Development of an amperometric sulfite biosensor based on SO(x)/PBNPs/PPY modified ITO electrode

A sulfite oxidase (SO(x)) (EC 1.8.3.1) purified from Syzygium cumini leaves was immobilized onto prussian blue nanoparticles/polypyrrole composite (PBNPs/PPY) electrodeposited onto the surface of indium tin oxide (ITO) electrode. An amperometric sulfite biosensor was fabricated using SO(x)/PBNPs/PPY/ITO electrode as working electrode, Ag/AgCl as standard and Pt wire as auxiliary electrode connected through a potentiostat. The working electrode was characterized by Fourier transform infrared (FTIR) spectroscopy, cyclic voltammetry (CV), scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS) before and after immobilization of SO(x). The biosensor showed optimum response within 2s, when operated at 20mVs(-1) in 0.1M Tris-HCl buffer, pH 8.5 and at 35°C. Linear range and minimum detection limit were 0.5-1000μM and 0.12μM (S/N=3) respectively. There was good correlation (r=0.99) between red wine samples sulfite value by standard DTNB method and the present method. The sensor was evaluated with 97% recovery of added sulfite in red wine samples and 2.2% and 4.3% within and between batch coefficients of variation respectively. The sensor was employed for determination of sulfite level in red and white wine samples. The enzyme electrode was used 200 times over a period of 3 months when stored at 4°C.     

An amperometric biosensor based on laccase immobilized onto MnO2NPs/cMWCNT/PANI modified Au electrode

A method is described for construction of an amperometric biosensor for detection of phenolic compounds based on covalent immobilization of laccase (Lac) onto manganese dioxide nanoparticles (MnO(2)NPs) decorated carboxylated multiwalled carbon nanotubes (cMWCNTs)/PANI composite electrodeposited onto a gold (Au) electrode through N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry. The modified electrode was characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The biosensor showed optimum response at pH 5.5 (0.1M sodium acetate buffer) and 35°C, when operated at 0.3 V vs. Ag/AgCl. Linear range, response time, detection limit were 0.1-10 μM (lower concentration range) and 10-500 μM (higher concentration range), 4s and 0.04 μM, respectively. Biosensor measured total phenolic content in tea leaves extract. The enzyme electrode was used 150 times over a period of 5 months.     

An amperometric biosensor based on laccase immobilized onto Fe(3)O(4)NPs/cMWCNT/PANI/Au electrode for determination of phenolic content in tea leaves extract

A method is described for the construction of an amperometric biosensor for detection of phenolic compounds based on covalent immobilization of laccase onto iron oxide nanoparticles (Fe(3)O(4)NPs) decorated carboxylated multiwalled carbon nanotubes (cMWCNTs)/polyaniline (PANI) composite electrodeposited onto a gold (Au) electrode. The modified electrode was characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The biosensor showed optimum response within 3s at pH 6.0 (0.1M sodium acetate buffer) and 35°C, when operated at 0.3V vs. Ag/AgCl. Linear range, detection limit were 0.1-10μM (lower concentration range) and 10-500μM (higher concentration range), and 0.03μM respectively. The sensor measured total phenolic content in tea leaves extract. The enzyme electrode lost 25% of its initial activity after its 150 uses over a period of 4 months, when stored at 4°C.     

Immobilization of creatininase, creatinase and sarcosine oxidase on iron oxide nanoparticles/chitosan-g-polyaniline modified Pt electrode for detection of creatinine

