Amperometric determination of serum total cholesterol with nanoparticles of cholesterol esterase and cholesterol oxidase

We describe the preparation of glutaraldehyde cross-linked and functionalized cholesterol esterase nanoparticles (ChENPs) and cholesterol oxidase nanoparticles (ChOxNPs) aggregates and their co-immobilization onto Au electrode for improved amperometric determination of serum total cholesterol. Transmission electron microscope (TEM) images of ChENPs and ChOxNPs showed their spherical shape and average size of 35.40 and 56.97 nm, respectively. Scanning electron microscope (SEM) studies of Au electrode confirmed the co-immobilization of enzyme nanoparticles (ENPs). The biosensor exhibited optimal response at pH 5.5 and 40 °C within 5 s when polarized at +0.25 V versus Ag/AgCl. The working/linear range of the biosensor was 10–700 mg/dl for cholesterol. The sensor showed high sensitivity and measured total cholesterol as low as 0.1 mg/dl. The biosensor was evaluated and employed for total cholesterol determination in sera of apparently healthy and diseased persons. The analytical recovery of added cholesterol was 90%, whereas the within-batch and between-batch coefficients of variation (CVs) were less than 2% and less than 3%. There was a good correlation (r = 0.99) between serum cholesterol values as measured by the standard enzymic colorimetric method and the current method. The initial activity of ENPs/working electrode was reduced by 50% during its regular use (200 times) over a period of 60 days when stored dry at 4 °C.     

Sensitive estimation of total cholesterol in blood using Au nanowires based micro-fluidic platform

Determination of cholesterol level in blood is important in clinical applications. In this work, modified Au nanowires-electrochemical biosensor based on MEMS micro-fluidic platform is proposed for estimating total cholesterol in blood. This sensor consists of "aligned" Au nanowires as working electrode, platinum counter electrode deposited on the silicon platform and Ag/AgCl (3M KCl) reference electrode. The "aligned" Au nanowires are immobilized with cholesterol oxidase and cholesterol esterase using specific covalent chemistry. Further, Au nanowires promotes better electron transfer between the enzymes and electrodes, because of their large surface to volume ratio, small diffusion time, large electrical conductivity and their aligned nature. The modified Au nanowires showed a stable calibration line and a quasi-linear relationship between cholesterol level and current response in the range of 1-6 mM (in steps of 1 mM over the baseline blood serum). The sensitivity of the modified electrode was found to be about 69 nA/mM with good storage and interference stability.     

Mercaptoethylpyrazine promoted electrochemistry of redox protein and amperometric biosensing of uric acid

Electrochemistry of microperoxidase-11 (MPx-11) anchored on the mixed self-assembled monolayer (SAM) of 2-(2-mercaptoethylpyrazine) (PET) and 4,4'-dithiodibutyric acid (DTB) on gold (Au) electrode and the biosensing of uric acid (UA) is described. MPx-11 has been covalently anchored on the mixed SAM of PET and DTB on Au electrode. MPx-11 on the mixed self-assembly exhibits reversible redox response characteristic of a surface confined species. The heterocyclic ring of PET promotes the electron transfer between the electrode and the redox protein. The apparent standard rate constant kapps obtained for the redox reaction of MPx-11 on the mixed monolayer is approximately 2.15 times higher than that on the single monolayer of DTB modified electrode. MPx-11 efficiently mediates the electrocatalytic reduction of H2O2. MPx-11 electrode is highly sensitive to H2O2 and it shows linear response for a wide concentration range. The electrocatalytic activity of the MPx-11 electrode is combined with the enzymatic activity of uricase (UOx) to fabricate uric acid biosensor. The bienzyme assembly is highly sensitive towards UA and it could detect UA as low as 2 microM at the potential of -0.1 V. The biosensor shows linear response with a sensitivity of 3.4+/-0.08 nA cm(-2) microM(-1). Ascorbate (AA) and paracetamol (PA) do not significantly interfere in the amperometric sensing of UA.     

Purification and some properties of cholesterol oxidase from Schizophyllum commune with covalently bound flavin.

