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.     

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).     

DNA entrapped polypyrrole-polyvinyl sulfonate film for application to electrochemical biosensor

Double-stranded calf thymus (dsCT)-DNA was electrochemically entrapped into polypyrrole-polyvinyl sulfonate (PPy-PVS) films deposited onto indium tin oxide (ITO) coated glass plates. These dsCT-DNA entrapped PPy-PVS/ITO films were characterized using cyclic voltammetry, UV-visible, Fourier transform infrared (FT-IR), scanning tunneling microscopy (STM), and electrochemical impedance measurements. Attempts made to use these dsCT-DNA entrapped PPy-PVS/ITO films for detection of 2-aminoanthracene (0.001-6.0 ppm) and 3-chlorophenol (0.01-55.0 ppm) revealed a response time of 30s and a shelf life of approximately 25 weeks when stored under desiccated conditions at 25 degrees C. The addition of salts such as Ca(2+) (250 ppm), Mg(2+) (200 ppm), Cl(-) (1560 ppm), and Na(+) (150 ppm) ions contained in water does not affect the observed amperometric response of the disposable dsCT-DNA entrapped PPy-PVS film-based electrochemical biosensor.     

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.     

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).     

Application of electrochemically prepared polypyrrole-polyvinyl sulphonate films to DNA biosensor

Double stranded calf thymus deoxyribonucleic acid (DNA) was physisorbed onto polypyrrole-polyvinyl sulphonate (PPY-PVS) films electrochemically deposited onto indium-tin-oxide (ITO) coated glass plates. These DNA immobilized PPY-PVS films optimized for various conditions, such as polymerization potential, pH of buffer, DNA concentration and scan rate were characterized using Fourier-transform infrared (FT-IR) spectroscopy, atomic force microscopy (AFM) and cyclic voltammetry (CV) techniques, respectively. The amperometric response studies of these DNA/PPY-PVS electrodes were carried out as a function of 2-aminoantharcene (2-AA, 0.01-20 ppm) and o-chlorophenol (OCP, 0.1-30 ppm) concentration, respectively at 25 degrees C. The observed amperometric current arising due to oxidation of guanine in the DNA/PPY-PVS films decreased linearly with the increase in the concentration of 2-AA and OCP. It has been revealed that 10 ppm of 2-AA is sufficient to reduce the observed guanine oxidation peak current by approximately -95+/-10% as compared to the reported values. A 25 ppm of OCP was capable enough to reduce the guanine oxidation current to zero. These DNA/PPY-PVS electrodes were found to have a shelf life of about 4 months when stored at 25 degrees C.     

Preparation of sulfonic-functionalized graphene oxide as ion-exchange material and its application into electrochemiluminescence analysis

Graphene oxide (GO) obtained from chemical oxidation of flake graphite was derivatized with sulfonic groups to form sulfonic-functionalized GO (GO-SO(3)(-)) through four sulfonation routes: through amide formation between the carboxylic group of GO and amine of sulfanilic acid (AA-GO-SO(3)(-)), aryl diazonium reaction of sulfanilic acid (AD-GO-SO(3)(-)), amide formation between the carboxylic group of GO and amine of cysteamine and oxidation by H(2)O(2) (CA-GO-SO(3)(-)), and alkyl diazonium reaction of cysteamine and oxidation by H(2)O(2) (CD-GO-SO(3)(-)). Results of Fourier transform infrared spectroscopy and X-ray photoelectrospectrocopy showed that -SO(3)(-) groups were attached onto GO. Thermo gravimetric analysis showed that derivatization with sulfonic groups improved thermo stability of GO. X-ray diffraction results indicated that GO-SO(3)(-) had more ordered π-π stacking structure than the original GO. GO-SO(3)(-) and cationic polyelectrote, poly (diallyldimethylammoniumchloride) (PDDA) were adsorbed at indium tin oxide (ITO) glass surface through layer-by-layer assembling to form (GO-SO(3)(-)/PDDA)(n)/ITO multilayers. After tris-(2,2'-bipyridyl) ruthenium (II) dichloride (Ru(bpy)(3)(2+)) was incorporated into the multilayers, the obtained Ru(bpy)(3)(2+)/(GO-SO(3)(-)/PDDA)(n)/ITO electrodes can be used as electrochemiluminescence sensors for detection of organic amine with high sensitivity (limit of detection of 1 nM) 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.     

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.     

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.     

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.     

