Amperometric choline biosensors based on multi-wall carbon nanotubes and layer-by-layer assembly of multilayer films composed of Poly(diallyldimethylammonium chloride) and choline oxidase

A layer-by-layer deposition technique combined with Multi-wall carbon nanotubes (MWCNTs) was employed for fabricating choline sensors. The terminals and side-walls were linked with oxygen-containing groups when MWCNTs were treated with concentrated acid mixtures. A film of MWCNTs was initially prepared on the platinum electrode surface. Based on the electrostatic interaction between positively charged polyallylamine (PAA) and negatively charged MWCNTs and poly(vinyl sulfate) (PVS), a polymer film of (PVS/PAA)₃ was alternately adsorbed on the modified electrode continuously to be used as a permselective layer. Then poly(diallyldimethylammonium) (PDDA) and choline oxidase(ChOx) multilayer films were assembled layer-by-layer on the pretreated electrode, so an amplified biosensor toward choline was constructed. The choline sensor showed a linear response range of 5 × 10^? 7 to 1 × 10^? 4 M with a detection limit of 2 × 10^? 7 M estimated at a signal-to-noise ratio of 3, and a sensitivity of 12.53 μA/mM with a response time of 7.6 s in the presence of MWCNTs. Moreover, it exhibited excellent reproducibility, long-term stability as well as good suppression of interference. This protocol could be used to immobilize other enzymes for biosensor fabrication.     

Electrodeposition of chitosan-glucose oxidase biocomposite onto Pt-Pb nanoparticles modified stainless steel needle electrode for amperometric glucose biosensor

A glucose biosensor was fabricated by electrodepositing chitosan (CS)-glucose oxidase(GOD) biocomposite onto the stainless steel needle electrode (SSN electrode) modified by Pt–Pb nanoparticles (Pt–Pb/SSN electrode). Firstly, Pt–Pb nanoparticles were deposited onto the SSN electrode and then CS-GOD biocomposite was co-electrodeposited onto the Pt–Pb/SSN electrode in a mixed solution containing p-benzoquinone (p-BQ), CS and GOD. The electrochemical results showed that the Pt–Pb nanoparticles can accelerate the electron transfer and improve the effective surface area of the SSN electrode. As a result, the detection range of the proposed biosensor was from 0.03 to 9 mM with a current sensitivity of 0.4485 μA/mM and a response time of 15 s. The Michaelis constant value was calculated to be 4.9837 mM. The cell test results indicated that the electrodes have a low cytotoxicity. This work provided a suitable technology for the fabrication of the needle-type glucose biosensor.     

Biopolymer-clay nanoparticles composite system (Chitosan-laponite) for electrochemical sensing based on glucose oxidase

Nanocomposite matrix based on chitosan/laponite was successfully utilized to construct a new type of amperometric glucose biosensor. This hybrid material combined the merits of organic biopolymer, chitosan, and synthesized inorganic clay, laponite. Glucose oxidase (GOD) immobilized in the material maintained its activity well as the usage of glutaraldehyde was avoided. The composite films were characterized by Fourier transform infrared (FT-IR). The parameters affecting the fabrication and experimental conditions of biosensors were optimized. The sensitivity of the proposed biosensor (33.9 mA M^− 1 cm^− 2) permitted the determination of glucose in the concentration range of 1 × 10^− 6–5 × 10^− 5 M with a detection limit of 0.3 μM based on S/N = 3. The apparent Michaelis–Menten constant (K_M^app) for the sensor was found to be 15.8 mM.     

Carbon nanotube/chitosan/gold nanoparticles-based glucose biosensor prepared by a layer-by-layer technique

A new amperometric glucose biosensor was constructed, based on the immobilization of glucose oxidase (GOx) with cross-linking in the matrix of chitosan on a glassy carbon electrode, which was modified by layer-by-layer assembled carbon nanotube (CNT)/chitosan (CHIT)/gold nanoparticles (GNp) multilayer films. With the increasing of CNT/CHIT/GNp layers, the response current to H₂O₂ was changed regularly and the response current reached a maximum value when the number of CNT/CHIT/GNp layers was 8. The assembling process of multilayer films was simple to operate. With GOx as an enzyme model, a new glucose biosensor was fabricated. The excellent electocatalytic activity and special structure of the enzyme electrode resulted in good characteristics. The linear range was 6 × 10^? 6 ～ 5 × 10^? 3 M, with a detection limit of 3 × 10^? 6 M estimated at a signal-to-noise ratio of 3, fast response time (less than 6 s). Moreover, it exhibited good reproducibility and stability.     

