Single-wall carbon nanotube-based voltammetric sensor and biosensor

The pH-sensitive property of the single-wall carbon nanotube modified electrode based on the electroactive group on the single-wall carbon nanotube was explored by differential pulse voltammetry technique. In pH range 1-13 investigated in Britton-Robinson (B-R) buffer, the anodic peak shifted negatively along with the increase of pH exhibiting a reversible Nernstian response. Experiments were carried out to investigate the response of the single-wall carbon nanotube (SWNT) modified electrode to analytes associated with pH change. The response behavior of the modified electrode to ammonia was studied as an example. The potential response could reach equilibrium within 5 min. The modified electrode had good operational stability. Voltammetric urease and acetylcholinesterase biosensors were constructed by immobilizing the enzymes with sol-gel hybrid material. The maximum potential shift could reach 0.130 and 0.220 V for urea and acetylthiocholine, respectively. The methods for preparing sensor and biosensor were simple and reproducible and the range of analytes could be extended to substrates of other hydrolyases and esterases. This broadened the biosensor application of carbon nanotube in electrochemical area.     

Enzyme-nanoparticle conjugates at oil-water interface for amplification of electrochemical immunosensing

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

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.     

Glucose biosensor prepared by glucose oxidase encapsulated sol-gel and carbon-nanotube-modified basal plane pyrolytic graphite electrode

A new glucose biosensor has been fabricated by immobilizing glucose oxidase into a sol-gel composite at the surface of a basal plane pyrolytic graphite (bppg) electrode modified with multiwall carbon nanotube. First, the bppg electrode is subjected to abrasive immobilization of carbon nanotubes by gently rubbing the electrode surface on a filter paper supporting the carbon nanotubes. Second, the electrode surface is covered with a thin film of a sol-gel composite containing encapsulated glucose oxidase. The carbon nanotubes offer excellent electrocatalytic activity toward reduction and oxidation of hydrogen peroxide liberated in the enzymatic reaction between glucose oxidase and glucose, enabling sensitive determination of glucose. The amperometric detection of glucose is carried out at 0.3 V (vs saturated calomel electrode) in 0.05 M phosphate buffer solution (pH 7.4) with linear response range of 0.2-20 mM glucose, sensitivity of 196 nA/mM, and detection limit of 50 microM (S/N=3). The response time of the electrode is < 5s when it is stored dried at 4 degrees C, the sensor showed almost no change in the analytical performance after operation for 3 weeks. The present carbon nanotube sol-gel biocomposite glucose oxidase sensor showed excellent properties for the sensitive determination of glucose with good reproducibility, remarkable stability, and rapid response and in comparison to bulk modified composite biosensors the amounts of enzyme and carbon nanotube needed for electrode fabrication are dramatically decreased.     

An electrochemical biosensor for 3-hydroxybutyrate detection based on screen-printed electrode modified by coenzyme functionalized carbon nanotubes

3-Hydroxybutyrate, one of the main blood ketone bodies, has been considered as a critical indicator for diagnosis of diabetic ketoacidosis. Biosensors designed for detection of 3-hydroxybutyrate with advantages of precision, easiness and speedy performance have attracted increasing attention. This study attempted to develop a 3-hydroxybutyrate dehydrogenase-based biosensor in which single-walled carbon nanotubes (SWCNT) was used in order to immobilize the cofactor, NAD⁺, on the surface of screen-printed electrode. The formation of NAD⁺–SWCNT conjugates was assessed by electrochemistry and electron microscopy. Cyclic voltammetry was used to analyze the performance of this biosensor electrochemically. The considerable shelf life and reliability of the proposed biosensor to analyze real sample was confirmed by this method. The reduction in the over potential of electrochemical oxidation of NADH to ?0.15 V can be mentioned as a prominent feature of this biosensor. This biosensor can detect 3-hydroxybutyrate in the linear range of 0.01–0.1 mM with the low detection limit of 0.009 mM. Simultaneous application of screen-printed electrode and SWCNT has made the biosensor distinguished which can open new prospects for detection of other clinically significant metabolites.     

