Rapid Detection of Legionella pneumophila in Drinking Water, Based on Filter Immunoassay and Chronoamperometric Measurement.

Legionella is a pathogenic bacterium, ubiquitous in freshwater environments and able to colonise man-made water systems from which it can be transmitted to humans during outbreaks. The prevention of such outbreaks requires a fast, low cost, automated and often portable detection system. In this work, we present a combination of sample concentration, immunoassay detection, and measurement by chronoamperometry. A nitrocellulose microfiltration membrane is used as support for both the water sample concentration and the Legionella immunodetection. The horseradish peroxidase enzymatic label of the antibodies permits using the redox substrate 3,3',5,5'-Tetramethylbenzidine to generate current changes proportional to the bacterial concentration present in drinking water. Carbon screen-printed electrodes are employed in the chronoamperometric measurements. Our system reduces the detection time: from the 10 days required by the conventional culture-based methods, to 2-3 h, which could be crucial to avoid outbreaks. Additionally, the system shows a linear response (R2 value of 0.99), being able to detect a range of Legionella concentrations between 101 and 104 cfu·mL-1 with a detection limit (LoD) of 4 cfu·mL-1.

Development of an integrated method of concentration and immunodetection of bacteria.

The microbial quality of water is a key aspect to avoid environmental and public health problems. The low pathogen concentration needed to produce a disease outbreak makes it essential to process large water volumes and use sensitive and specific methods such as immunoassays for its detection. In the present work, we describe the development of a device based on microfiltration membranes to integrate the concentration and the immunodetection of waterborne bacteria. A microfiltration membrane treatment protocol was designed to reduce the non-specific binding of antibodies, for which different blocking agents were tested. Thus, the proof of concept of the microbial detection system was also carried out using Escherichia coli as the bacterial pathogen model. E. coli suspensions were filtered through the membranes at 0.5 mL s-1, and the E. coli concentration measurements were made by absorbance, at 620 nm, of the resultant product of the enzymatic reaction among the horseradish peroxidase (HRP) bonded to the antibody, and the substrate 3,3',5,5'-tetramethylbenzidine (TMB). The results showed that the homemade concentration system together with the developed membrane treatment protocol is able to detect E. coli cells with a limit of detection (LoD) of about 100 CFU in 100 mL. Graphical abstract Scheme of the integrated method of concentration and immunodetection of bacteria.

Miniaturized metal oxide pH sensors for bacteria detection.

It is well known that the metabolic activity of some microorganisms results in changes of pH of the culture medium, a phenomenon that can be used for detection and quantification of bacteria. However, conventional glass electrodes that are commonly used for pH measurements are bulky, fragile and expensive, which hinders their application in miniaturized systems and encouraged to the search for alternatives. In this work, two types of metal oxide pH sensors have been tested to detect the metabolic activity of the bacterium Escherichia coli (E. coli). These pH sensors were produced on silicon chips with platinum metal contacts, onto which thin layers of IrOx or Ta2O5 were incorporated by two different methods (electrodeposition and e-beam sputtering, respectively). In order to facilitate measurement in small sample volumes, an Ag/AgCl pseudo-reference was also screen-printed in the chip and was assayed in parallel to an external Ag/AgCl reference electrode. As it is shown, the developed sensors generated results indistinguishable from those provided by a conventional glass pH-electrode but could be operated in significantly smaller sample volumes. After optimization of the detection conditions, the metal oxide sensors are successfully applied for detection of increasing concentrations of viable E. coli, with detection of less than 10(3)cfu mL(-1) in undiluted culture medium in just 5h.

Dual chronoamperometric detection of enzymatic biomarkers using magnetic beads and a low-cost flow cell.

In this work we report on the production of a low cost microfluidic device for the multiplexed electrochemical detection of magneto bioassays. As a proof of concept, the device has been used to detect myeloperoxidase (MPO), a cardiovascular biomarker. With this purpose, two bioassays have been optimized in parallel onto magnetic beads (MBs) for the simultaneous detection of MPO endogenous peroxidase activity and quantification of total MPO. Since the two bioassays produced signals of different magnitude for each concentration of MPO tested, two detection strategies have been compared, which entailed registering steady state currents (Iss) under substrate flow, and measuring the peak currents (Ip) produced in a stopped flow approach. As it will be shown, appropriate tuning of the detection and flow conditions can provide extremely sensitive detection, but also allow simultaneous detection of assays or parameters that would produce signals of different orders of magnitude when measured by a single detection strategy. In order to demonstrate the feasibility of the detection strategy reported, a dual MPO mass and activity assay has been finally applied to the study of 10 real plasma samples, allowing patient classification according to the risk of suffering a cardiovascular event.

Electrochemical biosensing of non-electroactive targets using ferrocene-labeled magnetic particles and CNT wiring.

We show that target binding onto ferrocene-modified magnetic microparticles (MP-Fc) promotes physical sheltering of the labels. This can be measured electrochemically by CNT wiring, which enhances ten-fold the signals registered compared to direct detection of the MPs alone. As a proof of concept, detection of detergents and antibodies is accomplished. In these preliminary experiments, random binding of 0.01% Tween 20 onto MP-Fc was detectable both voltametrically and impedimetrically after a 2 min incubation. Furthermore, affinity capture of 4 µg mL(-1) of biotinylated antibody by streptavidin MP-Fc could be measured in less than 30 min and even in the presence of 1 mg mL(-1) of BSA.

