Covalent immobilization of cholesterol oxidase on self-assembled gold nanoparticles for highly sensitive amperometric detection of cholesterol in real samples

A novel scheme for the fabrication of gold nanoparticle modified cholesterol oxidase based bioelectrode is presented and its application potential for cholesterol biosensor is investigated. The fabrication procedure is based on the deposition of gold nanoparticles on the 1,6-hexanedithiol modified gold electrode, functionalization of the surface of deposited gold nanoparticles with carboxyl groups using 11-mercaptoundecanoic acid and then covalent immobilization of cholesterol oxidase on the surface of gold nanoparticle film using the N-ethyl-N'-(3-dimethylaminopropyl carbodimide) and N-hydroxysuccinimide ligand chemistry. The assembly process of the bioelectrode is investigated using atomic force microscopy, cyclic voltammetry and electrochemical impedance spectroscopy. The gold nanoparticle film on the electrode surface provided an environment for the enhanced electrocatalytic activities and thus resulted in enhanced analytical response. The resulting bioelectrode is further applied to the amperometric detection of cholesterol and exhibited a linear response to cholesterol in the range of 0.04-0.22 mM with a detection limit of 34.6 μM, apparent Michaelis-Menten constant (K(m)(app)) of 0.062 mM and a high sensitivity of 9.02 μA mM(-1). The fabricated bioelectrode is successfully used for the selective determination of cholesterol in human serum samples.     

Development of DNA electrochemical biosensor based on covalent immobilization of probe DNA by direct coupling of sol-gel and self-assembly technologies

A new procedure for fabricating deoxyribonucleic acid (DNA) electrochemical biosensor was developed based on covalent immobilization of target single-stranded DNA (ssDNA) on Au electrode that had been functionalized by direct coupling of sol-gel and self-assembled technologies. Two siloxanes, 3-mercaptopropyltrimethoxysiloxane (MPTMS) and 3-glycidoxypropyltrimethoxysiloxane (GPTMS) were used as precursors to prepare functionally self-assembly sol-gel film on Au electrode. The thiol group of MPTMS allowed assembly of MPTMS sol-gel on gold electrode surface. Through co-condensation between silanols, GPTMS sol-gel with epoxide groups interconnected into MPTMS sol-gel and enabled covalent immobilization of target NH(2)-ssDNA through epoxide/amine coupling reaction. The concentration of MPTMS and GPTMS influenced the performance of the resulting biosensor due to competitive sol-gel process. The linear range of the developed biosensor for determination of complementary ssDNA was from 2.51 x 10(-9) to 5.02 x 10(-7)M with a detection limit of 8.57 x 10(-10)M. The fabricated biosensor possessed good selectivity and could be regenerated. The covalent immobilization of target ssDNA on self-assembled sol-gel matrix could serve as a versatile platform for DNA immobilization and fabrication of biosensors.     

Methyl parathion hydrolase based nanocomposite biosensors for highly sensitive and selective determination of methyl parathion

This article reports the fabrication of a nanocomposite biosensor for the sensitive and specific detection of methyl parathion. The nanocomposite sensing film was prepared via the formation of gold nanoparticles on silica particles, mixing with multiwall carbon nanotubes and subsequent covalent immobilization of methyl parathion hydrolase. The composite of the individual materials was finely tuned to offer the sensing film with high specific surface area and high conductivity. A significant synergistic effect of nanocomposites on the biosensor performance was observed in biosensing methyl parathion. The square wave voltammetric responses displayed well defined peaks, linearly proportional to the concentrations of methyl parathion in the range from 0.001 μg mL?1 to 5.0 μg mL?1 with a detection limit of 0.3 ng mL?1. The application of this biosensor in the analysis of spiked garlic samples was also evaluated. The proposed protocol can be used as a platform for the simple and fast construction of biosensors with good performance for the determination of enzyme-specific electroactive species.     

Amperometric glucose biosensor based on single-walled carbon nanohorns

The biosensing application of single-walled carbon nanohorns (SWCNHs) was demonstrated through fabrication of an amperometric glucose biosensor. The biosensor was constructed by encapsulating glucose oxidase in the Nafion-SWCNHs composite film. The cyclic voltammograms for glucose oxidase immobilized on the composite film displayed a pair of well-defined and nearly symmetric redox peaks with a formal potential of -0.453 V. The biosensor had good electrocatalytic activity toward oxidation of glucose. To decrease detection potential, ferrocene monocarboxylic acid was used as a redox mediator. The mediated glucose biosensor shows a linear range from 0 to 6.0 mM. The biosensor shows high sensitivity (1.06 microA/mM) and stability, and can avoid the commonly coexisted interference. Because of impressive properties of SWCNHs, such as high purity and high surface area, SWCNHs and their composites are expected to be promising material for biomolecular immobilization and biosensing applications.     