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.     

Cholesterol amperometric biosensor based on cytochrome P450scc

A screen-printed enzyme electrode based on flavocytochrome P450scc (RfP450scc) for amperometric determination of cholesterol has been developed. A one-step method for RfP450scc immobilization in the presence of glutaraldehyde or by entrapment of enzyme within a hydrogel of agarose is discussed. The sensitivity of the biosensor based on immobilization procedures of flavocytochrome P450scc by glutaric aldehyde is 13.8 nA microM(-1) and the detection limit is 300 microM with a coefficient of linearity 0.98 for cholesterol in the presence of sodium cholate as detergent. The detection limits and the sensitivity of the agarose-based electrode are 155 microM and 6.9 nA microM(-1) with a linearity coefficient of 0.99. For both types of electrodes, the amperometric response to cholesterol in the presence of detergent was rather quick (1.5-2 min).     

Acetylecholinesterase-based biosensor electrodes for organophosphate pesticide detection. II. Immobilization and stabilization of acetylecholinesterase

The dry and wet stability of Drosophila acetylcholinesterase non-covalently immobilized onto polyethyleneimine modified screen-printed carbon electrodes was improved when compared to non-immobilized acetylcholinesterase, and acetylcholinesterase covalently immobilized onto dialdehyde and polyethyleneimine modified electrodes. Stabilizer mixtures were characterized for additional stabilization of acetylcholinesterase during storage in the dry state, with dextran-sulphate/sucrose and polygalacturonic acid/sucrose mixtures proving highly effective for long-term storage of biosensor electrodes.     

Development of a carbon nanotube paste electrode osmium polymer-mediated biosensor for determination of glucose in alcoholic beverages

This paper describes a new amperometric biosensor for glucose monitoring. The biosensor is based on the activity of glucose dehydrogenase (GDH) and diaphorase (DI) co-immobilized with NAD(+) into a carbon nanotube paste (CNTP) electrode modified with an osmium functionalized polymer. This mediator was demonstrated to shuttle the electron transfer between the immobilized diaphorase and the CNTP electrode, thus, showing a good electrocatalytic activity towards NADH oxidation at potentials around +0.2V versus Ag|AgCl, where interfering reactions are less prone to occur. The biosensor exhibits a detection limit of 10 micromol L(-1), linearity up to 8 x 10(-4) mol L(-1), a sensitivity of 13.4 microA cm(-2)mmol(-1)L(-1), a good reproducibility (R.S.D. 2.1%, n=6) and a stability of about 1 week when stored dry at 4 degrees C. Finally, the proposed biosensor was applied for the determination of glucose in different samples of sweet wine and validated with a commercial spectrophotometric enzymatic kit.     

一种新的检测黄曲霉毒素B1的酶生物传感器的制作

本文报道了一种新的检测黄曲霉毒素B1的生物传感器,该传感器以开管的多壁纳米碳管固定化黄曲霉毒素氧化还原酶制作传感电极检测黄曲霉毒素B1,其线性范围达到0.16μM-3.2μM,当把特异性的黄曲霉毒素B1抗体与黄曲霉毒素氧化还原酶通过多壁纳米碳管共固定化制作修饰电极,传感器的检测限提高到16nM,灵敏度提高了10倍。用这种方法制作黄曲霉毒素酶生物传感器,使黄曲霉毒素酶生物传感器向实用化迈进了一步。     

Development of a screen-printed amperometric biosensor for the determination of L-lactate dehydrogenase level.

