Composite film of carbon nanotubes and chitosan for preparation of amperometric hydrogen peroxide biosensor

A new amperometric biosensor for hydrogen peroxide was developed based on cross-linking horseradish peroxidase (HRP) by glutaraldehyde with multiwall carbon nanotubes/chitosan (MWNTs/chitosan) composite film coated on a glassy carbon electrode. MWNTs were firstly dissolved in a chitosan solution. Then the morphology of MWNTs/chitosan composite film was characterized by field-emission scanning electron microscopy. The results showed that MWNTs were well soluble in chitosan and robust films could be formed on the surface. HRP was cross-linked by glutaraldehyde with MWNTs/chitosan film to prepare a hydrogen peroxide biosensor. The enzyme electrode exhibited excellent electrocatalytic activity and rapid response for H₂O₂ in the absence of a mediator. The linear range of detection towards H₂O₂ (applied potential: −0.2 V) was from 1.67 × 10⁻⁵ to 7.40 × 10⁻⁴ M with correction coefficient of 0.998. The biosensor had good repeatability and stability for the determination of H₂O₂. There were no interferences from ascorbic acid, glucose, citrate acid and lactic acid.     

Electrodeposition of gold nanoparticles on indium/tin oxide electrode for fabrication of a disposable hydrogen peroxide biosensor

A novel disposable third-generation hydrogen peroxide (H₂O₂) biosensor based on horseradish peroxidase (HRP) immobilized on the gold nanoparticles (AuNPs) electrodeposited indium tin oxide (ITO) electrode is investigated. The AuNPs deposited on ITO electrode were characterized by UV-vis, SEM, and electrochemical methods. The AuNPs attached on the ITO electrode surface with quasi-spherical shape and the average size of diameters was about 25 nm with a quite symmetric distribution. The direct electron chemistry of HRP was realized, and the biosensor exhibited excellent performances for the reduction of H₂O₂. The amperometric response to H₂O₂ shows a linear relation in the range from 8.0 μmol L⁻¹ to 3.0 mmol L⁻¹ and a detection limit of 2 μmol L⁻¹ (S/N = 3). The value of HRP immobilized on the electrode surface was found to be 0.4 mmol L⁻¹. The biosensor indicates excellent reproducibility, high selectivity and long-term stability.     

Bilayer lipid membrane biosensor with enhanced stability for amperometric determination of hydrogen peroxide

In this paper, a polydopamine (PDA) film is electropolymerized on the surface of bilayer lipid membrane (BLM) which is immobilized with horseradish peroxidase (HRP). The coverage of the PDA film on HRP/BLM electrode is monitored by electrochemical impedance spectroscopy (EIS). The electrocatalytic reduction of H₂O₂ at the PDA/HRP/BLM electrode is studied by means of cyclic voltammetry (CV). The biosensor has a fast response to H₂O₂ of less than 5 s and an excellent linear relationship is obtained in the concentration range from 2.5 × 10⁻⁷ to 3.1 × 10⁻³ mol L⁻¹, with a detection limit of 1.0 × 10⁻⁷ mol L⁻¹ (S/N = 3). The response current of BLM/HRP/PDA biosensor retains 84% of its original response after being stored in 0.1 mol L⁻¹ pH 7.0 PBS at 4 °C for 3 weeks. The selectivity, repeatability, and storage stability of PDA/HRP/BLM biosensor are greatly enhanced by the coverage of polydopamine film on BLM.     

Preparation and characterization of Prussian blue nanowire array and bioapplication for glucose biosensing

Prussian blue nanowire array (PBNWA) was prepared via electrochemical deposition with polycarbonate membrane template for effective modification of glassy carbon electrode. The PBNWA electrode thus obtained was demonstrated to have high-catalytic activity for the electrochemical reduction of hydrogen peroxide in neutral media. This enabled the PBNWA electrode to show rapid response to H₂O₂ at a low potential of −0.1 V over a wide range of concentrations from 1 × 10⁻⁷ M to 5 × 10⁻² M with a high sensitivity of 183 μA mM⁻¹ cm⁻². Such a low-working potential also substantially improved the selectivity of the PBNWA electrode against most electroactive species such as ascorbic acid and uric acid in physiological media. A detection limit of 5 × 10⁻⁸ M was obtained using the PBNWA electrode for H₂O₂, which compared favorably with most electroanalysis procedures for H₂O₂. A biosensor toward glucose was then constructed with the PBNWA electrode as the basic electrode by crosslinking glucose oxidase (GOx). The glucose biosensor allowed rapid, selective and sensitive determination of glucose at −0.1 V. The amperometric response exhibited a linear correlation to glucose concentration through an expanded range from 2 × 10⁻⁶ M to 1 × 10⁻² M, and the response time and detection limit were determined to be 3 s and 1 μM, respectively.     

