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.     

Poly-3-hexyl thiophene Langmuir-Blodgett films for application to glucose biosensor

Langmuir-Blodgett films of poly(3-hexyl thiophene) have been prepared by simultaneous entrapment of glucose oxidase and transferred onto the indium-tin-oxide coated glass plates. The films have been characterized by FTIR spectroscopy and detailed response studies have been performed with respect to glucose concentration, temperature, and storage time. These P3HT/SA/GOX electrodes have been utilized for glucose estimation from 100-500 mg/dL by amperometric method. The electrodes have been found to have sensitivity of 0.75 nA/mg/dL detection limit of 50 mg/dL and shelf life of about 75 days at 4 degrees C.     

Ultrasensitive DNA hybridization biosensor based on polyaniline

Ultrasensitive DNA hybridization biosensor based on polyaniline (PANI) electrochemically deposited onto Pt disc electrode has been fabricated using biotin-avidin as indirect coupling agent to immobilize single-stranded 5'-biotin end-labeled polydeoxycytidine (BdC) probes and 5'-biotin end-labeled 35 base-long oligonucleotide probe (BdE) to detect complementary target, using both direct electrochemical oxidation of guanine and redox electroactive indicator methylene blue (MB), respectively. These polyaniline-based disc electrodes have been characterized using differential pulse voltammetry (DPV), Fourier transform infrared spectroscopy (FT-IR), impedance measurements and scanning electron microscopy (SEM) techniques, respectively. Compared to direct electrochemical oxidation of guanine, hybridization detection using MB results in the enhanced detection limit by about 100 times. These DNA immobilized PANI electrodes have hybridization response time of about 60 s.     

Nickel oxide microfibers immobilized onto electrode by electrospinning and calcination for nonenzymatic glucose sensor and effect of calcination temperature on the performance

Nickel oxide microfibers (NiO-MFs) were directly immobilized onto the surface of fluorine tin oxide (FTO) electrode by electrospinning and calcination without using any immobilization matrix for nonenzymatic glucose sensor. Morphology and structure of NiO-MFs were characterized by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and X-ray diffraction pattern (XRD). The electrochemical and electrocatalytic performances of the NiO-MFs modified electrodes prepared at different calcination temperatures ranging from 300 to 500°C were evaluated by cyclic voltammetry (CV). The CV results have demonstrated that NiO-MFs modified electrode prepared at 300°C displayed distinct increase in electrocatalytic activity toward the oxidation of glucose, which is explored to develop an amperometric nonenzymatic glucose sensor. The NiO-MFs prepared at 300°C based amperometric nonenzymatic glucose sensor has ultrasensitive current (1785.41 μA mM(-1) cm(-2)) response and low detection limit of 3.3×10(-8) M (signal/noise ratio (S/N)=3), which are among the best values reported in literature. Additionally, excellent selectivity and stability have also been obtained.     

Kinetics of release of serotonin from isolated secretory granules. I. Amperometric detection of serotonin from electroporated granules.

We developed a method for measuring the efflux of 5-hydroxytryptamine (5-HT, serotonin) from isolated intact granules of the mast cell of the beige mouse. This method combines electroporation of the vesicle membrane with amperometric detection of 5-HT. A single secretory granule is placed between two platinum electrodes (distance approximately 100 microm) and positioned adjacent (<1 microm) to a carbon fiber microelectrode. A short (approximately 30 micros) high-intensity voltage pulse (electric field of approximately 5 kV/cm) is delivered to the electrodes to trigger the mechanical breakdown of the granule membrane, which activates the release of 5-HT. We observed concurrent swelling of the granule matrix with the oxidation of 5-HT at the carbon fiber electrode (overpotential + 650 mV). Similar to the release of secretory products during exocytosis, the oxidation current exhibits a spike-like time course with a noninstantaneous rising phase (time between onset of current and maximum flux, t(max)) with approximately 25% of the molecules released during this period. When the current reaches its maximum, the granule matrix attains its maximum swollen state. We found that the rising phase depends on the initial cross-sectional area of the granule (t(max) approximately 21r2) and reflects the time required for membrane rupture. The average t(1/2)spike of the amperometric spikes was found to be approximately 150 ms, which is 3-7 times faster than the t(1/2) measured during cellular exocytosis.     