Immobilization of glucose oxidase on electrodeposited nickel oxide nanoparticles: direct electron transfer and electrocatalytic activity

For the first time glucose oxidase (GOx) was successfully co-deposited on nickel-oxide (NiO) nanoparticles at a glassy carbon electrode. In this paper we present a simple fabrication method of biosensor which can be easily operated without using any specific reagents. Cyclic voltammetry was used for electrodeposition of NiO nanoparticle and GOx immobilization. The direct electron transfer of immobilized GOx displays a pair of well defined and nearly reversible redox peaks with a formal potential (E(0')) of -0.420 V in pH 7 phosphate buffer solution and the response shows a surface controlled electrode process. The surface coverage and heterogeneous electron transfer rate constant (k(s)) of GOx immobilized on NiO film glassy carbon electrode are 9.45 x 10(-13)mol cm(-2) and 25.2+/-0.5s(-1), indicating the high enzyme loading ability of the NiO nanoparticles and great facilitation of the electron transfer between GOx and NiO nanoparticles. The biosensor shows excellent electrocatalytical response to the oxidation of glucose when ferrocenmethanol was used as an artificial redox mediator. Furthermore, the apparent Michaelis-Menten constant 2.7 mM, of GOx on the nickel oxide nanoparticles exhibits excellent bioelectrocatalytic activity of immobilized enzyme toward glucose oxidation. In addition, this glucose biosensor shows fast amperometric response (3s) with the sensitivity of 446.2nA/mM, detection limit of 24 microM and wide concentration range of 30 microM to 5mM. This biosensor also exhibits good stability, reproducibility and long life time.     

Microstructured cystine dendrites-based impedimetric sensor for nucleic acid detection

We report results of the studies relating to the fabrication and characterization of novel biosensing electrode by covalent immobilization of DNA onto microstructural cystine (Cys) prepared by acoustic cavitation method. The TEM investigations of these structures reveal transformation of microstructured Cys from nanorods to dendritic structure under optimum conditions. The Cys dendrites (denCys) have been investigated by XRD, FT-IR, and SEM studies. These biosensing electrodes have been fabricated by immobilization of Escherichia coli (E. coli)-specific DNA probe onto the dendritic cystine. The results of the electrochemical impedance spectroscopy studies reveal that this nucleic acid sensor exhibits linear response to cDNA in the concentration range of 10(-6) to 10(-14) M with response time of 30 min. The biosensing characteristics show that the fabricated E. coli sensor can be reused about 4 times and is stable for ～4 weeks. The studies on cross-reactivity of the sensor for other water-borne pathogens like Salmonella typhimurium, Neisseria meningitides, and Klebsiella pneumonia reveal specificity of the bioelectrode for E. coli detection.     

Colorimetric paper bioassay by horseradish peroxidase for the detection of catechol and resorcinol in aqueous samples

Abstract

Phenolic compounds such as catechol and resorcinol are toxic and persistent pollutants in the aqueous environment. Detection procedures such as chromatographic and spectrophotometric methods are time-consuming and require sophisticated instruments with skilled manpower. Development of a simple, cost effective, portable and disposable paper based biosensor could be a better alternative to the conventional methods. The present study attempted to develop a paper based biosensor by immobilizing horseradish peroxidase enzyme to detect catechol and resorcinol in aqueous samples. Horseradish peroxidase catalyzes the oxidation of phenolic compounds to semiquinones, which on reaction with a chromogen, 3-methyl 2-benzothiazolinone hydrazine (MBTH) gives faint pink to red color depending on the compound and its concentration in the sample is the basis for biosensing application. Different methods of enzyme immobilization on filter paper like physical adsorption, covalent coupling, and polysaccharide entrapment were executed. The performance of the various enzyme immobilization methods was evaluated by analyzing the developed color intensity using ImageJ software. Entrapment technique is the most effective method of immobilizing enzyme on the filter paper that produces the highest color intensity with better stability. The visible limit of detection (LoD) was observed as 0.45?mM (50?mg/L) for catechol and 0.09?mM (10?mg/L) for resorcinol in aqueous samples.     

