Development of an Electrochemical Insulin Sensor Based on a High Affinity DNA Sequence Found in the Insulin-linked Polymorphic Region

The detection of pertinent biomarkers has the potential provide an early indication of disease progression before considerable damage has been incurred. A decrease in an individual’s sensitivity to insulin, which may be quantified as the ratio of insulin to glucose in the blood after a glucose pulse, has recently been reported as an early predictor of insulin-dependent diabetes mellitus. Routine measurement of insulin levels is therefore desirable in the care of diabetes-prone individuals. A rapid, simple, and reagentless method for insulin detection would allow for wide-spread screenings that provide earlier signs of diabetes onset. The aim of this thesis is to develop a folding-base electrochemical sensor for the detection of insulin. The sensor described herein consists of a DNA probe immobilized on a gold disc electrode via an alkanethiol linker and embedded in an alkanethiol self-assembled monolayer. The probe is labeled with a redox reporter, which readily transfers electrons to the gold electrode in the absence of insulin. In the presence of insulin, electron transfer is inhibited, presumably due to a binding-induced conformational or dynamic change in the DNA probe that significantly alters the electron-tunneling pathway. A 28-base segment of the insulin-linked polymorphic region that has been reported to bind insulin with high affinity serves as the capture element of the DNA probe. Three probe constructs that vary in their secondary structure and position of the redox label are evaluated for their utility as insulin-sensing elements on the electrochemical platform. The effects of probe modification on secondary structure are also evaluated using circular dichroism spectroscopy.

Quantification of terbinafine in pharmaceutical tablets using capillary electrophoresis with contactless conductivity detection and batch injection analysis with amperometric detection

Terbinafine hydrochloride (TerbHCl) is an allylamine derivative with fungicidal action, especially against dermatophytes. Different analytical methods have been reported for quantifying TerbHCl in different samples. These procedures require time-consuming sample preparation or expensive instrumentation. In this paper, electrochemical methods involving capillary electrophoresis with contactless conductivity detection, and amperometry associated with batch injection analysis, are described for the determination of TerbHCl in pharmaceutical products. In the capillary electrophoresis experiments, terbinafine was protonated and analyzed in the cationic form in less than 1 min. A linear range from 1.46 to 36.4 mu g mL(-1) in acetate buffer solution and a detection limit of 0.11 mu g mL(-1) were achieved. In the amperometric studies, terbinafine was oxidized at +0.85 V with high throughput (225 injection h(-1)) and good linear range (10-100 mu mol L-1). It was also possible to determine the antifungal agent using simultaneous conductometric and potentiometric titrations in the presence of 5% ethanol. The electrochemical methods were applied to the quantification of TerbHCl in different tablet samples; the results were comparable with values indicated by the manufacturer and those found using titrimetry according to the Pharmacopoeia. The electrochemical methods are simple, rapid and an appropriate alternative for quantifying this drug in real samples. (C) 2012 Elsevier B.V. All rights reserved.

Evaluation of Solid-Phase Microextraction using a Polythiophene Film and Liquid Chromatography with Spectrophotometric Detection for the Determination of Antidepressants in Plasma Samples

Polythiophene (PTh) phase electropolymerized on the stainless steel wire was evaluated as solid-phase microextraction (SPME), and analysis by liquid chromatography with spectrophotometric detection (LC-UV) for determination of new-generation antidepressants, selective serotonin reuptake inhibitors (SSRIs) (citalopram, paroxetine, fluoxetine and sertraline), in plasma samples. The influence of electropolymerization variables (scan rate, potential range and scan cycles) was evaluated on SPME performance. The SPME variables (extraction time, temperature, matrix pH, ionic strength and desorption procedure), as well as the influence of plasma proteins on sorption mechanisms were also evaluated. The SPME/LC-UV method developed for determination of antidepressants in plasma sample presented a linear range between the limit of quantification (LOQ, 200-250 ng mL(-1)) to 4000 ng mL(-1), and interday precision with coefficient of variation (CV) ranged from 11 to 15%. The proposed method can be a useful tool for the determination of antidepressants in human plasma samples in urgent toxicological analysis after the accidental or suicidal intake of higher doses of medications.

