A Laccase/Chitosan‐Lambda‐Carrageenan Based Voltammetric Biosensor for Phenolic Compound Detection

In this contribution, a new concept of voltammetric catechol biosensor, based on the encapsulation of laccase (LAC) in a chitosan+lambda‐carrageenan (CHIT+CAR) polyelectrolyte complex (PEC) employing a simple coacervation process is presented. Chitosan (CHIT) was prepared from α‐chitin extracted from shrimp shells and lambda‐carrageenan (CAR) was extracted from red algae, both polysaccharides and PEC being characterized using FTIR spectrometry and electrochemistry. Cyclic voltammetry was utilized to determine the analytical features of the laccase (LAC) biosensor for catechol detection. The linear range was from 10^?20 M to 10^?14 M with a sensitivity of 1.55 mA/p[catechol] and a limit of detection of 3×10^?21 M.The laccase biosensor exhibits good repeatability (RSD 2.38 %) and stability (four weeks). The developed biosensor was tested by applying it to the evaluation of the total polyphenolic content in natural oil samples.     

Bioelectroanalytical Determination of Rutin Based on bi‐Enzymatic Sensor Containing Iridium Nanoparticles in Ionic Liquid Phase Supported in Clay

A matrix comprising iridium nanoparticles and 1‐butyl‐3‐methylimidazolium tetrafluoroborate ionic liquid (Ir‐BMI.BF₄) supported in montmorillonite (MMT) was obtained through an efficient incorporation process. This modified clay matrix (Ir‐BMI.BF₄‐MMT) was used for the immobilization of the enzymes laccase (LAC) and polyphenol oxidase (PPO) and employed in the construction of a bi‐enzymatic biosensor for determination of rutin by square‐wave voltammetry. Under optimized conditions, the analytical curve showed a linear range for rutin concentrations from 9.17×10^?8 to 3.10×10^?6 mol L^?1 with a detection limit of 3.09×10^?8 mol L^?1. The method was successfully applied to the determination of rutin content in pharmaceutical samples.     

Amperometric Biosensor for the Determination of Phenols Using a Crude Extract of Sweet Potato (Ipomoea Batatas (L.) Lam.)

Abstract

An amperometric biosensor for the determination of phenols is proposed using a crude extract of sweet potato (Ipomoea batatas (L.) Lam.) as an enzymatic source of polyphenol oxidase (PPO; tyrosinase; catechol oxidase; EC 1.14.18.1). The biosensor is constructed by the immobilization of sweet potato crude extract with glutaraldehyde and bovine serum albumin onto an oxygen membrane. This biosensor provides a linear response for catechol, pyrogallol, phenol and p-cresol in the concentration ranges of 2.0×10^?5-4.3×10^?4mol L^?1, 2.0×10^?5-4.3×10^?4 mol L^?1, 2.0×10^?5-4.5×10^?4 mol L^?1 and 2.0×10^?5-4.5×10^?4mol L^?1, respectively. The response time was about 3–5 min for the useful response range, and the lifetime of this electrode was excellent for fifteen days (over 220 determinations for each enzymatic membrane). Application of this biosensor for the determination of phenols in industrial wastewaters is presented.     

Determination of Catechol by a Novel Laccase Biosensor Based on Zinc-Oxide Sol-Gel

A biosensor was fabricated by incorporating laccase in a ZnO sol-gel with chitosan as a matrix for the determination of catechol. The ZnO nanoparticles were characterized by X-ray diffraction and atomic force microscopy. The conductivity of the chitosan/ZnO/glassy carbon electrode film was investigated by alternating current impedance. The biosensor was employed to monitor the reduction of catechol, and the peak current increased linearly with concentration between 1.0 × 10^?6 and 1.0 × 10^?4 mole per liter with a limit of detection of 2.9 × 10^?7 mole per liter. The laccase biosensor exhibited good stability, reproducibility, and some selectivity.     

