Fabrication of a Highly Sensitive Glucose Biosensor Based on Immobilization of Osmium Complex and Glucose Oxidase onto Carbon Nanotubes Modified Electrode

In this research a novel osmium complex was used as electrocatalyst for electroreduction of oxygen and H₂O₂ in physiological pH solutions. Electroless deposition at a short period of time (60 s), was used for strong and irreversible adsorption of 1,4,8,12‐tetraazacyclotetradecane osmium(III) chloride (Os(III)LCl₂) ClO₄ onto single‐walled carbon nanotubes (SWCNTs) modified GC electrode. The modified electrode shows a pair of well defined and reversible redox couple, Os(IV)/Os(III) at wide pH range (1–8). The glucose biosensor was fabricated by covering a thin film of glucose oxidase onto CNTs/Os‐complex modified electrode. The biosensor can be used successfully for selective detection of glucose based on the decreasing of cathodic peak current of oxygen. The fabricated biosensor shows high sensitivity, 826.3 nA μM^?1cm^?2, low detection limit, 56 nM, fast response time <3 s and wide calibration range 1.0 μM–1.0 mM. The biosensor has been successfully applied to determination of glucose in human plasma. Because of relative low applied potential, the interference from electroactive existing species was minimized, which improved the selectivity of the biosensor. The apparent Michaelis‐Menten constant of GOx on the nanocomposite, 0.91 mM, exhibits excellent bioelectrocatalytic activity of immobilized enzyme toward glucose oxidation. Excellent electrochemical reversibility, high stability, technically simple and possibility of preparation at short period of time are of great advantages of this glucose biosensor.     

Development of Quantum Dots Modified Acetylcholinesterase Biosensor for the Detection of Trichlorfon

Poly (N‐vinyl‐2‐pyrrolidone) (PVP)‐capped CdS quantum dots (QCdS‐PVP) was synthesized with CdCl₂ and Na₂S in the presence of PVP. QCdS‐PVP has been used for the immobilization and stabilization of the acetylcholinesterase (AChE). The electrocatalytic activity of QCdS‐PVP leads to a greatly improved electrochemical detection of the enzymatically generated thiocholine product, and higher sensitivity and stability. The GCE/QCdS‐PVP/AChE biosensor was used for the detection of organophosphate pesticides (OPs), such as trichlorfon. The sensor performance, including pH and inhibition time, was optimized with respect to operating conditions. Under the optimal conditions, the biosensor was used to measure as low as 12 ppb trichlorfon with a 5‐min inhibition time.     

Sol‐Gel‐Derived Biosensor Based on Plant Tissue: The Inhibitory Effect of Atrazine on Polyphenol Oxidase Activity for Determination of Atrazine

This work presents a sol‐gel based biosensor for atrazine determination which has been obtained by introducing the enzyme polyphenol oxidase from apple tissue in a sol‐gel matrix. Apple tissue acts as a molecular recognition element. Atrazine is an inactive compound electrochemically; redox coupling of dopamine was used for studying atrazine behavior. Atrazine was determined by monitoring the inhibition power of polyphenol oxidase activity. The measurements were performed in 0.1 M KH₂PO₄‐NaOH buffer (pH 7.5). The effect of various experimental parameters such as pH, concentration of buffer, concentration of dopamine, incubation time and matrix composition has been investigated for optimum analytical performance. The biosensor consisted of 10.3% (w/w) of apple tissue. The bioelectrode exhibits a linear response for dopamine and atrazine concentrations in the range of 5.66 × 10^?6?2.27 × 10^?3M and 1 × 10^?5 ?1 × 10^?4 M with a detection limit of 4.2 × 10^?6 and 5.5 × 10^?6 M, respectively. A correlation coefficient of 0.9945 and a relative standard deviation (R.S.D.) of 3.29% for dopamine, 0.9944 and 3.69% for a trazine were achieved.     