Chitosan/polyaniline hybrid conducting biopolymer base impedimetric immunosensor to detect Ochratoxin-A

Chitosan (CS)-polyaniline (PANI) hybrid conducting biopolymer film was obtained on indium-tin-oxide (ITO) electrode using electrochemical polymerization process. Fourier transform infrared (FT-IR) spectra of PANI-CS had showed covalent and hydrogen binding between PANI and CS molecules. Electrochemical impedance spectroscopy (EIS) measurements had showed low charge transfer resistance (R(CT)) of PANI-CS and PANI. Successive rabbit antibody (IgGs) immobilization on PANI-CS, CS and PANI matrixes surface were confirmed with FT-IR and EIS measurements. Ochratoxin-A (OTA) interaction with IgGs had increased R(CT) values and showed linear response up to 10 ng/mL OTA concentration in electrolyte. Relative change in R(CT) was higher in PANI-CS due to higher proportion of carboxylic and hydroxyl functionalities at PANI-CS matrix surfaces. The absolute sensitivity of PANI, CS, and PANI-CS were 16+/-6, 22+/-9 and 53+/-8 Omega mL/ng, respectively derived from slope of linear response up to 10 ng/mL with 1 ng/mL minimum detection limit.     

Electrophoretic fabrication of chitosan-zirconium-oxide nanobiocomposite platform for nucleic acid detection

The present work describes electrophoretic fabrication of nanostructured chitosan-zirconium-oxide composite (CHIT-NanoZrO(2)) film (180 nm) onto indium-tin-oxide (ITO)-coated glass plate. This nanobiocomposite film has been explored as immobilization platform for probe DNA specific to M. Tuberculosis as model biomolecule to investigate its sensing characteristics. It is revealed that pH-responsive behavior of CHIT and its cationic skeleton is responsible for the movement of CHIT-NanoZrO(2) colloids toward cathode during electrophoretic deposition. The FT-IR, SEM, TEM, and EDX techniques have been employed for the structural, morphological, and composition analysis of the fabricated electrodes. The morphological studies clearly reveal uniform inter-linking and dispersion of hexagonal nanograins of ZrO(2) (30-50 nm) into the chitosan matrix, resulting in homogeneous nanobiocomposite formation. Electrochemical response measurements of DNA/CHIT-NanoZrO(2)/ITO bioelectrode, carried out using cyclic voltammetry and differential pulse voltammetry, reveal that this bioelectrode can specifically detect complementary target DNA up to 0.00078 μM with sensitivity of 6.38 × 10(-6) AμM(-1).     

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.     

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.     

Fabrication and photoelectric response of poly(allylamine hydrochloride)/PM thin films by layer-by-layer deposition technique

Thin films of poly(allylamine hydrochloride) (PAH) and bacteriorhodopsin (bR) embedded in purple membrane (PM) have been prepared by layer-by-layer (LBL) self-assembly technique. The results obtained by UV-Vis spectroscopy and atomic force microscopy (AFM) analysis showed that the biological activity of bR was preserved and PM fragments could be well oriented onto the ITO substrate. A photo-electrochemical cell with the structure of ITO/(PAH/PM)(n)/electrolyte (0.5M KCl)/Pt was fabricated and studied. The photocurrent peaks of (PAH/PM)(6) corresponding to light-on and light-off were about 200 and 100 nA/cm(2), respectively, with the former enhanced 30% higher than that of the reference films made of (PDAC/PM)(6).     

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.     

Glucose biosensor based on platinum microparticles dispersed in nano-fibrous polyaniline

A sensitive and selective amperometric glucose biosensor based on platinum microparticles dispersed in nano-fibrous polyaniline (PANI) was investigated. Poly (m-phenylenediamine) (PMPD), which was employed as an anti-interferent barrier and a protective layer to platinum microparticles, was deposited onto platinum-modified PANI in the presence of glucose oxidase. The morphology of PANI, Pt/PANI and PMPD-GOD/Pt/PANI were investigated by scanning electron microscopy. The results show that PANI has a nano-fibrous morphology. The enzyme electrode exhibits excellent response performance to glucose with linear range from 2 x 10(-6) to 12 x 10(-3) M and fast response time within 7s. Due to the selective permeability of PMPD, the enzyme electrode also shows good anti-interference to uric acid and ascorbic acid. The Michaelis-Menten constant km and the maximum current density imax of the enzyme electrode were 9.34 x 10(-3) M and 917.43 microA cm(-2), respectively. Furthermore, this glucose biosensor also has good stability and reproducibility.     

Cholesterol biosensor based on N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane self-assembled monolayer

Cholesterol oxidase (ChOx) has been covalently immobilized onto two-dimensional self-assembled monolayer (SAM) of N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (AEAPTS) deposited on the indium-tin oxide (ITO) coated glass plates using N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) chemistry. These ChO x/AEAPTS/ITO bioelectrodes are characterized using contact angle (CA) measurements, UV-visible spectroscopy, atomic force microscopy (AFM), electrochemical impedance technique, and Fourier transform infrared (FT-IR) technique. The covalently immobilized ChOx-modified AEAPTS bioelectrodes are used for the estimation of cholesterol in solution using UV-visible technique. These cholesterol sensing bioelectrodes show linearity as 50 to 500 mg/dl for cholesterol solution, detection limit as 25mg/dl, sensitivity as 4.499 x 10(-5) Abs (mg/dl)(-1), K(m) value as 58.137 mg/dl (1.5mM), apparent enzyme activity as 1.81 x 10(-3) U cm(-2), shelf life of approximately 10 weeks, and electrode reusability as 10 times.     

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.