Polyaniline/carbon nanotubes platform for sexually transmitted disease detection

Polyaniline/carbon nanotubes composite (PANI‐CNT) electrochemically deposited onto indium‐tin‐oxide (ITO) coated glass plate has been utilized for Neisseria gonorrhoeae detection by immobilizing 5′‐amino‐labeled Neisseria gonorrhoeae probe (aDNA) using glutaraldehyde as a cross‐linker. PANI‐CNT/ITO and aDNA‐Glu‐PANI‐CNT/ITO electrodes have been characterized using scanning electron microscopy (SEM), Fourier Transform Infrared (FT‐IR) spectroscopy, cyclic voltammetry (CV), and differential pulse voltammetry (DPV). This bioelectrode can be used to detect N. gonorrhoeae using methylene blue (MB) as redox indicator with response time of 60 s and stability of about 75 days when stored under refrigerated conditions. DPV studies reveal that this bioelectrode can detect complementary DNA concentration from 1 × 10^?6 M to 1 × 10^?17 M with detection limit of 1.2 × 10^?17 M. Further, this bioelectrode (aDNA‐Glu‐PANI‐CNT/ITO) exhibits specificity toward N. gonorrhoeae species and shows negative response with non‐Neisseria gonorrhoeae Neisseria species (NgNS) and other gram negative bacteria (GNB). Copyright © 2010 John Wiley & Sons, Ltd.     

Nucleic acid sensor for insecticide detection

Nucleic acid sensor based on polyaniline (PANI) has been fabricated by covalently immobilizing double stranded calf thymus (dsCT) DNA onto perchlorate (ClO(-) (4))-doped PANI film deposited onto indium-tin-oxide (ITO) glass plate using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) chemistry. These dsCT-DNA-PANI-ClO(4)/ITO and PANI-ClO(4)/ITO electrodes have been characterized using square wave voltammetry, electrochemical impedance, scanning electron microscopy (SEM) and Fourier-transform-infrared (FTIR) measurements. This disposable dsCT-DNA-PANI-ClO(4)/ITO bioelectrode, stable for about 4 months, can be used to detect cypermethrin (0.005 ppm) and trichlorfon (0.01 ppm) in 30 and 60 s, respectively.     

Iron oxide nanoparticles-chitosan composite based glucose biosensor

Iron oxide (Fe(3)O(4)) nanoparticles prepared using co-precipitation method have been dispersed in chitosan (CH) solution to fabricate nanocomposite film on indium-tin oxide (ITO) glass plate. Glucose oxidase (GOx) has been immobilized onto this CH-Fe(3)O(4) nanocomposite film via physical adsorption. The size of the Fe(3)O(4) nanoparticles estimated using X-ray diffraction (XRD) pattern and transmission electron microscopy (TEM) has been found to be approximately 22 nm. The CH-Fe(3)O(4) nanocomposite film and GOx/CH-Fe(3)O(4)/ITO bioelectrode have been characterized using UV-visible and Fourier transform infrared (FTIR) spectroscopic and scanning electron microscopy (SEM) techniques, respectively. This GOx/CH-Fe(3)O(4)/ITO nanocomposite bioelectrode has response time of 5s, linearity as 10-400 mgdL(-1) of glucose, sensitivity as 9.3 microA/(mgdLcm(2)) and shelf life of about 8 weeks under refrigerated conditions. The value of Michaelis-Menten (K(m)) constant obtained as 0.141 mM indicates high affinity of immobilized GOx towards the substrate (glucose).     

Chitosan encapsulated quantum dots platform for leukemia detection

We report results of the studies relating to electrophoretic deposition of nanostructured composite of chitosan (CS)-cadmium-telluride quantum dots (CdTe-QDs) onto indium-tin-oxide coated glass substrate. The high resolution transmission electron microscopic studies of the nanocomposite reveal molecular level coating of the CdTe-QDs with CS molecules in the colloidal dispersion medium. This novel composite platform has been explored to fabricate an electrochemical DNA biosensor for detection of chronic myelogenous leukemia (CML) by immobilizing amine terminated oligonucleotide probe sequence containing 22 base pairs, identified from BCR-ABL fusion gene. The results of differential pulse voltammetry reveal that this nucleic acid sensor can detect as low as 2.56 pM concentration of complementary target DNA with a response time of 60s. Further, the response characteristics show that this fabricated bioelectrode has a shelf life of about 6 weeks and can be used for about 5-6 times. The results of experiments conducted using clinical patient samples reveal that this sensor can be used to distinguish CML positive and the negative control samples.     