Carbon Nanotubes‐Based Amperometric Cholesterol Biosensor Fabricated Through Layer‐by‐Layer Technique

A carbon nanotubes‐based amperometric cholesterol biosensor has been fabricated through layer‐by‐layer (LBL) deposition of a cationic polyelectrolyte (PDDA, poly(diallyldimethylammonium chloride)) and cholesterol oxidase (ChOx) on multi‐walled carbon nanotubes (MWNTs)‐modified gold electrode, followed by electrochemical generation of a nonconducting poly(o‐phenylenediamine) (PPD) film as the protective coating. Electrochemical impedance measurements have shown that PDDA/ChOx multilayer film could be formed uniformly on MWNTs‐modified gold electrode. Due to the strong electrocatalytic properties of MWNTs toward H₂O₂ and the low permeability of PPD film for electroacitve species, such as ascorbic acid, uric acid and acetaminophen, the biosensor has shown high sensitivity and good anti‐interferent ability in the detection of cholesterol. The effect of the pH value of the detection solution on the response of the biosensor was also investigated. A linear range up to 6.0 mM has been observed for the biosensor with a detection limit of 0.2 mM. The apparent Michaelis‐Menten constant and the maximum response current density were calculated to be 7.17 mM and 7.32 μA cm^?2, respectively.     

Fast and Effective Turn‐on Paper‐based Phosphorescence Biosensor for Detection of Glucose in Serum

In this study, a turn‐on paper‐based optical analytical system with a rapid, sensitive and quantitative response for glucose was developed. The luminescence sensing material, crystalline iridium(III)‐Zn(II) coordination polymers, or Ir‐Zn_e, was grown electrochemically on stainless steel mesh and then deposited on filter paper. This sensing substrate was subsequently built up under glucose oxidase encapsulated in hydrogel and then immobilized on egg membrane with the layer‐by‐layer method. Once the glucose solution was dropped onto the paper, the oxygen content was depleted simultaneously with a concomitant increase in the phosphorescence of Ir‐Zn_e. The detection limit for glucose was 0.05 mM. The linear dynamic range for the determination of glucose was 0.05–8.0 mM with a correlation coefficient (R²) of 0.9956 (y=68.11 [glucose]?14.72). The response time was about 0.12 s, and the sample volume was less than 5 μL. The effects of mesh size, buffer concentration, pH, enzyme concentration, temperature, and interference, and the stability of the biosensor, have also been studied in detail. Finally, the biosensor was successfully applied to the determination of glucose in human serum.     

Electrogenerated Chemiluminescence Ethanol Biosensor Based on Carbon Nanotube‐Titania‐Nafion Composite Film

Mesoporous titania‐Nafion composite doped with carbon nanotube (CNT) has been used for the immobilization of tris(2,2′‐bipyridyl)ruthenium(II) (Ru(bpy)₃²⁺) and alcohol dehydrogenase on an electrode surface to yield a highly sensitive and stable electrogenerated chemiluminescence (ECL) ethanol biosensor. The presence of CNT in the composite film increases not only the sensitivity of the ECL biosensor but also the long‐term stability of the biosensor. The present biosensor responds linearly to ethanol in the wide concentration ranges from 1.0×10^?5 M to 1.0×10^?1 M with a detection limit of 5.0×10^?6 M (S/N=3). The present ECL ethanol biosensor exhibited higher ECL response compared to that obtained with the ECL biosensor based on the corresponding composite without CNT. The present CNT‐based ECL biosensor showed good long‐term stability with 75% of its initial activity retained after 2 weeks of storage in 50 mM phosphate buffer at pH 7.0.     

