Ciara K. O'Sullivan

Novel electrochemical immunosensors for seafood toxin analysis

The current work describes the optimisation of a screen-printed electrode (SPE) system for measurement of a variety of seafood toxins, such as okadaic acid, brevetoxin, domoic acid and tetrodotoxin. A disposable screen-printed carbon electrode coupled with amperometric detection of p-aminophenol at +300 mV vs. Ag/AgCl, produced by the label, alkaline phosphatase, was used for signal measurement. ELISA was primarily used to develop all toxin systems, prior to transferring to SPE. The sensors incorporate a relevant range for toxin detection, by which humans become ill, with detection limits achieved at SPE to the order of ng ml (-1) (ppb) or lower in some cases. The SPE system is simple and cost-effective due to their disposable nature, and analysis time is complete in 30 min. In addition, analyses can be achieved outside of a laboratory environment allowing for in-field measurements. Recovery experiments on selected toxins using the relevant working ranges highlighted the functionality of these systems yielding a +/-10% deviation for the true value.

Electron Permeable Self-Assembled Monolayers of Dithiolated Aromatic Scaffolds on Gold for Biosensor Applications

Self-assembled monolayers (SAMs) of thiolated compounds are formed by the spontaneous chemisorption of thiolate groups on metal surfaces. In biosensors, they are most commonly used to covalently immobilize a biorecognition molecule onto the surface of the transducer, thus offering the possibility of controlling the orientation, distribution, and spacing of the sensing element while reducing nonspecific interactions. In this paper, self-assembled monolayers of dithiolated derivatives of 3,5-dihydroxybenzyl alcohol containing carboxyl and hydroxyl end groups have been prepared on gold surfaces and characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Impedance measurements revealed that SAM formation is essentially completed after 3-5 h of exposure by observing the successive blocking of the faradic response of ferricyanide anion due to the adsorption of the dithiol molecules. The surface coverage of these molecules, estimated by reductive desorption experiments, was in the range of (1.1-2.8) x 10-10 mol/cm2. To demonstrate the potential of the dithiol SAM, a model system for detection of a tumor marker, prostate-specific antigen (PSA), was developed. The carboxyl groups of the SAM were succinimide-activated, and an anti-PSA antibody was covalently immobilized via amide bonds. The modified SAM was used for the label-free detection of prostate-specific antigen using EIS with a detection limit of 9 ng/mL. The results described here demonstrate that this kind of dithiol-modified SAM can be used as supports in electrochemical biosensors and the results are explained in terms of the structural features of these dithiols.

Electrochemical immunosensor for detection of celiac disease toxic gliadin in foodstuff.

Celiac disease is a gluten-sensitive enteropathy that affects as much as 1% of the population. Patients with celiac disease should maintain a lifelong gluten-free diet, in order to avoid serious complications and consequences. It is essential to have methods of analysis to reliably control the contents of gluten-free foods, and there is a definitive need for an assay that is easy to use, and can be used on site, to facilitate the rapid testing of incoming raw materials or monitoring for gluten contamination, by industries generating gluten-free foods. Here, we report on the development of an electrochemical immunosensor exploiting an antibody raised against the putative immunodominant celiac disease epitope, for the measurement of gliadin content and potential celiac toxicity of a foodstuff. To develop the gliadin immunosensor, we explored the use of two surface chemistries, based on the use of dithiols, 22-(3,5-bis((6-mercaptohexyl)oxy)phenyl)-3,6,9,12,15,18,21-heptaoxadocosanoic acid (1) and 1,2-dithiolane-3-pentanoic acid (thioctic acid) (2), for anchoring of the capture antibody. The different surface chemistries were evaluated in terms of time required for formation of self-assembled monolayers, stability, susceptibility to nonspecific binding, reproducibility, and sensitivity. The thioctic acid self-assembled monolayer took more than 100 h to attain a stable surface and rapidly destabilized following functionalization with capture antibody, while the heptaoxadocosanoic acid surface rapidlyformed (less than 3 h) and was stable for at least 5 days, stored at room temperature, following antibody immobilization. Both surface chemistries gave rise to highly sensitive immunosensors, with detection limits of 5.5 and 11.6 ng/mL being obtained for 1 and 2, respectively, with nonspecific binding of just 2.7% of the specific signal attained. The immunosensors were extremely reproducible, with RSD of 5.2 and 6.75% obtained for 1 and 2 (n = 5, 30 ng/mL), respectively. Finally, the immunosensor was applied to the analysis of commercial gluten-free and gluten-containing raw and processed foodstuffs, and excellent correlation achieved when its performance compared to that of an ELISA.