Highly sensitive and selective uric acid biosensor based on RF sputtered NiO thin film

Present study highlights the importance of RF sputtered NiO thin film deposited on platinum coated glass substrate (NiO/Pt/Ti/glass) as a potential matrix for the realization of highly sensitive and selective uric acid biosensor. Uricase has been immobilized successfully onto the surface of NiO matrix by physical adsorption technique. The prepared bioelectrode (uricase/NiO/Pt/Ti/glass) is utilized for sensing uric acid using the cyclic voltammetry and UV visible spectroscopy techniques. The bioelectrode is found to exhibit highly efficient sensing response characteristics with high sensitivity of 1278.48 μA/mM; good linearity of 0.05-1.0 mM, and very low Michaelis-Menten constant (k(m)) of 0.17 mM indicating high affinity of uricase towards the analyte. The enhanced response is due to the development of NiO matrix with good electron transport property and nanoporous morphology for effective loading of enzyme with preferred orientation.     

Immobilization and direct electrochemistry of glucose oxidase on a tetragonal pyramid-shaped porous ZnO nanostructure for a glucose biosensor

A tetragonal pyramid-shaped porous ZnO (TPSP-ZnO) nanostructure is used for the immobilization, direct electrochemistry and biosensing of proteins. The prepared ZnO has a large surface area and good biocompatibility. Using glucose oxidase (GOD) as a model, this shaped ZnO is tested for immobilization of proteins and the construction of electrochemical biosensors with good electrochemical performances. The interaction between GOD and TPSP-ZnO is examined by using AFM, N(2) adsorption isotherms and electrochemical methods. The immobilized GOD at a TPSP-ZnO-modified glassy carbon electrode shows a good direct electrochemical behavior, which depends on the properties of the TPSP-ZnO. Based on a decrease of the electrocatalytic response of the reduced form of GOD to dissolved oxygen, the proposed biosensor exhibits a linear response to glucose concentrations ranging from 0.05 to 8.2mM with a detection limit of 0.01mM at an applied potential of -0.50V which has better biosensing properties than those from other morphological ZnO nanoparticles. The biosensor shows good stability, reproducibility, low interferences and can diagnose diabetes very fast and sensitively. Such the TPSP-ZnO nanostructure provides a good matrix for protein immobilization and biosensor preparation.     

Enzyme integrated silicate-Pt nanoparticle architecture: a versatile biosensing platform

A novel 3-D nanoarchitectured platform based on Pt nanoparticles (nPts) is developed for the sensing of sub-nanomolar levels of hydrogen peroxide and for the fabrication of amperometric biosensor for uric acid, cholesterol and glucose. The nPts have been immobilized on the thiol functional group containing sol-gel silicate 3-D network derived from 3-mercaptopropyltrimethoxysilane (MPTS). The nanoparticles on the 3-D architecture have size distribution between 7 and 10nm. The nPts on the platform efficiently catalyze the oxidation of H(2)O(2) at the potential of +0.45 V in the absence of enzymes and redox mediators. This nanoarchitectured platform is highly sensitive and can detect H(2)O(2) at sub-nanomolar levels (0.1 nM) in neutral solution. The nanoarchitectured platform does not suffer from interference due to other common easily oxidizable interfering agents. Excellent reproducibility, long-term storage and operational stability are observed. This platform is used to determine H(2)O(2) concentration in rainwater and for the fabrication of biosensors. Amperometric oxidase-based biosensing platforms are developed by integrating the enzymes and nPts with the silicate network for the sensing of uric acid cholesterol and glucose. The enzyme encapsulated 3-D architecture retains the enzymatic activity and efficiently detects enzymatically generated H(2)O(2) without any interference. These biosensors are stable and show excellent sensitivity and fast response time. A linear response was obtained for a wide concentration range of all analytes. The practical utilization of the biosensor for the measurement of uric acid, cholesterol and glucose in serum sample is demonstrated. The biological sample analysis was validated with clinical laboratory measurements.     

Functionalized nanopipettes: toward label-free, single cell biosensors

Nanopipette technology has been proven to be a label-free biosensor capable of identifying DNA and proteins. The nanopipette can include specific recognition elements for analyte discrimination based on size, shape, and charge density. The fully electrical read-out and the ease and low-cost fabrication are unique features that give this technology an enormous potential. Unlike other biosensing platforms, nanopipettes can be precisely manipulated with submicron accuracy and used to study single cell dynamics. This review is focused on creative applications of nanopipette technology for biosensing. We highlight the potential of this technology with a particular attention to integration of this biosensor with single cell manipulation platforms.     

