applied potential of -0.1 V by the amperometric method.     

碳纳米管传感器的研究进展

综述了不同功能碳纳米管传感器(微型碳纳米管气体离子传感器、无线被动碳纳米管气体传感器、碳纳米管化学和力学传感器、碳纳米管阵列生物传感器、碳纳米管温度和风速传感器、碳纳米管神经毒气传感器)的制备、结构特点、性能和发展方向.     

Carbon nanotube/teflon composite electrochemical sensors and biosensors

The fabrication and attractive performance of carbon nanotube (CNT)/Teflon composite electrodes, based on the dispersion of CNT within a Teflon binder, are described. The resulting CNT/Teflon material brings new capabilities for electrochemical devices by combining the advantages of CNT and "bulk" composite electrodes. The electrocatalytic properties of CNT are not impaired by their association with the Teflon binder. The marked electrocatalytic activity toward hydrogen peroxide and NADH permits effective low-potential amperometric biosensing of glucose and ethanol, respectively, in connection with the incorporation of glucose oxidase and alcohol dehydrogenase/NAD(+) within the three-dimensional CNT/Teflon matrix. The accelerated electron transfer is coupled with minimization of surface fouling and surface renewability. These advantages of CNT-based composite devices are illustrated from comparison to their graphite/Teflon counterparts. The influence of the CNT loading upon the amperometric and voltammetric data, as well as the electrode resistance, is examined. SEM images offer insights into the nature of the CNT/Teflon surface. The preparation of CNT/Teflon composites overcomes a major obstacle for creating CNT-based biosensing devices and expands the scope of CNT-based electrochemical devices.     

Preparation of RF sputtered AZO/Au thin film hydrogen peroxide sensitive electrode for utilization as a biosensor

In this study, hydrogen peroxide (H₂O₂) sensitive Al doped ZO(AZO)/Au thin film electrode has been developed for the utilization as a biosensor. A preferred c-axis oriented AZO/Au thin film was deposited on quartz substrate by RF magnetron sputtering at room temperature. Structural, morphological and optical properties of the AZO film were analyzed by X-ray diffraction, atomic force microscopy and photoluminescence. The sensor performance was characterized by electrochemical analysis device. The sensibility of prepared thin film electrodes to H₂O₂ was studied. The dependence of amperometric response current on the glucose and cholesterol concentrations was also investigated.     

Microseparation chips for performing multienzymatic dehydrogenase/oxidase assays: simultaneous electrochemical measurement of ethanol and glucose

This report describes a new "lab-on-a-chip" protocol integrating on-line precolumn biocatalytic reactions of multiple (oxidase and dehydrogenase) enzymes and substrates with effective capillary electrophoresis microseparations and amperometric detection. The operation of the new oxidase/dehydrogenase reaction/separation microchip is illustrated for the simultaneous measurement of glucose and ethanol in connection to the corresponding glucose oxidase and alcohol dehydrogenase reactions, respectively. The enzymatic reactions generate hydrogen peroxide and NADH species that are separated (on the basis of their different charges) and detected amperometrically at the end-column thick-film detector. A driving voltage of 2000 V results in peroxide and NADH migration times of 74 and 230 s, respectively. Operating the gold-coated carbon detector at +1.0 V allows simultaneous anodic detection of both reaction products. Factors influencing the reaction, separation, and detection processes are examined and optimized. The applicability of the new multienzyme assay to wine samples is illustrated.     

Electrocatalytic detection of NADH and glycerol by NAD(+)-modified carbon electrodes

The electrochemical oxidation of the adenine moiety in NAD+ and other adenine nucleotides at carbon paste electrodes gives rise to redox-active products which strongly adsorb on the electrode surface. Carbon paste electrodes modified with the oxidation products of NAD+ show excellent electrocatalytic activity toward NADH oxidation, reducing its overpotential by about 400 mV. The rate constant for the catalytic oxidation of NADH, determined by rotating disk electrode measurements and extrapolation to zero concentration of NADH, was found to be 2.5 x 10(5) M-1 s-1. The catalytic oxidation current allows the amperometric detection of NADH at an applied potential of +50 mV (Ag/AgCl) with a detection limit of 4.0 x 10(-7) M and linear response up to 1.0 x 10(-5) M NADH. These modified electrodes can be used as amperometric transducers in the design of biosensors based on coupled dehydrogenase enzymes and, in fact, we have designed an amperometric biosensor for glycerol based on the glycerol dehydrogenase (GlDH) system. The enzyme GlDH and its cofactor NAD+ were co-immobilized in a carbon paste electrode using an electropolymerized layer of nonconducting poly(o-phenylenediamine) (PPD). After partial oxidation of the immobilized NAD+, the modified electrode allows the amperometric detection of the NADH enzymatically obtained at applied potential above 0 V (Ag/AgCl). The resulting biosensor shows a fast and linear response to glycerol within the concentration range of 1.0 x 10(-6)-1.0 x 10(-4) M with a detection limit of 4.3 x 10(-7) M. The amperometric response remains stable for at least 3 days. The biosensor was applied to the determination of glycerol in a plant-extract syrup, with results in good agreement with those for the standard spectrophotometric method.     

