Amperometric biosensor based on a site-specific immobilization of acetylcholinesterase via affinity bonds on a nanostructured polymer membrane with integrated multiwall carbon nanotubes

Acetylcholinesterase (AChE) was immobilized on chemically modified poly-(acrylonitrile-methyl-methacrylate-sodium vinylsulfonate) membranes in accordance with three different methods, the first of which involved random enzyme immobilization via glutaraldehyde, the second one—site-specific enzyme immobilization via glutaraldehyde and Concanavalin A (Con A) and the third method—modified site-specific enzyme immobilization via glutaraldehyde in the presence of a mixture of multiwall carbon nanotubes and albumin (MWCNs + BSA), glutaraldehyde and Con A. Preliminary tests for the activity of immobilized AChE were carried out using these three methods. The third method was selected as the most efficient one for the immobilization of AChE and the prepared enzyme carriers were used for the construction of amperometric biosensors for the detection of acetylthiocholine (ATCh).A five level three factorial central composite design was chosen to determine the optimal conditions for the enzyme immobilization with three critical variables: concentration of enzyme, Concanavalin A and MWCNs. The design illustrated that the optimum values of the factors influencing the amperometric current were C_E: 70 U mL⁻¹; C_{Con A}: 1.5 mg mL⁻¹ and C_MWCN: 11 mg mL⁻¹, with an amperometric current 0.418 μA. The basic amperometric characteristics of the constructed biosensor were investigated. A calibration plot was obtained for a series of ATCh concentrations ranging from 5 to 400 μM. A linear interval was detected along the calibration curve from 5 to 200 μM. The correlation coefficient for this concentration range was 0.995. The biosensor sensitivity was calculated to be 0.065 μA μM⁻¹ cm⁻². The detection limit with regard to ATCh was calculated to be 0.34 μM. The potential application of the biosensor for detection and quantification of organophosphate pesticides was investigated as well. It was tested against sample solutions of Paraoxon. The biosensor detection limit was determined to be 1.39 × 10⁻¹² g L⁻¹ of Paraoxon, as well as the interval (10⁻¹¹ to 10⁻⁸ g L⁻¹) within which the biosensor response was linearly dependant on the Paraoxon concentration. Finally the storage stability of the enzyme carrier was traced for a period of 120 days. After 30-day storage the sensor retained 76% of its initial current response, after 60 days—68% and after 120 days—61%.     

Amperometric ascorbic acid sensor based on doped ferrites nanoparticles modified glassy carbon paste electrode

In this study, a novel electrochemical sensor for quantification of ascorbic acid with amperometric detection in physiological conditions was constructed. For this purpose, cobalt and nickel ferrites were synthesized using microwave and ultrasound assistance, characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and X-ray powder diffraction (XRPD), and used for modification of glassy carbon paste electrode (GCPE). It was shown that introducing these nanoparticles to the structure of GCPE led to increasing analytical performance. Co ferrite modified GCPE (CoFeGCPE) showed better characteristics toward ascorbic acid sensing. The limit of detection (LOD) obtained by sensor was calculated to be 0.0270 mg/L, with linear range from 0.1758 to 2.6010 mg/L. This sensor was successfully applied for practical analysis, and the obtained results demonstrated that the proposed procedure could be a promising replacement for the conventional electrode materials and time-consuming and expensive separation methods.     

Dual-mode potential oscillations on an immobilized acetylcholinesterase membrane system

We have studied by numerical stimulations the effects of varying external salt concentration on a multibarrier kinetic model of a synthetic membrane system bearing AChE and BSA. For certain parameters, we find a spiky high frequency modulation imposed on a bursting low frequency carrier. In the absence of added salt, oscillations with only one mode arise, consistent with the experimental result of Friboulet and Thomas. From this study, we suggest how periodic ionic permeability changes of ions may arise in excitable membranes.     