Cholesterol oxidase [EC 1.1.3.6] from Schizophyllum commune was purified by an affinity chromatography using 3-O-succinylcholesterol-ethylenediamine (3-cholesteryl-3-[2-aminoethylamido]propionate) Sepharose gels. The resulting preparation was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 53,000 by SDS-gel electrophoresis and 46,000 by sedimentation equilibrium. The enzyme contained 483 amino acid residues as calculated on the basis of the molecular weight of 53,000. The enzyme consumed 60 mumol of O2/min per mg of protein with 1.3 mM cholesterol at 37 degrees C. The enzyme showed the highest activity with cholesterol; 3 beta-hydroxysteroids, such as dehydroepiandrosterone, pregnenolone, and lanosterol, were also oxidized at slower rates. Ergosterol was not oxidized by the enzyme. The Km for cholesterol was 0.33 mM and the optimal pH was 5.0. The enzyme is a flavoprotein which shows a visible absorption spectrum having peaks at 353 nm and 455 nm in 0.1 M acetate buffer, pH 4.0. The spectrum was characterized by the hypsochromic shift of the second absorption peak of the bound flavin. The bound flavin was reduced on anaerobic addition of a model substrate, dehydroepiandrosterone. Neither acid not heat treatment released the flavin coenzyme from the enzyme protein. The flavin of the enzyme could be easily released from the enzyme protein in acid-soluble form as flavin peptides when the enzyme protein was digested with trypsin plus chymotrypsin. The mobilities of the aminoacyl flavin after hydrolysis of the flavin peptides on thin layer chromatography and high voltage electrophoresis differed from those of free FAD, FMN, and riboflavin. A pKa value of 5.1 was obtained from pH-dependent fluorescence quenching process of the aminoacyl flavin. AMP was detected by hydrolysis of the flavin peptides with nucleotide pyrophosphatase. The results indicate strongly that cholesterol oxidase from Schizophyllum commune contains FAD as the prothetic group, which is covalently linked to the enzyme protein. The properties of the bound FAD were comparable to those of N (1)-histidyl FAD.     

Electrochemical studies of glucose oxidase immobilized on glutathione coated gold nanoparticles

Glutathione (L-gamma-glutamyl-L-cysteinyl-L-glycine; GSH) forms a surface monolayer on gold nanoparticles by tethering via sulfur bonds (Au:GSH). In the present study, glucose oxidase (GOx; EC 1.1.3.4) was immobilized by covalent chemical coupling reactions on to Au:GSH nanoparticles and the enzyme coupled nanoparticles formed a stable colloid (stable for several weeks) in water. The immobilized enzyme was investigated for electrochemical characteristics to monitor the FAD (prosthetic group of the GOx) redox potentials. Various concentrations of substrate (glucose) were added to check the oxidation characteristics. It was observed that with increase in substrate concentrations, the oxidation rate increased proportionally with the current. The present study demonstrated that GOx was effectively coupled to the gold nanoparticle (Au:GSH). The coupled nanoparticle system could be used in a potential biosensor application. Similarly, other enzymes (e.g., horseradish peroxidase) could be immobilized to the Au:GSH nanoparticles via the peptide arm (GSH) to achieve the desired characteristics needed for a specific application in biosensor.     

A comparison of different strategies for the construction of amperometric enzyme biosensors using gold nanoparticle-modified electrodes

A comparison of the analytical performances of several enzyme biosensor designs, based on the use of different tailored gold nanoparticle-modified electrode surfaces, is discussed. Glucose oxidase (GOx) and the redox mediator tetrathiafulvalene were coimmobilized in all cases by crosslinking with glutaraldehyde. The biosensor designs tested were based on the use of (i) colloidal gold (Au(coll)) bound on cysteamine (Cyst) monolayers self-assembled on a gold disk electrode (AuE) and (ii) glassy carbon electrodes (GCEs) modified with electrodeposited gold nanoparticles (nAu). The results obtained with these designs were compared with those provided by a GOx/Cyst-AuE and a GOx/MPA-AuE. In the second case (ii), configurations based on direct immobilization of GOx on nAu (GOx/nAu-GCE) or on Cyst or MPA self-assembled monolayers (SAMs) previously bound on gold nanoparticles (GOx/Cyst-nAu-GCE or GOx/MPA-nAu-GCE, respectively) were compared. The analytical characteristics of glucose calibration plots and the kinetic parameters of the enzyme reaction were compared for all of the biosensors tested. The GOx/Au(coll)-Cyst-AuE design showed a sensitivity for glucose determination higher than that achieved with GOx/Cyst-AuE and GOx/Au(coll)-Cyst/Cyst-AuE and similar to that achieved with GOx/MPA-AuE. Moreover, the useful lifetime of one single GOx/Au(coll)-Cyst-AuE was 28 days, remarkably longer than that of the other GOx biosensor designs.     