Carbon nanotube-based biosensors for DNA structure characterization

The possibility of DNA detection using electrodes modified with carbon nanotubes (CNTs) was studied. CNTs facilitate the electrochemical oxidation of DNA guanine nucleotide, which allows direct detection of DNA on a modified electrode. Electrochemical properties of DNA depend on its secondary structure and molecular weight. Denaturation of native DNA improves the adsorption of biopolymer on CNTs and results in an increase in DNA oxidation current on the modified electrode. A similar effect is observed after ultrasonic shearing of DNA or its treatment with Fenton’s reagent due to the fragmentation of biopolymer. Our results demonstrate the feasibility of biosensors based on CNT-modified electrodes for the direct detection and characterization of DNA and DNA damaging factors.     

Enzyme immunosensors based on electropolymerized polytyramine modified electrodes

Highly sensitive amperometric enzyme immunosensors for human immunoglobulin G (IgG) were prepared on the basis of electrogenerated polytyramine (PTy, tyramine = p-(2-aminoethyl)-phenol) modified electrodes. Properties of PTy films changed depending on electrolysis conditions. On the basis of the found properties of the films, an effective IgG sensor was prepared: a PTy film was formed first from an acid solution on a Pt electrode, and the surface was further covered with a PTy film from an alkaline methanol solution to give a PTy doubly coated electrode on which anti-IgG was then immobilized. This electrode provided a large surface area with little non-specific adsorption of proteins. By means of the competitive enzyme immunoassay technique using glucose oxidase (GOD) labeled IgG conjugates, IgG was determined in the concentration range of c. 10 pg/ml-1 mg/ml from the oxidation current of H2O2 generated by the enzyme (GOD) reaction using the above IgG sensor. Also, an anti-IgG immobilized electrode, prepared by using a Pt electrode singly covered with a PTy film from an alkaline methanol solution, acted as an effective IgG sensor with a detection limit for IgG of c. 100 pg/ml.     

Modified carbon paste electrodes for flow injection amperometric determination of isocitrate dehydrogenase activity in serum

A carbon paste electrode modified with the adsorbed products of the electrochemical oxidation of adenosine triphosphate is described. The electrode was applied to the amperometric electrocatalytic detection of the reduced form of both nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate. The catalytic oxidation current shows a linear dependence on the concentration of the reduced form of nicotinamide adenine dinucleotide up to 1x10(-4)M, with a detection limit of 5x10(-9)M. Modified carbon paste electrodes were coated with an electrogenerated film of nonconducting poly(o-phenylenediamine) to obtain a stable amperometric response for at least 150h. In addition to static measurements, determination of both reduced cofactors was carried out in a flow injection analysis system with a thin-layer amperometric detection cell. The electrocatalytic monitoring of reduced nicotinamide adenine dinucleotide phosphate was applied to flow injection measurement of isocitrate dehydrogenase activity in serum. The results were in good agreement with those for the standard spectrophotometric test kit. The proposed method consumed less time and reagents and provided better precision than the standard method.     

Lithographically defined 3D nanoporous nonenzymatic glucose sensors

Nonenzymatic glucose oxidation is demonstrated on highly faceted palladium nanowflower-modified porous carbon electrodes fabricated by interference lithography. Varying electrodeposition parameters were used to control the final shape and morphology of the deposited nanoparticles on the 3D porous carbon which showed a 12 times increase in the electrochemically active surface area over analogous planar electrodes. Extremely fast amperometric glucose responses (achieving 95% of the steady state limiting current in less than 5s) with a linear range from 1 to 10mM and a detection limit of 10 μM were demonstrated. The unusual surface properties of the pyrolyzed photoresist films produced strongly adhered palladium crystal structures that were stable for hundreds of cycles towards glucose oxidation without noticeable current decay.     