Aptamer conformational switch as sensitive electrochemical biosensor for potassium ion recognition

We report the first use of an electrochemical aptasensor for selective potassium recognition, based on a conformational change, affording an electric signal transduced electrochemically by square wave voltammetry or electrochemical impedance spectroscopy.

Amperometric immunosensor for detection of celiac disease toxic gliadin based on Fab fragments.

Immunosensor sensitivity is strongly dependent on the density of free active epitopes per surface area, which could be achieved via well-oriented immobilization of antibody fragments as bioreceptor molecules. Here, we report on the development of an electrochemical gliadin immunosensor based on the spontaneous self-assembly of antigliadin Fab fragments (CDC5-Fab) on Au surfaces. The analytical performance of this immunosensor is compared with a similar containing whole CDC5 antibodies previously modified with thiol groups (CDC5-SH) as the recognition element. Fab fragments were generated by reduction of the disulfide bond of F(ab)(2) fragments obtained by bromelain digestion of CDC5 antibody. Surface plasmon resonance (SPR) was used to evaluate the degree of immobilization and recognition ability of immobilized CDC5-Fab and CDC5-SH on gold surfaces. The studied surface chemistries were evaluated in terms of time required for SAM formation, stability, susceptibility to nonspecific interactions, and sensitivity using surface plasmon resonance, electrochemical impedance spectroscopy (EIS), and amperometry. CDC5-Fab formed a stable monolayer on gold after 15 min and retained >90% of antigen recognition ability after 2 months of storage at 4 degrees C. Detection of gliadin of Fab modified electrodes was evaluated by impedance and amperometry. Labeless impedimetric detection achieved a LOD of 0.42 microg/mL while the amperometric immunosensor based on Fab fragments showed a highly sensitive response with an LOD of 3.29 ng/mL. The Fab based immunosensor offers the advantages of being highly sensitive, easy, and rapid to prepare, with a low assay time.

Layer-by-Layer Biomolecular Assemblies for Enzyme Sensors, Immunosensing, and Nanoarchitectures

Abstract Layer-by-Layer (LbL) assemblies are multi-modular constructions created on solid substrate mainly via electrostatic interactions between polyanions and polycations. The LbL approach can be used for the inter-layer immobilization of biomolecules with enhanced properties for biocatalytic and immunosensing applications, and to create nanoarchitectures for drug encapsulation and release systems. This LbL approach is generic and provides a strategy to rationally design the properties of immobilized films. The following review reports on the LbL state-of-the-art, characterization techniques, applications in biosensing, and nanoarchitectures.

DNA Aptamers against the Lup an 1 Food Allergen

Using in vitro selection, high affinity DNA aptamers to the food allergen Lup an 1, ß-conglutin, were selected from a pool of DNA, 93 bases in length, containing a randomised sequence of 49 bases. ß-conglutin was purified from lupin flour and chemically crosslinked to carboxylated magnetic beads. Peptide mass fingerprinting was used to confirm the presence of the ß-conglutin. Single stranded DNA was generated from the randomised pool using T7 Gene 6 Exonuclease and was subsequently incubated with the magnetic beads and the captured DNA was released and amplified prior to a further round of Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Evolution was monitored using enzyme linked oligonucleotide assay and surface plasmon resonance. Once a plateau in evolution was reached, the isolated DNA sequences were cloned and sequenced. The consensus motif was identified via alignment of the sequences and the affinities of these sequences for immobilised ß-conglutin were determined using surface plasmon resonance. The selected aptamer was demonstrated to be highly specific, showing no cross-reactivity with other flour ingredients or with other conglutin fractions of lupin. The secondary structures of the selected aptamers were predicted using m-fold. Finally, the functionality of the selected aptamers was demonstrated using a competitive assay for the quantitative detection of ß-conglutin. . Future work will focus on structure elucidation and truncation of the selected sequences to generate a smaller aptamer for application to the analysis of the Lup an 1 allergen in foodstuffs.