A comparative study of the electrochemical oxidation of the herbicide tebuthiuron using boron-doped diamond electrode

The thiadiazolylurea derivative tebuthiuron (TBH) is commonly used as an herbicide even though it is highly toxic to humans. While various processes have been proposed for the removal of organic contaminants of this type from wastewater, electrochemical degradation has shown particular promise. The aim of the present study was to investigate the electrochemical degradation of TBH using anodes comprising boron-doped (5000 and 30000 ppm) diamond (BDD) films deposited onto Ti substrates operated at current densities in the range 10-200 mA cm(-2). Both anodes removed TBH following a similar pseudo first-order reaction kinetics with k(ap)p close to 3.2 x 10(-2) min(-1). The maximum mineralization efficiency obtained was 80%. High-pressure liquid chromatography with UV-VIS detection established that both anodes degraded TBH via similar intermediates. Ion chromatography revealed that increasing concentrations of nitrate ions (up to 0.9 ppm) were formed with increasing current density, while the formation of nitrite ions was observed with both anodes at current densities >= 150 mA cm(-2). The BDD film prepared at the lower doping level (5000 ppm) was more efficient in degrading TBH than its more highly doped counterpart. This unexpected finding may be explained in terms of the quantity of impurities incorporated into the diamond lattice during chemical vapor deposition. (C) 2012 Elsevier Ltd. All rights reserved.

Real-time electrochemical determination of phenolic compounds after benzene oxidation

A sensitive electrochemical sensor was successfully developed on multi-walled carbon nanotubes (MWCNT) and cobalt phthalocyanine (CoPc) modified glassy carbon electrode (GC), and used to detect byproducts formed after the electrolysis of benzene. The GC/MWCNT/CoPc electrode was applied in the detection of phenolic compounds using square wave voltammetry (SWV). The proposed sensor exhibited a sequence in the sensitivity of the tested phenols: catechol > hydroquinone > resorcinol > phenol and 1,4-benzoquinone. The detection limits for individual phenols were also calculated: catechol (15.62 mu g L-1), hydroquinone (17.91 mu g L-1), resorcinol (46.12 mu g L-1), phenol (58.83 mu g L-1) and 1,4-benzoquinone (13.75 mu g L-1). The proposed sensor was successfully applied in the determination of the total amount of phenols formed after the benzene oxidation, and the obtained results were in full agreement with those from the HPLC procedure. (C) 2012 Elsevier B.V. All rights reserved.

ELECTROCHEMICAL OXIDATION OF THE HERBICIDE TEBUTHIURON USING DSA (R)-TYPE ELECTRODE

ELECTROCHEMICAL OXIDATION OF THE HERBICIDE TEBUTHIURON USING DSA (R)-TYPE ELECTRODE. Tebuthiuron (TBH) is a herbicide widely used in different cultures and known for its toxic effects. Electrochemical methods are promising for removing pollutants such as pesticides. This study showed the degradation of TBH using a DSA (R) anode operated at current densities of 50 to 200 mA cm(-2). Removal presented pseudo-first order kinetics while high-pressure liquid chromatography (UV detection) showed two peaks, ascribed to degradation intermediates. The maximum percentage of total organic carbon removed was 12.9%. Ion chromatography revealed that higher concentrations of nitrate and nitrite ions formed with increasing current density.

Carbon nanotubes modified with antimony nanoparticles: A novel material for electrochemical sensing

In this study, a novel material for the electrochemical determination of bisphenol A using a nanocomposite based on multi-walled carbon nanotubes modified with antimony nanoparticles has been investigated. The morphology, structure, and electrochemical performance of the nanocomposite electrodes were characterised by field emission gun scanning electron microscopy, energy-dispersive X-ray spectroscopy, and cyclic voltammetry. A scan rate study and electrochemical impedance spectroscopy showed that the bisphenol A oxidation product is adsorbed on nanocomposite electrode surface. Differential pulse voltammetry in phosphate buffer solution at pH 6, allowed the development of a method to determine bisphenol A levels in the range of 0.5-5.0 mu mol L-1, with a detection limit of 5.24 nmol L-1 (1.19 mu g L-1). (C) 2012 Elsevier Ltd. All rights reserved.

