Evaluation of an Ag85B Immunosensor with Potential for Electrochemical Mycobacterium Tuberculosis Diagnostics

Tuberculosis remains a major global health concern, especially in the developing world, and monitoring/early detection of the disease relies on low cost technologies that provide rapid and accurate results. Mycobacterium tuberculosis is the responsible bacterial pathogen and it is currently estimated by the World Health Organisation (WHO), that one quarter of the world’s population, mainly in the developing world, is infected with TB. The overall aim of this work was to advance a screening electrochemical sensor for label free detection of Ag85B, a member of the Antigen 85 complex - major secretary protein of M. Tuberculosisand biomarker for disease. An indirect ELISA Ag85B assay was optimised with capture antibody and antigen levels determined via a checkerboard titration (0.625μg.ml-1 and 2.5 μg.ml-1 respectively). Following assay development, crosslinking of the bioreceptor Anti-Ag85B onto electrochemically deposited gold nanoparticle (AuNP) modified carbon electrodes was achieved and Ag85B binding successfully evaluated electrochemically via cyclic voltammetry. Following each modification step, ΔEp of a redox probe was monitored and overall results show that GCE/AuNP/anti-Ag85B electrochemical transducers are a viable method for Ag85B detection, capable of measuring antigen levels \u3c 2.5 μg.mL-1

Disposable Printed Lateral Flow Electrochemical Immunosensors for Human Cardiac Troponin T

Here we report an electrochemical ELISA approach for cardiac Troponin T (cTnT) determination based on a lateral flow membrane with underlying screen printed electrodes (\u3c;100 Ω /cm 2 ). The thick film transducer was modified with the anti-cardiac Troponin T antibody via physisorption and the electrochemical performance of the immunosensor was evaluated using cyclic voltammetry. The capture antibody coated immunoelectrode employed for electrochemical determination of Troponin T antigen used a sandwich assay format with horseradish peroxidase conjugated signaling antibodies held in the track of a lateral flow sensor strip. A simple two step procedure realized signal acquisition within \u3c;20 min (total assay time). The lateral flow electrochemical immunosensor response resulted in a calibration curve with linear response (0-700 ng/ml cTnT) with limit of detection of 0.15 ng/ml

Design and development of some amperometric biosensors based on enzymes, antibodies and plant tissue

Amperometric biosensors for various drugs and metabolites have been developed, based on immobilised enzymes in both aqueous and organic media. Chapter 1 serves as a general review of the field of biosensors, including both an overview of recognition methods together with the techniques involved in transduction. The remainder of the thesis is divided into four experimental chapters. The first of these describes the development of some amperometric enzyme electrodes for certain drugs and metabolites in aqueous solution. Various immobilisation methods, including use of polymers such as Nafion and Eastman AQ55D, gelatine membranes, entrapment of the enzyme within electropolymerised films, together with direct adsorption, have been employed. Discriminative permselective films were found to show great effectiveness in excluding interfering compounds commonly found in serum. Application to the clinical and in vivo fields are discussed. Limits of detection down to the jiM level were found. Fast wash times permitted application to flowing streams with frequencies of up to 200 samples per hour, with good precision. The second experimental chapter deals with the construction of biosensors for inhibiting compounds in the organic phase. A study of the kinetics of tyrosinase at the rotating disc electrode in non-aqueous media is also described. Reactions which are impossible in aqueous media due to kinetic or thermodynamic constraints become possible in non-aqueous solvents. The recent discovery that biocatalysts can function in extreme environments such as organic solvents has important implications for the implementation of biosensor technology in formerly inaccessible environments. It also extends the number of detectable analytes to include poorly water soluble organic species in the petrochemical, food and environmental areas. Chapter 4 deals with the electrochemistry of the anticancer drug 7-OH-coumarin and the development of an immunosensor for this compound. The final experimental chapter deals with the detection of some metal ions using a plant tissue modified carbon paste electrode, and includes speciation studies of copper