一种基于壳聚糖固定葡萄糖氧化酶的葡萄糖生物传感器的研究

本文选用生物相容性好的壳聚糖作为基体材料，使其与戊二醛交联成网状结构包埋葡萄糖氧化酶制成电化学传感器。这种壳聚糖膜不仅可以减小葡萄糖氧化酶的流失，而且能为酶提供了适宜的微环境。用红外光谱、紫外光谱及透射电镜对膜的形态和性质进行了表征。实验结果表明该传感器具有很快的响应速度，很好的稳定性和重现性，能选择性地催化葡萄糖并测定其浓度。该传感器的制备方法简单，成本低，于冰箱中放置两周信号保持在90％以上，对葡萄糖测量的线性范围为1×10^-5 - 3.4×10^-3mol•L^-1，当信噪比为3:1时检测限为5×10^-6mol•L^-1。     

Acetone extracted propolis as a novel membrane and its application in phenol biosensors: the case of catechol

A novel biosensor for catechol has been constructed by immobilizing polyphenol oxidase (PPO) into acetone-extracted propolis (AEP) composite modified with gold nanoparticles (GNPs) and attached to multiwalled carbon nanotube (MWCNTs) on a gold electrode surface. The propolis for AEP was obtained from honeybee colonies. Under the optimum conditions, this method could be successfully used for the amperometric determination of catechol within a concentration range of 1 × 10^?6 to 5 × 10^?4?M, with a detection limit of 8 × 10^?7?M (S/N = 3). The effects of pH and operating potential are also explored to optimize the measurement conditions. The best response was obtained at pH?5, while an optimum ratio of signal-to-noise (S/N) was obtained at ?20?mV (versus Ag/AgCl), which was selected as the applied potential for the amperometric measurements. All subsequent experiments were performed at pH?5. Cyclic voltammetry and electrochemical impedance spectroscopy was used to characterize the PPO/CNTs/GNPs/AEP/Au biosensor. The biosensor also exhibited good selectivity, stability, and reproducibility.     

Determination of Catechol by a Laccase Biosensor Based on Silica-Modified Zirconia Nanoparticles

A promising nanotechnological material, zirconia nanoparticles modified with SiO₂, was used as a medium for the immobilization of laccase to construct a novel biosensor that exhibits sensitive amperometric response to catechol in 0.1 mol · L^?1 phosphate buffer (pH 6.0) using cyclic voltammetry. The linear response to catechol was from 1.0 × 10^?6 to 1.0 × 10^?4 mol · L^?1 and the detection limit was 3.5 × 10^?7 mol · L^?1 at a signal-to-noise ratio of 3. The biosensor exhibited good stability, precision, and few interferences.     

Simultaneous Determination of Hydroquinone and Catechol at a Glassy Carbon Electrode Modified with Multiwall Carbon Nanotubes

A simply and high selectively electrochemical method for simultaneous determination of hydroquinone and catechol has been developed at a glassy carbon electrode modified with multiwall carbon nanotubes (MWNT). It was found that the oxidation peak separation of hydroquinone and catechol and the oxidation currents of hydroquinone and catechol greatly increase at MWNT modified electrode in 0.20 M acetate buffer solution (pH 4.5). The oxidation peaks of hydroquinone and catechol merge into a large peak of 302 mV (vs. Ag/AgCl, 3 M NaCl) at bare glassy carbon electrode. The two corresponding well‐defined oxidation peaks of hydroquinone in the presence of catechol at MWNT modified electrode occur at 264 mV and 162 mV, respectively. Under the optimized condition, the oxidation peak current of hydroquinone is linear over a range from 1.0×10^?6 M to 1.0×10^?4 M hydroquinone in the presence of 1.0×10^?4 M catechol with the detection limit of 7.5×10^?7 M and the oxidation peak current of catechol is linear over a range from 6.0×10^?7 M to 1.0×10^?4 M catechol in the presence of 1.0×10^?4 M hydroquinone with the detection limit of 2.0×10^?7 M. The proposed method has been applied to simultaneous determination of hydroquinone and catechol in a water sample with simplicity and high selectivity.     

Layered Zirconium(IV) Aminoethylphosphonate Intercalated with Horseradish Peroxidase for an Amperometric Biosensor

Layered zirconium(IV) aminoethylphosphonate (ZrAEP) have been used as matrices for immobilization of horseradish peroxidase (HRP) to fabricate enzyme electrode for an amperometric biosensor. The biocompatible HRP–ZrAEP films were fabricated on gold electrode surface by electro‐co‐deposition method. The morphology of the HRP–ZrAEP composite was characterized by scanning electron microscopy (SEM). UV–vis spectroscopy indicated that the intercalated HRP retained its native structure after incorporation in the ZrAEP. The immobilized HRP at the HRP–ZrAEP films exhibited good electro catalytic responses to the reduction of hydrogen peroxide. The response time of the biosensor was less than 3 s, and the linear range is from 2.5 × 10^?6 to 3.22 × 10^?3 M, with a detection limit of 7.0 × 10^?7 M (S/N = 3). The Michaelis–Menten constant (K^app_M) value is estimated to be 2.21 mM. In addition, the obtained biosensor possesses high sensitivity, good stability and reproducibility.     