Development of an enzymeless electroanalytical method for the indirect detection of creatinine in urine samples

The aim of this study is to develop a new enzymeless electroanalytical method for the indirect quantification of creatinine from urine sample. This method is based on the electrochemical monitoring of picrate anion reduction at a glassy carbon electrode in an alkaline medium before and after it has reacted with creatinine (Jaffe's reaction). By using the differential pulse voltammetry technique under the optimum experimental conditions (step potential, amplitude potential, reaction time, and temperature), a linear analytical curve was obtained for concentrations of creatinine ranging from 1 to 80 mu mol L-1, with a detection limit of 380 nmol L-1. This proposed method was used to measure creatinine in human urine without the interference of most common organic species normally present in biological fluids (e.g., uric acid, ascorbic acid, glucose, and phosphocreatinine). The results obtained using urine samples were highly similar to the results obtained using the reference spectrophotometric method (at a 95% confidence level). (C) 2012 Elsevier B.V. All rights reserved.

Evaluation of the detection and quantification limits in electroanalysis using two popular methods: application in the case study of paraquat determination

In this work, the reduction reaction of paraquat herbicide was used to obtain analytical signals using electrochemical techniques of differential pulse voltammetry, square wave voltammetry and multiple square wave voltammetry. Analytes were prepared with laboratory purified water and natural water samples (from Mogi-Guacu River, SP). The electrochemical techniques were applied to 1.0 mol L-1 Na2SO4 solutions, at pH 5.5, and containing different concentrations of paraquat, in the range of 1 to 10 mu mol L-1, using a gold ultramicroelectrode. 5 replicate experiments were conducted and in each the mean value for peak currents obtained -0.70 V vs. Ag/AgCl yielded excellent linear relationships with pesticide concentrations. The slope values for the calibration plots (method sensitivity) were 4.06 x 10(-3), 1.07 x 10(-2) and 2.95 x 10(-2) A mol(-1) L for purified water by differential pulse voltammetry, square wave voltammetry and multiple square wave voltammetry, respectively. For river water samples, the slope values were 2.60 x 10(-3), 1.06 x 10(-2) and 3.35 x 10(-2) A mol(-1) L, respectively, showing a small interference from the natural matrix components in paraquat determinations. The detection limits for paraquat determinations were calculated by two distinct methodologies, i.e., as proposed by IUPAC and a statistical method. The values obtained with multiple square waves voltammetry were 0.002 and 0.12 mu mol L-1, respectively, for pure water electrolytes. The detection limit from IUPAC recommendations, when inserted in the calibration curve equation, an analytical signal (oxidation current) is smaller than the one experimentally observed for the blank solution under the same experimental conditions. This is inconsistent with the definition of detection limit, thus the IUPAC methodology requires further discussion. The same conclusion can be drawn by the analyses of detection limits obtained with the other techniques studied.

Optical chemical sensors using polythiophene derivatives as active layer for detection of volatile organic compounds

Several studies on polythiophene gas sensors, based mainly on electrochemical and gravimetric principles can be found in the literature. However, other principles of gas detection, such as optical and thermal, are still little studied. Optical sensing is suitable for remote detection and offers great versatility at low cost. Here,we report on the use of thin films of seven polythiophene derivatives as active layer in optical sensors for the detection of six volatile organic compounds (n-hexane, toluene, tetrahydrofuran, chloroform, dichloromethane and methanol) and water vapor, in concentration range of 500-30,000 ppm. The results showed that it is possible to use different polythiophene derivatives to differentiate VOCs by optical sensing. Differentiation can be performed based on the presence or not of response to an analyte and the sensitivity value of the sensors for the analytes. Another important feature is the lack of the effect of humidity on the response of most films, which could be a major drawback in the application of these sensors. (C) 2011 Elsevier B.V. All rights reserved.

Glass/PDMS hybrid microfluidic device integrating vertically aligned SWCNTs to ultrasensitive electrochemical determinations

This communication reports a promising platform for rapid, simple, direct, and ultrasensitive determination of serotonin. The method is related to integration of vertically aligned single-walled carbon nanotubes (SWCNTs) in electrochemical microfluidic devices. The required microfabrication protocol is simple and fast. In addition, the nanomaterial influenced remarkably the obtained limit-of-detection (LOD) values. Our system achieved a LOD of 0.2 nmol L-1 for serotonin, to the best of our knowledge one of the lowest values reported in the literature.