Amperometric determination of total phenolic content in wine by laccase immobilized onto silver nanoparticles/zinc oxide nanoparticles modified gold electrode

Tyrosine sulfation is a ubiquitous posttranslational modification that regulates extracellular protein-protein interactions, intracellular protein transportation modulation, and protein proteolytic process. However, identifying tyrosine sulfation sites remains a challenge due to the lability of sulfation sequences. In this study, we developed a method called PredSulSite that incorporates protein secondary structure, physicochemical properties of amino acids, and residue sequence order information based on support vector machine to predict sulfotyrosine sites. Three types of encoding algorithms-secondary structure, grouped weight, and autocorrelation function-were applied to mine features from tyrosine sulfation proteins. The prediction model with multiple features achieved an accuracy of 92.89% in 10-fold cross-validation. Feature analysis showed that the coil structure, acidic amino acids, and residue interactions around the tyrosine sulfation sites all contributed to the sulfation site determination. The detailed feature analysis in this work can help us to understand the sulfation mechanism and provide guidance for the related experimental validation. PredSulSite is available as a community resource at http://www.bioinfo.ncu.edu.cn/inquiries_PredSulSite.aspx.     

Electrochemical sensor based on gold nanoparticles fabricated molecularly imprinted polymer film at chitosan-platinum nanoparticles/graphene-gold nanoparticles double nanocomposites modified electrode for detection of erythromycin

A molecularly imprinted electrochemical sensor was fabricated based on gold electrode decorated by chitosan-platinum nanoparticles (CS-PtNPs) and graphene-gold nanoparticles (GR-AuNPs) nanocomposites for convenient and sensitive determination of erythromycin. The synergistic effects of CS-PtNPs and GR-AuNPs nanocomposites improved the electrochemical response and the sensitivity of the sensor. The molecularly imprinted polymers (MIPs) were prepared by HAuCl(4), 2-mercaptonicotinic acid (MNA) and erythromycin. Erythromycin and MNA were used as template molecule and functional monomer, respectively. They were first assembled on the surface of GR-AuNPs/CS-PtNPs/gold electrode by the formation of Au-S bonds and hydrogen-bonding interactions. Then the MIPs were formed by electropolymerization of HAuCl(4), MNA and erythromycin. The sensor was characterized by cyclic voltammetry (CV), scanning electron microscope (SEM), UV-visible (UV-vis) absorption speactra and amperometry. The linear range of the sensor was from 7.0×10(-8)mol/L-9.0×10(-5)mol/L, with the limit of detection (LOD) of 2.3×10(-8)mol/L (S/N=3). The sensor showed high selectivity, excellent stability and good reproducibility for the determination of erythromycin, and it was successfully applied to the detection of erythromycin in real spiked samples.     

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

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

Amperometric biosensor based on coentrapment of enzyme and mediator by gold nanoparticles on indium-tin oxide electrode

A disposable pseudo-mediatorless amperometric biosensor has been fabricated for the determination of hydrogen peroxide (H2O2). In the current study, an indium-tin oxide (ITO) electrode was modified with thiol functional group by (3-mercaptopropyl)trimethoxysilane. The stable nano-Au-SH monolayer (AuS) was then prepared through covalent linking of gold nanoparticles and thiol groups on the surface of the ITO. The horseradish peroxidase (HRP) and tetramethyl benzidine (TMB) were finally coentrapped by the colloidal gold nanoparticles. The immobilized TMB was used as an electron transfer mediator that displayed a surface-controlled electrode process at a scan rate of less than 50mV/s. The biosensor was characterized by photometric and electrochemical measurements. The results showed that the prepared AuS monolayer not only could steadily immobilize HRP but also could efficiently retain HRP bioactivity. Parameters affecting the performance of the biosensor, including the concentrations of the immobilized TMB and HRP, the pH value, and the reaction temperature, were optimized. Under the optimized experimental conditions, H(2)O(2) could be determined in a linear calibration range from 0.005 to 1.5mM with a correlation coefficient of 0.998 (n=14) and a detection limit of 1microM at a signal/noise ratio of 3. The proposed method provides a new alternative to develop low-cost biosensors by using ITO film electrodes from industrial mass production.     

An amperometric immunosensor for osteoproteogerin based on gold nanoparticles deposited conducting polymer

An amperometric immunosensor was fabricated for the detection of osteoproteogerin (OPG) by covalently immobilizing a monoclonal OPG antibody (anti-OPG) onto the gold nanoparticles (AuNPs) deposited functionalized conducting polymer (5,2′:5′,2″-terthiophene-3′-carboxylic acid). AuNPs were electrochemically deposited onto the conducting polymer using cyclic voltammetry. The particle size of deposited AuNPs was controlled by varying the scan rate and was characterized by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The immobilization of anti-OPG was also confirmed using XPS. The principle of immunosensor was based on a competitive immunoassay between free-OPG and labeled-OPG for the active sites of anti-OPG. HRP was used as a label that electrochemically catalyzes the H₂O₂ reduction. The catalytic reduction was monitored amperometrically at −0.4 V vs. Ag/AgCl. The immunosensor showed a linear range between 2.5 and 25 pg/ml and the detection limit was determined to be 2 pg/ml. The proposed immunosensor was successfully applied for real human samples to detect OPG.     