Amperometric glucose biosensor based on gold-deposited polyvinylferrocene film on Pt electrode

The preparations and performances of the novel amperometric biosensors for glucose based on immobilized glucose oxidase (GOD) on modified Pt electrodes are described. Two types of modified electrodes for the enzyme immobilization were used in this study, polyvinylferrocene (PVF) coated Pt electrode and gold deposited PVF coated Pt electrode. A simple method for the immobilization of GOD enzyme on the modified electrodes was described. The enzyme electrodes developed in this study were called as PVF-GOD enzyme electrode and PVF-Au-GOD enzyme electrode, respectively. The amperometric responses of the enzyme electrodes were measured at constant potential, which was due to the electrooxidation of enzymatically produced H2O2. The electrocatalytic effects of the polymer, PVF, and the gold particles towards the electrooxidation of the enzymatically generated H2O2 offers sensitive and selective monitoring of glucose. The biosensor based on PVF-Au-GOD electrode has 6.6 times larger maximum current, 3.8 times higher sensitivity and 1.6 times larger linear working portion than those of the biosensor based on PVF-GOD electrode. The effects of the applied potential, the thickness of the polymeric film, the amount of the immobilized enzyme, pH, the amount of the deposited Au, temperature and substrate concentration on the responses of the biosensors were investigated. The optimum pH was found to be pH 7.4 at 25 degrees C. Finally the effects of interferents, stability of the biosensors and applicability to serum analysis of the biosensor were also investigated.     

Impedimetric immunosensors based on the conjugated polymer PPV

In the work reported here, we investigated the interaction between the semiconducting polymer MDMO-PPV and antibodies against the fluorescent dyes fluorescein isothiocyanate (FITC) and Cy5. The antibodies are adsorbed physically onto thin polymer films on gold electrodes, as seen in AFM images of these films. By tuning the antibody concentration, the contact angle of distilled water with the film can be made to vary between 95 degrees and 50 degrees, showing that different surface densities of antibody can be obtained. That these biosensor films specifically bind their antigenic fluorescent molecules from PBS buffer solution is demonstrated by confocal fluorescence microscopy. Specific antigen-antibody recognition is demonstrated by lack of cross-sensitivity between the two antibodies and their antigens. In a biosensor prototype based on differential impedance spectroscopy, these polymer films show a clear response to 1 ppb antigen solution, with a time constant of 2-3 min.     

NO(2) Sensor which uses immobilized nitrite oxidizing bacteria

A biosensor consisting of immobilized nitrite oxidizing bacteria and an oxygen electrode has been developed for the amperometric determination of NO(2) (nitrogen dioxide) gas. The response time for the determination of NO(2) was within 3 min. A linear relationship was observed between the current decrease and the NO(2) concentration below 255 ppm. The minimum concentration for the determination of NO(2) was 0.51 ppm. The current decrease was reproducible within +/-4% of the relative error. The selectivity of the microbial sensor for NO(2) was satisfactory. The current output of the sensor was almost constant for more than 24 days and 400 assays.     

Immobilization of urease on poly(N-vinyl carbazole)/stearic acid Langmuir-Blodgett films for application to urea biosensor

Urease was immobilized in mixed monolayers of poly(N-vinyl carbazole) (PNVK) and stearic acid (SA) formed at an air-water interface. The monolayers were transferred onto indium-tin-oxide (ITO) coated glass plates using Langmuir-Blodgett (LB) film deposition technique. Urease immobilized on PNVK/SA LB films, characterized using FTIR and UV-visible spectroscopy, was found to exhibit increased stability over a wide pH (6.5-8.5) and temperature (25-50 degrees C) range. Potentiometric measurements on these urease electrodes were carried out using an ammonium ion analyzer. Two values for K(m)(app) were obtained at lower and higher concentrations of substrate urea.     