Commercial enzymes, creatininase (CA) from Pseudomonas sp, creatinase (CI) from Pseudomonas sp, sarcosine oxidase (SO) from Bacillus sp were co-immobilized onto iron oxide nanoparticles/chitosan-graft-polyaniline (Fe(3)O(4)-NPs/CHIT-g-PANI) composite film electrodeposited on surface of Pt electrode through glutaraldehyde coupling. Transmission electron microscopy (TEM) was used for characterization of Fe(3)O(4)-NPs. A creatinine biosensor was fabricated using Enzymes/Fe(3)O(4)-NPs/CHIT-g-PANI/Pt electrode as working electrode, Ag/AgCl as reference electrode and Pt wire as auxiliary electrode. The enzyme electrode was characterized by cyclic voltammetry (CV), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopic and electrochemical impedance spectroscopy (EIS). The biosensor exhibited an optimum response within 2s at pH 7.5 and 30 °C, when polarized at 0.4V vs Ag/AgCl. The electrocatalytic response showed a linear dependence on creatinine concentration ranging from 1 to 800 μM. The sensitivity of the biosensor was 3.9 μA μM(-1) cm(-2), with a detection limit of 1 μM (S/N=3). Apparent Michaelis-Menton (K(m)) value for creatinine was 0.17 mM. The biosensor showed only 10% loss in its initial response after 120 uses over 200 days, when stored at 4 °C. The biosensor measured creatinine in the serum of apparently healthy persons which correlated well with a standard colorimetric method (r=0.99).     

Development of electrochemical sulfite biosensor based on SOX/PBNPs/PPY modified Au electrode

A sulfite oxidase (SO_X) (EC 1.8.3.1) purified from Syzygium cumini leaves was immobilized onto Prussian blue nanoparticles/polypyrrole (PBNPs/PPY) nanocomposite film electrodeposited onto the surface of gold (Au) electrode. An electrochemical sulfite biosensor was fabricated using SO_X/PBNPs/PPY/Au electrode as working electrode, Ag/AgCl as standard electrode and Pt wire as auxiliary electrode connected through a potentiostat. The working electrode was characterized by Fourier Transform infrared (FTIR) spectroscopy, cyclic voltammetry (CV), scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS) at different stages of its construction. The biosensor showed optimum response within 2 s, when operated at 20 mV s⁻¹ in 0.1 M Tris–HCl buffer, pH 8.0 and at 30 °C. Linear range and minimum detection limit were 0.5–1000 μM and 0.1 μM (S/N = 3) respectively. The sensor was evaluated with 95.0% recovery of added sulfite in red wine samples and 1.9% and 3.3% within and between batch coefficients of variation respectively. There was a good correlation (r = 0.96) between red wine samples sulfite value by standard DTNB method and the present method. The sensor was employed for determination of sulfite level in red, white and rose wine samples. The enzyme electrode was used 300 times over a period of 4 months, when stored at 4 °C.     

Immobilization of lysine oxidase on a gold–platinum nanoparticles modified Au electrode for detection of lysine

A commercial lysine oxidase (LyOx) from Trichoderma viride was immobilized covalently onto gold nanoparticles (AuNPs) and platinum nanoparticles (PtNPs) electrodeposited onto Au electrode using 3-aminopropyltriethoxy silane (3-APTES) and glutaraldehyde cross linking chemistry. A lysine biosensor was fabricated using LyOx/3-APTES/AuNPs-PtNPs/Au 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), Fourier transform infrared (FTIR) spectroscopy, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The cumulative effect of AuNPs and PtNPs showed excellent electrocatalytic activity at low applied potential for detection of H₂O₂, a product of LyOx reaction. The sensor showed its optimum response within 4 s, when polarized at 0.2 V vs. Ag/AgCl in 0.1 M phosphate buffer, pH 7.5 at 30 °C. The linear range and detection limit of the sensor were 1.0–600 μM and 1.0 μM (S/N = 3), respectively. Biosensor measured lysine level in sera, milk and amino acid tablet, which correlated well with those by standard HPLC method. The enzyme electrode lost 50% of its initial activity after 200 uses over a period of 4 months.     

Construction of an amperometric TG biosensor based on AuPPy nanocomposite and poly (indole-5-carboxylic acid) modified Au electrode