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.     

二茂铁衍生物电子传递剂键合的溶胶-凝胶葡萄糖生物传感器

The sol-gel derived glucose biosensor was developed, and the sol-gel membrane was organically modified by N-（3-triethoxysilylpropyl）-ferrocenylmethylamine （FcSi） as sol-gel precursor to make electrochemical biosensor. The structure of biosensor was sol-gel/FcSi＋GOx/GC type （glucose oxidase, GOx）. The ferrocene mediator was chemically immobilized to the silane network, and GOx was entrapped to the sol-gel glass network. Therefore, these structures prevented mediator leakage and retained the enzyme activity. Additionally, pH of electrolyte, temperature effects, and interference of positive substances with biosensor were investigated. And the electrochemical performance of biosensor was studied by amperometry. The results indicated that the linear range, detection limit. and response slope of biosensor was 2.00×10^-4-1.57×10^-3 mol·L^-1, 2.0×10^-4 mol·L^-1 and 5.06×10^5 nA·mol^- 1·L, respectively.     

Laccase-Nafion Based Biosensor for the Determination of Polyphenolic Secondary Metabolites

A laccase-based biosensor was developed by specific enzyme adsorption on screen-printed working electrodes of DROPSENS cells, and stabilized with Nafion 0.1% membrane. The electrode was characterized with respect to response time, sensitivity, linear range, detection limit, pH dependence, interferences, and long-term stability. The tested substrates were catechol, rosmarinic acid, caffeic acid, chlorogenic acid, and gallic acid. The optimized biosensor proved the following characteristic performances: the apparent Michaelis Menten calculated considering rosmarinic acid substrate 8.3 × 10^?6 mol L^?1 (r = 0.995, n = 6); the dynamic range of biosensor response for rosmarinic acid 7 × 10^?7 ? 1.5 × 10^?6 mol L^?1; the detection limit for rosmarinic acid 1.19 × 10^?7 mol L^?1 (RSD = 1.08%, n = 3). It was noticed that the biosensor reaches systematically 90% to 94.3% from the response obtained by LC-DAD-ESI-MS for real samples.     

CuO‐Doped Mesoporous Silica Hybrid for Rapid and Sensitive Amperometric Detection of Phenolic Compounds

This work constructed an amperometric biosensing platform using CuO doped mesoporous silica hybrid (CuO/SBA‐15) as a carrier. The CuO/SBA‐15 showed a pair of redox peaks of Cu^2+/0. Upon immobilization of tyrosinase on the hybrid, the resulting biosensor exhibited a rapid (<0.5 s) and sensitive amperometric response to phenolic compounds under the optimized conditions. The linear response to catechol ranged from 1.2×10^?9 to 3.0×10^?5 M. The activation energy for enzymatic reaction was calculated to be 26.6 kJ mol^?1. The apparent Michaelis–Menten constants of the enzyme electrode were estimated to be 54.6, 145, 17.0, 74.8 and 633 µM for catechol, phenol, p‐cresol, m‐cresol and dopamine hydrochloride, respectively. The metal oxide doped mesoporous silica hybrid exhibited excellent performance for construction of new biosensors.     

A Nitric Oxide Biosensor Based on Myoglobin Adsorbed on Multi‐Walled Carbon Nanotubes

Myoglobin (Myb) of horse heart is incorporated on multi‐walled carbon nanotubes (MWNTs) and immobilized at a glassy carbon (GC) electrode surface. Its electrochemical behavior and enzyme activity are characterized by employing electrochemical methods. The results indicate that MWNTs can obviously promote the direct electron transfer between Myb and electrode, and that the Myb on MWNTs behaves as an enzyme‐like activity towards the electrochemical reduction of nitric oxide (NO). Accordingly, an unmediated NO biosensor is constructed. Experimental results reveal that the peak current related to NO is linearly proportional to its concentration in the range of 2.0×10^?7–4.0×10^?5 mol/L. The detection limit is estimated to be 8.0×10^?8 mol/L. Considering a relative standard deviation of 2.1% in seven independent determinations of 1.0×10^?5 mol/L NO, this biosensor shows a good reproducibility. The biosensor based on Myb/MWNTs modified electrode can be used for the rapid determination of trace NO in aqueous solution with a good stability, nice selectivity and easy construction.     