ENFET glucose biosensor produced with mesoporous silica microspheres

The biosensors based on ENFETs for direct glucose oxidase (GOD) concentration analysis have been fabricated. Mesoporous silica (MS) microspheres of different pore sizes were used as the substrates for immobilizing enzyme to construct ENFETs. The MS microspheres show significantly improved enzyme immobilization capacity. The glucose sensitive ENFET based on the modification of the gate surface of ion-sensitive field-effect transistors (ISFETs) with MS microspheres and glucose oxidase (GOD) shows obviously enhanced sensitivity and extended lifetime compared with the conventional ones. MS microspheres of different pore sizes were investigated and the sensor with remodeled MS microspheres which experienced the pore expanding treatment show better performance than untreated MS microspheres. The fabricated sensors have a linear range of 0.25–2.0 mM, and a detection limit of ca. 0.10 mM. The influence of buffer concentration, ionic strength and pH was discussed. The biosensor also has good stability and reproducibility.     

Fabrication and characterization of platinum black and mesoporous platinum electrodes for in-vivo and continuously monitoring electrochemical sensor applications

Most electrochemical biosensors are disposable due to enzymes that are living creatures. Thus, these are limited to use in in-vivo and continuously monitoring biosensor system applications. The mesoporous (pores with a size of 2-50 nm) platinum (Pt) structure formed on a rod-shaped Pt microelectrode was reported for developments glucose sensors without any enzymes. In this paper, plane Pt electrode (non-treated), Pt black electrode, and mesoporous Pt electrode are fabricated and characterized on a silicon substrate in order to check their usability as enzymeless sensing electrodes for in-vivo and continuously monitoring electrochemical biosensors integrated with silicon CMOS read-out circuitry. The Pt black electrode with rough surface was fabricated by using an electrodeposition technique with hexachloroplatinic acid hydrate (HCPA) solutions. The proposed mesoporous Pt electrode with approximately 3 nm in pore diameter was fabricated by using an electrodeposition technique with nonionic surfactant octaethylene glycol monohexadecyl ether (C₁₆EO₈) and HCPA. The measured current responses at 40 mM glucose solution of the fabricated plane Pt, Pt black, and mesoporous Pt electrodes are approximately 12.4 nA/mm², 2.1 μA/mm², and 2.8 μA/mm², respectively. These data indicate that the mesoporous Pt electrode is much more sensitive than the other Pt electrodes and has strong potential for enzymeless electrochemical sensor applications.     

The preparation of amperometric xanthine sensors based on multilayer thin films containing xanthine oxidase

An amperometric xanthine biosensor was prepared by immobilizing xanthine oxidase (XOx) on the surface of a platinum (Pt) disk electrode. The Pt electrode was first covered with a polyelectrolyte multilayer (PEM) film composed of poly(allylamine hydrochloride) (PAH) and poly(vinyl sulfate) (PVS) to block the Pt surface from the access of uric acid, a reaction product of the XOx-catalyzed oxidation reaction of xanthine, because uric acid can be electrochemically oxidized on the Pt electrode to induce interference. The PEM film-covered Pt electrode was further modified with a XOx-containing multilayer film composed of XOx and poly(dimethyldiallylammonium chloride) (PDDA). The xanthine biosensors thus prepared can be used successfully for detecting xanthine in the concentration range of 3–300 μM. The output current of the sensors depended on the number of the XOx/PDDA layers in the film. The PAH/PVS layer was effective to eliminate the uric acid interference.     