Direct electrochemistry of hemoglobin immobilized on gold electrode by Langmuir-Blodgett technique

In this research, we reported a novel method of forming hemoglobin (Hb)-linoleic acid (LA) Langmuir-Blodgett (LB) monolayer by spreading Hb solution directly onto the subphase covered with a layer of LA. This method is suitable for preparing electrochemical devices with protein-lipid LB film because almost no protein adsorbed on electrode surface before protein-lipid film transferred from air-water interface to electrode, which ensured better electrode activity. The compressibility of Hb-LA monolayer was used to character the phase transition during compression process. Optimal experimental conditions were obtained by analyzing pressure-time, pressure-area and pressure-compressibility curves. The direct electrochemistry of Hb, which was immobilized on Au electrode surface incorporated with LA layer by LB method, was investigated using cyclic voltammetry for the first time. The electrode modified with Hb-LA LB film holds high electrochemical activity and shows a fast direct electron transfer of Hb. Redox peak currents increased linearly with the increase of scan rate, indicating a surface-controlled electrode process. The electron transfer rate constant was 2.68+/-0.45 s-1. As a target of this research, this work provides a new way to prepare biomimetic film and biosensor.     

Interfacial electron transfer of glucose oxidase on poly(glutamic acid)-modified glassy carbon electrode and glucose sensing

The interfacial electron transfer of glucose oxidase (GOx) on a poly(glutamic acid)-modified glassy carbon electrode (PGA/GCE) was investigated. The redox peaks measured for GOx and flavin adenine dinucleotide (FAD) are similar, and the anodic peak of GOx does not increase in the presence of glucose in a mediator-free solution. These indicate that the electroactivity of GOx is not the direct electron transfer (DET) between GOx and PGA/GCE and that the observed electroactivity of GOx is ascribed to free FAD that is released from GOx. However, efficient electron transfer occurred if an appropriate mediator was placed in solution, suggesting that GOx is active. The PGA/GCE-based biosensor showed wide linear response in the range of 0.5–5.5 mM with a low detection limit of 0.12 mM and high sensitivity and selectivity for measuring glucose.     

Fabrication of polymeric electron-transfer mediator/enzyme hydrogel multilayer on an Au electrode in a layer-by-layer process

The layer-by-layer (LBL) construction of an enzyme electrode covered with a multilayer structure alternately composed of a polymeric electron transfer mediator and a polymer-modified enzyme was examined. Poly(2-methacryloyloxyethyl phosphorylcholine-co-p-vinylphenylboronic acid-co-vinylferrocene) (PMVF) was synthesized and used as a polymeric electron transfer mediator. Glucose oxidase (GOx) was selected as a model enzyme and poly(vinyl alcohol) (PVA) chains were bound to the GOx (GOx-PVA) under mild conditions. The PMVF and PVA formed a gel spontaneously through a selective reaction between phenylboronic acid units and hydroxyl groups in both polymers. Using the spin coating technique, a repeating PMVF/GOx-PVA multilayer was fabricated on the surface of an Au electrode. The thickness of each PMVF/GOx-PVA layer was around 5.8 nm, corresponding to the dimensions of GOx. The electrochemical performance of the electrode was evaluated in glucose concentration measurement. The oxidation current of glucose by GOx was measured at 0.38 V (vs. Ag/AgCl), verifying that ferrocene units in the PMVF of the hydrogel electrically wired the immobilized GOx. Moreover, the current increased with the number of PMVF/GOx-PVA layers. That is, both intermolecular electron transfer between each individual layer and the presence of a freely diffusing substrate in the hydrogel were achieved. We conclude that a LBL structure constructed from PMVF and a PVA-modified enzyme is effective for use in developing bioelectronic devices that employ enzyme molecules.     