Amperometric sensing of ascorbic acid using a disposable screen-printed electrode modified with electrografted o-aminophenol film

Electrode modification by electrochemical reduction of diazonium salts of different aryl derivatives is useful for catalytic, analytical and biotechnological applications. A simple and sensitive method for the electrocatalytic detection of ascorbic acid using disposable screen-printed carbon electrodes modified with an electrografted o-aminophenol film, via the electrochemical reduction of its in situ prepared diazonium salts in aqueous solution, is presented. The performance of two commercial SPEs as substrates for grafting of diazonium films has been compared and the grafting process optimized with respect to deposition time and diazonium salt concentration, with the modified surfaces being characterised using cyclic voltammetry. The functionalised screen-printed electrodes demonstrated an excellent electrocatalytic activity towards the oxidation of ascorbic acid shifting the overpotential from 298 and 544 mV to 160 and 244 mV, respectively vs. Ag/AgCl. DC amperometric measurements were carried out for the quantitative determination of ascorbic acid using the modified electrodes. The catalytic oxidation peak current was linearly dependent on the ascorbic acid concentration in the range of 2-20 microM, with a correlation coefficient 0.998, and a limit of detection of 0.86 microM was obtained with an excellent reproducibility (RSD% = 1.98, n = 8). The functionalised screen-printed electrodes exhibited notable surface stability, and were used as a simple and precise disposable sensor for the selective determination of ascorbic acid.

Detection of Antigliadin Autoantibodies in Celiac Patient Samples Using a Cyclodextrin-Based Supramolecular Biosensor

Celiac disease is a condition associated with the ingestion of gluten by genetically susceptible individuals. Measurement of serum antigliadin antibodies is a diagnostic tool also used as a means of monitoring a patients compliance to a gluten-free diet. In this work, we demonstrate the applicability of an electrochemical supramolecular platform based on cyclodextrin-modified gold surfaces to detect antigliadin antibodies in real serum samples. Several support layer-biorecognition element combinations were tested in order to maximize the electrochemical response, and the assay was optimized in terms of incubation times and resistance to nonspecific interactions. The developed supramolecular biosensor was then applied to the amperometric detection of antigliadin IgA and IgG autoantibodies in real samples of celiac disease patients under follow-up treatment; the results were compared with a commercial enzyme linked immunosorbent assay (ELISA) test, and an excellent correlation was observed between both methods.

Design and testing of a packaged microfluidic cell for the multiplexed electrochemical detection of cancer markers

We present the rapid prototyping of electrochemical sensor arrays integrated to microfluidics towards the fabrication of integrated microsystems prototypes for point‐of‐care diagnostics. Rapid prototyping of microfluidics was realised by high‐precision milling of polycarbonate sheets, which offers flexibility and rapid turnover of the desired designs. On the other hand, the electrochemical sensor arrays were fabricated using standard photolithographic and metal (gold and silver) deposition technology in order to realise three‐electrode cells comprising gold counter and working electrodes as well as silver reference electrode. The integration of fluidic chips and electrode arrays was realised via a laser‐machined double‐sided adhesive gasket that allowed creating the microchannels necessary for sample and reagent delivery. We focused our attention on the reproducibility of the electrode array preparation for the multiplexed detection of tumour markers such as carcinoembryonic antigen and prostate‐specific antigen as well as genetic breast cancer markers such as estrogen receptor‐α, plasminogen activator urokinase receptor, epidermal growth factor receptor and erythroblastic leukemia viral oncogene homolog 2. We showed that by carefully controlling the electrode surface pre‐treatment and derivatisation via thiolated antibodies or short DNA probes that the detection of several key health parameters on a single chip was achievable with excellent reproducibility and high sensitivity.