Covalent immobilization of cholesterol oxidase on self-assembled gold nanoparticles for highly sensitive amperometric detection of cholesterol in real samples

A novel scheme for the fabrication of gold nanoparticle modified cholesterol oxidase based bioelectrode is presented and its application potential for cholesterol biosensor is investigated. The fabrication procedure is based on the deposition of gold nanoparticles on the 1,6-hexanedithiol modified gold electrode, functionalization of the surface of deposited gold nanoparticles with carboxyl groups using 11-mercaptoundecanoic acid and then covalent immobilization of cholesterol oxidase on the surface of gold nanoparticle film using the N-ethyl-N'-(3-dimethylaminopropyl carbodimide) and N-hydroxysuccinimide ligand chemistry. The assembly process of the bioelectrode is investigated using atomic force microscopy, cyclic voltammetry and electrochemical impedance spectroscopy. The gold nanoparticle film on the electrode surface provided an environment for the enhanced electrocatalytic activities and thus resulted in enhanced analytical response. The resulting bioelectrode is further applied to the amperometric detection of cholesterol and exhibited a linear response to cholesterol in the range of 0.04-0.22 mM with a detection limit of 34.6 μM, apparent Michaelis-Menten constant (K(m)(app)) of 0.062 mM and a high sensitivity of 9.02 μA mM(-1). The fabricated bioelectrode is successfully used for the selective determination of cholesterol in human serum samples.     

Molecularly imprinted polyaniline film for ascorbic acid detection

Molecularly imprinted polyaniline (PANI) film (～ 100 nm thick) has been electrochemically fabricated onto indium-tin-oxide (ITO) coated glass plate using ascorbic acid (AA) as template molecule. Fourier transform infra-red spectroscopy, scanning electron microscopy, cyclic voltammetry and differential pulse voltammetry (DPV) studies indicate the presence of AA in PANI matrix, which also acts as a dopant for PANI. Further, the AA selective molecularly imprinted PANI electrode (AA-MI-PANI/ITO) has been developed via over-oxidation of AA doped PANI electrode which leads to the removal of AA moieties from PANI film. The response studies using DPV technique have revealed that this molecularly imprinted AA-MI-PANI/ITO electrode can detect AA in the range of 0.05-0.4 mM with detection limit of 0.018 mM and sensitivity of 1.2 × 10(-5) AmM(-1). Interestingly, this AA-MI-PANI/ITO electrode shows excellent reusability, selectivity and stability.     

Polyaniline-carbon nanotube composite film for cholesterol biosensor

Nanocomposite film composed of polyaniline (PANI) and multiwalled carbon nanotubes (MWCNT), prepared electrophoretically onto indium tin oxide (ITO)-coated glass plate, was used for covalent immobilization of cholesterol oxidase (ChOx) via N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) chemistry. Results of linear sweep voltammetric measurements reveal that ChOx/PANI-MWCNT/ITO bioelectrode can detect cholesterol in the range of 1.29 to 12.93 mM with high sensitivity of 6800 nA mM⁻¹ and a fast response time of 10 s. Photometric studies for ChOx/PANI-MWCNT/ITO bioelectrode indicate that it is thermally stable up to 45 °C and has a shelf life of approximately 12 weeks when stored at 4 °C. The results of these studies have implications for the application of this interesting matrix (PANI-MWCNT) toward the development of other biosensors.     