Solution and solid phase electrochemical behaviour of [Os(bpy)3]3[P2W18O62]

[Os(bpy)3]3[P2W18O62] has been synthesised and characterised by elemental analysis,spectroscopic (UV/Vis, IR spectroscopy) and electrochemical techniques. In 0.1M Bu4NPF6DMSO the complex shows a series of redox couples associated with the Os3+/2+ and bipyridine ligands of the cationic [Os(bpy)3]2+ moiety and the tungsten-oxo framework of the associated Dawson parent heteropolyanion, [P2W18O62]6-. At this electrolyte concentration the Os3+ redox form of the complex was seen to adsorb onto the electrode surface. When the electrolyte concentration is lowered to 0.01M Bu4NPF6 in addition to the Os3+/2+ redox couple,the redox process associated with the [P2W18O62]8-/7- couple also exhibited properties indicating surface based processes were present. Electroactive films of the complex were formed on carbon macroelectrodes by the redox switching of the transition metal within the complex. Voltammetric investigations into the film’s behaviour in a range of buffer solutions(pH 2.0, 4.5 and 7.0) were performed. The films were found to possess better stability in acidic pH and the same pH dependence for the tungsten-oxo framework of the heteropolyanions as in solution. Solid state electrochemical measurements on mechanically attached microparticles of the complex were performed, with the effect of both the nature and concentration of the aqueous electrolyte on this behaviour being investigated. Upon redox switching between the Os2+/3+ redox states there is an associated insertion/expulsion of anions from/to the solution phase. Scanning electron micrographs of these solid state films were attained before and after redox cycling

Electrochemical enzyme-linked immunosorbent assay (e-ELISA) for parasitic nematode: Ostertagia ostertagi (brown stomach worm) infections in dairy cattle

A sensitive electrochemical immunoassay (e-ELISA) has been developed for the detection of the gastrointestinal parasitic nematode Ostertagia ostertagi (brown stomach worm) in infected and control serum samples. An antigen-indirect immunoassay format was employed to detect the presence of O. ostertagi antibodies, coupled with an anti-species monoclonal horseradish peroxidase (HRP) conjugate. ABTS (2,2\u27-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) and TMB (3,3\u27,5,5\u27-tetramethylbenzidine/hydrogen peroxide) were investigated as both chromogenic visualising reagents for optical ELISA and electroactive substrates for electrochemical ELISA in the HRP catalysed oxidation reaction. Coulometry was applied for the detection of O. ostertagi antibodies (via TMB electrochemistry) and compared with the commercial optical ELISA (ABTS based SVANOVIR® O. ostertagi-Ab ELISA kit). Cost-effective in-house sensors were designed and fabricated using polyester and chemical adhesive materials with the aid of stencil printing and laser machining techniques. The performance of the electrochemical ELISA and sensor was evaluated by investigating redox mediators (ABTS vs. TMB), stop solutions (sodium dodecyl sulfate vs. sulfuric acid) and incubation times (150 min vs. 70 min vs. 25 min). For a total assay incubation time of 70 minutes, the TMB/H2SO4 based e-ELISA was able to differentiate between positive (P) and negative (N) control serum samples, with a P/N70 control ratio 1.6 times higher than that of optical ELISA (TMB/H2SO4 combination) and 2.9 times higher than that of the commercial ELISA kit (ABTS/SDS combination). Furthermore, the e-ELISA approach is quicker and required only 25 min (total incubation time) with even better response (P/N25 = 14.7), which is approximately 4-fold higher than the optical immunoassay (P/N25 = 3.8). The proposed e-ELISA is specific (selective Ab-Ag interactions) and highly sensitive - capable of detecting up to 16-fold dilutions of a positive control serum sample. The electrochemical ELISA approach has the potential for rapid sample screening in a portable, disposable format, contributing to the quest for effective prevention and control of parasitic Ostertagia ostertagi infections in cattle