Photovoltaic behavior of chlorophyll a and beta-carotene in Langmuir films

The photovoltaic properties of chlorophyll a and beta-carotene Langmuir films and Langmuir films of a mixure of chlorophyll a and beta-carotene at different molar ratios were investigated. SnO2-optically transparent electrodes were used as a support. It was shown that the film photovoltage value depends on surface pressure and the total film thickness (the number of layers deposited onto SnO2-optically transparent electrodes). Fifteen layers (SP = 35 mH/m) of chlorophyll and 7-10 layers (SP = 20 mH/m) of beta-carotene give rise to a maximal photovoltage of 140 and 270 mV (at a shunt resistance of 10(7) Ohm), correspondingly. It was found that the photovoltage values in films of the carotene-chlorophyll mixture increase with the carotene concentration. The photovoltage value of a film containing 80-90% of carotene exceeds that of single-component pigment films prepared under the same deposition conditions.     

Amperometric protein sensor - fabricated as a polypyrrole, poly-aminophenylboronic acid bilayer

An approach to the design of electrodes for the production of sensors, which show significant changes to the passage of current in response to the concentration of target protein molecules, is presented. Screen-printed platinum electrodes, modified with two separately applied conducting polymer layers, have been developed as a potential route to forming cheap disposable protein sensors. To achieve a heightened response for the target molecules, an initial layer of polypyrrole was formed on the electrode's surface by electro-deposition. This composite was then employed as a substrate for the subsequent electro-deposition of a relatively thin 'sensing layer' of poly-aminophenylboronic acid. Cyclic voltammetry (CV) of the prepared films revealed an excursion in the current versus potential curve in the anodic phase at approximately 0.0 to +0.2V. It was clearly shown that the introduction of proteins into the CV cell resulted in a measurable decrease in the passage of current in buffered aqueous media. Measured current reductions observed on introducing lysozyme (10ppm) into the test solution were 2.3x10(-6)A for an electrode formed with a poly-aminophenylboronic acid layer on platinum, and 1.75x10(-5)A for a composite electrode formed with poly-aminophenylboronic acid on a polypyrrole coated platinum substrate. The introduction of the competing analytes, dl adrenaline or dopamine, at concentrations typically found in human urine, had little effect on the sensor's response. Additionally, the sensing system was able to maintain a response to added target proteins with as much as 2vol.% urine in the test solution. Using the electrodes in high concentrations of competing physiological analytes, they were able to respond to protein concentrations as low as 0.5ppm in buffered solutions containing urea at a concentration representative of human urine (17,000ppm), which additionally contained glucose (1000ppm).     

Electrochemical detection of catecholamine release using planar iridium oxide electrodes in nanoliter microfluidic cell culture volumes

Release of neurotransmitters and hormones by calcium regulated exocytosis is a fundamental cellular/molecular process that is disrupted in a variety of psychiatric, neurological, and endocrine disorders. Therefore, this area represents a relevant target for drug and therapeutic development, efforts that will be aided by novel analytical tools and devices that provide mechanistically rich data with increased throughput. Toward this goal, we have electrochemically deposited iridium oxide (IrOx) films onto planar thin film platinum electrodes (20 μm×300 μm) and utilized these for quantitative detection of catecholamine release from adrenal chromaffin cells trapped in a microfluidic network. The IrOx electrodes show a linear response to norepinephrine in the range of 0-400 μM, with a sensitivity of 23.1±0.5 mA/M mm(2). The sensitivity of the IrOx electrodes does not change in the presence of ascorbic acid, a substance commonly found in biological samples. A replica molded polydimethylsiloxane (PDMS) microfluidic device with nanoliter sensing volumes was aligned and sealed to a glass substrate with the sensing electrodes. Small populations of chromaffin cells were trapped in the microfluidic device and stimulated by rapid perfusion with high potassium (50mM) containing Tyrode's solution at a flow rate of 1 nL/s. Stimulation of the cells produced a rapid increase in current due to oxidation of the released catecholamines, with an estimated maximum concentration in the cell culture volume of ~52 μM. Thus, we demonstrate the utility of an integrated microfluidic network with IrOx electrodes for real-time quantitative detection of catecholamines released from small populations of chromaffin cells.     

