Francesc Xavier Muñoz

Detection of Escherichia coli and Salmonella typhimurium using interdigitated microelectrode capacitive immunosensors: The importance of transducer geometry

This paper presents an immunosensing system to detect Escherichia coli and Salmonella based on electrochemical impedance spectroscopy at interdigitated electrode structures. Our results show the importance of good electrode design in the final detection limit. Four different structures have been fabricated and functionalized. Biotinylated polyclonal antibodies have been immobilized on neutravidin-coated chips, and BSA has been used to avoid nonspecific adsorption. The immunosensor may be said to be capacitive since it is that part of the impedance used to monitor the presence of bacteria in phosphate buffer solution samples. Detection limits around 10(4)-10(5) cells mL(-1) have been reached using chips featuring interdigitated structures of less than 10 microm wide and 1.5 mm long. In both cases, the detection limits of the corresponding ELISA assays, using the same antibodies, was 1 order of magnitude higher (10(5)-10(6) cells mL(-1)). The analysis time, including sensor preparation was less than 5 h.

On-Chip Electric Field Driven Electrochemical Detection Using a Poly(dimethylsiloxane) Microchannel with Gold Microband Electrodes

An external electric field driven in-channel detection technique for on-chip electrochemical detection in micro fabricated devices is described based on a microfluidic system containing an array of 20 microband electrodes. It is shown that an external electric field induces a potential difference between two gold microband electrodes in a poly(dimethylsiloxane) (PDMS) microchannel, and that this enables the electrochemical detection of electroactive species such as ascorbic acid and Fe(CN) 6 (4-). The results, which are supported by simulations of the behavior of the microband electrodes in the microfluidic system, show that the induced potential difference between the electrodes can be controlled by altering the external electric field or by using different microbands in the microband array. As the obtained currents depend on the concentrations of electroactive species in the flowing solution and the detection can be carried out anywhere within the channel without interference of the external electric field, the present approach significantly facilitates electrochemical detection in capillary electrophoresis. This approach consequently holds great promise for application in inexpensive portable chip-based capillary electrophoresis (CE) devices.

Magnetic entrapment for fast, simple and reversible electrode modification with carbon nanotubes: Application to dopamine detection

Carbon nanotubes (CNT) have been exploited for an important number of electroanalytical and sensing purposes. Specifically, CNT incorporation to an electrode surface coating increases its roughness and area, provides electrocatalytic activity towards a variety of molecules, and improves electron transfer. This modification is generally based on the irreversible deposition of CNT on surface. Nevertheless, CNT are highly porous materials that might promote molecule non-specific adsorption and/or electrodeposition, which could induce sample-to-sample cross-contamination and affect measurement specificity and reproducibility. This drawback has been often circumvented by combining CNT with charged polymers able to repel molecules of opposed charge. We demonstrate that single-walled CNT (SWCNT) have a strong tendency to non-specifically adsorb onto the surface of protein-coated magnetic particles (MP). Magnetic capture of those MP generates CNT coentrapment and allows extremely fast, simple and reversible production of SWCNT electrodes. We have exploited this phenomenon for the production of modified screen-printed electrodes (MP/CNT-SPE), which have been characterized by Scanning Electron Microscopy. The surface has been additionally optimized by evaluating the electrochemical performance of SPE modified with different amounts and proportions of MP and CNT. The modified devices have then been used for dopamine detection. MP/CNT-SPE generated improved assay sensitivity, lower limit of detection, and up to 500% higher current signals than bare electrodes. Magnetic entrapment is proposed as a promising strategy for the fast, simple and reversible generation of nanostructured electrodes of enhanced performance within a few minutes and electrode re-utilisation by simple magnet removal and surface washing.

Transient storage of electrical charge in biofilms of Shewanella oneidensis MR-1 growing in a microbial fuel cell.