Hydrogen peroxide and peracetic acid determination in waste water using a reversible reagentless biosensor

During the reversible reaction between peroxidase (HRP) and peroxides, several peroxidase intermediate species, showing different molecular absorption spectra, are formed which can be used for their determination. On this basis, a reversible reagentless optical biosensor based on HRP for hydrogen peroxide and peracetic acid determinations has been developed. The biosensor (which can be used for at least 3 months and/or more than 200 measurements) is prepared by HRP entrapment in a polyacrylamide gel matrix. A mathematical model (in which optical, kinetic and transport aspects are considered) relating the measured absorbance with the analyte concentration is also presented. Both peroxides show similar responses in the sensor film. Under the recommended working conditions, the biosensor shows linear response ranges from 6 × 10⁻⁷ to 1.0 × 10⁻⁴ M using FIA mode, and from 2 × 10⁻⁷ to 1.5 × 10⁻⁵ M using continuous mode for both peroxides; the precision, expressed as R.S.D., is about 4%. This biosensor has been applied for peroxide determination in waste water samples previously treated with peroxides.     

One-step construction of reagentless biosensor based on chitosan-carbon nanotubes-nile blue-horseradish peroxidase biocomposite formed by electrodeposition

A simple and controllable electrodeposition approach was established for one-step construction of novel reagentless biosensors by in situ formation of chitosan-carbon nanotubes-nile blue-horseradish peroxidase (CS-CNTs-NB-HRP) biocomposite film on electrode surface. The mediator effect of NB, conducting performance of CNTs and the biocompatible microenvironment of CS were combined by such one-step non-manual process. NB could interact with CNTs and resulted in good dispersion of CNTs-NB nanocomposites in aqueous solution. Cyclic voltammetry measurements demonstrated that electrons were efficiently shuttled between HRP and the electrode mediated by NB. The developed reagentless biosensor exhibited a fast amperometric response for the determination of H₂O₂ and 95% of the steady-state current was obtained within 2 s. The linear response of the reagentless biosensor for the determination of H₂O₂ ranged from 1.0 × 10⁻⁶ to 2.4 × 10⁻⁴ mol l⁻¹ with a detection limit of 1.2 × 10⁻⁷ mol l⁻¹. The biosensor exhibited high reproducibility and long-time storage stability. The as-prepared biosensor also showed effective anti-interference capability. The ease of the one-step non-manual technique and the promising feature of the biocomposite could serve as a versatile platform for fabricating electrochemical biosensors.     

Amperometric biosensor for hydrogen peroxide based on coimmobilized horseradish peroxidase and methylene green in ormosils matrix with multiwalled carbon nanotubes

A novel amperometric biosensor for the analytical determination of hydrogen peroxide was developed. The fabrication of the biosensor was based on the coimmobilization of horseradish peroxidase (HRP), methylene green (MG) and multiwalled carbon nanotubes within ormosils; 3-aminopropyltrimethoxysilane (APTMOS), 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane (ETMOS) and phenyltrimethoxysilane (PHTMOS). APTMOS determined the hydrophilicity/hydrophobicity of the ormosils and PHTMOS and ETMOS increased the physical and mechanical strength of the ormosil matrix. The ormosil modified electrodes were characterized with SEM, UV-vis spectroscopy and electrochemical methods. Cyclic voltammetry and amperometric measurements demonstrated the MG coimmobilized with HRP in this way, displayed good stability and could efficiently shuttle electrons between immobilized enzyme and electrode, and MWCNTs facilitated the electrocatalytic reduction of H₂O₂ at reduced over potential. The Micheaelis constant of the immobilized HRP was 1.8 mM, indicating a high affinity of the HRP to H₂O₂ without loss of enzymatic activity in ormosil matrix. The prepared biosensor had a fast response of H₂O₂, less than 10 s, and excellent linear range of concentration from 5 × 10⁻⁷ to 2 × 10⁻⁵ M with the detection limit of 0.5 μM (S/N = 3) under the optimum conditions. At the same time, the influence of solution pH, effect of enzyme amount, steady-state applied potential and temperature on the biosensor were investigated. The enzyme electrode retained about 90% of its initial activity after 30 days of storage in a dry state at 4 °C. The preparation of the developed biosensor was convenient and showed high sensitivity with good stability.     