A novel nanobiocomposite based glucose biosensor using neutral red functionalized carbon nanotubes

The performance of a new glucose biosensor based on the combination of biocatalytic activity of glucose oxidase (GOx) with the electrocatalytic properties of CNTs and neutral red (NR) for the determination of glucose is described. This sensor is comprised of a multiwalled carbon nanotubes (MWNTs) conduit functionalized with NR and Nafion (Nf) as a binder and glucose oxidase as a biocatalyst. Neutral red was covalently immobilized on carboxylic acid groups of the CNTs via carbodiimide reaction. The functionalized MWNTs were characterized by microscopic, spectroscopic and thermal methods. The MWNT-NR-GOx-Nf nanobiocomposite was prepared by mixing the GOx solution with NR functionalized CNTs followed by mixing homogeneously with Nafion. The performance of the MWNT-NR-GOx-Nf nanobiocomposite modified electrode was examined by electrochemical impedance spectroscopy and cyclic voltammetry. The catalytic reduction of hydrogen peroxide liberated from the enzymatic reaction of glucose oxidase upon glucose with NR functionalized CNTs leads to the selective detection of glucose. The excellent electrocatalytic activity and the influence of nanobiocomposite film result in good characteristics such as low potential detection of glucose with a large determination range from 1 x 10(-8) to 1 x 10(-3)M with a detection limit of 3 x 10(-9)M glucose, a short response time (with 4s), good stability and anti-interferent ability. The improved electrocatalytic activity and stability made the MWNT-NR-GOx-Nf nanobiocomposite biosensor system a potential platform to immobilize different enzymes for other bioelectrochemical applications.     

Electrodeposition of polypyrrole-multiwalled carbon nanotube-glucose oxidase nanobiocomposite film for the detection of glucose

A nanobiocomposite film consisted of polypyrrole (PPy), functionalized multiwalled carbon nanotubes (cMWNTs), and glucose oxidase (GOx) were electrochemically synthesized by electrooxidation of 0.1M pyrrole in aqueous solution containing appropriate amounts of cMWNTs and GOx. Potentiostatic growth profiles indicate that the anionic cMWNTs is incorporated within the growing PPy-cMWNTs nanocomposite for maintaining its electrical neutrality. The morphology of the PPy-cMWNTs nanocomposite was characterized by scanning electron microscopy (SEM). The PPy-cMWNTs nanocomposite was deposited homogeneously onto glassy carbon electrode. The amperometric responses vary proportionately to the concentration of hydrogen peroxide at the PPy-cMWNTs nanocomposite modified electrode at an operating potential of 0.7V versus Ag/AgCl (3M). The results indicate that the electroanalytical PPy-cMWNTs-GOx nanobiocomposite film was highly sensitive and suitable for glucose biosensor based on GOx function. The GOx concentration within the PPy-cMWNTs-GOx nanobiocomposite and the film thickness are crucial for the performance of the glucose biosensor. The amperometric responses of the optimized PPy-cMWNTs-GOx glucose biosensor (1.5 mgmL(-1) GOx, 141 mCcm(-2) total charge) displayed a sensitivity of 95 nAmM(-1), a linear range up to 4mM, and a response time of about 8s.     

Polyaniline Langmuir-Blodgett film based aptasensor for ochratoxin A detection

Ochratoxin A (OTA) produced by Aspergillus Ochraceus and Penicillium verrucosum is a very dangerous toxin due to its toxic effects in human beings and its presence in a wide range of food products and cereals. A Langmuir-Blodgett (polyaniline (PANI)-stearic acid (SA)) film based highly sensitive and robust impedimetric aptasensor has been developed for ochratoxin A (OTA) detection. DNA Aptamer (Apt-DNA) specific to OTA has been covalently immobilized onto mixed Langmuir-Blodgett (LB) monolayer comprising of PANI-SA deposited onto indium tin-oxide (ITO) coated glass plates. This Apt-DNA/PANI-SA/ITO aptaelectrode has been characterized using scanning electron microscopy, Fourier transform-infrared spectroscopy, contact angle measurements, cyclic voltammetry and electrochemical impedance spectroscopy, respectively. The Apt-DNA/PANI-SA/ITO aptasensor shows detection of OTA by electrochemical impedance spectroscopy in the linear range of 0.0001 μg/ml (0.1 ng/ml) to 0.01 μg/ml (10 ng/ml) and 1 μg/ml-25 μg/ml with detection limit of 0.1 ng/ml in 15 min. The Apt-DNA/PANI-SA/ITO aptasensor can be reused ～13 times. The binding or affinity constant (K(a)) of aptamer with OTA, calculated using Langmuir adsorption isotherm, is found be 1.21×10(7) M(-1).     