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.     

Integrated nanoparticle-biomolecule systems for biosensing and bioelectronics

The similar dimensions of biomolecules such as enzymes, antibodies or DNA, and metallic or semiconductor nanoparticles (NPs) enable the synthesis of biomolecule-NP hybrid systems where the unique electronic, photonic and catalytic properties of NPs are combined with the specific recognition and biocatalytic properties of biomolecules. The unique functions of biomolecule-NP hybrid systems are discussed with several examples: (i) the electrical contacting of redox enzymes with electrodes is the basis for the development of enzymatic electrodes for amperometric biosensors or biofuel cell elements. The reconstitution of the apo-glucose oxidase or apo-glucose dehydrogenase on flavin adenine dinucleotide (FAD)-functionalized Au NPs (1.4 nm) associated with electrodes, or on pyrroloquinoline quinone (PQQ)-functionalized Au NPs (1.4 nm) associated with electrodes, respectively, yields electrically contacted enzyme electrodes. The aligned, reconstituted enzymes on the electrode surfaces reveal effective electrical contacting, and the glucose oxidase and glucose dehydrogenase reveal turnover rates of 5000 and 11,800 s(-1), respectively. (ii) The photoexcitation of semiconductor nanoparticles yields fluorescence with a wavelength controlled by the size of the NPs. The fluorescence functions of semiconductor NPs are used to develop a fluorescence resonance energy transfer (FRET) assay for nucleic acids, and specifically, for analyzing telomerase activity in cancer cells. CdSe-ZnS NPs are functionalized by a primer recognized by telomerase, and this is elongated by telomerase extracted from HeLa cancer cells in the presence of dNTPs and Texas-red-functionalized dUTP. The dye integrated into the telomers allows the FRET process that is intensified as telomerization proceeds. Also, the photoexcited electron-hole pair generated in semiconductor NPs is used to generate photocurrents in a CdS-DNA hybrid system associated with an electrode. A redox-active intercalator, methylene blue, was incorporated into a CdS-duplex DNA monolayer associated with a Au electrode, and this facilitated the electron transfer between the electrode and the CdS NPs. The direction of the photocurrent was controlled by the oxidation state of the intercalator. (iii) Biocatalysts grow metallic NPs, and the absorbance of the NPs provides a means to assay the biocatalytic transformations. This is exemplified with the glucose oxidase-induced growth of Au NPs and with the tyrosinase-stimulated growth of Au NPs, in the presence of glucose or tyrosine, respectively. The biocatalytic growth of the metallic NPs is used to grow nanowires on surfaces. Glucose oxidase or alkaline phosphatase functionalized with Au NPs (1.4 nm) acted as 'biocatalytic inks' for the synthesis of metallic nanowires. The deposition of the Au NP-modified glucose oxidase, or the Au NP-modified alkaline phosphatase on Si surfaces by dip-pen nanolithography led to biocatalytic templates, that after interaction with glucose/AuCl4- or p-aminophenolphosphate/Ag+, allowed the synthesis of Au nanowires or Ag nanowires, respectively.     

Study of carbon nanotube modified biosensor for monitoring total cholesterol in blood

A carbon nanotube modified biosensor for monitoring total cholesterol in blood was studied. This sensor consists of a carbon working electrode and a reference electrode screen-printed on a polycarbonate substrate. Cholesterol esterase, cholesterol oxidase, peroxidase and potassium ferrocyanide were immobilized on the screen-printed carbon electrodes. Multi-walled carbon nanotubes (MWCN) were added to prompt electron transfer. Experimental results show that the carbon nanotube modified biosensor offers a reliable calibration profile and stable electrochemical properties.     

