Biosensor based on acetylcholinesterase immobilized onto layered double hydroxides for flow injection/amperometric detection of organophosphate pesticides

We developed a highly sensitive flow injection/amperometric biosensor for the detection of organophosphate pesticides (OPs) using layered double hydroxides (LDHs) as the immobilization matrix of acetylcholinesterase (AChE). LDHs provided a biocompatible microenvironment to keep the bioactivity of AChE, due to the intrinsic properties of LDHs (such as a regular structure, good mechanical, chemical and thermal stabilities, and swelling properties). By integrating the flow injection analysis (FIA) with amperometric detection, the resulting AChE-LDHs modified electrode greatly catalyzed the oxidation of the enzymatically generated thiocholine product, and facilitated the detection automation, thus increasing the detection sensitivity. The analytical conditions for the FIA/amperometric detection of OPs were optimized by using methyl parathion (MP) as a model. The inhibition of MP was proportional to its concentration ranging from 0.005 to 0.3μgmL(-1) and 0.3 to 4.0μgmL(-1) with a detection limit 0.6ngmL(-1) (S/N=3). The developed biosensor exhibited good reproducibility and acceptable stability.     

Xanthine oxidase/laponite nanoparticles immobilized on glassy carbon electrode: Direct electron transfer and multielectrocatalysis

In this work, colloidal laponite nanoparticles were further expanded into the design of the third-generation biosensor. Direct electrochemistry of the complex molybdoenzyme xanthine oxidase (XnOx) immobilized on glassy carbon electrode (GCE) by laponite nanoparticles was investigated for the first time. XnOx/laponite thin film modified electrode showed only one pair of well defined and reversible cyclic voltammetric peaks attributed to XnOx–FAD cofactor at about −0.370 V vs. SCE (pH 5). The formal potential of XnOx–FAD/FADH₂ couple varied linearly with the increase of pH in the range of 4.0–8.0 with a slope of −54.3 mV pH⁻¹, which indicated that two-proton transfer was accompanied with two-electron transfer in the electrochemical reaction. More interestingly, the immobilized XnOx retained its biological activity well and displayed an excellent electrocatalytic performance to both the oxidation of xanthine and the reduction of nitrate. The electrocatalytic response showed a linear dependence on the xanthine concentration ranging from 3.9 × 10⁻⁸ to 2.1 × 10⁻⁵ M with a detection limit of 1.0 × 10⁻⁸ M based on S/N = 3.     

Selective and sensitive biosensor for theophylline based on xanthine oxidase electrode

Milk and microbial xanthine oxidases (XOs) were used for the construction of amperometric enzyme electrodes. Substrate specificity differences of these enzymes were studied. Of the two enzymes, only the microbial XO was found to oxidize theophylline, but not theobromine and caffeine. The substrate specificity of microbial XO was affected by pH, where the optimum for xanthine was 5.5, while for theophylline it was in the range from 6.5 to 8.5. The theophylline biosensor showed a low detection limit of 2 x 10(-7) M and signal linearity up to 5 x 10(-5) M. The sensitivity of the microbial XO electrode to theophylline could be selectively eliminated by immersion in alkaline phosphate solution, thus allowing for the construction of a blank electrode for differential measurements. The feasibility of this approach has been demonstrated by the determination of free (unbound) and total theophylline in blood samples. The biosensor exhibited good operational (>6 h) and shelf (>3 months) stability when trehalose was used as a stabilizer of the biocatalytic layer.     

Electrochemical characterization and application of azurin-modified gold electrodes for detection of superoxide

A novel biosensor for superoxide radical (O(2)(*-)) detection based on Pseudomonas aeruginosa azurin immobilized on gold electrode was designed. The rate constant of azurin reduction by O(2)(*-) was found to be 10(5)M(-1)s(-1) in solution and five times lower, i.e., 0.2 x 10(5)M(-1)s(-1), for azurin coupled to gold by 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP). The electron transfer rate between the protein and the electrode ranged from 2 to 6s(-1). The sensitivity of this biosensor to O(2)(*-) was 6.8 x 10(2)Am(-2)M(-1). The response to the interference substances, such as uric acid, H(2)O(2), and dimethylsulfoxide was negligible below 10 microM. The electrode was applied in three O(2)(*-) generating systems: (i) xanthine oxidase (XOD), (ii) potassium superoxide (KO(2)), and (iii) stimulated neutrophil granulocytes. The latter was compared with luminol-amplified chemiluminescence. The biosensor responded to O(2)(*-) in all three environments, and the signals were antagonized by superoxide dismutase.     