Enzymatic glucose biosensor based on CeO2 nanorods synthesized by non-isothermal precipitation

Cerium oxide nanorods (CeO(2) NRs) were synthesized without templates through a low cost and simple non-isothermal precipitation method. The structure and morphology of CeO(2) NRs were characterized by X-ray diffraction and transmission electron microscopy. The CeO(2) NRs films, deposited on indium tin oxide (ITO)-coated glass substrates through electrophoretic deposition, were used for the immobilization of glucose oxidase (GOx). Field emission scanning electron microscopy, Fourier transform infrared spectroscopy, cyclic voltammetry, and electrochemical impedance spectroscopy were used to characterize the CeO(2) NRs/ITO and GOx/CeO(2) NRs/ITO electrodes. The GOx/CeO(2) NRs/ITO electrode exhibits a linear range for the detection of glucose from 2 to 26 mM (correlation coefficient: 0.99) at 1-2s response time. Biosensor sensitivity is 0.165 μA mM(-1) cm(-2) with 100 μM detection limit. The anti-interference ability of the biosensor was also examined. The mediator-less application of CeO(2) NRs for glucose sensing was demonstrated.     

A novel hydrogen peroxide biosensor based on the immobilization of hemoglobin on three-dimensionally ordered macroporous (3DOM) gold-nanoparticle-doped titanium dioxide (GTD) film

The three-dimensionally ordered macroporous gold-nanoparticle-doped titanium dioxide (3DOM GTD) film was modified on the indium-tin oxide (ITO) electrode surface. Hemoglobin (Hb) has been successfully immobilized on the 3DOM GTD film and the fabrication process was characterized by Raman and UV-vis spectra. The results indicated that the Hb immobilized on the film retained its biological activity and the secondary structure of Hb was not destroyed. The direct electrochemistry and electrocatalysis of Hb immobilized on this film have been investigated. The Hb/3DOM GTD/ITO electrode exhibited two couples of redox peaks corresponding to the Hb intercalated in the mesopores and adsorbed on the external surface of the film with the formal potential of -0.20 and -0.48 V in 0.1M PBS (pH7.0), respectively. The Hb/3DOM GTD/ITO electrode exhibits an excellent eletrocatalytic activity, a wide linear range for H(2)O(2) from 5.0 μM to 1.0mM with a limit of detection of 0.6μM, high sensitivity (144.5 μA mM(-1)), good stability and reproducibility. Compared with the TiO(2) nanoneedles modified electrode, the GTD modified electrode has higher sensitivity and response peak current. The 3DOM GTD provided a good matrix for bioactive molecules immobilization, suggesting it has the potential use in the fields of H(2)O(2) biosensors.     

Quaternary ammonium functionalized clay film electrodes modified with polyphenol oxidase for the sensitive detection of catechol

Naturally occurring Cameroonian smectite clay has been grafted with trimethylpropylammonium (TMPA) groups and the resulting organoclay has been deposited onto a glassy carbon electrode surface as a suitable immobilization matrix for polyphenol oxidase (PPO). High sensitivity of the electrochemical device to catechol biosensing can be achieved when the enzyme was impregnated within the organoclay film subsequent to its deposition due to favorable electrostatic interaction between PPO and the TMPA-clay layer. The bioelectrode preparation method was also compatible with the use of a mediator (i.e., ferrocene) and the best performance was obtained with a three-layer configuration made of glassy carbon coated with a first layer of ferrocene (Fc), which was then covered with the PPO-impregnated TMPA-clay layer, and finally overcoated with an enzyme-free TMPA-clay film acting as a protecting overlayer to avoid leaching of the biomolecule in solution. The electrochemical behavior of the modified film electrodes was first characterized by cyclic voltammetry and, then, they were evaluated for the amperometric biosensing of the model analyte catechol in batch conditions and in flow injection analysis. Various experimental parameters likely to influence the biosensor response have been investigated, including the electrode preparation mode (composition configuration, thickness), the usefulness of a mediator, the operating potential and pH of the medium, as well as the advantageous features of the TMPA-clay in comparison to related film electrodes based on non-functionalized clays. The organoclay was found to provide a favorable environment to enzyme activity and the multilayer configuration of the film electrode to provide a biosensor with good characteristics, such as an extended linear range for catechol detection (2 x 10(-8) to 1.2 x 10(-5)M) and a detection limit in the nanomolar range (9 x 10(-9)M).     