We attempted to develop a screen-printed biosensor for the amperometric determination of L-lactate dehydrogenase (LDH) level on the basis of NAD(+)/NADH-dependent dehydrogenase reaction. The printing ink for the working electrode consisted of L-lactate, NAD(+), composite polymer of hydroxyethyl cellulose with ethylene glycol, 3,4-dihydroxybenzaldehyde (3,4-DHB) as an electron transferring mediator, and graphite as the conducting material. The 3,4-DHB was electropolymerized on the carboneous working electrode by potential cycling between -200 and +300 mV vs. Ag/AgCl reference electrode. Through the electrocatalytic reaction with immobilized 3,4-DHB, the NADH generated by the LDH reaction could be efficiently oxidized at lower potential than the unmodified carbon electrode. The analytical performance of the electrode was characterized in terms of linear sensing range and detection limit for LDH. The response from the developed biosensor was linear up to 500 U/l of LDH, and the detection limit of 50 U/l was observed at the signal-to-noise ratio of 3.     

Development of urinary albumin immunosensor based on colloidal AuNP and PVA

The development of immunosensors with high sensitivity and specificity in detecting the pathogenic or physiologically relevant molecules in the body, offers a powerful opportunity in early diagnosis and treatment of diseases. In this study, we developed a new competitive immunosensor with employing antibody (Ab) labeled AuNP (Ab-AuNP) and PVA modified screen-printed carbon electrode (SPCE) surface to detect the urine albumin. Field emission scanning electron microscopy (FE-SEM) of modified electrode showed a suitable and stable attachment between HSA antigen- mAb and AuNP. Cyclic voltammetric (CV) method demonstrated that modification process was well performed. Electrochemical measurements including differential pulse voltammetry (DPV) and square wave voltammetry (SWV) were employed for quantitative antigen detection. The electrochemical measurements performed with other proteins mixed with samples demonstrated a high specificity and selectivity for this biosensor in detecting the HSA. In optimal conditions, the immunosensor could detect HSA in a high linear range (from 2.5 to 200 μg/mL) with a low detection limit of 25 ng/mL. This new strategy could be improved and applied to detect the other antigen.     

Comparison of amperometric biosensors fabricated by palladium sputtering, palladium electrodeposition and Nafion/carbon nanotube casting on screen-printed carbon electrodes

Different strategies, including palladium electrodeposition (Pd(CV)), Pd sputtering (Pd(S)) and Nafion-solubilized carbon nanotube casting (Nafion/CNT), were used to modify screen-printed carbon electrodes (SPCEs) for the fabrication of amperometric enzyme biosensors. The electrochemical properties of the bare and modified SPCEs and the optimal conditions for surface modification were determined. The electrochemical response of the bare SPCE to H(2)O(2) under the potential of 0.3 V could be improved about 100-fold by Pd modification by electrodeposition or sputtering. By contrast, the electrochemical response of the bare SPCE was enhanced by only about 11-fold by Nafion/CNT casting. Moreover, the Pd(CV)-SPCEs exhibited better reproducibility of electrochemical response (a relative standard deviation (R.S.D.)<6.0%) than freshly prepared Pd(S)-SPCEs (R.S.D.>10%). The glucose biosensor fabricated from Pd-modified electrodes could be stored for up to 108 days without loosing significant activity. The Pd(CV)-SPCE also showed very reliable signal characteristics upon 50 consecutively repeated measurements of ascorbic acid. The electrocatalytic detection of the Pd-SPCE was combined with additional advantages of resistance to surface fouling and hence good stability. In conclusion, this study demonstrated that deposition of Pd thin film on SPCEs by electrodeposition or sputtering provided superior enhancement of electrochemical properties compared to Nafion/CNT-SPCEs. Despite their high electrochemical response, Pd(S)-SPCEs required an activation process to improve stability and Pd(CV)-SPCEs suffered from poor between electrode reproducibility.     