Amperometric biosensor based on a nanoporous ZrO2 matrix

A biosensor was investigated based on the use of ZrO₂ sol-gel matrix for enzyme immobilization in the mild condition. This bioceramic zirconia alcogel has been prepared by the novel alcohothermal route with a cheap inorganic salt Zr(NO₃)₄·5H₂O with several desirable features including a large surface area (about 460 m² g⁻¹) as well as pore volume and a well-developed textural mesoporosity, and horseradish peroxidase was selected as a model enzyme. The results of transmission electron microscopy (TEM) and BET measurement of the substrate showed that the as-prepared zirconia matrix has an advantageous microenvironment and large surface area available for high enzyme loading. The parameters affecting both the entrapment of enzyme and the biosensor response were optimized. The resulting biosensor exhibited high sensitivity of 111 μA mM⁻¹ for hydrogen peroxide over a wide range of concentrations from 2.5×10⁻⁷ to 1.5×10⁻⁴ mol l⁻¹, quick response of less than 10 s and good stability over 3 months.     

Development of hydrogen peroxide biosensor based on in situ covalent immobilization of horseradish peroxidase by one-pot polysaccharide-incorporated sol-gel process

A simple and reliable one-pot approach was established for the development of a novel hydrogen peroxide (H₂O₂) biosensor based on in situ covalent immobilization of horseradish peroxidase (HRP) into biocompatible material through polysaccharide-incorporated sol-gel process. Siloxane with epoxide ring and trimethoxy anchor groups was applied as the bifunctional cross-linker and the inorganic resource for organic-inorganic hybridization. The reactivity between amine groups and epoxy groups allowed the covalent incorporation of HRP and the functional biopolymer, chitosan (CS) into the inorganic polysiloxane network. Some experimental variables, such as mass ratio of siloxane to CS, pH of measuring solution and applied potential for detection were optimized. HRP covalently immobilized in the hybrid matrix possessed high electrocatalytic activity to H₂O₂ and provided a fast amperometric response. The linear response of the as-prepared biosensor for the determination of H₂O₂ ranged from 2.0 × 10⁻⁷ to 4.6 × 10⁻⁵ mol l⁻¹ with a detection limit of 8.1 × 10⁻⁸ mol l⁻¹. The apparent Michaelis-Menten constant was determined to be 45.18 μmol l⁻¹. Performance of the biosensor was also evaluated with respect to possible interferences. The fabricated biosensor exhibited high reproducibility and storage stability. The ease of the one-pot covalent immobilization and the biocompatible hybrid matrix serve as a versatile platform for enzyme immobilization and biosensor fabricating.     

Ordered mesoporous polyaniline film as a new matrix for enzyme immobilization and biosensor construction

Ordered mesoporous polyaniline film has been fabricated by electrodepositing from the hexagonal lyotropic liquid crystalline (LCC). Horseradish peroxidase (HRP), as a symbol biomolecule, was successfully immobilized on the film to construct a new kind of hydrogen peroxide biosensor. The biosensor combined the advantages of the good conductivity of polyaniline and the higher surface area of the ordered mesoporous film. Polyaniline could be served as a wire to relay electron between HRP and the electrode. The high surface area of the film supplied more sites for HRP immobilization, therefore increased the catalytic activity of the biosensor. The ordered mesoporous character of the film increased the rate of mass transport, which resulted in the improvement of sensor response and linearity. The biosensor displayed excellent electrocatalytic response to the detection of H₂O₂ in a concentration range from 1.0 μM to 2.0 mM with a detection limit of 0.63 μM. Good reproducibility, stability, high precision, wide linearity and low detection limit were assessed for the biosensor.     