Electrochemically fabricated polypyrrole nanofiber-modified electrode as a new electrochemical DNA biosensor

A new biosensor employing immobilized DNA on a nano-structured conductive polymer fixed onto a platinum electrode is presented. Upon optimization of synthesis parameters, polypyrrole nanofibers, 30-90 nm in diameter, were synthesized in an aqueous media by the electropolymerization of pyrrole using normal pulse voltammetry (NPV). The nanofiber film was investigated by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Double-stranded DNA was physisorbed onto the PPy nanofiber films. Various parameters, including the pH and DNA concentration, were optimized. The DNA immobilized on the nanofiber films was characterized using differential pulse voltammetry (DPV) and Fourier-transform infrared (FTIR) spectroscopy. Using DPV to study the interaction of spermidine with DNA, a binding constant (K) value of 4.08 x 10(5)+/-0.05 M(-1) was obtained. For the determination of spermidine, the proposed method exhibited a good dynamic range, correlation coefficient (0.05-1.0 microM and 0.9983, respectively) and a low detection limit (0.02 microM), although Ca(2+) ions were found to electrostatically bind to DNA and weaken the spermidine-DNA interaction.     

Microstructured cystine dendrites-based impedimetric sensor for nucleic acid detection

We report results of the studies relating to the fabrication and characterization of novel biosensing electrode by covalent immobilization of DNA onto microstructural cystine (Cys) prepared by acoustic cavitation method. The TEM investigations of these structures reveal transformation of microstructured Cys from nanorods to dendritic structure under optimum conditions. The Cys dendrites (denCys) have been investigated by XRD, FT-IR, and SEM studies. These biosensing electrodes have been fabricated by immobilization of Escherichia coli (E. coli)-specific DNA probe onto the dendritic cystine. The results of the electrochemical impedance spectroscopy studies reveal that this nucleic acid sensor exhibits linear response to cDNA in the concentration range of 10(-6) to 10(-14) M with response time of 30 min. The biosensing characteristics show that the fabricated E. coli sensor can be reused about 4 times and is stable for ～4 weeks. The studies on cross-reactivity of the sensor for other water-borne pathogens like Salmonella typhimurium, Neisseria meningitides, and Klebsiella pneumonia reveal specificity of the bioelectrode for E. coli detection.     

Highly sensitive and selective uric acid biosensor based on RF sputtered NiO thin film

Present study highlights the importance of RF sputtered NiO thin film deposited on platinum coated glass substrate (NiO/Pt/Ti/glass) as a potential matrix for the realization of highly sensitive and selective uric acid biosensor. Uricase has been immobilized successfully onto the surface of NiO matrix by physical adsorption technique. The prepared bioelectrode (uricase/NiO/Pt/Ti/glass) is utilized for sensing uric acid using the cyclic voltammetry and UV visible spectroscopy techniques. The bioelectrode is found to exhibit highly efficient sensing response characteristics with high sensitivity of 1278.48 μA/mM; good linearity of 0.05-1.0 mM, and very low Michaelis-Menten constant (k(m)) of 0.17 mM indicating high affinity of uricase towards the analyte. The enhanced response is due to the development of NiO matrix with good electron transport property and nanoporous morphology for effective loading of enzyme with preferred orientation.     

Chitosan-iron oxide nano-composite platform for mismatch-discriminating DNA hybridization for Neisseria gonorrhoeae detection causing sexually transmitted disease

Electrochemically fabricated nano-composite film of chitosan (CH)-iron oxide (Fe(3)O(4)) has been used to detect gonorrhoea, a sexually transmitted disease (STD) via immobilization of biotinylated probe DNA (BDNA) using avidin-biotin coupling for rapid and specific (mismatch-discriminating) DNA hybridization. The presence of Fe(3)O(4) nanoparticles (～18nm) increases the electro-active surface area of the nano-biocomposite that provides desirable environment for loading of DNA with better conformation leading to increased electron transfer kinetics between the medium and electrode. The differential pulse voltammetric (DPV) studies have been conducted using BDNA/avidin/CH-Fe(3)O(4)/ITO electrode owing to the reduction of the methylene blue (MB) indicator and investigate electron transfer between MB moieties and electrode for one and two-bases mismatch. This STD biosensor is found to have a detection limit (1 × 10(-15)M) and a wide dynamic range (from 1 × 10(-16)M to 1 × 10(-6)M) using the complementary target DNA. In addition, the sensing system can be utilized to accurately discriminate complementary sequence from mismatch sequences.     