An Electrochemical Evaluation of Novel Ferrocene Derivatives for Glutamate and Liver Biomarker Biosensing

Here, we present an evaluation of two new monosubstituted ferrocene (Fc) derivatives, 3-(1H-pyrrol-1-yl)propanamidoferrocene and 1-hydroxy-2-[2-(thiophen-3-yl)-ethylamino]ethylferrocene, as glutamate oxidase mediators, together with their preparation and characterisation. Taking into consideration the influence of the electronic effects of substituents on the redox potentials of the Fc species, two candidates with pyrrole or thiophene moieties were proposed for investigation. Film studies involved potential sweeping in the presence of pyrrole or 3,4-ethylenedioxythiophene monomers resulting in stable electroactive films with % signal loss upon cycling ranging from 1 to 7.82% and surface coverage (Γ) 0.47–1.15 × 10−9 mol/cm2 for films formed under optimal conditions. Construction of a glutamate oxidase modified electrode resulted in second-generation biosensing with the aid of both cyclic voltammetry and hydrodynamic amperometry, resulting in glutamate sensitivity of 0.86–1.28 μA/mM and Km (app) values over the range 3.67–5.01 mM. A follow-up enzyme assay for liver biomarker γ-glutamyl transpeptidase realised unmediated and mediated measurement establishing reaction and incubation time investigations and a realising response over/L γ-glutamyl transpeptidase with a sensitivity of 5 nA/UL−1

Synthesis, co-polymerisation and electrochemical evaluation of novel ferrocene-pyrrole derivatives

New ferrocene derivatives, ((4-(1H-pyrrol-1-yl)phenoxy)carbonyl) ferrocene (4) and (6-(4-(1H-pyrrol-1-yl)phenoxy)hexyl) ferrocene (7) were synthesised, characterised and electrochemically evaluated as redox active films formed via anodic oxidation with pyrrole. Thin film studies were conducted and films formed from both compounds resulted in a stable Fe II/III redox couple with Eo = 0.04V and 0.36V vs. Ag/Ag+ for (7) and (4) respectively. Both potential sweeping and chronocoulometry were employed for film formation with the former resulting in controllable, reproducible film deposition. Growth conditions and solution concentrations were varied in the case of both compounds in order to assess influence on electrochemical behavior. Surface coverage’s were of the order 10-8-10-9 mol cm-2, surface confined behavior (ip vs. n) was evident up to 0.2 V s-1 with semi-infinite diffusion (ip vs. n1/2) dominating at higher scan rates. Laviron theory was employed where possible for the determination of electron transfer co-efficient and rate constants

β-Methylumbelliferone Surface Modification and Permeability Investigations at PENTEL™ Graphite Electrodes

Electrochemical and micro-imaging analysis of a commercial graphite-composite material is presented following electro-oxidation with β-methylumbelliferone. Charge-transfer surface modification was observed for the graphite electrode, presumed to have arisen from adsorbed interfacial umbelliferone moieties. The molecular permeability of the new surface towards a range of similar, yet size-variable (23 Å3–136 Å3) molecular redox probes is discussed. Red-shift fluorescence in confocal microscopy offers further support for the presence of a surface-bound umbelliferone layer. An SEM-platinum profiling technique was used as an imaging tool to map the umbelliferone surface and size-distribution of electroactive sites

Enzymatic doped ionogels - new materials for inherently biocompatible molecular sensors

• The key challenges currently faced in lab-on-a-chip biochemical sensor developments are device reliability and power consumption. • Point-of-care (POC) glucose biosensors play an important role in the management of blood sugar levels in patients with diabetes. Glucose biosensors still account for approximately 85% of the current world market which is estimated to be worth $5 billon. • Ionic liquids (ILs) have evolved as a new type of solvent for biocatalysis, mainly due to their unique and tunable physical properties