Electrical detection of deoxyribonucleic acid hybridization based on carbon-nanotubes/nano zirconium dioxide/chitosan-modified electrodes

A novel and sensitive electrochemical DNA biosensor based on nanoparticles ZrO₂ and multi-walled carbon nanotubes (MWNTs) for DNA immobilization and enhanced hybridization detection is described. The MWNTs/nano ZrO₂/chitosan-modified glassy carbon electrode (GCE) was fabricated and oligonucleotides were immobilized to the GCE. The hybridization reaction on the electrode was monitored by differential pulse voltammetry (DPV) analysis using electroactive daunomycin as an indicator. Compared with previous DNA sensors with oligonucleotides directly incorporated on carbon electrodes, this carbon nanotube-based assay with its large surface area and good charge-transport characteristics increased DNA attachment quantity and complementary DNA detection sensitivity. The response signal increases linearly with the increase of the logarithm of the target DNA concentration in the range of 1.49 × 10⁻¹⁰ to 9.32 × 10⁻⁸ mol L⁻¹ with the detection limit of 7.5 × 10⁻¹¹ mol L⁻¹ (S/N = 3). The linear regression equation is I = 32.62 + 3.037 log C_DNA (mol L⁻¹) with a correlation coefficient value of 0.9842. This is the first application of carbon nanotubes combined with nano ZrO₂ to the fabrication of an electrochemical DNA biosensor with a favorable performance for the rapid detection of specific hybridization.     

Electrochemical sensing platform based on covalent immobilization of thionine onto gold electrode surface via diazotization-coupling reaction

A novel electrochemical sensing platform by modification of electroactive thionine (Th) onto gold electrode surface was constructed, which was realized by diazotization of 4-aminothiophenol (ATP) self-assembled monolayer, followed by coupling of Th with the diazonium group to form a covalent diazo bond. A pair of well-defined redox peaks of Th was observed in the cyclic voltammetric measurement. The resulting diazo-ATP monolayer displayed superior electrical conductivity, which contributed to the sensitive detection of hydrogen peroxide (H₂O₂). The immobilized Th also showed a remarkable stability, which may benefit from the π-π stacking force and the covalent diazo bond between diazo-ATP and Th molecules. Under the optimized experimental conditions, the current fabricated non-enzyme and reagentless sensor could show a rapid response to H₂O₂ within 3 s and a linear calibration plot ranged from 1.0 × 10⁻⁶ to 6.38 × 10⁻³ M with a detection limit of 6.7 × 10⁻⁷ M. The current fabrication strategy of electroactive interface is expected to be used as a versatile route for the immobilization of more electroactive molecules and offer more opportunities for the applications in electrochemical sensor, biosensor, electrocatalysis, etc.     

Bienzymatic amperometric biosensor for glucose based on polypyrrole/ceramic carbon as electrode material

A novel amperometric biosensor utilizing two enzymes, glucose oxidase (GOD) and horseradish peroxidase (HRP), was developed for the cathodic detection of glucose. The glucose biosensor was constructed by electrochemical formation of a polypyrrole (PPy) membrane in the presence of GOD on the surface of a HRP-modified sol-gel derived-mediated ceramic carbon electrode. Ferrocenecarboxylic acid (FCA) was used as mediator to transfer electron between enzyme and electrode. In the hetero-bilayer configuration of electrode, all enzymes were well immobilized in electrode matrices and showed favorable enzymatic activities. The amperometric detection of glucose was carried out at +0.16 V (versus saturated calomel reference electrode (SCE)) in 0.1 M phosphate buffer solution (pH 6.9) with a linear response range between 8.0×10⁻⁵ and 1.3×10⁻³ M glucose. The biosensor showed a good suppression of interference in the amperometric detection.     

A novel amperometric biosensor for the detection of nitrophenol

We report on a novel amperometric biosensor for detecting phenolic compounds based on the co-immobilization of horseradish-peroxidase (HRP) and methylene blue (MB) with chitosan on Au-modified TiO₂ nanotube arrays. The titania nanotube arrays were directly grown on a Ti substrate using anodic oxidation first; a gold thin film was then coated onto the TiO₂ nanotubes by an argon plasma technique. The morphology and composition of the fabricated Au-modified TiO₂ nanotube arrays were characterized by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). Cyclic voltammetry and amperometry were used to study the proposed electrochemical biosensor. The effect of pH, applied electrode potential and the concentration of H₂O₂ on the sensitivity of the biosensor have been systemically investigated. The performance of the proposed biosensor was tested using seven different phenolic compounds, showing very high sensitivity; in particular, the linearity of the biosensor for the detection of 3-nitrophenol was observed from 3 × 10⁻⁷ to 1.2 × 10⁻⁴ M with a detection limit of 9 × 10⁻⁸ M (based on the S/N = 3).     