Sensitive Detection of Organophosphorus Pesticides Using a Needle Type Amperometric Acetylcholinesterase-based Bioelectrode. Thiocholine Electrochemistry and Immobilised Enzyme Inhibition

An acetylcholinesterase (AChE) based amperometric bioelectrode for a selective detection of low concentrations of organophosphorus pesticides has been developed. The amperometric needle type bioelectrode consists of a bare cavity in a PTFE isolated Pt-Ir wire, where the AChE was entrapped into a photopolymerised polymer of polyvinyl alcohol bearing styrylpyridinium groups (PVA-SbQ). Cyclic voltammetry, performed at Pt and AChE/Pt disk electrodes, confirmed the irreversible, monoelectronic thiocholine oxidation process and showed that a working potential of +0.410 V vs. Ag/AgCl, KCl sat was suitable for a selective and sensitive amperometric detection of thiocholine. The acetylthiocholine detection under enzyme kinetic control was found in the range of 0.01-0.3 U cm ^?2 of immobilised AChE. The detection limit, calculated for an inhibition ratio of 10%, was found to reach 5 μM for dipterex and 0.4 μM for paraoxon. A kinetic analysis of the AChE-pesticide interaction process using Hanes-Woolf or Lineweaver-Burk linearisations and secondary plots allowed identification of the immobilised enzyme inhibition process as a mixed one (non/uncompetitive) for both dipterex and paraoxon. The deviation from classical Michaelis Menten kinetics induced from the studied pesticides was evaluated using Hill plots.     

One-step electrochemically co-assembled redox-active [Ru(bpy)(2)(tatp)](2+)-BSA-SWCNTs hybrid film for non-redox protein biosensors

A redox-active [Ru(bpy)(2)(tatp)](2+)-BSA-SWCNTs (bpy=2,2'-bipyridine, tatp=1,4,8,9-tetra-aza-triphenylene, BSA=bovine serum albumin, SWCNTs=single-walled carbon nanotubes) hybrid film is fabricated on an indium-tin oxide (ITO) electrode via one-step electrochemical co-assembly approach. BSA is inherently dispersive and therefore served as the linking mediator of SWCNTs, which facilitate the redox reactions of [Ru(bpy)(2)(tatp)](2+) employed as a reporter of BSA. The evidences from differential pulse voltammetry, cyclic voltammetry, scanning electron microscope, emission spectroscopy and fluorescence microscope reveal that the [Ru(bpy)(2)(tatp)](2+)-BSA-SWCNTs hybrid can be electrochemically co-assembled on the ITO electrode, showing two pairs of well-defined Ru(II)-based redox waves. Furthermore, the electrochemical co-assembly of the [Ru(bpy)(2)(tatp)](2+)-BSA-SWCNTs hybrid is found to be strongly dependent on the simultaneous presence of BSA and SWCNTs, indicating a good linear response to BSA in the range from 6 to 50mgL(-1). The results from this study provide an electrochemical co-assembly method for the development of non-redox protein biosensors.     

Amperometric phenol biosensor based on laponite clay-chitosan nanocomposite matrix

A novel strategy to fabricate an amperometric biosensor for phenol determination based on chitosan/laponite nanocomposite matrix was described. The composite film was used to immobilize PPO on the surface of a glassy carbon electrode. Chitosan was utilized to improve the analytical performance of the pure clay-modified bioelectrode. The biosensor exhibited a series of properties: good affinity to its substrate (the apparent Michaelis-Menten constant for the sensor was found to be 0.16 mM), high sensitivity (674 mA M(-1)cm(-2) for catechol) and remarkable long-term stability in storage (it retains 88% of the original activity after 60 days). In addition, optimization of the biosensor construction as well as effects of experimental variables such as pH, operating potential and temperature on the amperometric response of the sensor were discussed.