Current output of microbial fuel cells (MFCs) depends on a number of engineering variables mainly related to the design of the fuel cell reactor and the materials used. In most cases the engineering of MFCs relies on the premise that for a constant biomass, current output correlates well with the metabolic activity of the cells. In this study we analyze to what extent, MFC output is also affected by the mode of operation, emphasizing how discontinuous operation can affect temporal patterns of current output. The experimental work has been carried out with Shewanella oneidensis MR-1, grown in conventional two-chamber MFCs subject to periodic interruptions of the external circuit. Our results indicate that after closure of the external circuit, current intensity shows a peak that decays back to basal values. The result suggests that the MFC has the ability to store charge during open circuit situations. Further studies using chronoamperometric analyses were carried out using isolated biofilms of Shewanella oneidensis MR-1 developed in a MFC and placed in an electrochemistry chamber in the presence of an electron donor. The results of these studies indicate that the amount of excess current over the basal level released by the biofilm after periods of circuit disconnection is proportional to the duration of the disconnection period up to a maximum of approximately 60 min. The results indicate that biofilms of Shewanella oneidensis MR-1 have the ability to store charge when oxidizing organic substrates in the absence of an external acceptor.

Membraneless glucose/O2 microfluidic enzymatic biofuel cell using pyrolyzed photoresist film electrodes

Biofuel cells typically yield lower power and are more difficult to fabricate than conventional fuel cells using inorganic catalysts. This work presents a glucose/O2 microfluidic biofuel cell (MBFC) featuring pyrolyzed photoresist film (PPF) electrodes made on silicon wafers using a rapid thermal process, and subsequently encapsulated by rapid prototyping techniques into a double-Y-shaped microchannel made entirely of plastic. A ferrocenium-based polyethyleneimine polymer linked to glucose oxidase (GOx/Fc-C6-LPEI) was used in the anode, while the cathode contained a mixture of laccase, anthracene-modified multi-walled carbon nanotubes, and tetrabutylammonium bromide-modified Nafion (MWCNTs/laccase/TBAB-Nafion). The cell performance was studied under different flow-rates, obtaining a maximum open circuit voltage of 0.54 ± 0.04 V and a maximum current density of 290 ± 28 μA cm(-2) at room temperature under a flow rate of 70 μL min(-1) representing a maximum power density of 64 ± 5 μW cm(-2). Although there is room for improvement, this is the best performance reported to date for a bioelectrode-based microfluidic enzymatic biofuel cell, and its materials and fabrication are amenable to mass production.

Gold immuno-functionalisation via self-assembled monolayers: Study of critical parameters and comparative performance for protein and bacteria detection

Surface functionalisation is of extreme importance in assay and biosensor development because it ensures the selective capture and detection of the targets of interest. In the present report, we compare the performance of several gold functionalisation strategies/chemistries, based on SAM self-assembly and Ab conjugation, for protein and bacteria detection. The first part of the work summarises the optimisation of the various protocols considered. Their efficiency was initially evaluated in terms of reduction of biomolecule non-specific adsorption and specific detection competence impairment, using as a model-target an enzyme-labelled protein. With this purpose, the effect of several parameters, such as thiomolecule length and concentration, self-assembly time and temperature, polymer incorporation, or Ab conjugation strategy was determined. The three best performing strategies consisted of antibody (Ab) conjugation to self-assembled monolayers (SAM) containing mercaptoundecanoic acid alone, or conjugated to either long-chain hydrophilic diamines or CM-dextran. In the three cases, results demonstrated that Abs had been successfully incorporated and remained functional for protein detection. Nevertheless, as showed in the second part of the work, we demonstrate for the first time that these chemistries can be inadequate for bacteria detection. The possible reasons and implications will be discussed. Ab physisorption is proposed as a cost-effective gold immuno-functionalisation strategy alternative to SAM-based Ab incorporation for bacteria detection.

Biosensing at disk microelectrode arrays. Inter-electrode functionalisation allows formatting into miniaturised sensing platforms of enhanced sensitivity