An electrochemical gas sensor based on paper supported room temperature ionic liquids

A sensitive and fast-responding membrane-free amperometric gas sensor is described, consisting of a small filter paper foil soaked with a room temperature ionic liquid (RTIL), upon which three electrodes are screen printed with carbon ink, using a suitable mask. It takes advantage of the high electrical conductivity and negligible vapour pressure of RTILs as well as their easy immobilization into a porous and inexpensive supporting material such as paper. Moreover, thanks to a careful control of the preparation procedure, a very close contact between the RTIL and electrode material can be achieved so as to allow gaseous analytes to undergo charge transfer just as soon as they reach the three-phase sites where the electrode material, paper supported RTIL and gas phase meet. Thus, the adverse effect on recorded currents of slow steps such as analyte diffusion and dissolution in a solvent is avoided. To evaluate the performance of this device, it was used as a wall-jet amperometric detector for flow injection analysis of 1-butanethiol vapours, adopted as the model gaseous analyte, present in headspace samples in equilibrium with aqueous solutions at controlled concentrations. With this purpose, the RTIL soaked paper electrochemical detector (RTIL-PED) was assembled by using 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl) imide as the wicking RTIL and printing the working electrode with carbon ink doped with cobalt(II) phthalocyanine, to profit from its ability to electrocatalyze thiol oxidation. The results obtained were quite satisfactory (detection limit: 0.5 mu M; dynamic range: 2-200 mu M, both referring to solution concentrations; correlation coefficient: 0.998; repeatability: +/- 7% RSD; long-term stability: 9%), thus suggesting the possible use of this device for manifold applications.

Surface-plasmon optical and electrochemical characterization of biofunctional surface architectures

The development and characterization of biomolecule sensor formats based on the optical technique Surface Plasmon Resonance (SPR) Spectroscopy and electrochemical methods were investigated. The study can be divided into two parts of different scope. In the first part new novel detection schemes for labeled targets were developed on the basis of the investigations in Surface-plamon Field Enhanced Spectroscopy (SPFS). The first one is SPR fluorescence imaging formats, Surface-plamon Field Enhanced Fluorescence Microscopy (SPFM). Patterned self assembled monolayers (SAMs) were prepared and used to direct the spatial distribution of biomolecules immobilized on surfaces. Here the patterned monolayers would serve as molecular templates to secure different biomolecules to known locations on a surface. The binding processed of labeled target biomolecules from solution to sensor surface were visually and kinetically recorded by the fluorescence microscope, in which fluorescence was excited by the evanescent field of propagating plasmon surface polaritons. The second format which also originates from SPFS technique, Surface-plamon Field Enhanced Fluorescence Spectrometry (SPFSm), concerns the coupling of a fluorometry to normal SPR setup. A spectrograph mounted in place of photomultiplier or microscope can provide the information of fluorescence spectrum as well as fluorescence intensity. This study also firstly demonstrated the analytical combination of surface plasmon enhanced fluorescence detection with analyte tagged by semiconducting nano- crystals (QDs). Electrochemically addressable fabrication of DNA biosensor arrays in aqueous environment was also developed. An electrochemical method was introduced for the directed in-situ assembly of various specific oligonucleotide catcher probes onto different sensing elements of a multi-electrode array in the aqueous environment of a flow cell. Surface plasmon microscopy (SPM) is utilized for the on-line recording of the various functionalization steps. Hybridization reactions between targets from solution to the different surface-bound complementary probes are monitored by surface-plasmon field-enhanced fluorescence microscopy (SPFM) using targets that are either labeled with organic dyes or with semiconducting quantum dots for color-multiplexing. This study provides a new approach for the fabrication of (small) DNA arrays and the recording and quantitative evaluation of parallel hybridization reactions. In the second part of this work, the ideas of combining the SP optical and electrochemical characterization were extended to tethered bilayer lipid membrane (tBLM) format. Tethered bilayer lipid membranes provide a versatile model platform for the study of many membrane related processes. The thiolipids were firstly self-assembled on ultraflat gold substrates. Fusion of the monolayers with small unilamellar vesicles (SUVs) formed the distal layer and the membranes thus obtained have the sealing properties comparable to those of natural membranes. The fusion could be monitored optically by SPR as an increase in reflectivity (thickness) upon formation of the outer leaflet of the bilayer. With EIS, a drop in capacitance and a steady increase in resistance could be observed leading to a tightly sealing membrane with low leakage currents. The assembly of tBLMs and the subsequent incorporation of membrane proteins were investigated with respect to their potential use as a biosensing system. In the case of valinomycin the potassium transport mediated by the ion carrier could be shown by a decrease in resistance upon increasing potassium concentration. Potential mediation of membrane pores could be shown for the ion channel forming peptide alamethicin (Alm). It was shown that at high positive dc bias (cis negative) Alm channels stay at relatively low conductance levels and show higher permeability to potassium than to tetramethylammonium. The addition of inhibitor amiloride can partially block the Alm channels and results in increase of membrane resistance. tBLMs are robust and versatile model membrane architectures that can mimic certain properties of biological membranes. tBLMs with incorporated lipopolysaccharide (LPS) and lipid A mimicking bacteria membranes were used to probe the interactions of antibodies against LPS and to investigate the binding and incorporation of the small antimicrobial peptide V4. The influence of membrane composition and charge on the behavior of V4 was also probed. This study displays the possibility of using tBLM platform to record and valuate the efficiency or potency of numerous synthesized antimicrobial peptides as potential drug candidates.