A method is described for construction of an amperometric triglyceride (TG) biosensor based on covalent co-immobilization of lipase, glycerol kinase and glycerol-3-phosphate oxidase onto gold polypyrrole nanocomposite decorated poly indole-5-carboxylic acid electrodeposited on the surface of a gold electrode. The enzyme electrode was characterized by transmission electron microscopy, scanning electron microscopy, electrochemical impedance studies, Fourier transform infrared spectroscopy and cyclic voltammetry. Biosensor showed optimum response within 4 s at pH 6.5 and 35 °C, when polarized at +0.1 V against Ag/AgCl. There was a linear relationship between sensor response and triolein concentration in the range 50–700 mg/dl. Biosensor was employed for determination of TG in serum. Detection limit of the biosensor was 20 mg/dl. Biosensor was evaluated with 91–95 % recovery of added triolein in sera and 4.14 and 5.85 % within and between batch coefficients of variation, respectively. There was a good correlation (r = 0.99) between sera TG values by standard method (Enzymic colorimetric) and the present method. The biosensor was unaffected by a number of serum substances at their physiological concentration. Biosensor lost 50 % of its initial activity after its 100 uses over 7 months, when stored at 4 °C.     

Silver nanoparticles/multiwalled carbon nanotube/polyaniline film for amperometric glutathione biosensor

A new silver nanoparticles (AgNPs)/carboxylated multiwalled carbon nanotubes (c-MWCNT)/polyaniline (PANI) film has been synthesized on Au electrode using electrochemical techniques. The enzyme glutathione oxidase (GSHOx) (EC 1.8.3.3) was immobilized covalently on the surface of AgNPs/c-MWCNT/PANI/Au electrode to construct the glutathione biosensor. The modified electrode was characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and Fourier transform infrared (FTIR) spectrophotometry. The biosensor showed optimum response within 4s at +0.4V vs. Ag/AgCl, pH 6.0 and 35 °C, with a linear working range of 0.3-3500 μM and a detection limit of 0.3 μM. The glutathione biosensor was employed for measurement of glutathione content in hemolysated erythrocyte (RBC). The sensor was evaluated with 97.77% and 99.16% recovery of added glutathione in hemolysated RBC and 2.4% and 6.3% within and between batch coefficients of variation (CVs) respectively. The enzyme electrode lost 50% of its initial activity after 300 uses over a period of 3 months, when stored at 4 °C. The biosensor has the advantages over earlier biosensors in terms of greater stability, lower response time and no interference by a number of RBC hemolysate substances.     

Polyphenol biosensor based on laccase immobilized onto silver nanoparticles/multiwalled carbon nanotube/polyaniline gold electrode

Laccase purified from Ganoderma sp. was immobilized covalently onto electrochemically deposited silver nanoparticles (AgNPs)/carboxylated multiwalled carbon nanotubes (cMWCNT)/polyaniline (PANI) layer on the surface of gold (Au) electrode. A polyphenol biosensor was fabricated using this enzyme electrode (laccase/AgNPs/cMWCNT/PANI/Au electrode) as the working electrode, Ag/AgCl as the reference electrode, and platinum (Pt) wire as the auxiliary electrode connected through a potentiostat. The biosensor showed optimal response at pH 5.5 (0.1 M acetate buffer) and 35 °C when operated at a scan rate of 50 mV s⁻¹. Linear range, response time, and detection limit were 0.1–500 μM, 6 s, and 0.1 μM, respectively. The sensor was employed for the determination of total phenolic content in tea, alcoholic beverages, and pharmaceutical formulations. The enzyme electrode was used 200 times over a period of 4 months when stored at 4 °C. The biosensor has an advantage over earlier enzyme sensors in that it has no leakage of enzyme during reuse and is unaffected by the external environment due to the protective PANI microenvironment.     

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.     

Construction of amperometric uric acid biosensor based on uricase immobilized on PBNPs/cMWCNT/PANI/Au composite

A chitosan-glutaraldehyde crosslinked uricase was immobilized onto Prussian blue nanoparticles (PBNPs) absorbed onto carboxylated multiwalled carbon nanotube (c-MWCNT) and polyaniline (PANI) layer, electrochemically deposited on the surface of Au electrode. The nanohybrid-uricase electrode was characterized by scanning electron microscopic (SEM), Fourier transform infrared spectroscopy (FTIR) and cyclic voltammetry. An amperometric uric acid biosensor was fabricated using uricase/c-MWCNT/PBNPs/Au electrode as working electrode, Ag/AgCl as standard and Pt wire as auxiliary electrode connected through a potentiostat. The biosensor showed optimum response within 4 s at pH 7.5 and 40 °C, when operated at 0.4 V vs. Ag/AgCl. The linear working range for uric acid was 0.005-0.8 mM, with a detection limit of 5 μM. The sensor was evaluated with 96% recovery of added uric acid in sera and 4.6 and 5.4% within and between batch of coefficient of variation respectively and a good correlation (r = 0.99) with standard enzymic colorimetric method. This sensor measured uric acid in real serum samples. The sensor lost only 37% of its initial activity after its 400 uses over a period of 7 months, when stored at 4 °C.     