One-step construction of biosensor based on chitosan-ionic liquid-horseradish peroxidase biocomposite formed by electrodeposition

A simple and controllable electrodeposition approach was established for one-step construction of hydrogen peroxide (H(2)O(2)) biosensors by in situ formation of chitosan-ionic liquid-horseradish peroxidase (CS-IL-HRP) biocomposite film on electrode surface. A highly porous surface with orderly three-dimensional network was revealed by scanning electron microscopy (SEM) investigation. The biocomposite provided improved conductivity and biocompatible microenvironment. The developed biosensor exhibited a fast amperometric response for the determination of H(2)O(2) and 95% of the steady-state current was obtained within 2s. The linear response of the developed biosensor for the determination of H(2)O(2) ranged from 6.0x10(-7) to 1.6x10(-4)M with a detection limit of 1.5x10(-7)M. Performance of the biosensor was evaluated with respect to possible interferences and a good selectivity was revealed. The fabricated biosensor exhibited high reproducibility and long-time storage stability. The ease of the one-step non-manual technique and the promising feature of biocomposite could serve as a versatile platform for the fabrication of electrochemical biosensors.     

Amperometric determination of choline released from rat submandibular gland acinar cells using a choline oxidase biosensor

A choline (CHO) biosensor based on the determination of H(2)O(2) generated at the electrode surface by the enzyme choline oxidase (CHOx) was developed. The biosensor consisted of CHOx retained onto a horseradish peroxidase (HRP) immobilized solid carbon paste electrode (sCPE). The HRPsCPE contained the molecule phenothiazine as redox mediator and CHOx was physically retained on the electrode surface using a dialysis membrane. Several parameters have been studied such as, mediator amount, influence of applied potential, etc. The CHO measurements were performed in 0.1 M phosphate buffer, pH 7.4. Amperometric detection of CHO was realized at an applied potential of 0.0 mV vs Ag/AgCl. The response is linear over the concentration range 5.0x10(-7)-7.0x10(-5) M, with a detection limit of 1.0x10(-7) M. This biosensor was used to detect choline released from phosphatidylcholine (PC) by phospholipase D (PLD) in isolated rat salivary gland cells stimulated by a purinergic agonist (ATP).     

Electrochemical study of a new methylene blue/silicon oxide nanocomposition mediator and its application for stable biosensor of hydrogen peroxide

A novel organic-inorganic nanocomposite of methylene blue (MB) and silicon oxide was synthesized and characterized by TEM, FTIR, and UV-vis. The as-prepared material was able to transfer the electron of the MB to electrode and was different from other SiO2 spheres structurally. It can be used as mediator to construct a biosensor with horseradish peroxidase (HRP) coimmobilized in the gelatine matrix and cross-linked with formaldehyde. The resulting biosensor exhibited fast amperometric response and good stability to hydrogen peroxide (H2O2). The linear range for H2O2 determination was from 1 x 10(-5) to 1.2 x 10(-3) M, with a detection limit of 4 x 10(-6) M based on S/N = 3. Moreover, the lifetime is more than 3 months under dry conditions at 4 degrees C.     

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.     

Construction and application of an amperometric xanthine biosensor based on zinc oxide nanoparticles-polypyrrole composite film

Zinc oxide nanoparticles (ZnO-NPs) were synthesized from zinc nitrate by simple and efficient method in aqueous media at 55°C without any requirement of calcinations step. A mixture of ZnO-NPs and pyrrole was eletropolymerized on Pt electrode to form a ZnO-NPs-polypyrrole (PPy) composite film. Xanthine oxidase (XOD) was immobilized onto this nanocomposite film through physiosorption. The ZnO-NPs/polypyrrole/Pt electrode was characterized by Fourier transform infrared (FTIR), cyclic voltammetry (CV), X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and electrochemical impedance spectroscopy (EIS) before and after immobilization of XOD. The XOD/ZnO-NPs-PPy/Pt electrode as working electrode, Ag/AgCl as reference electrode and Pt wire as auxiliary electrode were connected through a potentiostat to construct a xanthine biosensor. The biosensor exhibited optimum response within 5s at pH 7.0, 35°C and linearity from 0.8 μM to 40 μM for xanthine with a detection limit 0.8 μM (S/E=3). Michaelis Menten constant (K(m)) for xanthine oxidase was 13.51 μM and I(max) 0.071 μA. The biosensor measured xanthine in fish meat and lost 40% of its initial activity after its 200 uses over 100 days, when stored at 4°C.     