Mesoporous silica hollow sphere (MSHS) for the bioelectrochemistry of horseradish peroxidase

In this work, novel mesoporous silica hollow spheres (MSHS) were chosen as an immobilization matrix, to construct a mediator-free third-generation HRP biosensor. UV-vis spectroscopy revealed that horseradish peroxidase (HRP) entrapped in MSHS could retain its native structure. FTIR spectroscopy and nitrogen adsorption-desorption isotherms indicated that HRP are intercalated into the mesopores. The direct electron transfer of HRP entrapped in MSHS was observed. A pair of stable and well-defined redox peaks of HRP with a formal potential of about −0.150 V (vs. Ag/AgCl) in 0.1 M pH 7.0 phosphate-buffered solution (PBS) were obtained. The biosensor exhibited a fast amperometric response to H₂O₂ with a linear range of 3.9 × 10⁻⁶ to 1.4 × 10⁻⁴ M (R = 0.997, N = 20). The detection limit was 1.2 × 10⁻⁶ M based S/N = 3.     

Inhibition biosensor for determination of nicotine

An amperometric nicotine inhibition biosensor has been substantially simplified and used for determination of nicotine in tobacco sample. Besides the use of single enzyme choline oxidase to replace bienzyme, the use of 1,4-benzoquinone as an electron mediator makes it possible to avoid the use of oxygen or hydrogen peroxide sensor as the internal transducer. Choline oxidase was immobilized on the carbon paste electrode through cross-linking with bovine serum albumin (BSA) by glutaraldehyde. In the presence of choline oxidase and its endogenous cofactor flavin-ademine dinneleotide (FAD), choline was oxidized into betaine while FAD was reduced to FADH₂ which subsequently reduced 1,4-benzoquinone into hydroquinone. The later was finally oxidized at a relatively low potential of +450 mV versus saturated calomel electrode (SCE). Nicotine inhibits the activity of enzyme with an effect of decreasing of oxidation current. The experimental conditions were optimized. The electrode has a linear response to choline within 1.25×10⁻⁴ to 1.25×10⁻³ mol l⁻¹. The nicotine measurements were carried out in 0.067 mol l⁻¹phosphate buffer of pH 7.4 at an applied potential of 450 mV versus SCE. The electrode provided a linear response to nicotine over a concentration range of 2.0×10⁻⁵ to 9.2×10⁻⁴ mol l⁻¹ with a detection limit of 1.0×10⁻⁵ mol l⁻¹. The system was applied to the determination of nicotine in tobacco samples.     

An intimately bonded titanate nanotube–polyaniline–gold nanoparticle ternary composite as a scaffold for electrochemical enzyme biosensors

In this work, titanate nanotubes (TNTs), polyaniline (PANI) and gold nanoparticles (GNPs) were assembled to form a ternary composite, which was then applied on an electrode as a scaffold of an electrochemical enzyme biosensor. The scaffold was constructed by oxidatively polymerising aniline to produce an emeraldine salt of PANI on TNTs, followed by gold nanoparticle deposition. A novel aspect of this scaffold lies in the use of the emeraldine salt of PANI as a molecular wire between TNTs and GNPs. Using horseradish peroxidase (HRP) as a model enzyme, voltammetric results demonstrated that direct electron transfer of HRP was achieved at both TNT-PANI and TNT-PANI-GNP-modified electrodes. More significantly, the catalytic reduction current of H₂O₂ by HRP was ∼75% enhanced at the TNT-PANI-GNP-modified electrode, compared to that at the TNT-PANI-modified electrode. The heterogeneous electron transfer rate constant of HRP was found to be ∼3 times larger at the TNT-PANI-GNP-modified electrode than that at the TNT-PANI-modified electrode. Based on chronoamperometric detection of H₂O₂, a linear range from 1 to 1200 μM, a sensitivity of 22.7 μA mM⁻¹ and a detection limit of 0.13 μM were obtained at the TNT-PANI-GNP-modified electrode. The performance of the biosensor can be ascribed to the superior synergistic properties of the ternary composite.     