Amperometric biosensor for hydrogen peroxide based on horseradish peroxidase onto gold nanowires and TiO2 nanoparticles

An electrochemical biosensor for determination of hydrogen peroxide (H₂O₂) was fabricated, based on the electrostatic immobilization of horseradish peroxidase (HRP) with one-dimensional gold nanowires (Au NWs) and TiO₂ nanoparticles (nano-TiO₂) on a gold electrode. The nano-TiO₂ can give a biocompatible microenvironment and compact film, and the Au NWs can provide fast electron transferring rate and greatly add the amount of HRP molecules immobilized on the electrode surface. Au NWs were characterized by ultraviolet–visible spectra and transmission electron microscope. The electrode modification process was probed by cyclic voltammetry and electrochemical impedance spectroscopy. Chronoamperometry was used to study the electrochemical performance of the resulting biosensor. Under optimal conditions, the linear range for the determination of H₂O₂ was from 2.3 × 10⁻⁶ to 2.4 × 10⁻³ M with a detection limit of 7.0 × 10⁻⁷ M (S/N = 3). Moreover, the proposed biosensor showed superior stability and high sensitivity.     

Amperometric ATP biosensor based on polymer entrapped enzymes

A dual enzyme electrode for the detection of adenosine-5'-triphosphate (ATP) at physiologically relevant pH levels was developed by co-immobilization of the enzymes glucose oxidase (GOD) and hexokinase (HEX) using pH-shift induced deposition of enzyme containing polymer films. Application of a simple electrochemical procedure for the co-immobilization of the enzymes at electrode surfaces exhibits a major improvement of sensitivity, response time, reproducibility, and ease of fabrication of ATP biosensors. Competition between glucose oxidase and hexokinase for the substrate glucose involving ATP as a co-substrate allows the determination of ATP concentrations. Notable control on the immobilization process enables fabrication of micro biosensors with a diameter of 25 microm. The presented concept provides the technological basis for a new generation of fast responding, sensitive, and robust biosensors for the detection of ATP at physiological pH values with a detection limit of 10 nmol l(-1).     

Label-free antibody-antigen binding detection by optical sensor array based on surface-synthesized gold nanoparticles

Gold nanoparticles grown in situ from printed seed particles on a glass substrate have been fabricated into a biosensor array. The light-scattering properties of the resulting surfaces show sensitivity to changes in the local refractive index. Each array spot is functionalized with fibrinogen or bovine serum albumin and scattered radiation is used to monitor the refractive index change on label-free binding of the antibodies to their antigens from whole blood antiserum. Data were collected real-time and the association rate constants for the specific antibody-antigen binding were derived from a kinetic analysis. The minimum antibody concentration detection sensitivity is of 100 nM.     

Amperometric biosensor based on tyrosinase immobilized on a boron-doped diamond electrode

A novel method has been developed to immobilize tyrosinase onto the surface of boron-doped diamond (BDD) electrode. The hydrogen-terminated BDD (HBDD) surface was first functionalized by photochemically linking vinyl groups of allylamine, producing covalently linked amine-terminated active BDD (ABDD) surface. Then the tyrosinase was immobilized onto the ABDD surface by carbodiimide coupling reaction. The amperometric response was measured as a function of concentration of phenolic compounds in 0.1M phosphate buffer solution (pH 6.5). The tyrosinase-modified ABDD electrode gave a linear response range of 1-175, 1-200 and 1-200 microM and sensitivity of 80.0, 181.4 and 110.0 mA M(-1)cm(-2) for phenol, p-cresol, 4-chlorophenol, respectively. Moreover, selective detection of dopamine (DA) in the presence of ascorbic acid (AA) has been demonstrated with the tyrosinase-modified ABDD electrode. Linearity was observed within the range of 5-120 microM. The above enzyme electrode could maintain 90% of its original activity after intermittent use for 1 month when storing in a dry state at 4 degrees C.     