A Third‐Generation Hydrogen Peroxide Biosensor Based on Horseradish Peroxidase Immobilized in Carbon Nanotubes/ SBA‐15 Film

A new third‐generation biosensor for H₂O₂ assay was developed on the basis of the immobilization of horseradish peroxidase (HRP) in a nanocomposite film of carbon nanotubes (CNTs)‐SBA‐15 modified gold electrode. The biological activity of HRP immobilizing in the composite film was characterized by UV‐vis spectra. The HRP immobilized in the nanocomposite matrix displayed excellent electrocatalytic activity to the reduction of H₂O₂. The effects of the experimental variables such as solution pH and working potential were investigated using steady‐state amperometry. Under the optimal conditions, the resulting biosensor showed a linear range from 1 µM to 7 mM and a detection limit of 0.5 µM (S/N=3). Moreover, the stability and reproducibility of this biosensor were evaluated with satisfactory results.     

A New Amperometric Biosensor Based on HRP/Nano-Au/L-Cysteine/Poly（o-Aminobenzoic acid）-Membrane-Modified Platinum Electrode for the Determination of Hydrogen Peroxide

The third generation amperometric biosensor for the determination of hydrogen peroxide （H2O2） has been described. For the fabrication of biosensor, o-aminobenzoic acid （oABA） was first electropolymerized on the surface of platinum （Pt） electrode as an electrostatic repulsion layer to reject interferences. Horseradish peroxidase （HRP） absorbed by nano-scaled particulate gold （nano-Au） was immobilized on the electrode modified with polymerized o-aminobenzoic acid （poABA） with L-cysteine as a linker to prepare a biosensor for the detection of H2O2. Amperometric detection of H2O2 was realized at a potential of ＋20 mV versus SCE. The resulting biosensor exhibited fast response, excellent reproducibility and sensibility, expanded linear range and low interferences. Temperature and pH dependence and stability of the sensor were investigated. The optimal sensor gave a linear response in the range of 2.99×10^-6 to 3.55×10^-3 mol·L^-1 to H2O2 with a sensibility of 0.0177 A·L^-1·mol^-1 and a detection limit （S/N = 3） of 4.3×10^-7 mol·L^-1. The biosensor demonstrated a 95% response within less than 10 s.     

Novel Protocol for Covalent Immobilization of Horseradish Peroxidase on Gold Electrode Surface

A novel protocol for immobilization of horseradish peroxidase (HRP) onto diazonium functionalized screen‐printed gold electrode (SPGE) has been successfully developed. This protocol involved 1) electrochemical reduction of p‐nitrophenyl diazonium salts synthesized in situ in acidic aqueous solution to graft a layer of p‐nitrophenyl on SPGE, 2) electrochemical reduction of the nitro groups to convert to amines, 3) chemical reaction with nitrous acid to transform the amine to diazonium derivative and 4) chemical coupling of the enzyme with the diazonium group to form a covalent diazo bond. The fabricated biosensor showed the direct electrochemistry of HRP and displayed electrocatalytic activity towards the reduction of hydrogen peroxide (H₂O₂) without any mediator. The biosensor exhibited fast amperometric response to H₂O₂. The catalytic current increased with increasing H₂O₂ concentration from 5 μM to 30 μM and the detection limit of the biosensor was 2 μM. The biosensor exhibited acceptable sensitivity, good reproducibility and long‐term stability.     

Amperometric Biosensor for Hydrogen Peroxide,Using Supramolecularly Immobilized Horseradish Peroxidase on the β‐Cyclodextrin‐Coated Gold Electrode

Horseradish peroxidase, previously modified with 1‐adamantane moieties, was supramolecularly immobilized on gold electrodes coated with perthiolated β‐cyclodextrin. The functionalized electrode was employed for the construction of an amperometric biosensor device for hydrogen peroxide using 1 mM hydroquinone as electrochemical mediator. The biosensor exhibited a fast amperometric response (6 s) and a good linear response toward H₂O₂ concentration between 12 μM and 450 μM. The biosensor showed a sensitivity of 1.02 mA/M cm², and a very low detection limit of 5 μM. The electrode retained 97% of its initial electrocatalytic activity after 30 days of storage at 4 ⁰C in 50 mM sodium phosphate buffer, pH 7.0.     