Electrochemical study of ferrocenemethanol-modified layered double hydroxides composite matrix: application to glucose amperometric biosensor

A novel amperometric glucose sensor based on co-immobilization of ferrocenemethanol (MeOHFc) and glucose oxidase (GOD) in the layered double hydroxides (LDHs) was described. MeOHFc immobilized in LDHs played effectively the role of an electron shuttle and allowed the detection of glucose at 0.25 V (versus SCE), with dramatically reduced interference from easily oxidizable constituents. The sensor (LDHs/MeOHFc/GOD) exhibited a relatively fast response (response time was about 5s), low detection limit (3 microM), and high sensitivity (ca. 60 mA M(-1)cm(-2)) with a linear range of 6.7 x 10(-6) to 3.86 x 10(-4)M of glucose. Apparent Michaelis-Menten constant was calculated to be 2.25 mM.     

Amperometric hydrogen peroxide biosensor with sol-gel/chitosan network-like film as immobilization matrix

A new type of sol-gel/organic hybrid composite material based on the cross-linking of natural polymer chitosan with (3-aoryloxypropyl) dimethoxymethylsilane was developed for the fabrication of an amperometric H(2)O(2) biosensor. The composite film was used to immobilize horseradish peroxidase (HRP) on a gold disk electrode. The properties of sol-gel/chitosan and sol-gel/chitosan-HRP films have been carefully characterized by atomic force microscopy and Fourier transform infrared. By using fluorescent label, a protein density on sol-gel/chitosan has been calculated to be 3.14 x 10(12) moleculescm(-2). With the aid of catechol mediator, the biosensor had a fast response of less than 2 s with linear range of 5.0 x 10(-9)-1.0 x 10(-7) mol l(-1) and a detection limit of 2 x 10(-9) mol l(-1). Its current response shows a typical Michaelis-Menten mechanism. The apparent Michaelis-Menten constant K(M)(app) is found to be 1.30 micromol l(-1). The activation energy for enzymatic reaction is calculated to be 8.22 kJ mol(-1). The biosensor retained approximately 75% of its original activity after about 60 days of storage in a phosphate buffer at 4 degrees C.     

Calcium carbonate nanoparticles: a host matrix for the construction of highly sensitive amperometric phenol biosensor

We reported on the utilization of a novel attractive nanoscaled calcium carbonate (nano-CaCO(3))-polyphenol oxidase (PPO) biocomposite to create a highly responsive phenol biosensor. The phenol sensor could be easily achieved by casting the biocomposite on the surface of glassy carbon electrode (GCE) via the cross-linking step by glutaraldehyde. The special three-dimensional structure, porous morphology, hydrophilic and biocompatible properties of the nano-CaCO(3) matrix resulted in high enzyme loading, and the enzyme entrapped in this matrix retained its activity to a large extent. The proposed PPO/nano-CaCO(3) exhibited dramatically developed analytical performance such as such as a broad determination range (6 x 10(-9) -2 x 10(-5)M), a short response time (less than 12 s), high sensitivity (474 mA M(-1)), subnanomolar detection limit (0.44 nM at a signal to noise ratio of 3) and good long-term stability (70% remained after 56 days). In addition, effects of pH value, applied potential, temperature and electrode construction were investigated and discussed.     