A sensitive DNA biosensor based on a facile sulfamide coupling reaction for capture probe immobilization

A novel DNA biosensor was fabricated through a facile sulfamide coupling reaction. First, the versatile sulfonic dye molecule of 1-amino-2-naphthol-4-sulfonate (AN-SO₃⁻) was electrodeposited on the surface of a glassy carbon electrode (GCE) to form a steady and ordered AN-SO₃⁻ layer. Then the amino-terminated capture probe was covalently grafted to the surface of SO₃⁻-AN deposited GCE through the sulfamide coupling reaction between the amino groups in the probe DNA and the sulfonic groups in the AN-SO₃⁻. The step-by-step modification process was characterized by electrochemistry and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Using Ru(NH₃)₆³⁺ as probe, the probe density and the hybridization efficiency of the biosensor were determined to be 3.18 × 10¹³ strands cm⁻² and 86.5%, respectively. The hybridization performance of the biosensor was examined by differential pulse voltammetry using Co(phen)₃^3+/2+ (phen = 1,10-phenanthroline) as the indicator. The selectivity experiments showed that the biosensor presented distinguishable response after hybridization with the three-base mismatched, non-complementary and complementary sequences. Under the optimal conditions, the oxidation peak currents of Co(phen)₃^3+/2+ increased linearly with the logarithm values of the concentration of the complementary sequences in the range from 1.0 × 10⁻¹³ M to 1.0 × 10⁻⁸ M with a regression coefficient of 0.9961. The detection limit was estimated to be 7.2 × 10⁻¹⁴ M based on 3σ.     

A sensitive mediator-free tyrosinase biosensor based on an inorganic-organic hybrid titania sol-gel matrix

A novel inorganic-organic hybrid titania sol-gel nanocomposite film was prepared to fabricate a sensitive tyrosinase biosensor for the amperometric detection of trace phenolic compounds without additional electron mediators. Acetylacetone worked as a complexing ligand to chelate with Ti atom in the synthesis process, and the pH of the titania solution could be adjusted to the value which was optimum for retaining tyrosinase activity and such a membrane was stably attached on to the surface of a glassy carbon electrode (GCE). This titania matrix could supply a good environment for enzyme loading, which resulted in a high sensitivity of 15.78 μA μM⁻¹ cm⁻² for monitoring phenols with a detection limit of 1×10⁻⁸ M at a signal-to-noise ratio of 3. The TiO₂ sol-gel derived biosensor exhibited a fast response less than 10 s and a good stability for more than 2 months.     

Hyphenation of ionic liquid albumin glassy carbon biosensor or protein label-free sensor with differential pulse stripping voltammetry for interaction studies of human serum albumin with fenoprofen enantiomers

A new biosensor or protein label-free sensor composed of 1-butyl-3-methylimidazolium hexafluorophosphates (BMIMPF₆)-human serum albumin (HSA) film on glassy carbon electrode (GCE) was produced. Unfortunately, the native proteins themselves are often unstable in physiological conditions. Here, we introduced conjugation with ionic liquid (IL) such as BMIMPF₆ which improved the stability and binding affinity of protein onto GCE. A rapid, simple and reliable method for the chiral discrimination and real time protein binding studies of fenoprofen enantiomers with HSA was developed by hyphenating ionic liquid albumin glassy carbon (ILAGC) biosensor with differential pulse cathodic stripping voltammetry under physiological conditions. The electrochemical behavior of chiral fenoprofen was monitored by cyclic voltammetry, from which large response was obtained from l-fenoprofen. The surface coverage of fenoprofen enantiomers was calculated by double potential-step chronocoulometry. The binding constants of chiral fenoprofen with HSA were estimated to be 3.2 × 10⁵ ± 0.3 L mol⁻¹ and 0.8 × 10⁴ ± 0.4 L mol⁻¹ for l- and d-fenoprofen, respectively giving acceptable precision (SD ≤ 0.4) and good agreement with the literature values. The competitive interactions of ibuprofen with fenoprofen enantiomers–HSA were studied giving a significant decreasing in the binding degrees of analytes to HSA. The reciprocal competitive experiments indicated that l-fenoprofen replaced d-fenoprofen from HSA. The proposed electrochemical biosensor holds great potential for chiral discrimination and real time binding studies of drugs with protein.