Electrochemical biosensor based on integrated assembly of dehydrogenase enzymes and gold nanoparticles

Development of a highly sensitive nanostructured electrochemical biosensor based on the integrated assembly of dehydrogenase enzymes and gold (Au) nanoparticle is described. The Au nanoparticles (AuNPs) have been self-assembled on a thiol-terminated, sol-gel-derived, 3-D, silicate network and enlarged by hydroxylamine seeding. The AuNPs on the silicate network efficiently catalyze the oxidation of NADH with a decrease in overpotential of approximately 915 mV in the absence of any redox mediator. The surface oxides of AuNP function as an excellent mediator, and a special inverted "V" shape voltammogram at less positive potential was observed for the oxidation of NADH. The AuNP self-assembled sol-gel network behaves like a nanoelectrode ensemble. The nanostructured electrode shows high sensitivity (0.056 +/- 0.001 nA/nM) toward NADH with an amperometric detection limit of 5 nM. The electrode displays excellent operational and storage stability. A novel methodology for the fabrication of a NADH-dependent dehydrogenase biosensor based on the integration of dehydrogenase enzyme and AuNPs with the silicate network is developed. The enzymatically generated NADH is, in turn, electrocatalytically detected by the AuNPs on the silicate network. The integrated assembly has been successfully used for the amperometric biosensing of lactate and ethanol at a potential of -5 mV. The biosensor is very stable and highly sensitive, and it has a fast response time. The excellent performance validates the integrated assembly as an attractive sensing element for the development of new dehydrogenase biosensors.     

Reversible fluorescence quenching in carbon nanotubes for biomolecular sensing

Biosensing applications of single-walled carbon nanotubes have been demonstrated in solid-state device structures. Bioanalyte sensing schemes based on coupling of reversible nanotube fluorescence quenching to redox reactions paired to enzymatic peroxide generation have also been pursued. Here we show a new approach to highly sensitive nanotube-based optical sensing. Single-walled carbon nanotubes interacting with dye-ligand conjugates--a redox-active dye molecule that is covalently bound to a biological receptor ligand (such as biotin in this case)--showed fluorescence quenching. Further interaction between the receptor ligand on the conjugates and target analytes (avidin in this case) induced the recovery of the quenched fluorescence, forming the basis of the sensing scheme. Nanomolar sensitivity was attained with high specificity for the target analyte. This is a versatile approach because a wide range of conjugation possibilities exists between the potential receptors and redox quenchers.     

用单壁纳米碳管制备葡萄糖生物传感器的研究

以单壁纳米碳管为代表材料,对利用纳米碳管制备葡萄糖生物传感器中纳米碳管的作用和纳米碳管修饰电极的方法、酶的固定化方法及电极种类等因素对传感器性能的影响进行了研究.研究结果表明,纳米碳管的加入能有效地改善传感器的电化学性能,利用二茂铁和单壁纳米碳管共同修饰电极所制得的传感器的性能要好于仅用单壁纳米碳管修饰电极制得的传感器.在酶的固定化方法中,戊二醛交联法要略好于明胶包埋法;而利用铂电极制备出的生物传感器对葡萄糖的响应电流要明显高于利用金电极和玻碳电极制备出的生物传感器.这些结论对于开发纳米碳管在生物传感领域及生命科学相关领域的应用有参考价值.     