Energetics of OCP1-OCP2 complex formation

OCP1 and OCP2, the most abundant proteins in the cochlea, are putative subunits of an SCF E3 ubiquitin ligase. Previous work has demonstrated that they form a heterodimeric complex. The thermodynamic details of that interaction are herein examined by isothermal titration calorimetry. At 25 degrees C, addition of OCP1 to OCP2 yields an apparent association constant of 4.0 x 10(7) M(-1). Enthalpically-driven (DeltaH=-35.9 kcal/mol) and entropically unfavorable (-TDeltaS=25.5 kcal/mol), the reaction is evidently unaccompanied by protonation/deprotonation events. DeltaH is strongly dependent on temperature, with DeltaC(p)=-1.31 kcal mol(-1) K(-1). Addition of OCP2 to OCP1 produces a slightly less favorable DeltaH, presumably due to the requirement for dissociation of the OCP2 homodimer prior to OCP1 binding. The thermodynamic signature for OCP1/OCP2 complex formation is inconsistent with a rigid-body association and suggests that the reaction is accompanied by a substantial degree of folding.     

Mechanisms of DNA damage by chromium(V) carcinogens.

Reactions of bis(2-ethyl-2-hydroxy-butanato)oxochromate(V) with pUC19 DNA, single-stranded calf thymus DNA (ss-ctDNA), a synthetic oligonucleotide, 5'-GATCTATGGACTTACTTCAAGGCCGGGTAATGCTA-3' (35mer), deoxyguanosine and guanine were carried out in Bis-Tris buffer at pH 7.0. The plasmid DNA was only nicked, whereas the single-stranded DNA suffered extensive damage due to oxidation of the ribose moiety. The primary oxidation product was characterized as 5-methylene-2-furanone. Although all four bases (A, C, G and T) were released during the oxidation process, the concentration of guanine exceeds the other three. Orthophosphate and 3'-phosphates were also detected in this reaction. Likewise, the synthetic oliogomer exhibits cleavage at all bases with a higher frequecncy at G sites. This increased cleavage at G sites was more apparent after treating the primary oxidation products with piperidine, which may indicate base oxidation as well. DNA oxidation is shown to proceed through a Cr(V)-DNA intermediate in which chromium(V) is coordinated through the phosphodiester moiety. Two alternative mechanisms for DNA oxidation by oxochromate(V) are proposed to account for formation of 5-methylene-2-furanone, based on hydrogen abstraction or hydride transfer from the C1' site of the ribose followed by hydration and two successive beta-eliminations. It appears that phosphate coordination is a prerequisite for DNA oxidation, since no reactions between chromium(V) and deoxyguanosine or guanine were observed. Two other additional pathways, hydrogen abstraction from C4' and guanine base oxidation, are also discussed.     

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.     

Proton NMR investigation of the nucleosome core particle: evidence for regions of altered hydrogen bonding

Novel 1H nuclear magnetic resonance (NMR) resonances, arising from exchangeable protons and centered at approximately 11.2 and 10.1 parts per million (ppm), have been observed in the low-field spectrum (10-15 ppm) of the chicken erythrocyte core particle [145 +/- 2 base pairs (bp)]. These peaks are located upfield from the normal adenine-thymine (A-T) and guanine-cytosine (G-C) imino peaks characteristic of B-form deoxyribonucleic acid (DNA) and are not observed in free DNA under identical conditions. The appearance of the new peaks is ionic strength dependent and temperature-reversible below 75 degrees C. At 25 degrees C, the upfield peak area represents 5% of the DNA base pairs (7 bp), while between 45 and 55 degrees C, the area increases to 18%, affecting approximately 25 bp. Area increases in the upfield resonances result in a complementary decrease in the A-T and G-C imino peaks found between 12 and 14 ppm. We believe these novel proton signals represent a histone-induced DNA conformational change which involves localized alteration of base pairing in the core particle.