Development of acetylcholinesterase biosensor based on CdTe quantum dots/gold nanoparticles modified chitosan microspheres interface

In this paper, a novel acetylcholinesterase (AChE) biosensor was constructed by modifying glassy carbon electrode with CdTe quantum dots (QDs) and excellent conductive gold nanoparticles (GNPs) though chitosan microspheres to immobilize AChE. Since GNPs have shown widespread use particularly for constructing electrochemical biosensors through their high electron-transfer ability, the combined AChE exhibited high affinity to its substrate and thus a sensitive, fast and cheap method for determination of monocrotophos. The combination of CdTe QDs and GNPs promoted electron transfer and catalyzed the electro-oxidation of thiocholine, thus amplifying the detection sensitivity. This novel biosensing platform based on CdTe QDs-GNPs composite responded even more sensitively than that on CdTe QDs or GNPs alone because of the presence of synergistic effects in CdTe-GNPs film. The inhibition of monocrotophos was proportional to its concentration in two ranges, from 1 to 1000ngmL(-1) and from 2 to 15mugmL(-1), with a detection limit of 0.3ngmL(-1). The proposed biosensor showed good precision and reproducibility, acceptable stability and accuracy in garlic samples analysis.     

Amperometric third-generation hydrogen peroxide biosensor based on the immobilization of hemoglobin on multiwall carbon nanotubes and gold colloidal nanoparticles

A convenient and effective strategy for preparation nanohybrid film of multi-wall carbon nanotubes (MWNT) and gold colloidal nanoparticles (GNPs) by using proteins as linker is proposed. In such a strategy, hemoglobin (Hb) was selected as model protein to fabricate third-generation H2O2 biosensor based on MWNT and GNPs. Acid-pretreated, negatively charged MWNT was first modified on the surface of glassy carbon (GC) electrode, then, positively charged Hb was adsorbed onto MWNT films by electrostatic interaction. The {Hb/GNPs}n multilayer films were finally assembled onto Hb/MWNT film through layer-by-layer assembly technique. The assembly of Hb and GNPs was characterized with cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and transmission electron microscopy (TEM). The direct electron transfer of Hb is observed on Hb/GNPs/Hb/MWNT/GC electrode, which exhibits excellent electrocatalytic activity for the reduction of H2O2 to construct a third-generation mediator-free H2O2 biosensor. As compared to those H2O2 biosensors only based on carbon nanotubes, the proposed biosensor modified with MWNT and GNPs displays a broader linear range and a lower detection limit for H2O2 determination. The linear range is from 2.1x10(-7) to 3.0x10(-3) M with a detection limit of 8.0x10(-8) M at 3sigma. The Michaelies-Menten constant KMapp value is estimated to be 0.26 mM. Moreover, this biosensor displays rapid response to H2O2 and possesses good stability and reproducibility.     

Third-generation superoxide anion sensor based on superoxide dismutase directly immobilized by sol-gel thin film on gold electrode

A third-generation biosensor for superoxide anion (O(2)*-) was developed based on superoxide dismutase (SOD) immobilized by thin silica-PVA sol-gel film on gold electrode surface. A rapid and direct electron transfer of SOD in the thin sol-gel film at the gold electrode was realized without any mediators or promoters. The characterization of the SOD electrodes showed a quasi-reversible electrochemical redox behavior with a formal potential of 80 + 5 mV (versus SCE) in 50 mmol l(-1) phosphate buffer solution (PBS), pH 7.0. The heterogeneous electron transfer rate constant was evaluated to be about 2.1s(-1). The anodic and cathodic transfer coefficients are 0.6 and 0.4, respectively. Based on biomolecular recognition for specific reactivity of SOD toward O(2)*- the SOD electrode was applied to a sensitive and selective measurement of O(2)*- with the low operation potential (-0.15 V versus SCE) in phosphate buffer solution, pH 7.0. The amperometric response was proportional to O(2)*- concentration in the range of 0.2-1.6 micromol l(-1) and the detection limit was 0.1 micromol l(-1) at a signal-to-noise ration of 3. The preparation of SOD electrode is easy and simple. The uniform porous structure of the silica-PVA sol-gel matrix results in a fast response rate of immobilized SOD and is very efficient for stabilizing the enzyme activity.     