Ultrasensitive electrochemical detection of Bacillus thuringiensis transgenic sequence based on in situ Ag nanoparticles aggregates induced by biotin-streptavidin system

A novel electrochemical biosensor was developed for detecting short DNA oligonucleotide of Bacillus thuringiensis (Bt) transgenic sequence based on Ag nanoparticle aggregates. To fabricate this DNA biosensor, the thiol-modified capture DNA (cDNA) was first anchored on gold (Au) electrode, and then the target DNA (tDNA) was hybridized with the immobilized cDNA. Subsequently, the probe DNA (pDNA) functionalized by biotinylated Ag nanoparticle was associated with the fixed tDNA, and the single Ag nanoparticle label was obtained (cited as SAg label). Finally, dissociative biotinylated Ag nanoparticle was bound to the resultant biotinylated SAg label assembled on Au electrode by virtue of bridge molecule streptavidin (SA) through biotin-SA specific interaction, which could lead to in situ aggregate of Ag nanoparticles on Au electrode and induce a novel tag including multiple Ag nanoparticles (cited as MAg tag). The novel tag exhibited excellent electroactive property in the solid-state Ag/AgCl process and was successfully applied to Bt transgenic sequence assay. A detection limit of 10 fM was achieved, which was improved by three orders of magnitude as compared to the SAg label. Furthermore, this novel DNA biosensor demonstrated a good selectivity towards tDNA.     

Electrophoretic transfer protein zymography

A mixture of commercial creatinine amidohydrolase (CA), creatine amidinohydrolase (CI), and sarcosine oxidase (SO) was coimmobilized covalently via N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry onto carboxylated multiwalled carbon nanotube (c-MWCNT)/polyaniline (PANI) nanocomposite film electrodeposited over the surface of a platinum (Pt) electrode. A creatinine biosensor was fabricated using enzyme/c-MWCNT/PANI/Pt as working electrode, Ag/AgCl as reference electrode, and Pt wire as auxiliary electrode connected through potentiostat. The enzyme electrode was characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and electrochemical impedance spectroscopy (EIS). The biosensor detected creatinine levels as low as 0.1 μM, estimated at a signal-to-noise ratio of 3, within 5 s at pH 7.5 and 35 °C. The optimized biosensor showed a linear response range of 10 to 750 μM creatinine with sensitivity of 40 μA/mM/cm². The fabricated biosensor was successfully employed for determination of creatinine in human serum. The biosensor showed only 15% loss in its initial response after 180 days when stored at 4 °C.     

Immobilization of rat brain acetylcholinesterase on porous gold-nanoparticle-CaCO₃ hybrid material modified Au electrode for detection of organophosphorous insecticides

An acetylcholinesterase (AChE) purified from rat brain was immobilized onto gold nanoparticles (AuNPs) assembled on the surface of porous calcium carbonate (CaCO₃) microsphere. The resulting AChE-AuNPs-CaCO₃ bioconjugate was mounted on the surface of Au electrode with the help of silica sol-gel matrix to prepare the working electrode. This electrode was connected to Ag/AgCl (3 M/saturated KCl) as standard and Pt wire as an auxiliary electrode through a potentiostat to construct an organophosphorus (OP) biosensor. The biosensor was based on inhibition of AChE by OP compounds/insecticides. The biosensor showed optimum response at pH 7.0, 30 °C, when polarized at +0.2 V. Two OP compounds, malathion and chlorpyrifos could be detected in the range of 0.1-100 nM and 0.1-70 nM, respectively at 2.0-3.0% inhibition level of AChE. The sensor was reactivated by immersing it in 0.1 mM 2-pyridine aldoxime for 10 min. The detection limit of the sensor was 0.1 nM for both malathion and chlorpyrifos. The biosensor exhibited good reusability (50 times without considerable loss) and storage stability (50% within 60 days, when stored at 4 °C).     