Wiring of PQQ-dehydrogenases

The performance of pyrroloquinoline quinone (PQQ) dependent alcohol dehydrogenase (ADH) and two types of PQQ-glucose dehydrogenases in solution and when immobilized on the carbon paste electrodes modified with ferrocene derivatives is investigated. The immobilization of ADH consisting of PQQ and four hemes improves its stability up to 10 times. Both PQQ and heme moieties are involved in the electron transport from substrate to electrode. The ferrocene derivatives improve the electron transport 10-fold. Membrane-bound alcohol dehydrogenase from Gluconobacter sp. 33, intracellular soluble glucose dehydrogenase from Acinetobacter calcoaceticus L.M.D. 79.41 (s-GDH), and the membrane-bound enzyme (m-GDH) from Erwinia sp. 34-1 were purified and investigated. Soluble and membrane-bound PQQ-glucose dehydrogenases display different behavior during the immobilization on the modified carbon electrodes. The immobilization of s-GDH leads to a decrease in both stability and substrate specificity of the enzyme. This suggests that PQQ dissociates from the enzyme active center and operates as a free-diffusing mediator. The rate-limiting step of the process is likely the loading of PQQ onto the apo-enzyme. The immobilization of m-GDH leads to its substantial stabilization and improves the substrate specificity. The nature of m-GDH binding to the electrode surface is presumably similar to the binding to the cell membrane through its anchor-subunit. The enzyme operates as an enzyme and mediator complex.     

Immobilization of rat brain acetylcholinesterase on ZnS and poly(indole-5-carboxylic acid) modified Au electrode for detection of organophosphorus insecticides

A novel, highly sensitive amperometric biosensor for detection of organophosphorus (OP) compounds has been constructed, based on rat brain acetylcholinesterase (AChE) immobilized onto nanocomposite of ZnS-nanoparticles (ZnSNPs) and poly(indole-5-carboxylic acid) electrodeposited on Au electrode. In the presence of acetylthiocholine chloride (ATCl) as a substrate, ZnSNPs promoted electron transfer reactions at a lower potential and catalyzed electrochemical oxidation of enzymatically formed thiocholine, thus increasing detection sensitivity. Under optimum conditions (phosphate buffer, pH 7.5 and 30°C), the inhibition of AChE by malathion and chlorpyrifos was proportional to their concentrations in the range, 0.1-50nM and 1.5-40nM, respectively. The biosensor determined malathion and chlorpyrifos in spiked tap water samples with a acceptable accuracy (95-100%). The enzyme electrode had long-storage stability (50% retention of initial activity within 2 months, when stored at 4°C).     

Reconstitution of apoglucose oxidase with FAD conjugates for biosensoring of progesterone

The reconstitution of Aspergillus niger apoglucose oxidase (apoGOx) with FAD conjugates for biosensoring of progesterone was investigated. ApoGOx prepared by partial unfolding of the protein under acidic conditions consisted of reconstitutable monomers (50+/-10%), reconstitutable dimers (20+/-10%) and irreversibly aggregated oligomers (30+/-20%). Incubation of monomeric apoGOx with FAD or N(6)-(6-aminohexyl)-FAD (ahFAD) restored glucose oxidase (GOx) activity and induced dimerization with stoichiometric incorporation of FAD. N(6)-(6-aminohexyl)-FAD progesterone conjugates also induced dimerization. However, holoenzyme reconstitution required relatively high concentrations of apoprotein and was dependent on the type of conjugate. Restoration to 25-50% of the original enzyme activity was obtained. Binding of the FAD-progesterone conjugates might hinder the closure of a protein lid needed for dimer formation. Our results illustrate the prospects of FAD conjugates in sensitive detection of progesterone in biological matrices in a biosensor based on the recombination of apoGOx with progesterone-conjugated FAD.     

Determination of glucose oxidase immobilised as monolayer onto a flat surface

An assay to estimate the amount of glucose oxidase immobilised as a monolayer onto a flat surface is reported. This method is based on the electrochemical detection of the flavin adenine dinucleotide (FAD) cofactor released by the immobilised enzyme in acid solutions. FAD concentration in the acid solution was measured by amperometry, using a flow injection analysis (FIA) system equipped with a wall-jet electrode, and with a sensitivity of (9.2+/-2.0)x10(-2) nA/nM. By this method, the amount of glucose oxidase molecules present in a monolayer deposited on a silanised glass slide was easily detected, in which the detection limit is more than one order of magnitude lower than the maximum loading of the surface with an ordered monolayer of glucose oxidase.     