Chronoamperometric magneto immunosensor for myeloperoxidase detection in human plasma based on a magnetic switch produced by 3D laser sintering.

In this work, an amperometric immunosensor for detection of myeloperoxidase (MPO) in human plasma is reported. Detection is based on the immobilization of anti-MPO antibodies onto magnetic beads (MBs). Following MPO immunocapture and washing steps, MBs are transferred to a customized modular detector device produced by 3D laser sintering. This tool integrates electrodes, electrical connectors, and a novel magnetic switch, whose functioning is founded on the vertical displacement of a permanent magnet. In this way, magnetic switching makes possible the confinement of MBs over the working electrode for electrochemical detection, followed by the release of MBs for electrode washing and reutilization. Notably, electrochemical detection is based on the endogenous MPO activity, which reduces reagent consumption and assay time compared to sandwich assays using enzyme-labeled antibodies. After optimization, the assay could be completed in 45 min and displayed a linear response between 0.9 and 60 ng mL(-1) for MPO and a limit of detection of 0.4 ng mL(-1). The real applicability of this approach is demonstrated by the ability to carry out the successful analysis of MPO in human plasma samples. Furthermore, the results allowed the classification of patients into three groups at risk of suffering cardiac events (i.e., low, medium, or high) and correlated well with data provided by a commercially available standardized method.

Voltammetric discrimination of skatole and indole at disposable screen printed electrodes.

In this work, we study the electrochemical behaviour of skatole, one of the compounds responsible for the offensive smell in pork meat that is known as boar taint, at different metal and carbon electrodes. We then demonstrate for the first time that skatole and indole, the main electroactive interferent potentially present in real samples, can be discriminated and separately quantified using cheap and disposable screen printed electrodes (SPE). This implies significant progress compared to the colorimetric method reported by Mortensen in 1983.

Electrochemical detection of testosterone by use of three-dimensional disc-ring microelectrode sensing platforms: application to doping monitoring.

Testosterone is one of the androgenic steroid hormones, the consumption of which is considered doping in most sports. Here, we present powerful 3D sensing platforms using novel disc-ring microelectrode array devices and exploit them for the competitive immunosensing of testosterone. Each device contains a microelectrode array that consists of a large number of individual microdiscs and is used as the substrate for immunofunctionalization and assay performance. One micrometer above it, a second microelectrode array, this time consisting of microrings, is used as the working electrode for electrochemical monitoring. The physical separation of these two functions allows the incorporation of relatively thick biocomponent layers during immunofunctionalization of the microdiscs without negatively affecting electrochemical detection at the rings. Moreover, it permits electrochemical activation of the latter immediately before substrate addition and hence enables optimal electrode performance. The optimized assay showed a linear range between 0.01 and 10 ng/mL and a limit of detection of 12.5 pg/mL testosterone with detection times of 45 min.

Improved bacteria detection by coupling magneto-immunocapture and amperometry at flow-channel microband electrodes.

This paper describes the first immunosensing system reported for the detection of bacteria combining immunomagnetic capture and amperometric detection in a one-step sandwich format, and in a microfluidic environment. Detection is based on the electrochemical monitoring of the activity of horseradish peroxidase (HRP), an enzyme label, through its catalysis of hydrogen peroxide (H(2)O(2)) in the presence of the mediator hydroquinone (HQ). The enzymatic reaction takes place in an incubation micro-chamber where the magnetic particles (MPs) are confined, upstream from the working electrode. The enzyme product is then pumped along a microchannel, where it is amperometrically detected by a set of microelectrodes. This design avoids direct contact of the biocomponents with the electrode, which lowers the risk of electrode fouling. The whole assay can be completed in 1h. The experiments performed with Escherichia coli evidenced a linear response for concentrations ranging 10(2)-10(8) cell ml(-1), with a limit of detection of 55 cells ml(-1) in PBS, without pre-enrichment steps. Furthermore, 100 cells ml(-1) could be detected in milk, and with negligible interference by non-target bacteria such as Pseudomonas.

Amperometric detection of Enterobacteriaceae in river water by measuring ß-galactosidase activity at interdigitated microelectrode arrays.

Two simple methodologies are compared for the detection of faecal contamination in water using amperometry at gold interdigitated microelectrodes. They rely on the detection of ß-galactosidase (ß-gal) by redox cycling amperometry of the p-aminophenol (PAP) produced by the enzyme from the 4-aminophenyl ß-d-galactopyranoside (PAPG) substrate. The use of phages as specific agents for the release of the bacteria-enclosed enzyme allowed the detection of 6×10(5) CFU mL(-1)Escherichia coli in 2 h without any pre-enrichment or preconcentration steps. Better limits of detection were achieved for the second strategy in the absence of phages. In this case, bacteria were enriched in the presence of both ß-d-1-thiogalactopyranoside (IPTG) and substrate but in the absence of phages. Under such experimental conditions, 5×10(4) CFU mL(-1) E. coli could be detected after 2 h of incubation, while 7 h of incubation were enough to detect down to 10 CFU mL(-1) in river water samples. This represents a straightforward one-step method for the detection of faecal contamination that can be conducted in a single working day with minimal sample manipulation by the user.