A high-performance glucose biosensor based on monomolecular layer of glucose oxidase covalently immobilised on indium-tin oxide surface

In this paper, a mediatorless amperometric glucose biosensor based on direct covalent immobilisation of monomolecular layer of glucose oxidase (GOx) on a semiconducting indium-tin oxide (ITO) is demonstrated. The abundance of surface hydroxyl functional group of ITO allows it to be used as a suitable platform for direct covalent immobilisation of the enzyme for sensor architecture. The anodic current corresponding to electrochemical oxidation of the enzymatic product, hydrogen peroxide, at a sputtered Pt electrode at 0.500 V (vs. SCE) was obtained as the sensor signal. It was found that the biosensor based on the direct immobilisation scheme shows a fast biosensor response, minimum interference from other common metabolic species and ease of biosensor miniaturisation. A linear range of 0-10 mM of glucose was demonstrated, which exhibits a high sensitivity as far as performance per immobilised GOx molecule is concerned. A detection limit as low as 0.05 mM and long-term stability were observed. Even more important, the biosensor design allows fabrication through a dry process. These characteristics make it possible to achieve mass production of biosensor compatible with the current electronic integrated circuit manufacturing technologies.     

A new impedimetric biosensor utilizing VEGF receptor-1 (Flt-1): early diagnosis of vascular endothelial growth factor in breast cancer

A new impedimetric biosensor, based on the use of vascular endothelial growth factor receptor-1 (VEGF-R1), was developed for the determination of vascular endothelial growth factor (VEGF). VEGF-R1 was immobilized through covalent coupling with 3-mercaptopropionic acid which formed a self-assembled monolayer on gold electrodes. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy techniques were employed to characterize the immobilization process and to detect VEGF. To successfully construct the biosensor current, experimental parameters were optimized. Kramers-Kronig Transform was performed on the experimental impedance data. The obtained results provided a linear response range from 10 to 70 pg/mL human VEGF. The applicability of the developed biosensor in the determination of VEGF in a spiked artificial human serum sample was experienced, yielding average recovery of 101%, in that order, with an average relative deviation value less than 5%.     

A novel and simple biomolecules immobilization method: electro-deposition ZrO2 doped with HRP for fabrication of hydrogen peroxide biosensor

For the first time, a very novel and simple immobilization method for fabrication of hydrogen peroxide biosensor was reported in this paper. The biocompatible composite HRP-ZrO(2) thin films were synthesized on gold electrode surface based on electro-deposition zirconia doped with horseradish peroxidase (HRP) by cyclic voltammetry scanning in KCl solution containing ZrO(2) and HRP. The fabricated process of biosensor was characterized by electrochemical impedance spectroscopy (EIS) and the surface topography of the prepared films was imaged by atomic force microscope (AFM). The HRP in HRP-ZrO(2) thin films kept its bioactivity and exhibited excellent electrocatalytical response to the reduction of H(2)O(2). Experimental conditions influencing the biosensor performance such as pH, potential were optimized. The resulting biosensor (HRP-ZrO(2)/Au electrode) showed a linear response to H(2)O(2) over a concentration range from 0.02 to 9.45mM with a detection limit of 2muM based on a signal-to-noise ratio of 3 under optimized conditions. The apparent Michaelis-Menten constant (K(M)(app)) was evaluated to be 8.01mM, which indicated the HRP in HRP-ZrO(2) thin films kept its native bioactivity and had high affinity for H(2)O(2). Moreover, the proposed biosensor showed high sensitivity, good reproducibility and long-term stability. What is more, this immobilization methodology widened biosensor application in biomolecules immobilization and could further develop for other protein and biomolecules immobilization.     

Development of an amperometric immunosensor for detection of staphylococcal enterotoxin type A in cheese

This paper reports a novel electrochemical immunosensor for the sensitive detection of staphylococcal enterotoxin A (SEA) based on self-assembly monolayer (SAM) and protein A immobilization on gold electrode. Three different methods of protein A immobilization were tested: physical adsorption, cross-linking using glutaraldehyde and covalent binding after activation with N-hydroxysuccinimide (NHS)/N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) on cysteamine-modified gold electrode. The EDC/NHS method for protein A immobilization was selected to lead development of the biosensor. The coating steps of the surface modification were characterized by cyclic voltammetry and the biosensor response by chronoamperometry. The advantages of the immunosensor were exposed in its high sensitivity and specificity. The proposed amperometric immunosensor was successfully used for determination of SEA in contaminated and non-contaminated cheese samples with excellent responses.     