Development of a nanocomposite system and its application in biosensors construction

The present work describes the development of a nanocomposite system and its application in construction of a new amperometric biosensor applied in the determination of total polyphenolic content from propolis extracts. The nanocomposite system was based on covalent immobilization of laccase on functionalized indium tin oxide nanoparticles and it was morphologically and structural characterized. The casting of the developed nanocomposite system on the surface of a screen-printed electrode was used for biosensor fabrication. The analytical performance characteristics of the settled biosensor were determined for rosmarinic acid, caffeic acid and catechol (as laccase specific substrate). The linearity was obtained in the range of 1.06×10^?6 ? 1.50×10^?5 mol L^?1 for rosmarinic acid, 1.90×10^?7 ? 2.80×10^?6 mol L^?1 for caffeic acid and 1.66×10^?6 ? 7.00×10^?6 mol L^?1 for catechol. A good sensitivity of amperometric biosensor 141.15 nA µmol^?1 L^?1 and fair detection limit 7.08×10^?8 mol L^?1 were obtained for caffeic acid. The results obtained for polyphenolic content of propolis extracts were compared with the chromatographic data obtained by liquid-chromatography with diode array detection.      

Development of Electrochemical Biosensor Based on Nanostructured Carbon Materials for Paracetamol Determination

This work describes the development of a biosensor for paracetamol (PAR) determination based on a glassy carbon electrode (GCE) modified with multiwalled carbon nanotubes (MWCNT) and laccase enzyme (LAC), which was immobilized by means of covalent crosslinking using glutaraldehyde. Voltammetric investigations were carried out by cyclic voltammetry (CV), differential pulse voltammetry (DPV) and square wave voltammetry (SWV). The biosensor was characterized by Scanning Electron Microscope (SEM) and Fourier Transform Infrared Spectroscopy (FT‐IR). The results showed that the use of MWCNT/LAC composite increased the sensor sensitivity, compared to bare glassy carbon electrode. Factors affecting the voltammetric signals such as pH, ionic strength, scan rate and interferents were assessed. Linear range, limit of detection (LOD) and limit of quantitation (LOQ) obtained were 10–320 μmol L^?1, 7 μmol L^?1 and 10 μmol L^{? 1}, respectively. The developed biosensor was successfully applied to PAR determination in urine and pharmaceutical formulations samples, with recovery varying from 99.96 to 106.20 % in urine samples and a relative standard deviation less than 1.04 % for PAR determination in pharmaceutical formulations. Therefore, the MWCNT‐LAC/GCE exhibits excellent sensitivity and can be used to PAR determination as a viable alternative in clinical analyzes and quality control of pharmaceutical formulations, through a simple, fast and inexpensive methodology.     

Amperometric Biosensor for Evaluation of Competitive Cholinesterase Inhibition by the Reactivator HI‐6

Abstract

Amperometric biosensors containing enzymes butyrylcholinesterase or acetylcholinesterase were prepared. The biosensors were employed for studying of cholinesterase reactivator: HI‐6. Competitions between HI‐6 and acetylthiocholine as enzyme substrate were used for determination of IC₅₀ value. Biosensors with butyrylcholinesterase from human serum determined IC₅₀ as (1.00±0.02)×10^?6 M; the biosensor with acetylcholinesterase from human erythrocytes performance provided IC₅₀ (3.31±0.13)×10^?6 M, the one with human recombinant acetylcholinesterase (2.00±0.06)×10^?6 M and finally biosensor with acetylcholinesterase from electric eel (6.17±0.17)×10^?6 M when 5 mM acetylthiocholine as substrate was used. We are encouraged to consider presented biosensors as a very useful for evaluation of newly prepared cholinesterase reactivators.     