An H2O2 Biosensor Based on Immobilization of Horseradish Peroxidase Labeled Nano‐Au in Silica Sol‐Gel/Alginate Composite Film

Abstract

A novel approach to assemble an H₂O₂ amperometric biosensor was introduced. The biosensor was constructed by entrapping horseradish peroxidase (HRP) labeled nano‐scaled particulate gold (nano‐Au) (HRP‐nano‐Au electrostatic composite) in a new silica sol‐gel/alginate hybrid film using glassy carbon electrode as based electrode. This suggested strategy fully merged the merits of sol‐gel derived inorganic‐organic composite film and the nano‐Au intermediator. The silica sol‐gel/alginate hybrid material can improve the properties of conventional sol‐gel material and effectively prevent cracking of film. The entrapment of HRP in the form of HRP‐nano‐Au can not only factually prevent the leaking of enzyme out of the film but also provide a favorable microenvironment for HRP. With hydroquinone as an electron mediator, the proposed HRP electrode exhibited good catalytic activity for the reduction of H₂O₂. The parameters affecting both the qualities of sol‐gel/alginate hybrid film and the biosensor response were optimized. The biosensor exhibited high sensitivity of 0.40 Al mol^?1 cm^?2 for H₂O₂ over a wide linear range of concentration from 1.22×10^?5 to 1.46×10^?3 mol L^?1, rapid response of <5 s and a detection limit of 0.61×10^?6 mol L^?1. The enzyme electrode has remarkable stability and retained 86% of its initial activity after 45 days of storage in 0.1 mol L^?1 Tris‐HCl buffer solutions at pH 7.     

Electrochemical Biosensor for Organophosphate Pesticides and Huperzine‐A Detection Based on Pd Wormlike Nanochains/Graphitic Carbon Nitride Nanocomposites and Acetylcholinesterase

A new electrochemical biosensor was developed for organophosphorus pesticides (OPs) and huperzine‐A (hupA) detection based on Pd wormlike nanochains/graphitic carbon nitride (Pd WLNCs/g‐C₃N₄) nanocomposites and acetylcholinesterase (AChE). The morphologies and components of the nanocomposites were analyzed by transmission electron microscopy (TEM) and X‐ray photoelectron spectroscopy (XPS). The Pd WLNCs/g‐C₃N₄ nanocomposites could effectively immobilize enzymes and promote the signal amplification. Under the optimum condition, the proposed biosensor displayed well performance. The linear response ranges for the determination of OPs and hupA were 1.00 nM to 14.96 µM and 3.89 nM to 20.80 µM, respectively. The corresponding detection limits were 0.33 nM and 1.30 nM (S/N=3). Meanwhile, the biosensor owned good reproducibility and stability, and could also be applied to analyze practical samples, which would be a new hopeful method for pesticide analysis.     

Direct Electrochemistry and Bioelectrocatalysis of Microperoxidase‐11 Immobilized on Chitosan‐Graphene Nanocomposite

A bioelectrochemical platform has been constructed for the direct electron transfer and biosensing purposes of microperoxidase‐11 (MP‐11) immobilized on the chitosan dispersed multilayer graphene nanocomposite. The immobilized MP‐11 at the modified gold electrode displays a well‐defined and quasireversible redox peaks, with a formal potential of ?0.38 V/SCE in a buffer solution (pH 7.0). MP‐11 absorbed on the electrode surface exhibits high electrocatalytic activity toward the reduction of both oxygen and hydrogen peroxide and also shows good analytical performance for the amperometric detection of H₂O₂ with a linear range from 2.5 to 135 μM. These results indicate the graphene modified electrode might be used as a third generation biosensor for H₂O₂ detection.     