Controlled fabrication of gold nanoparticles biomediated by glucose oxidase immobilized on chitosan layer-by-layer films

The control of size and shape of metallic nanoparticles is a fundamental goal in nanochemistry, and crucial for applications exploiting nanoscale properties of materials. We present here an approach to the synthesis of gold nanoparticles mediated by glucose oxidase (GOD) immobilized on solid substrates using the Layer-by-Layer (LbL) technique. The LbL films contained four alternated layers of chitosan and poly(styrene sulfonate) (PSS), with GOD in the uppermost bilayer adsorbed on a fifth chitosan layer: (chitosan/PSS)₄/(chitosan/GOD). The films were inserted into a solution containing gold salt and glucose, at various pHs. Optimum conditions were achieved at pH 9, producing gold nanoparticles of ca. 30 nm according to transmission electron microscopy. A comparative study with the enzyme in solution demonstrated that the synthesis of gold nanoparticles is more efficient using immobilized GOD.     

Multilayer membranes via layer-by-layer deposition of glucose oxidase and Au nanoparticles on a Pt electrode for glucose sensing

We prepared multilayer membranes by the layer-by-layer deposition of glucose oxidase (GOx) and Au nanoparticles (5, 10, or 50 nm φ) on sensor substrates, such as a Pt electrode and a quartz glass plate, to prepare glucose sensors. The enzyme activity of GOx remained even in alternate assemblies, and the activity increased with the increasing number of depositions. The apparent K_m values of the deposited GOx were 28–32 mM, while a reported value in a solution is 33 mM. These results suggest that Au nanoparticles can be used as binders for the deposition of GOx without significant change in the affinity between GOx and glucose.     

壳聚糖凝胶材料固定葡萄糖氧化酶制电极的研究

以壳聚糖为载体研究凝胶法固定葡萄糖氧化酶制电极。试验研究了载体壳聚糖的降解性；交联剂戊二醛的浓度、用量；电极的载酶量等固定化条件对所组建的传感器性能的影响。通过影响规律的分析、优化固定化条件的研究，找出了根据壳聚糖溶液粘度适当调整交联剂成二醛的用量和铂丝在酶膜母液中浸涂时间，克服壳聚糖的降解性对酶电极性能的影响，建立了制备性能相近的GOD传感器的方法。     

Glucose biosensor from covalent immobilization of chitosan-coupled carbon nanotubes on polyaniline-modified gold electrode

An amperometric glucose biosensor was prepared using polyaniline (PANI) and chitosan-coupled carbon nanotubes (CS-CNTs) as the signal amplifiers and glucose oxidase (GOD) as the glucose detector on a gold electrode (the Au-g-PANI-c-(CS-CNTs)-GOD biosensor). The PANI layer was prepared via oxidative graft polymerization of aniline from the gold electrode surface premodified by self-assembled monolayer of 4-aminothiophenol. CS-CNTs were covalently coupled to the PANI-modified gold substrate using glutaradehyde as a bifunctional linker. GOD was then covalently bonded to the pendant hydroxyl groups of chitosan using 1,4-carbonyldiimidazole as the bifunctional linker. The surface functionalization processes were ascertained by X-ray photoelectron spectroscopy (XPS) analyses. The field emission scanning electron microscopy (FESEM) images of the Au-g-PANI-c-(CS-CNTs) electrode revealed the formation of a three-dimensional surface network structure. The electrode could thus provide a more spatially biocompatible microenvironment to enhance the amount and biocatalytic activity of the immobilized enzyme and to better mediate the electron transfer. The resulting Au-g-PANI-c-(CS-CNTs)-GOD biosensor exhibited a linear response to glucose in the concentration range of 1-20 mM, good sensitivity (21 μA/(mM·cm(2))), good reproducibility, and retention of >80% of the initial response current after 2 months of storage.     

ENFET glucose biosensor produced with dendrimer encapsulated Pt nanoparticles

The biosensors based on ENFETs for direct glucose concentration analysis have been fabricated by introducing dendrimer encapsulated Pt nanoparticles and glucose oxidase (GOx) via a layer-by-layer self-assembly method. The free amine groups located on each poly(amidoamine) dendrimer molecule were exploited to immobilize enzyme through covalent attachment. Depending on metal nanoparticles within dendrimers and biocompatibility of dendrimers, the fabricated glucose sensitive ENFET shows obviously enhanced sensitivity and extended lifetime compared with the conventional ones. The fabricated sensor has a linear range of 0.25–2.0 mM, and a detection limit of ca. 0.15 mM. The influence of buffer concentration, ionic strength and pH was discussed. The biosensor also has good stability, which response could be used for detecting of glucose samples at intervals for at least 1 month when it stored in dry state at 4 °C.     

