New system for the detection of Legionella pneumophila in water samples.

Waterborne pathogens are a global concern for public health worldwide. Despite continuing efforts to maintain water safety, water quality is still affected by deterioration and pollution. Legionella pneumophila colonizes man-made water systems and can infect humans causing Legionnaire's disease (LD), pneumonia. The prevention of LD is a public health issue and requires specific systems to control and detect these microorganisms. Culture plate is the only technique currently approved, but requires more than 10 days to obtain results. A rapid test that inform in hours about the presence of Legionella pneumophila in water samples will improve the control of this pathogen colonization. In order to control colonization by L. pneumophila we developed a membrane filter method to capture and immunodetect this microorganism in water samples. This membrane filter is used to retain the bacteria using a nitrocellulose disc inside a home-made cartridge. Subsequently we perform the immunodetection of the bacteria retained in the nitrocellulose (blocking, antibody incubation, washings and developing). On comparing our test with the gold-standard, the most important finding is the considerably reduction in time maintaining the same detection limit. This rapid test is easily automated for L. pneumophila detection allowing a comprehensive surveillance of L. pneumophila in water facilities and reducing the variability in the analyses due to the low need for manipulation. Moreover, corrective measures may be applied the same day of the analysis. This method considerably reduces the detection time compared with the conventional, gold-standard detection culture method that requires more than 10 days, being decisive to prevent outbreaks.

Antibody test for Legionella pneumophila detection.

Legionella pneumophila is responsible for Legionnaires' disease (LD). Its detection in both environmental and clinical samples is mainly performed by culture plate method which requires up to 10days to obtain results. Nowadays, there are commercial antibodies against this bacterium, but they have not been tested against all subgroups of L. pneumophila sg 1 or serogroups 1-16 or their cross-reactions with other non-Legionella bacteria. Indeed, many of these antibodies became available when only 8 serogroups of L. pneumophila had been described. We tested 7 antibodies and found that 2 (Mab 8/5 and OBT) specifically detected all the subgroups of L. pneumophila sg 1, one without cross-reactions (Mab8/5). Moreover, the LP3IIG2 antibody detected almost all serogroups tested with lower rates of cross-reactivity, resulting in a specific sensitive antibody for the detection of L. pneumophila. LP3IIG2 presented higher rate of cross-reactivity against respiratory non-Legionella isolates, thereby contraindicating its clinical applicability.

Impedimetric antimicrobial peptide-based sensor for the early detection of periodontopathogenic bacteria.

Peri-implantitis, an inflammation caused by biofilm formation, constitutes a major cause of implant failure in dentistry. Thus, the detection of bacteria at the early steps of biofilm growth represents a powerful strategy to prevent implant-related infections. In this regard, antimicrobial peptides (AMPs) can be used as effective biological recognition elements to selectively detect the presence of bacteria. Thus, the aim of the present study was to combine the use of miniaturized and integrated impedimetric transducers and AMPs to obtain biosensors with high sensitivity to monitor bacterial colonization. Streptococcus sanguinis, which is one of the most prevalent strains in the onset of periodontal diseases, was used as a model of oral bacteria. To this end, a potent AMP derived from human lactoferrin was synthesized and covalently immobilized on interdigitated electrode arrays (IDEA). X-ray photoelectron spectroscopy (XPS) and electrochemical impedance spectroscopy (EIS) were employed to optimize and characterize the method of immobilization. Noteworthy, the interaction of Streptococcus sanguinis with AMP-coated sensors provoked significant changes in the impedance spectra, which were univocally associated with the presence of bacteria, proving the feasibility of our method. In this regard, the developed biosensor permits to detect the presence of bacteria at concentrations starting from 10(1) colony forming units (CFU)mL(-1) in KCl and from 10(2) CFUmL(-1) in artificial saliva. Moreover, the system was devoid of cytotoxicity for human fibroblasts. These results indicate that the proposed approach can be effective in the detection of initial stages of biofilm formation, and may be useful in the early prevention and treatment of peri-implantitis.

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.

Design and fabrication of a COP-based microfluidic chip: chronoamperometric detection of Troponin T.

This work demonstrates the design and fabrication of an all cyclo-olefin polymer based microfluidic device capable of capturing magnetic beads and performing electrochemical detection in a series of gold electrodes. The size of chip is of a microscope slide and features six independent measuring cells for multianalyte detection purposes. The aim of this work is to show that rapid prototyping techniques can be instrumental in the development of novel bioassays, particularly in clinical diagnosis applications. We show the successful determination of troponin-T, a cardiac disease marker, in the clinically relevant range of 0.05-1.0 ng/mL. This methodology achieves a detection limit of 0.017 ng/mL in PBS solutions, and is capable of detecting less than 1 ng/mL in a 1:50 human serum dilution.

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.