A Simple Layer‐by‐Layer Assembly Strategy for a Reagentless Biosensor Based on a Nanocomposite of Methylene Blue‐Multiwalled Carbon Nanotubes

A simple layer‐by‐layer (LBL) assembly strategy was established for constructing a novel reagentless biosensor based on a nanocomposite of methylene blue multiwalled carbon nanotubes (MB‐MWNTs). A nanocomposite of MB‐MWNTs was obtained by direct premixing and possessed good dispersion in barbital‐HCl buffer. Through electrostatic interactions, the nanocomposite of MB‐MWNTs could alternately be assembled with horseradish peroxidase (HRP) on the Au electrode modified with precursor films. UV/Vis spectra and scanning electron microscopy (SEM) were applied to reveal the formation of the nanocomposite of MB‐MWNTs. The LBL assembly process was also verified by electrochemical impedance spectroscopy (EIS). The MB is a well‐established mediator and efficiently facilitated the electron shuttle between the HRP and the electrode, as demonstrated by the cyclic voltammetry (CV) measurements. The as‐prepared reagentless biosensor exhibited a fast response for the determination of hydrogen peroxide (H₂O₂) and reached 95% of the steady‐state current within 3 s. It was found that the linear response range of the reagentless biosensor for H₂O₂ was from 4.0 μM to 3.78 mM with a detection limit of 1.0 μM and a sensitivity of 22.5 μA mM⁻¹. The biosensor exhibited a high reproducibility and stability.     

HPLC‐UV Detection for Analysis of p‐Benzoquinone Dioxime and p‐Nitrosophenol,and Chromatographic Fingerprint Applied in Quality Control of Industrial p‐Benzoquinone Dioxime

Abstract

A reliable reversed‐phase high performance liquid chromatographic (HPLC) method has been developed for simultaneous determination of p‐benzoquinone dioxime (BQD) and its related impurity p‐nitrosophenol (NSP). Separation was achieved on a Kromasil C₁₈ column by using methanol‐water‐NH₄Ac‐NH₃ solution (pH=7.0, 50 mM) (30/50/20, v/v/v) as the mobile phase, and detection was operated by UV absorption at a wavelength of 305 nm. The method was seen to have good linearity, accuracy, and precision for the concentration range and to be an attractive choice for the quality control of BQD for industrial use. Moreover, the HPLC‐UV‐vis fingerprint of BQD has been established, and successfully applied to quality control of industrial BQD in laboratories of some rubber factories in China. Chromatographic fingerprints of intermediates would become an effective strategy for accelerating the progress of fine chemical industry.     

Development of Quercetin Biosensor Through Immobilizing Laccase in a Modified β‐Cyclodextrin Matrix Containing Ag Nanoparticles in Ionic Liquid

A biosensor containing Ag nanoparticles in ionic liquid (IL) 1‐butyl‐3‐methylimidazolium bis(trifluoromethylsulfonyl)imide (BMI.Tf₂N) and laccase (Lac) immobilized on β‐cyclodextrin modified with epichlorohydrin (β‐CDEpi) was developed for quercetin determination. Lac catalyses the oxidation of quercetin to quinone, which is then reduced on the biosensor surface and the resulting current was investigated by square‐wave voltammetry. The β‐CDEpi support was analyzed by scanning electron microscopy and Ag‐BMI.Tf₂N by transmission electron microscopy and X‐ray diffraction. The linear range (0.499–7.407 μM) and low detection limit (0.037±0.004 μM) show that the proposed biosensor is suitable for quercetin determination in real samples.     