Uric acid determination in the presence of ascorbic acid using self-assembled submonolayer of dimercaptothiadiazole-modified gold electrodes

This article reports the determination of uric acid (UA) in the presence of ascorbic acid (AA) using a self-assembled submonolayer of heteroaromatic dithiol, 2,5-dimercapto-1,3,4-thiadiazole (DMcT), on gold (Au) electrode. Submonolayer to multilayers of DMcT can be prepared on Au electrode by varying the soaking time of Au electrode in 1mM aqueous solution of DMcT. The formation of submonolayer, monolayer, and multilayers of DMcT on Au electrode was confirmed from its reductive desorption measurements and electrochemical blocking behavior toward ferricyanide. Interestingly, submonolayer of DMcT separates the voltammetric signal of UA from AA by 210 mV, whereas monolayer and multilayers of DMcT fail to separate them. The voltammetric signals of AA and UA are highly stable and reproducible at submonolayer of DMcT. Fast electron transfer, weak hydrogen bonding interactions with AA and UA, and prevention of fouling effect caused by oxidized product of AA can be achieved at submonolayer of DMcT, and thus it successfully separates the voltammetric signals of AA and UA. The practical application of the current system is demonstrated by measuring the concentration of UA in human urine samples without any treatment.     

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.     

Polyaniline protected gold nanoparticles based mediator and label free electrochemical cortisol biosensor

Polyaniline protected gold nanoparticles (PPAuNPs) were electrophoretically deposited onto a gold electrode, and utilized to fabricate an electrochemical cortisol biosensor. Cortisol specific monoclonal antibody (C-Mab) was covalently immobilized onto the surface of a PPAuNP/Au electrode using N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) chemistry. BSA was employed for blocking nonspecific adsorption on the electrode surface. PPAuNP formation and BSA/C-Mab/PPAuNP/Au electrode fabrication were characterized using transmission electron microscopy, atomic force microscopy and electrochemical impedance techniques, respectively. Cyclic voltammetry and differential pulse voltammetric techniques were used to determine the cortisol concentration in a phosphate buffer saline (PBS) solution. Results confirmed that the PPAuNP based electrode was stable during repeated scans and exhibited repeatable redox peaks. Further, the BSA/C-Mab/PPAuNP/Au electrode in the PBS buffer accurately detected cortisol in the range of 1 pM-100 nM with a sensitivity of 1.63 μAM(-1). The biosensor was found to be selective against BSA and 17-α-hydroxy progesterone. This research establishes the feasibility of using a PPAuNP based matrix for a label and mediator free electrochemical biosensor for cortisol, a stress biomarker.     

Electrodeposition of gold-platinum alloy nanoparticles on ionic liquid-chitosan composite film and its application in fabricating an amperometric cholesterol biosensor

An electrodeposition method was applied to form gold-platinum (AuPt) alloy nanoparticles on the glassy carbon electrode (GCE) modified with a mixture of an ionic liquid (IL) and chitosan (Ch) (AuPt-Ch-IL/GCE). AuPt nanoparticles were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM) and electrochemical methods. AuPt-Ch-IL/GCE electrocatalyzed the reduction of H(2)O(2) and thus was suitable for the preparation of biosensors. Cholesterol oxidase (ChOx) was then, immobilized on the surface of the electrode by cross-linking ChOx and chitosan through addition of glutaraldehyde (ChOx/AuPt-Ch-IL/GCE). The fabricated biosensor exhibited two wide linear ranges of responses to cholesterol in the concentration ranges of 0.05-6.2 mM and 6.2-11.2 mM. The sensitivity of the biosensor was 90.7 μA mM(-1) cm(-2) and the limit of detection was 10 μM of cholesterol. The response time was less than 7 s. The Michaelis-Menten constant (K(m)) was found as 0.24 mM. The effect of the addition of 1 mM ascorbic acid and glucose was tested on the amperometric response of 0.5 mM cholesterol and no change in response current of cholesterol was observed.