Electrochemical detection of quorum sensing signaling molecules by dual signal confirmation at microelectrode arrays.

n-Acyl homoserine lactones (AHLs) are produced by gram-negative bacteria to regulate gene expression in a cell density dependent manner. For instance, expression of virulence factors by pathogens such as Pseudomonas aeruginosa is induced only when a threshold concentration of AHLs is reached, which indicates that the bacterial population is big enough to promote infection. In this study, the indicator strain Agrobacterium tumefaciens NTL4 (pZLR4), which carries a ß-galactosidase (ß-gal) reporter gene under the control of a quorum sensing promoter, was used to develop an electrochemical biosensor to detect AHLs using the model n-(3-oxo)-dodecanoyl-L-homoserine lactone (oxo-C12-HSL), an AHL previously detected in cystic fibrosis patients infected with P. aeruginosa. The substrate 4-aminophenyl ß-D-galactopyranoside was used to detect ß-gal activity by cyclic voltammetry. Furthermore, simultaneous monitoring of substrate consumption and p-aminophenol production by ß-gal allowed on-chip result verification by dual-signal confirmation. The sensor exhibited high reproducibility and accurately detected oxo-C12-HSL in a low picomolar to low nanomolar range in spiked liquid cultures and artificial saliva, as well as AHLs naturally released by P. aeruginosa in culture supernatants. Moreover, detection took just 2 h, required no sample pretreatment or preconcentration steps, and was easier and faster than traditional methods.

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.

Integration of a zero dead-volume PDMS rotary switch valve in a miniaturised (bio)electroanalytical system.

This work features the design, fabrication and characterisation of a miniaturised electroanalytical lab on a chip that allows the performance of a complete bioassay, from the capture of magnetic particles through their functionalisation and sample incubation to the detection of electroactive reaction products. The system is built using mainly polymeric materials such as PMMA and PDMS and fast prototyping techniques such as milling and moulding. The system also includes a set of microelectrodes, photo-lithographed on a silicon chip. The novelty lies in the design of the rotary microvalve, which contains a microreactor so that various reaction and incubation steps can be carried out in isolation from the detection event with zero dead volume. This avoids contamination and fouling of the electrodes by proteins or other organic matter, and extends the useful lifetime of the detector. The system operation is demonstrated by a model example, consisting in the functionalisation of streptavidin-coated magnetic particles with biotinylated beta-galactosidase over periods ranging from 5 to 15 min, at which point the particles saturate. Although the system is intended for the development of enzyme-based electrochemical bioassays, the concept of its rotary microreactor can be applied more broadly.

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.

Why 'the bigger the better' is not always the case when utilising microelectrode arrays: high density vs. low density arrays for the electroanalytical sensing of chromium(VI).

We demonstrate, with the example of the electroanalytical sensing of chromium(vi) using ultra-microelectrode arrays, that a larger number of microelectrodes comprising an array do not necessarily provide improved electroanalytical performance. Using a low density array, which consists of 256 microdiscs where each microdisc comprising the array has a radius of 10 microns in a cubic arrangement separated from their nearest neighbour by 100 microns, the electroanalytical sensing of chromium(vi) is shown to be possible over the range 13-428 microM with a limit of detection of 3.4 microM readily achievable. Using a high density microelectrode, consisting of 2597 microdiscs where each microdisc has a radius of 2.5 microns in a hexagonal pattern which are separated from their nearest neighbour by 55 microns, the electroanalytical performance, in terms of linear range and sensitivity, is considerably lower going against the misconception that a high density array should produce a superior analytical response. The reason for this disparity is discussed and it is shown that the arrangement of the microelectrodes on the array is critical due to the interaction of diffusion zones between neighbouring electrodes allowing analysts to make informed decisions on the conscientious choice of microelectrode arrays.

Captavidin: a new regenerable biocomponent for biosensing?

Functionalisation of a biosensor's sensing surface with the appropriate biorecognition elements is essential for the correct performance of the biosensor. In this paper, we investigate by Surface Plasmon Resonance (SPR) if captavidin, a recently described biotin-binding regenerable protein, could be used to bind and release biotinylated biocomponents for the development of regenerable biosensors. In our experiments, biotinylated antibodies were successfully subjected to up to nine serial capture-release events from the captavidin-functionalised surface. Up to three consecutive captures were possible when a protein target had been subsequently added. On the other hand, biotinylated bacteria were also efficiently captured and released from the gold surface, suggesting also the suitability of captavidin for the development of whole-cell regenerable biosensors. Our results indicate that captavidin is a promising regenerable molecular tool that could be used during biosensor optimisation and validation, and that captavidin-modified surfaces could be fine-tuned into truly reusable sensors.

Microarrays of ring-recessed disk electrodes in transient generator-collector mode: theory and experiment.