An Electrochemical Biosensor with Dual Signal Outputs: Toward Simultaneous Quantification of pH and O2 in the Brain upon Ischemia and in a Tumor during Cancer Starvation Therapy

Herein, we develop a novel method for designing electrochemical biosensors with both current and potential signal outputs for the simultaneous determination of two species in a living system. Oxygen (O₂) and pH, simple and very important species, are employed as model molecules. By designing and synthesizing a new molecule, Hemin‐aminoferrocene (Hemin‐Fc), we create a single electrochemical biosensor for simultaneous detection and ratiometric quantification of O₂ and pH in the brain. The reduction peak current of the hemin group increases with the concentration of O₂ from 1.3 to 200.6 μm . Meanwhile, the peak potential positively shifts with decreasing pH from 8.0 to 5.5, resulting in the simultaneous determination of O₂ and pH. The Fc group can serve as an internal reference for ratiometric biosensing because its current and potential signals remain almost constant with variations of O₂ and pH. The developed biosensor has high temporal and spatial resolutions, as well as remarkable selectivity and accuracy, and is successfully applied in the real‐time quantification of O₂ and pH in the brain upon ischemia, as well as in tumor during cancer therapy.     

Amperometric Flow‐Biosensor for Cyanide Based on an Inhibitory Effect upon Bioelectrocatalytic Reduction of Oxygen by Peroxidase‐Modified Carbon‐Felt

A novel, simple and relative highly sensitive amperometric flow biosensor for cyanide was developed by using horseradish peroxidase (HRP)‐adsorbed carbon‐felt (CF), based on an inhibitory effect on the HRP‐catalyzed O₂ reduction. The HRP‐CF showed a sufficient bioelecrocatalytic activity for O₂ reduction in the potential region from 0 to ?0.5 V at pH 5.0, due to a direct electron transfer‐based O₂ reduction process via ferrous‐HRP and compound III. This HRP‐catalyzed O₂ reduction was reversibly inhibited by cyanide, which enabled to fabricate a novel and simple reagentless (i.e., no requirement of the ordinary substrate, H₂O₂, and the electron transfer mediators) flow‐biosensor for cyanide. When air‐saturated 0.1 M phosphate buffer (pH 5.0) was used as a carrier under the applied potential of ?0.2 V vs. Ag/AgCl, the steady‐state base‐current due to the HRP‐catalyzed O₂ reduction was reversibly inhibited by the cyanide injection (200 µL), resulting in peak‐shape current responses. The magnitude of the inhibition peak currents linearly increased with increasing concentrations of cyanide up to 1 µM, and the detection limit was found to be 0.04 µM (S/N=2). The apparent inhibition constant K_i′ was estimated to be 0.87 µM.     

Fabricating of Acetylcholine Biosensor by a Layer‐by‐layer Deposition Technique for Determining Trichlorfon

An acetylcholine (ACh) biosensor has been fabricated with bienzymes/poly(diallyldimethylammonium chloride) (PDDA) multilayer film‐modified platinum (Pt) electrodes by a layer‐by‐layer technique (LBL). The ACh biosensor was optimized and the properties are described. This ACh biosensor was used for the detection of organophosphate pesticide trichlorfon. The detection limits (found 0.001 μg/mL for trichlorfon) make it possible to detect the pollutants. This simple protocol of biosensor preparation, high sensitivity and stability are very promising for the determination of environmental pollutants in field conditions.     

Graphene/Poly(vinylferrocene) Composite Based Amperometric Biosensor for L‐lysine Determination

A novel and sensitive amperometric biosensor for L‐lysine determination based on a glassy carbon electrode (GCE) modified with graphene (GR) and redox polymer poly(vinylferrocene) (PVF) was constructed. L‐lysine‐α‐oxidase was immobilized onto the modified GCE by a glutaraldehyde/bovine serum albumin cross‐linking procedure. SEM, CV and EIS were used for the characterization of the surface morphology and stepwise fabrication processes of PVF/GR composite. Optimal composition of the biosensor and experimental conditions that affect the performance of the biosensor are discussed. The effect of buffer pH on biosensor response was studied in detail over a wide pH range. L‐lysine biosensor displayed a linear range of 9.9×10⁻⁷ ‐ 3.1×10⁻⁴ M with a low detection limit of 2.3×10⁻⁷ M and K_M ^app value of 0.4 mM. The L‐lysine biosensor was tested using pharmaceutical sample and cheese with satisfactory results.