ZnS nanoparticles electrodeposited onto ITO electrode as a platform for fabrication of enzyme-based biosensors of glucose

The electrochemical and photoelectrochemical biosensors based on glucose oxidase (GOD) and ZnS nanoparticles modified indium tin oxide (ITO) electrode were investigated. The ZnS nanoparticles were electrodeposited directly on the surface of ITO electrode. The enzyme was immobilized on ZnS/ITO electrode surface by sol–gel method to fabricate glucose biosensor. GOD could electrocatalyze the reduction of dissolved oxygen, which resulted in a great increase of the reduction peak current. The reduction peak current decreased linearly with the addition of glucose, which could be used for glucose detection. Moreover, ZnS nanoparticles deposited on ITO electrode surface showed good photocurrent response under illumination. A photoelectrochemical biosensor for the detection of glucose was also developed by monitoring the decreases in the cathodic peak photocurrent. The results indicated that ZnS nanoparticles deposited on ITO substrate were a good candidate material for the immobilization of enzyme in glucose biosensor construction.     

A highly-sensitive l-lactate biosensor based on sol-gel film combined with multi-walled carbon nanotubes (MWCNTs) modified electrode

A new type of amperometric l-lactate biosensor based on silica sol-gel and multi-walled carbon nanotubes (MWCNTs) organic–inorganic hybrid composite material was developed. The sol-gel film was used to immobilize l-lactate oxidase on the surface of glassy carbon electrode (GCE). MWCNTs were used to increase the current response and improve the performance of biosensor. The sol-gel film fabrication process parameters such as H₂O : TEOS and pH were optimized, Effects of some experimental variables such as applied potential, temperature, and pH on the current response of the biosensor were investigated. Analytical characteristics and dynamic parameters of the biosensors with and without MWCNTs in the hybrid film were compared, and the results showed that analytical performance of the biosensor could be improved greatly after introduction of the MWCNTs. Sensitivity, linear range, limit of detection (S / N = 3) were 2.097 μA mM^− 1, 0.3 to 1.5 mM, 0.8 × 10^− 3 mM for the biosensor without MWCNTs and 6.031 μA mM^− 1, 0.2 to 2.0 mM, 0.3 × 10^− 3 mM for the biosensor with MWCNTs, respectively. This method has been used to determine the l-lactate concentration in real human blood samples.     

Direct electron transfer and biosensing of glucose oxidase immobilized at multiwalled carbon nanotube-alumina-coated silica modified electrode

Investigations are reported regarding the direct electrochemical performance of glucose oxidase (GOD) immobilized on a film of multiwalled carbon nanotube-alumina-coated silica (MWCNT-ACS). The surface morphology of the GOD/MWCNT-ACS nanobiocomposite is characterized by scanning electron microscopy. In cyclic voltammetric response, the immobilized GOD displays a pair of well-defined redox peaks, with a formal potential (E°′) of ? 0.466 V versus Ag/AgCl in a 0.1 M phosphate buffer solution (pH 7.5) at a scan rate of 0.05 V s^? 1; also the electrochemical response indicates a surface-controlled electrode process. The dependence of formal potential on solution pH indicates that the direct electron transfer reaction of GOD is a reversible two-electron coupled with a two-proton electrochemical reaction process. The glucose biosensor based on the GOD/MWCNT-ACS nanobiocomposite shows a sensitivity of 0.127 A M^? 1 cm^? 2 and an apparent Michaelis–Menten constant of 0.5 mM. Furthermore, the prepared biosensor exhibits excellent anti-interference ability to the commonly co-existed uric acid and ascorbic acid.     