Preparation of poly(thionine) modified screen-printed carbon electrode and its application to determine NADH in flow injection analysis system

A poly(thionine) modified screen-printed carbon electrode has been prepared by an electrooxidative polymerization of thionine in neutral phosphate buffer. The modified electrodes are found to give stable and reproducible electrocatlytic responses to NADH and exhibit good stability. Several techniques, including cyclic voltammetry, X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM), have been employed to characterize the poly(thionine) film. Further, the modified screen-printed carbon electrode was found to be promising as an amperometric detector for the flow injection analysis (FIA) of NADH, typically with a dynamic range of 5-100 microM.     

Development of a disposable ethanol biosensor based on a chemically modified screen-printed electrode coated with alcohol oxidase for the analysis of beer

A disposable amperometric biosensor for the measurement of ethanol has been developed. It comprises a screen-printed carbon electrode doped with 5% cobalt phthalocyanine (CoPC-SPCE), and coated with alcohol oxidase; a permselective membrane on the surface acts as a barrier to interferents. The measurement of ethanol is based on the signal produced by H2O2, the product of the enzymatic reaction. Optimisation studies were performed using amperometry in stirred solution and the magnitude of the signal was found to be dependent on pH, enzyme loading, type of membrane and applied potential. The same technique was used to evaluate the biosensor for the determination of ethanol in samples. The results obtained compared well with the manufacturers specifications. In order to test the possibility of using the devices in the field, chronoamperometry was also used to determine ethanol in the same beer samples. The precision and recovery data again indicated that the biosensor should give reliable results under the conditions described.     

Electrodeposition of gold-platinum alloy nanoparticles on ionic liquid-chitosan composite film and its application in fabricating an amperometric cholesterol biosensor

An electrodeposition method was applied to form gold-platinum (AuPt) alloy nanoparticles on the glassy carbon electrode (GCE) modified with a mixture of an ionic liquid (IL) and chitosan (Ch) (AuPt-Ch-IL/GCE). AuPt nanoparticles were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM) and electrochemical methods. AuPt-Ch-IL/GCE electrocatalyzed the reduction of H(2)O(2) and thus was suitable for the preparation of biosensors. Cholesterol oxidase (ChOx) was then, immobilized on the surface of the electrode by cross-linking ChOx and chitosan through addition of glutaraldehyde (ChOx/AuPt-Ch-IL/GCE). The fabricated biosensor exhibited two wide linear ranges of responses to cholesterol in the concentration ranges of 0.05-6.2 mM and 6.2-11.2 mM. The sensitivity of the biosensor was 90.7 μA mM(-1) cm(-2) and the limit of detection was 10 μM of cholesterol. The response time was less than 7 s. The Michaelis-Menten constant (K(m)) was found as 0.24 mM. The effect of the addition of 1 mM ascorbic acid and glucose was tested on the amperometric response of 0.5 mM cholesterol and no change in response current of cholesterol was observed.     

Sensitive estimation of total cholesterol in blood using Au nanowires based micro-fluidic platform

Determination of cholesterol level in blood is important in clinical applications. In this work, modified Au nanowires-electrochemical biosensor based on MEMS micro-fluidic platform is proposed for estimating total cholesterol in blood. This sensor consists of "aligned" Au nanowires as working electrode, platinum counter electrode deposited on the silicon platform and Ag/AgCl (3M KCl) reference electrode. The "aligned" Au nanowires are immobilized with cholesterol oxidase and cholesterol esterase using specific covalent chemistry. Further, Au nanowires promotes better electron transfer between the enzymes and electrodes, because of their large surface to volume ratio, small diffusion time, large electrical conductivity and their aligned nature. The modified Au nanowires showed a stable calibration line and a quasi-linear relationship between cholesterol level and current response in the range of 1-6 mM (in steps of 1 mM over the baseline blood serum). The sensitivity of the modified electrode was found to be about 69 nA/mM with good storage and interference stability.     