Development of an amperometric ethanol biosensor based on a multiwalled carbon nanotube-Nafion-alcohol dehydrogenase nanobiocomposite

An amperometric biosensor for the determination of ethanol has been constructed. It comprises a multiwalled carbon nanotubes (MWNTs) conduit, a Nafion binder, and an alcohol dehydrogenase (ADH) function. The measurement of ethanol is based on the signal produced by beta-nicotinamide adenine dinucleotide (NADH), the product of the enzymatic reaction. The MWNTs are cylindrical with an outer diameter in the range 40-60 nm, an inner diameter in the range 2-5 nm, and a length of up to several micrometers. The homogeneity of the resulting nanobiocomposite film was characterized by field emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM). The performance of the MWNTs-Nafion-ADH nanobiocomposite modified glassy carbon electrode was examined using cyclic voltammetry and amperometry in presence of NADH and in the presence of ethanol. The electrocatalytic activity of MWNTs towards the oxidation of NADH has allowed an effective low-potential amperometric determination of ethanol. In the case of 6 mgmL(-1) ADH, the MWNTs-Nafion-ADH nanobiocomposite film displayed a sensitivity of 830 nAmM(-1), a linear range up to 0.1 mM, a detection limit of 3 microM, and a response time of about 4 s.     

Electrochemical DNA biosensor based on conducting polyaniline nanotube array

Most of the recent developments in ultrasensitive detection of nucleic acid are based on the gold nanoparticles and carbon nanotubes as a medium of signal amplification. Here, we present an ultrasensitive electrochemical nucleic acid biosensor using the conducting polyaniline (PANI) nanotube array as the signal enhancement element. The PANI nanotube array of a highly organized structure was fabricated under a well-controlled nanoscale dimension on the graphite electrode using a thin nanoporous layer as a template, and 21-mer oligonucleotide probes were immobilized on these PANI nanotubes. In comparison with gold nanoparticle- or carbon nanotube-based DNA biosensors, our PANI nanotube array-based DNA biosensor could achieve similar sensitivity without catalytic enhancement, purification, or end-opening processing. The electrochemical results showed that the conducting PANI nanotube array had a signal enhancement capability, allowing the DNA biosensor to readily detect the target oligonucleotide at a concentration as low as 1.0 fM (approximately 300 zmol of target molecules). In addition, this biosensor demonstrated good capability of differentiating the perfect matched target oligonucleotide from one-nucleotide mismatched oligonucleotides even at a concentration of 37.59 fM. This detection specificity indicates that this biosensor could be applied to single-nucleotide polymorphism analysis and single-mutation detection.     

Antibody covalent immobilization on carbon nanotubes and assessment of antigen binding

Controlling the covalent bonding of antibodies onto functionalized carbon nanotubes is a key step in the design and preparation of nanotube-based conjugates for targeting cancer cells. For this purpose, an anti-MUC1 antibody (Ab) is linked to both multi-walled (MWCNTs) and double-walled carbon nanotubes (DWCNTs) using different synthetic strategies. The presence of the Ab attached to the nanotubes is confirmed by gel electrophoresis and thermogravimetric analysis. Most importantly, molecular recognition of the antigen by surface plasmon resonance is able to determine similar Ab binding capacities for both Ab-DWCNTs and Ab-MWCNTs. These results are very relevant for the design of future receptor-targeting strategies using chemically functionalized carbon nanotubes.     

Electrochemical Oxidation of NADH at Highly Boron-Doped Diamond Electrodes

Conductive boron-doped chemical vapor-deposited diamond thin films, already known to have superior properties for general electroanalysis, including low background current and a wide potential window, are here shown to have additional advantages with respect to electrochemical oxidation of nicotinamide adenine dinucleotide (NADH), including high resistance to deactivation and insensitivity to dissolved oxygen. Cyclic voltammetry, amperometry, and the rotating disk electrode technique were used to study the reaction in neutral pH solution. Highly reproducible cyclic voltammograms for NADH oxidation were obtained at as-deposited diamond electrodes. The response was stable over several months of storage in ambient air, in contrast to glassy carbon electrodes, which deactivated within 1 h. The diamond electrode exhibited very high sensitivity for NADH, with an amperometric detection limit of 10 nM (S/N = 7). The response remained stable, even in the very low concentration range, for several months. In addition, interference effects due to ascorbic acid were minimal when the concentrations of NADH and ascorbic acid were comparable. An NADH-mediated dehydrogenese-based ethanol biosensor incorporating an unmodified diamond electrode is demonstrated. The present results indicate that diamond is a useful electrode material for the analytical detection of NADH, making it attractive for use in sensors based on enzyme-catalyzed reactions involving NADH as a cofactor.