Biosensor performance depends on the effective functionalisation of a transducer with suitable biorecognition elements. During functionalisation, surface blocking steps are normally carried out to avoid later binding of undesirable molecules and thus guarantee biosensor specificity. However, these blocking steps may be deleterious in electrochemical systems where transduction ultimately relies on electron transfer between the electrode and a redox species in solution. This work presents a novel approach to develop improved amperometric biosensing platforms using microfabricated disk microelectrode arrays, based on the functionalisation of the inert surface surrounding the active microdisks. These devices more than doubled assay sensitivity compared to conventional biosensors produced using the same arrays. This approach benefits from three advantages: the functionalisation of a broader surface, the possibility to activate the microelectrodes immediately before detection, and access to enhanced rates of mass transport to microelectrodes that improve device sensitivity. To demonstrate this, we first studied the electrochemical behaviour of tetramethylbenzidine (TMB) at gold disk microelectrode arrays, and then used TMB as the redox mediator for the amperometric biosensing of HRP/H(2)O(2). Down to 0.54pM H(2)O(2) or as little as 25pM HRP were detected within 5s of enzyme activity in just 10 microl of enzyme substrate solution. We postulate that microelectrode arrays may be used to develop novel electrochemical biosensing platforms that are faster and more sensitive than conventional biosensors.

Surface plasmon resonance assay for real-time monitoring of somatic coliphages in wastewaters.

ABSTRACT The surface plasmon resonance (SPR) technique is a well-established method for the measurement of molecules binding to surfaces and the quantification of binding constants between surface-immobilized proteins and proteins in solution. In this paper we describe an extension of the methodology to study bacteriophage-bacterium interactions. A two-channel microfluidic SPR sensor device was used to detect the presence of somatic coliphages, a group of bacteriophages that have been proposed as fecal pollution indicators in water, using their host, Escherichia coli WG5, as a target for their selective detection. The bacterium, E. coli WG5, was immobilized on gold sensor chips using avidin-biotin and bacteriophages extracted from wastewater added. The initial binding of the bacteriophage was observed at high concentrations, and a separate, time-delayed cell lysis event also was observed, which was sensitive to bacteriophage at low concentrations. As few as 1 PFU/ml of bacteriophage injected into the chamber could be detected after a phage incubation period of 120 min, which equates to an approximate limit of detection of around 102 PFU/ml. The bacteriophage-bacterium interaction appeared to cause a structural change in the surface-bound bacteria, possibly due to collapse of the cell, which was observed as an increase in mass density on the sensor chip. These results suggest that this methodology could be employed for future biosensor technologies and for quantification of the bacteriophage concentration.

Carbon Nanotube Wiring: A Tool for Straightforward Electrochemical Biosensing at Magnetic Particles

Here, we combine the unique properties of carbon nanotubes (CNTs) and magnetic particles (MPs) to develop a novel biosensing approach for the specific detection of electroactive labels and targets. The assay is based on label/target capture and concentration using MPs. It follows addition of CNTs, which adsorb onto the surface of the beads. The subsequent magnetic entrapment of the CNT/MP complexes onto an electrode allows straightforward electrochemical sensing of the MP surface by exploiting CNT wiring. As a proof of concept, the assay has been applied to detection of ferrocene labels, and to the specific immunodetection of dopamine in both artificial saline solutions and real sample matrixes. The results demonstrate the applicability of CNT as wiring tools for enzymeless and substrateless electrochemical biosensing.

Fast electrochemical detection of anti-HIV antibodies: Coupling allosteric enzymes and disk microelectrode arrays

Here a novel electrochemical method for the rapid detection of anti-HIV antibodies in serum is presented. The novelty lies in the combination of allosteric enzymes and coulometry to yield a fast, simple and reliable HIV diagnostic method. We have used a previously developed beta-galactosidase enzyme that is efficiently activated by anti-HIV antibodies directed against a major B-cell epitope of the gp41 glycoprotein. When these antibodies bind the enzyme, the 3D conformation changes positively affecting the performance of the active site and, consequently, the enzyme activity is stimulated. Using 4-aminophenyl beta-D-galactopyranoside (PAPG) as substrate yields p-aminophenol (PAP), which is reversibly oxidised at a very mild potential, ca. 0.37 V vs. Ag/AgCl over a range of electrode materials within the working pH range of beta-galactosidase. In the present case, photolithographically produced microelectrode arrays resulted in a detection limit of 4 microM for 4-aminophenol (PAP). The presence of anti-HIV antibodies results in enzyme activity increases above 50% which, combined with the sensitivity and response time afforded by the microelectrode arrays, allowed for the diagnosis of HIV in sera samples within an hour.