Electrochemical DNA biosensor for bovine papillomavirus detection using polymeric film on screen-printed electrode

A new electrochemical DNA biosensor for bovine papillomavirus (BPV) detection that was based on screen-printed electrodes was comprehensively studied by electrochemical methods of cyclic voltammetry (CV) and differential pulse voltammetry (DPV). A BPV probe was immobilised on a working electrode (gold) modified with a polymeric film of poly-L-lysine (PLL) and chitosan. The experimental design was carried out to evaluate the influence of polymers, probe concentration (BPV probe) and immobilisation time on the electrochemical reduction of methylene blue (MB). The polymer poly-L-lysine (PLL), a probe concentration of 1μM and an immobilisation time of 60min showed the best result for the BPV probe immobilisation. With the hybridisation of a complementary target sequence (BPV target), the electrochemical signal decreased compared to a BPV probe immobilised on the modified PLL-gold electrode. Viral DNA that was extracted from cattle with papillomatosis also showed a decrease in the MB electrochemical reduction, which suggested that the decreased electrochemical signal corresponded to a bovine papillomavirus infection. The hybridisation specificity experiments further indicated that the biosensor could discriminate the complementary sequence from the non-complementary sequence. Thus, the results showed that the development of analytical devices, such as a biosensor, could assist in the rapid and efficient detection of bovine papillomavirus DNA and help in the prevention and treatment of papillomatosis in cattle.     

Development of a sandwich format, amperometric screen-printed uric acid biosensor for urine analysis

A screen-printed carbon electrode (SPCE) incorporating the electrocatalyst cobalt phthalocyanine (CoPC), fabricated using a water-based ink formulation, has been investigated as the base transducer for a uric acid biosensor. A sandwich biosensor was fabricated by first depositing cellulose acetate (CA) onto this transducer (CoPC-SPCE), followed by uricase (UOX) and finally a polycarbonate (PC) membrane; this device is designated PC-UOX-CA-CoPC-SPCE. This biosensor was used in conjunction with chronoamperometry to optimize the conditions for the analysis of urine: temperature, 35°C; buffer, pH 9.2; ionic strength, 50 mM; uricase, 0.6 U; incubation time, 180 s. The proposed biosensor was applied to urine from a healthy subject. The precision determined on unspiked urine (n=6) was 5.82%. Urine was fortified with 0.225 mM UA, and the resulting precision and recovery were 4.21 and 97.3%, respectively. The linear working range of the biosensor was found to be 0.015 to 0.25 mM (the former represents the detection limit), and the sensitivity was calculated to be 2.10 μA/mM.     

Carbon nanotube/polysulfone screen-printed electrochemical immunosensor

The simple and efficient method for preparing sensitive carbon nanotube/polysulfone/RIgG immunocomposite is described. The membrane of the modified disposable screen-printed electrochemical immunosensor is based on phase inversion method. Carbon nanotube/polysulfone membrane acts both as reservoir of immunological material and transducer while offering high surface area, high toughness and mechanical flexibility. The comparison with graphite/polysulfone/RIgG immunosensors shows a much higher sensitivity for those prepared with carbon nanotubes coupled with polysulfone (PSf). The membrane was characterized by scanning electron microscopy/energy dispersive X-ray analysis (SEM/EDX), laser profilometer and by atomic force microscopy (AFM). The purity of the materials was evaluated by thermogravimetric analysis (TGA). The roughness value is doubled when MWCNTs are used instead of graphite into the PSf membranes and the incorporation of antibodies enhances the dispersion of the carbon with the polymeric membrane reducing the roughness in all cases. This biosensor was based on the competitive assay between free and labelled anti-RIgG for the available binding sites of immobilized rabbit IgG (RIgG). The RIgG was incorporated into the polysulfone membrane by a phase inversion method. Horse radish peroxidase (HRP) enzyme was used as label and hydroquinone as mediator. The detection limit for competitive assay was determined to be 1.66 microg/ml. the linear range of anti-RIgG from 2 to 5 microg/ml and the C(50) was found at 3.56 microg/ml. The sensitivity is five times higher for MWCNT than for graphite electrodes, showing lower unspecific adsorption.