Electrochemical studies of glucose oxidase immobilized on glutathione coated gold nanoparticles

Glutathione (L-gamma-glutamyl-L-cysteinyl-L-glycine; GSH) forms a surface monolayer on gold nanoparticles by tethering via sulfur bonds (Au:GSH). In the present study, glucose oxidase (GOx; EC 1.1.3.4) was immobilized by covalent chemical coupling reactions on to Au:GSH nanoparticles and the enzyme coupled nanoparticles formed a stable colloid (stable for several weeks) in water. The immobilized enzyme was investigated for electrochemical characteristics to monitor the FAD (prosthetic group of the GOx) redox potentials. Various concentrations of substrate (glucose) were added to check the oxidation characteristics. It was observed that with increase in substrate concentrations, the oxidation rate increased proportionally with the current. The present study demonstrated that GOx was effectively coupled to the gold nanoparticle (Au:GSH). The coupled nanoparticle system could be used in a potential biosensor application. Similarly, other enzymes (e.g., horseradish peroxidase) could be immobilized to the Au:GSH nanoparticles via the peptide arm (GSH) to achieve the desired characteristics needed for a specific application in biosensor.     

Ultrasensitive DNA sensor based on gold nanoparticles/reduced graphene oxide/glassy carbon electrode

We have designed a simple and novel electrochemical biosensor based on glassy carbon electrode (GCE) for DNA detection. GCE was modified with reduced graphene oxide (RGO) and gold nanoparticles (AuNPs) by the electrochemical method, which is helpful for immobilization of thiolated bioreceptors. The electrode modification processes were characterized by scanning electron microscopy (SEM) and electrochemical methods. Then a single-stranded DNA (ssDNA) probe for BRCA1 5382 insC mutation detection was immobilized on the modified electrode for a specific time. The experimental conditions, such as probe immobilization time and target DNA (complementary DNA) hybridization time and temperature with probe DNA, were optimized using electrochemical methods. The electrochemical response for DNA hybridization and synthesis was measured using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) methods. The calibration graph contains two linear ranges; the first part is in the range of 3.0 × 10⁻²⁰ to 1.0 × 10⁻¹² M, and the second segment part is in the range of 1.0 × 10⁻¹² to 1.0 × 10⁻⁷ M. The biosensor showed excellent selectivity for the detection of the complementary sequences from noncomplementary sequences, so it can be used for detection of breast cancer.     

Colorimetric detection of bisphenol A based on unmodified aptamer and cationic polymer aggregated gold nanoparticles

In this study, a colorimetric method was exploited to detect bisphenol A (BPA) based on BPA-specific aptamer and cationic polymer-induced aggregation of gold nanoparticles (AuNPs). The principle of this assay is very classical. The aggregation of AuNPs was induced by the concentration of cationic polymer, which is controlled by specific recognition of aptamer with BPA and the reaction of aptamer and cationic polymer forming “duplex” structure. This method enables colorimetric detection of BPA with selectivity and a detection limit of 1.50 nM. In addition, this colorimetric method was successfully used to determine spiked BPA in tap water and river water samples.     

Amperometric biosensors for detection of phenol using chemically modified electrodes containing immobilized bacteria

Eight strains of Pseudomonas were studied for development of phenol sensor. The immobilization of cells was performed by absorbing them on the working part of mediator-modified screen-printed electrodes (SPEs). Only three Pseudomonas strains were able to transfer electrons resulting from specific oxidation of phenol to the electrode by means of mediators; ferrocene, duroquinone and dimethyferrocene were successfully used with the strains 394 (p20), 74-III and 83-IV (working names), respectively. The lower limits for detection of phenol were 1 micro M for the strain 74-III and 10 micro M for the strain 83-IV and 394 (p20). Calibrations were obtained as the dependencies of logarithm of current changes (log deltaI) on logarithm of concentration (logC), log delta I vs. logC. Among all substrates tested (phenol, catechol, hydroquinone, ethanol, methanol, propanol, isopropanol, isobutanol, isoamylalcohol, acetate, glucose, xylose, vanillin, 2,4,6-trichlorphenol, 2,3,6-trichlorphenol, 4-hydroxy-3-methoxybenzoic acid, coumarin, pentafluorophenol), bacterial sensor demonstrated a good selectivity with respect to phenol and lower responses to catechol and hydroquinone (10-times lower). The dependence of signals on operating conditions was studied. The biosensor should be used during the day of preparation. The operational stability was satisfactory to perform up to 10 consecutive measurements. Low cost and very simple manufacturing procedure allow for bacterial sensor to be applied as disposable devices.