Development of a high analytical performance amperometric glucose biosensor based on glucose oxidase immobilized in a composite matrix: layered double hydroxides/chitosan

This paper aimed at showing the interest of the composite material based on layered double hydroxides (LDHs) and chitosan (CHT) as suitable host matrix likely to immobilize enzyme onto electrode surface for amperometric biosensing application. This hybrid material combined the advantages of inorganic LDHs and organic biopolymer, CHT. Glucose oxidase (GOD) immobilized in the composite material maintained its activity well as the usage of glutaraldehyde was avoided. The process parameters for the fabrication of the enzyme electrode and various experimental variables such as pH, applied potential and temperature, were explored for optimum analytical performance of the enzyme electrode. The enzyme electrode provided a linear response to glucose over a concentration range of 1 x 10(-6) to 3 x 10(-3) M with a high sensitivity of 62.6 mA M(-1) cm(-2) and a detection limit of 0.1 muM based on the signal-to-noise ratio of 3.     

An amperometric cholesterol biosensor based on multiwalled carbon nanotubes and organically modified sol-gel/chitosan hybrid composite film

A new type of amperometric cholesterol biosensor based on sol-gel chitosan/silica and multiwalled carbon nanotubes (MWCNTs) organic-inorganic hybrid composite material was developed. The hybrid composite film was used to immobilize cholesterol oxidase on the surface of Prussian blue-modified glass carbon electrode. Effects of some experimental variables such as enzyme loading, concentration of Triton X-100, pH, temperature, and applied potential on the current response of the biosensor were investigated. Analytical characteristics and dynamic parameters of the biosensors with and without MWCNTs in the hybrid film were compared, and the results show that analytical performance of the biosensor can be improved greatly after introduction of the MWCNTs. Response time, sensitivity, linear range, limit of detection (S/N=3), and apparent Michaelis-Menten constant Km are 25s, 0.54 microA mM(-1), 8.0 x 10(-6) to 4.5 x 10(-4) M, 4.0 x 10(-6) M, and 0.41 mM for the biosensor without MWCNTs and 13 s, 1.55 microA mM(-1), 4.0 x 10(-6) to 7.0 x 10(-4) M, 1.0 x 10(-6) M, and 0.24 mM for the biosensor with MWCNTs, respectively. The activation energy of the enzyme-catalyzed reaction was measured to be 42.6 kJ mol(-1). This method has been used to determine the free cholesterol concentration in real human blood samples.     

Amperometric determination of lysine using a lysine oxidase biosensor based on rigid-conducting composites

In this study, amperometric biosensors based on rigid conducting composites are developed for the determination of lysine. These lysine biosensors consist of chemically immobilized lysine oxidase membranes attached to either graphite-methacrylate or peroxidase-modified graphite-methacrylate electrodes. The enzymatic degradation of lysine releases hydrogen peroxide, which is the basis of the amperometric detection. The direct oxidation of hydrogen peroxide is monitored at +1000 mV with a graphite-methacrylate electrode, while with the peroxidase-modified electrode reductive detection is performed. In addition, for the peroxidase-modified biocomposite electrode, both direct electron transfer and hydroquinone-mediated detection are studied. For the lysine biosensor based on the hydroquinone-mediated peroxidase biocomposite, the linear range is up to 1.6 x 10(-4) M, the sensitivity 11300 microA/M, the repeatability 1.8%, the detection limit 8.2 x 10(-7) M and the response time t95% is 42 s. The proposed biosensors are used to determine lysine in pharmaceutical samples. Results are consistent with those obtained with the standard method.     

Hybrid material based on chitosan and layered double hydroxides: characterization and application to the design of amperometric phenol biosensor

A new type of amperometric phenol biosensor based on chitosan/layered double hydroxides organic-inorganic composite film was described. This hybrid material combined the advantages of organic biopolymer, chitosan, and inorganic layered double hydroxides. Polyphenol oxidase (PPO) immobilized in the material maintained its activity well as the usage of glutaraldehyde was avoided. The composite films have been characterized by Fourier transform infrared. The results indicated that PPO retained the essential feature of its native structure in the composite film. The enzyme electrode provided a linear response to catechol over a concentration range of 3.6 x 10(-9) to 4 x 10(-5) M with a sensitivity of 2750 +/- 52 mA M(-1) cm(-2) and a detection limit of 0.36 nM based on S/N = 3. The apparent Michaelis-Menten constant K(app)(M) for the sensor was found to be 0.13 mM. The activation energy for enzymatic reaction was calculated to be 27.6 kJ mol(-1). Furthermore, the biosensor exhibited excellent long-term stability and satisfactory reproducibility.     