Comparative study of conductometric glucose biosensor based on gold and on magnetic nanoparticles

The aim of this study was to show the feasibility and the performances of nanoparticle biosensing. A glucose conductometric biosensor was developed using two types of nanoparticles (gold and magnetic), glucose oxidase (GOD) being adsorbed on PAH (poly(allylamine hydrochloride)) modified nanoparticles, deposited on a planar interdigitated electrode (IDEs). The best sensitivities for glucose detection were obtained with magnetic nanoparticles (70 μM/mM and 3 μM of detection limit) compared to 45 μM/mM and 9 μM with gold nanoparticles and 30 μM/mM and 50 μM with GOD directly cross-linked on IDEs. When stored in phosphate buffer (20 mM, pH 7.3) at 4 °C, the biosensor showed good stability for more than 12 days.     

Electrochemical biosensor based on multi-walled carbon nanotubes and Au nanoparticles synthesized in chitosan

A new biosensor is prepared by cross-linking glucose oxidase (GOD) with glutaradehyde at the electrode combining Au nanoparticles (AuNP) with multi-walled carbon nanotubes (MWCNTs). Au nanoparticles-doped chitosan (CS) solution (AuNP-CS) is prepared by treating the CS solution followed by chemical reduction of Au (III) with NaBH4. MWCNTs are then dispersed in AuNP-CS solution. TEM, FT-IR, and UV-Vis show that the AuNP-CS solution is highly dispersed and stable. The synergistic effect between AuNP and CNTs of the AuNP-CNTs-CS material has been investigated by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and amperometric methods. The modified glassy carbon electrode (GCE) allows low-potential detection of H2O2 with high sensitivity and fast response time. With the immobilization of GOD, a biosensor has been constructed. In phosphate buffer solutions (PBS, pH 7.0), nearly free interference determination of glucose has been realized at 0.4 V(vs. Ag/AgCl/3.0 M KCI) with a wide linear range from 2.0 x 10(-5) to 1.5 x 10(-2) M and a fast response time within 5s. The biosensor has been used to determine glucose in human serum samples and the results are satisfactory.     

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 l-lactate biosensor based on l-lactate oxidase immobilized through silica sol–gel film on multi-walled carbon nanotubes/platinum nanoparticle modified glassy carbon electrode

Platinum nanoparticles (Ptnano) were used in combination with multi-walled carbon nanotubes (MWCNTs) for fabricating sensitivity-enhanced electrochemical l-lactate biosensor. The composite film of MWCNTs and Ptnano was dispersed on the surface of the glassy carbon electrode (GCE). l-lactate oxidase (LOD) was immobilized on MWCNTs/Ptnano/GCE surface by adsorption. The resulting LOD/MWCNTs/Ptnano electrode was covered by a thin layer of sol–gel to avoid the loss of LOD in determination and to improve the anti-interferent ability. Moreover, the sol–gel microenviroment contributes to both intensified stability and permselectivity. The cyclic voltammetry results indicated that MWCNTs/Ptnano catalyst displayed a higher performance than MWCNTs. Under the optimized conditions of applied potential 0.5 V, pH 6.4, room temperature, the proposed biosensor showed a large determination range (0.2–2.0 mM), a short response time (within 5 s), a high sensitivity (6.36 μA mM^{− 1}) and good stability (90% remains after 4 weeks). The fabricated biosensor had practically good selectivity against interferences. The results for whole blood samples measured by the present biosensor showed a good agreement with those measured by spectrophotometric method.     

Biosensor enhanced by glucose oxidase biomimetic membrane containing the platinum and silica nanoparticles

In this paper, glucose biosensor is fabricated with immobilization of glucose oxidase (GOx) in platinum and silica sol. The glucose biosensor combined with Pt and SiO₂ nanoparticles could make full use of the properties of nanoparticles. A set of experimental results indicates that the current response for the enzyme electrode containing platinum and silica nanoparticles increases from 0.32 µA cm^− 2 to 33 µA cm^− 2 in the solution of 10 mM β-D-glucose. The linear range is 3 × 10^− 5 to 3.8 × 10^− 3 M with a detection limit of 2 × 10^− 5 M at 3σ. The effects of the various volume ratios of Pt and SiO₂ sols with respect to the current response and the stability of the enzyme electrodes are studied.