Receptor elements for biosensors in two ways of methylotrophic yeast immobilization

Receptor elements for biosensors based on Hansenula polymorpha NCYC 495 ln yeast cells for ethanol assay were developed using two ways of cell immobilization, i.e., physical adsorption on a glass fiber membrane and covalent binding on a modified nitrocellulose membrane. The linear diapason of ethanol assays for a biosensor based on yeast cells adsorbed on glass fiber was 0.05–1.18; for a biosensor based on yeasts immobilized on a nitrocellulose membrane, 0.2–1.53 mM. Receptor elements based on sorbed cells possessed 2.5 times higher long-term stability. The time response was 1.5 times less for cells immobilized using DEAE-dextran and benzoquinone. The results of ethyl alcohol assays using biosensors based on cells immobilized via adsorption and covalent binding, as well as using the standard areometric method, had high correlation coefficients (0.998 and 0.997, respectively, for the two ways of immobilization). The results indicate the possibility to consider the described models of receptor elements for biosensors as prototypes for experimental samples for practical use.     

Fabrication of butyrylcholinesterase sensor using polyurethane-based ion-selective membranes.

A simple method of enzyme immobilization was investigated which is useful for fabrication of enzyme sensors based on polymeric ion-selective membranes. The enzyme membrane was built by coating a thin hydrophilic polyurethane (HPU) film directly mixed with an enzyme over an underlying polyurethane (PU)-based ion-selective membrane. This highly simple method of enzyme immobilization was applied to the fabrication of a potentiometric butyrylcholinesterase-based biosensor for the determination of organophosphorus pesticides. The enzyme was well entrapped within the HPU film and the intrinsic potentiometric response of the underlying ion-selective PU membrane was not influenced significantly by the outer HPU/enzyme membrane. The enzyme electrode was optimized by changing systematically the composition of the enzyme membrane to evaluate the effect of the changes on sensor response. The sensor was successfully applied to the analysis of paraoxon, an organophosphorus pesticide.     

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.     

Bio-smart hydrogels: co-joined molecular recognition and signal transduction in biosensor fabrication and drug delivery

Two classes of polymers that are currently receiving widespread attention in biosensor development are hydrogels and conducting electroactive polymers. The present study reports on the integration of these two materials to produce electroactive hydrogel composites that physically entrap enzymes within their matrices for biosensor construction and chemically stimulated controlled release. Enhanced biosensing capabilities of these membranes have been demonstrated in the fabrication of glucose, cholesterol and galactose amperometric biosensors. All biosensors displayed extended linear response ranges (10(-5)-10(-2) M), rapid response times (<60 s), retained storage stabilities of up to 1 year, and excellent screening of the physiological interferents ascorbic acid, uric acid, and acetaminophen. When the cross-linked hydrogel components of these composite membranes were prepared with the amine containing dimethylaminoethyl methacrylate monomer the result was polymeric devices that swelled in response to pH changes (neutral to acidic). Entrapment of glucose oxidase within these materials made them glucose-responsive through the formation of gluconic acid. When insulin was co-loaded with glucose oxidase into these "bio-smart" devices, there was a twofold increase in insulin release rate when the devices were immersed in glucose solutions. This demonstrates the potential of such systems to function as a chemically-synthesized artificial pancreas.     

A novel biosensor based on activation effect of thiamine on the activity of pyruvate oxidase

A biosensor based on pyruvate oxidase (POX) enzyme was developed for the investigation of the effect of thiamine (vitamin B(1)) molecule on the activity of the enzyme. The biosensor was prepared with a chemical covalent immobilization method on the dissolved oxygen (DO) probe by using gelatin and cross-linking agent, glutaraldehyde. POX catalyzes the degradation of pyruvate to acetylphosphate, CO(2) and H(2)O(2) in the presence of phosphate and oxygen. Thiamine is an activator for POX enzyme and determination method of the biosensor was based on this effect of thiamine on the activity of the enzyme. The biosensor responses showed increases in the presence of thiamine. Increases in the biosensor responses were related to thiamine concentration. Thiamine determination is based on the assay of the differences on the biosensor responses on the oxygenmeter in the absence and the presence of thiamine. The biosensor response depend linearly on thiamine concentration between 0.025 and 0.5 microM with 2 min response time. In the optimization studies of the biosensor the most suitable enzyme amount was found as 2.5 U cm(-2) and also phosphate buffer (pH 7.0; 50 mM) and 35 degrees C were obtained as the optimum working conditions. In the characterization studies of the biosensor some parameters such as activator and interference effects of some substances on the biosensor response and reproducibility were carried out.