Enzyme Immobilization and Direct Electrochemistry Based on a New Matrix of Phospholipid‐Monolayer‐Functionalized Graphene

A new nanocomposite material for enzyme immobilization and subsequent direct electrochemistry and electrocatalysis was developed by using 1,2‐dimyristoyl‐sn‐glycero‐3‐phospho‐(1‐rac‐glycerol)‐phospholipid‐monolayer‐membrane‐modified graphene (DMPG‐G). Microperoxidase‐11 (MP11) was chosen as a model enzyme to investigate the composite system. Owing to the improved conductivity and biocompatible microenvironment, MP11 that was immobilized in the matrix of the DMPG‐G nanocomposite (DMPG‐G‐MP11) effectively retained its native structure and bioactivity. DMPG‐G‐MP11‐modified glassy carbon electrode (DMPG‐G‐MP11/GCE) exhibited a pair of well‐defined quasi‐reversible redox peaks of MP11 and showed high electrocatalytic activity towards hydrogen peroxide (H₂O₂). The linear response of the developed biosensor for the determination of H₂O₂ ranged from 2.0×10^?6 to 4.5×10^?4 M with a detection limit of 7.2×10^?7 M . This biosensor exhibited high reproducibility and long‐term storage stability. The promising features of this biosensor indicate that these lipid–graphene nanocomposites are ideal candidate materials for the direct electrochemistry of redox proteins and that they could serve as a versatile platform for the construction of a third‐generation biosensor.     

Simultaneous Determination of Dihydroxybenzene Isomers at MWCNTs/β‐Cyclodextrin Modified Carbon Ionic Liquid Electrode in the Presence of Cetylpyridinium Bromide

Simultaneous determination of dihydroxybenzene isomers was investigated at a multi‐wall carbon nanotubes (MWCNTs)/β‐cyclodextrin composite modified carbon ionic liquid electrode in phosphate buffer solution (pH 7.0, 1/15 mol/L) in the presence of cationic surfactant cetylpyridinium bromide (CPB). With the great enhancement of surfactant CPB, the voltammetric responses of dihydroxybenzene isomers were more sensitive and selective. The oxidation peak potential of hydroquinone was about 0.024 V, catechol was about 0.140 V and resorcinol 0.520 V in differential pulse voltammetric (DPV) measurements, which indicated that the dihydroxybenzene isomers could be separated entirely. The electrode showed wide linear behaviors in the range of 1.2×10^?7–2.2×10^?3, 7.0×10^?7–1.0×10^?3, 2.6×10^?6–9.0×10^?4 mol/L for hydroquinone, catechol and resorcinol, respectively. And the detection limits of the three dihydroxybenzene isomers were 4.0×10^?8, 8.0×10^?8, 9.0×10^?7 mol/L, respectively. The proposed method could be applied to the determination of dihydroxybenzene isomers in artificial wastewater, and the recovery was from 97.4% to 104.2%.     

Disposable Amperometric Biosensor Based on Poly‐L‐lysine and Fe3O4 NPs‐chitosan Composite for the Detection of Tyramine in Cheese

An amperometric tyramine biosensor based on poly‐L‐lysine (PLL) and Fe₃O₄ nanoparticles (Fe₃O₄NP) modified screen printed carbon electrode (SPCE) was developed. PLL was formed on the SPCE by the electropolymerization of L‐lysine. Subsequently, Fe₃O₄NP suspension prepared in chitosan (CH) solution was casted onto the PLL/SPCE. Tyrosinase (Ty) enzyme was immobilized onto the modified Fe₃O₄?CH/PLL/SPCE and the electrode was coated with Nafion to fabricate the Ty/Fe₃O₄?CH/PLL/SPCE. Different techniques including scanning electron microscopy, chronoamperometry (i–t curve), cyclic voltammetry and electrochemical impedance spectroscopy were utilized to study the fabrication processes, electrochemical characteristics and performance parameters of the biosensor. The analytical performance of the tyramine biosensor was evaluated with respect to linear range, sensitivity, limit of detection, repeatability and reproducibility. The response of the biosensor to tyramine was linear between 4.9×10^?7–6.3×10^?5 M with a detection limit of 7.5×10^?8 M and sensitivity of 71.36 μA mM^?1 (595 μA mM^?1 cm^?2). The application of the developed biosensor for the determination of tyramine was successfully tested in cheese sample and mean analytical recovery of added tyramine in cheese extract was calculated as 101.2±2.1 %. The presented tyramine biosensor is a promising approach for tyramine analysis in real samples due to its high sensitivity, rapid response and easy fabrication.