The fabrication, characterization, and use of arrays of ring-recessed disk microelectrodes are reported. These devices are operated in generator-collector mode with a disk acting as the generator and the ring as the collector. We report experiments and simulations relating to time-of-flight experiments in which material electrogenerated at a disk is diffusionally transported to the ring. Analysis of the current transient measured at the latter when it is potentiostatted at a value to ensure diffusionally controlled "collection" is shown to sensitively reflect the diffusion coefficients of the species forming the redox couple being driven at the generator electrode. The method is applied to the ferrocene/ferrocenium couple in the room temperature ionic liquid [N(6, 2, 2, 2)][NTf(2)], and the results are found to agree with independent measurements.

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.

Investigating the concept of diffusional independence. Potential step transients at nano- and micro-electrode arrays: theory and experiment.

Microelectrode arrays find broad application in electroanalysis offering the enhanced sensitivity associated with microelectrodes, but with a high total current output. Such arrays are often constructed to make the electrodes 'diffusionally independent'. To emphasize that this is a time dependent property, a two-dimensional simulation, in conjunction with the diffusional domain approach, is used to model potential step transient currents at microelectrode arrays. Two types of array, hexagonal and cubic, are considered. In both cases the absolute (not relative) microelectrode separation distance has a significant effect on transient current. Three different regimes of transient current versus time can be observed at microelectrode arrays. At short times the transient response of isolated microelectrodes is seen, then at intermediate times the steady-state response of independent electrodes can be observed. At longer times planar diffusion to the entire array takes over. It follows that only at timescales corresponding to the first two regimes can the electrodes be considered as diffusionally independent. To verify the theory the potential step experiment is performed at a regularly spaced hexagonal iridium microdisk array. Theory is found to be in a good agreement with the experimental results.

Sensing bacteria but treating them well: determination of optimal incubation and storage conditions.

Bacteria detection in real samples often involves long and tedious methodologies such as culture enrichment, biochemical screening, and serological confirmation. In this context, the development of biosensors and quick assays for bacteria detection appears as fast growing fields. However, a detailed study of reports in these areas reveals the existence of important differences in bacteria storage, handling, and detection conditions, indicating that authors do not take advantage of the well-established procedures existing for classical techniques such as enzyme-linked immunosorbent assay (ELISA). In the current work, we exploit standard ELISA methodology to identify and study diverse parameters that can be critical along the different steps of bacteria detection and sensing. Among others, we studied in detail the effect of the bacterial strain used and the presence of detergent and glycerol in assay performance, as well as the effects of heat inactivation or storing conditions, on bacteria integrity and thus detectability. Finally, we describe the use of "ready-to-use" frozen bacterial pellets as an excellent alternative to the use of daily prepared fresh cultures during assay optimization and preparation of calibration standards. The results presented are also supported by an extensive bibliography search, giving shape to an important compilation of information that will be useful to authors working in a variety of methodologies and sensing formats.

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.

Enzyme shadowing: using antibody-enzyme dually-labeled magnetic particles for fast bacterial detection.

Using magnetic particles and immunoseparation for target recovery and detection has been reported to improve the performance and detection limits of traditional analytical methods. For example, magnetic immunocapture can be coupled to detection in a sandwich format using an antibody (Ab) labeled with a reporter molecule or enzyme. In this work we demonstrate that simultaneous incorporation of capture and reporter biocomponents onto the sensing surface is possible and provides assays that are extremely fast and easy to carry out. As a proof of concept, we have produced dually-labeled magnetic particles, simultaneously functionalized with antibody and reporter enzyme. Subsequent capture of Escherichia coli generates a shadowing effect on the particle surface and interferes with the activity of a number of enzyme units. The decrease in signal recorded is proportional to the bacterial concentration and is specific for the target microorganism, with a detection limit of 10(3)-10(4) cell mL(-1) in an assay of about one hour.

Immunofunctionalisation of gold transducers for bacterial detection by physisorption.

Functionalisation of the sensing surface is a key factor in immunosensor fabrication as it allows target-selective capture and prevents nonspecific adsorption of undesired components. Gold immunofunctionalisation using self-assembled monolayers (SAM) has been widely exploited to this end for the detection of small targets. However, we recently demonstrated that this strategy fails when detecting whole bacteria cells (Baldrich et al., Anal Bioanal Chem 390:1557-1562, 2008). We now investigate different physisorption-based alternatives using E. coli as the target organism. Our results demonstrate that physisorption generates the appropriate substrate for the specific detection of bacteria on gold surfaces, providing detection limits down to 10(5) cells mL(-1) in an ELISA-type colorimetric assay. Additionally, surface coverage is highly reproducible when assayed by impedance spectroscopy and the inter- and intra-assay coefficients of variation are below 10-15% in all cases. These surfaces were stable, retained functionality and did not suffer from significant biomolecule desorption after 10 days storage in PBS at 37 degrees C, hence confirming physisorption as a cheap, simple and efficient strategy for the detection of bacteria.

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.