Electrochemical surface modification of Single walled carbon nanotubes and graphene-based electrodes for (bio)sensing applications

Sensors are devices that have shown widespread use, from the detection of gas molecules to the tracking of chemical signals in biological cells. Single walled carbon nanotube (SWCNT) and graphene based electrodes have demonstrated to be an excellent material for the development of electrochemical biosensors as they display remarkable electronic properties and the ability to act as individual nanoelectrodes, display an excellent low-dimensional charge carrier transport, and promote surface electrocatalysis. The present work aims at the preparation and investigation of electrochemically modified SWCNT and graphene-based electrodes for applications in the field of biosensors. We initially studied SWCNT films and focused on their topography and surface composition, electrical and optical properties. Parallel to SWCNTs, graphene films were investigated. Higher resistance values were obtained in comparison with nanotubes films. The electrochemical surface modification of both electrodes was investigated following two routes (i) the electrografting of aryl diazonium salts, and (ii) the electrophylic addition of 1, 3-benzodithiolylium tetrafluoroborate (BDYT). Both the qualitative and quantitative characteristics of the modified electrode surfaces were studied such as the degree of functionalization and their surface composition. The combination of Raman, X-ray photoelectron spectroscopy, atomic force microscopy, electrochemistry and other techniques, has demonstrated that selected precursors could be covalently anchored to the nanotubes and graphene-based electrode surfaces through novel carbon-carbon formation.

Screen-printed electrode-based electrochemical detector coupled with in-situ ionic-liquid-assisted dispersive liquid–liquid microextraction for determination of 2,4,6-trinitrotoluene

A novel method is reported, whereby screen-printed electrodes (SPELs) are combined with dispersive liquid–liquid microextraction. In-situ ionic liquid (IL) formation was used as an extractant phase in the microextraction technique and proved to be a simple, fast and inexpensive analytical method. This approach uses miniaturized systems both in sample preparation and in the detection stage, helping to develop environmentally friendly analytical methods and portable devices to enable rapid and onsite measurement. The microextraction method is based on a simple metathesis reaction, in which a water-immiscible IL (1-hexyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide, [Hmim][NTf2]) is formed from a water-miscible IL (1-hexyl-3-methylimidazolium chloride, [Hmim][Cl]) and an ion-exchange reagent (lithium bis[(trifluoromethyl)sulfonyl]imide, LiNTf2) in sample solutions. The explosive 2,4,6-trinitrotoluene (TNT) was used as a model analyte to develop the method. The electrochemical behavior of TNT in [Hmim][NTf2] has been studied in SPELs. The extraction method was first optimized by use of a two-step multivariate optimization strategy, using Plackett–Burman and central composite designs. The method was then evaluated under optimum conditions and a good level of linearity was obtained, with a correlation coefficient of 0.9990. Limits of detection and quantification were 7 μg L−1 and 9 μg L−1, respectively. The repeatability of the proposed method was evaluated at two different spiking levels (20 and 50 μg L−1), and coefficients of variation of 7 % and 5 % (n = 5) were obtained. Tap water and industrial wastewater were selected as real-world water samples to assess the applicability of the method.