Development of disposable amperometric sulfur dioxide biosensors based on screen printed electrodes

The possibility of developing amperometric biosensors for the measurement of SO(2) in flowing gas streams has been examined. Screen-printed carbon electrodes (SPCEs) were tailored with the enzyme sulfite oxidase and cytochrome c and the response is generated through the resulting enzymatic and electrocatalytic reactions involving SO(3)(2-), formed when SO(2) gas is dissolved in the supporting electrolyte. Two methods of integrating the enzyme and cytochrome c with the SPCE were investigated. In one design (b-type biosensor), the components were mixed thoroughly with the same ink used to produce the SPCEs, then the modified ink was spread over the working electrode. In the second approach the bio-components were dissolved in the supporting electrolyte and simply deposited on top of the transducer (s-type biosensor). Both devices gave linear responses over the range 4--50 ppm but the sensitivity of the s-type was approximately twice that of the b-type biosensor. In addition, the time taken to reach 90% of the maximum response (t(90%)) was 110 s for the s-type biosensor compared with 200 s for the b-type biosensor. These studies illustrate the successful use of biosensors for the detection of sulfur dioxide at the relatively low potential of +0.3 V versus Ag.AgCl and should provide useful alternatives for decentralised environmental studies.     

An amperometric H2O2 biosensor based on cytochrome c immobilized onto nickel oxide nanoparticles/carboxylated multiwalled carbon nanotubes/polyaniline modified gold electrode

Cytochrome c was immobilized covalently onto nickel oxide nanoparticles/carboxylated multiwalled carbon nanotubes/polyaniline composite (NiO-NPs/cMWCNT/PANI) electrodeposited on gold (Au) electrode. An amperometric H₂O₂ biosensor was constructed by connecting this modified Au electrode along Ag/AgCl as reference and Pt wire as counter electrode to the galvanostat. The modified Au electrode was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM) and Fourier transform infra-red spectroscopy (FTIR). Cyclic voltammetric (CV) studies of the electrode at different stages demonstrated that the modified Au electrode had enhanced electrochemical oxidation of H₂O₂, which offered a number of attractive features to develop an amperometric biosensor based on split of H₂O₂. There was a good linear relationship between the current (mA) and H₂O₂ concentration in the range 3–700 μM. The sensor had a detection limit of 0.2 μM (S/N = 3) with a high sensitivity of 3.3 mA μM^?1 cm^?2. The sensor gave accurate and satisfactory results, when employed for determination of H₂O₂ in different fruit juices.     

An electrochemical biosensor for fructosyl valine for glycosylated hemoglobin detection based on core-shell magnetic bionanoparticles modified gold electrode

A high-performance amperometric fructosyl valine (FV) biosensor was developed, based on immobilization of fructosyl amino-acid oxidase (FAO) on core-shell magnetic bionanoparticles modified gold electrode. Chitosan was used to introduce amino groups onto the surface of core-shell magnetic bionanoparticles (MNPs). With FAO as an enzyme model, a new fructosyl valine biosensor was fabricated. The biosensor showed optimum response, when operated at 50 mVs(-1) in 0.1M potassium phosphate buffer, pH 7.5 and 35°C. The biosensor exhibited excellent sensitivity [the detection limit is down to 0.1mM for FV], fast response time (less than 4s), wide linear range (from 0 to 2mM). Analytical recovery of added FV was 95.00-98.50%. Within batch and between batch coefficients of variation were <2.58% and <5.63%, respectively. The enzyme electrode was used 250 times over 3 months, when stored at 4°C.     