Bienzyme amperometric biosensor using gold nanoparticle-modified electrodes for the determination of inulin in foods

A biosensor design involving coimmobilization of fructose dehydrogenase (FDH) and inulinase (INU) on a gold nanoparticle-cysteamine (Cyst) self-assembled monolayer (SAM)-modified gold electrode (Au(coll)-Cyst-AuE), for the determination of the carbohydrate inulin in foodstuffs, is reported. Tetrathiafulvalene (TTF), used as the mediator, was also coimmobilized by crosslinking with glutaraldehyde. INU catalyzes the hydrolysis of inulin, forming fructose that is detected through the fructose dehydrogenase system by the electrochemical oxidation of TTF at the bioelectrode. The variables involved in the preparation and performance of both the single enzyme FDH biosensor and the bienzyme inulin biosensor were optimized. The FDH-Au(coll)-Cyst-AuE biosensor exhibited rapid and sensitive response to fructose, allowing the obtention of improved analytical characteristics for the determination of fructose with respect to other FDH electrochemical biosensors. Moreover, the lifetime of this biosensor was 35 days. The bienzyme INU/FDH-Au(coll)-Cyst-AuE biosensor provided a calibration plot for inulin in the (5-100)x10(-6) M linear range, with a detection limit of 6.6 x 10(-7) mol L(-1). One single bienzyme biosensor responded within the control limits, set at +/-3x the standard deviation of the currents measured on the first day of use, for more than 5 months. Furthermore, the biosensor exhibited high selectivity with respect to other carbohydrates. The usefulness of the biosensor was evaluated by the rapid determination of inulin in food products involving minimization of the fructose interference.     

Hyphomicrobium bacterial electrode for determination of monomethyl sulfate

A biosensor consisting of physically entrapped monomethyl sulfate (methyl sulfate) degrading bacterium, Hyphomicrobium MS 219, and a combined glass electrode has been developed for the determination of methyl sulfate. The response of the bacterial electrode is linear between 2.5 x 10(-2)M and 6.3 x 10(-1)M methyl sulfate with an effective response to concentrations as low as 10(-3)M and as high as 1M methyl sulfate. The probe has an average slope of 8 mV per concentration decade over the linear range. Response times vary from 5 min in the linear range to 30 min at the detection limit. The sensor has a lifetime of at least 1 week and shows high selectivity to methyl sulfate in the absence of other growth substrates.     

Electrochemical detection of xanthine in fish meat by xanthine oxidase immobilized on carboxylated multiwalled carbon nanotubes/polyaniline composite film

A commercial xanthine oxidase (XOD) was immobilized covalently onto carboxylated multiwalled carbon nanotubes (c-MWCNT) and polyaniline (PANI) composite film electrodeposited on the surface of a Pt electrode, using N-ethyl-N′-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry. A xanthine biosensor was fabricated using XOD/c-MWCNT/PANI/Pt electrode as a working electrode, Ag/AgCl (3 M KCl) as standard electrode and Pt wire as auxiliary electrode connected through a potentiostat. The enzyme electrode was characterized by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectrophotometry and electrochemical impedance spectroscopy (EIS). The biosensor showed optimum response within 4 s at pH 7.0 and 35 °C, when polarized at 0.4 V. The optimized xanthine biosensor showed linear response range of 0.6–58 μM, with a detection limit of 0.6 μM (S/N = 3), and a correlation coefficient of 0.98. The biosensor was applied to determine xanthine in fish meat. The biosensor lost 50% of its initial activity after its 200 uses over a period of 100 days.     

Hydrogen peroxide biosensor based on microperoxidase-11 entrapped in lipid membrane

A highly catalytic activity microperoxidase-11 (MP-11) biosensor for H(2)O(2) was developed to immobilizing the heme peptide in didodecyldimethylammonium bromide (DDAB) lipid membrane. The enzyme electrode thus obtained responded to H(2)O(2) without electron mediator or promoter, at a potential of +0.10 V versus Agmid R:AgCl. A linear calibration curve is obtained over the range from 2.0 x 10(-5) to 2.4 x 10(-3) M. The biosensor responds to hydrogen peroxide in 15 s and has a detection limit of 8 x 10(-7) M (S/N=3) Providing a natural environment with lipid membrane for protein immobilization and maintenance of protein functions is a suitable option for the design of biosensors.