A nylon membrane based amperometric biosensor for polyphenol determination

A nylon membrane based amperometric biosensor employing banana fruit polyphenol oxidase (PPO) is presented for polyphenol detection. Nylon membrane was first activated and then coupled with chitosan. PPO was covalently attached to this membrane through glutaraldehyde coupling. The membrane bioconjugate was characterized by scanning electron microscopy (SEM) and Fourier Transform Infrared (FTIR) study and then mounted onto Au electrode using parafilm to construct a working electrode. Once assembled along with Ag/AgCl as reference and Pt as auxiliary electrode, the biosensor gave optimum response within 15 s at pH 7.5 and 30 °C, when polarized at +0.4 V. The response (in mA) was directly proportional to polyphenol concentration in the range 0.2–400 μM. The lower detection limit of the biosensor was 0.2 μM. The biosensor was employed for determination of polyphenols in tea, beverages and water samples. The enzyme electrode showed 25% decrease in initial activity after 150 reuses over 6 months, when stored at 4 °C.     

Low potential detection of NADH based on Fe₃O₄ nanoparticles/multiwalled carbon nanotubes composite: fabrication of integrated dehydrogenase-based lactate biosensor

Fe(3)O(4) magnetic nanoparticles were in situ loaded on the surface of multiwalled carbon nanotubes (MWCNTs) by a simple coprecipitation procedure. The resulting Fe(3)O(4)/MWCNTs nanocomposite brings new capabilities for electrochemical sensing by combining the advantages of Fe(3)O(4) magnetic nanoparticles and MWCNTs. It was found that Fe(3)O(4) has redox properties similar to those of frequently used mediators used for electron transfer between NADH and electrode. The cyclic voltammetric results indicated the ability of Fe(3)O(4)/MWCNTs modified GC electrode to catalyze the oxidation of NADH at a very low potential (0.0 mV vs. Ag/AgCl) and subsequently, a substantial decrease in the overpotential by about 650 mV compared with the bare GC electrode. The catalytic oxidation current allows the stable and selective amperometric detection of NADH at an applied potential of 0.0 mV (Ag/AgCl) with a detection limit of 0.3 μM and linear response up to 300 μM. This modified electrode can be used as an efficient transducer in the design of biosensors based on coupled dehydrogenase enzymes. Lactate dehydrogenase (LDH) and NAD(+) were subsequently immobilized onto the Fe(3)O(4)/MWCNTs nanocomposite film by covalent bond formation between the amine groups of enzyme or NAD(+) and the carboxylic acid groups of the Fe(3)O(4)/MWCNT film. Differential pulse voltammetric detection of lactate on Fe(3)O(4)/MWCNT/LDH/NAD(+) modified GC electrode gives linear responses over the concentration range of 50-500 μM with the detection limit of 5 μM and sensitivity of 7.67 μA mM(-1). Furthermore, the applicability of the sensor for the analysis of lactate concentration in human serum samples has been successfully demonstrated.     

Construction of d-amino acid biosensor based on d-amino acid oxidase immobilized onto poly (indole-5-carboxylic acid)/zinc sulfide nanoparticles hybrid film

d-Amino acid oxidase (DAAO) purified from goat kidney was immobilized covalently via N-ethyl-N-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry onto poly indole 5-carboxylic acid (Pin5-COOH)/zinc sulfide nanoparticles (ZnSNPs) hybrid film electrodeposited on surface of an Au electrode. A highly sensitive d-amino acid biosensor was constructed using this enzyme electrode as working electrode, Ag/AgCl as reference electrode, and Pt wire as auxiliary electrode connected through potentiostat. The biosensor showed optimum response within 3 s at pH 7.5 and 35 °C, when polarized at 0.15 V vs. Ag/AgCl. There was a linear relationship between biosensor response (mA) and d-alanine concentration in the range 0.001–2.0 mM. The sensitivity of the biosensor was 58.85 μA cm^?2 mM^?1 with a detection limit of 0.001 mM (S/N = 3). The enzyme electrode was used 120 times over a period of 2 months when stored at 4 °C. The biosensor has an advantage over earlier enzyme sensors that it has no leakage of enzyme during reuse and is unaffected by the external environment due to the protective layer of poly indole-5-carboxylic acid film. The biosensor was evaluated and employed for measurement of d-amino acid level in fruits and vegetables.