Eva Baldrich

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.

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.

Using magnetic beads and signal amplifiers to produce short and simple immunoassays: Application to MMP-9 detection in plasma samples

Magnetic beads (MB) and signal amplifiers, such as horseradish peroxidase polymers (poly-HRP), have been used before for the production of highly sensitive immunoassays. However, most of the examples reported previously entailed long and tedious multi-step procedures, which were not necessarily shorter or simpler than classical paths such as Enzyme-Linked Immunosorbent Assay (ELISA). Here, instead of exploiting the combination of MB and poly-HRP to ameliorate sensitivity, we show that they conform a powerful tool that can be used to shorten the incubation times, which allows optimizing extremely simple, fast and efficient immunoassays with minimal technical requirements. In order to do so, here we used the highly sensitive and specific pair of antibodies of a commercial ELISA kit to optimize a magneto-ELISA for the detection of matrix metallopeptidase 9 (MMP-9). Three signal amplifiers were then tested and the best performing one was implemented in the magneto-assay to shorten the incubation times and improve assay performance. As we show, the shortened magneto-assay could be carried out in about 35xa0min, which included two 5-min incubations, washing, and incubation with enzyme substrate for 20xa0min before colorimetric detection. Moreover, the quantification of MMP-9 provided by the shortened assay in 12 plasma samples collected from patients was comparable to that generated by the 5-h ELISA, which was 8.5 times longer.

Detection of plasma MMP-9 within minutes. Unveiling some of the clues to develop fast and simple electrochemical magneto-immunosensors

Magnetic beads (MB) have been extensively used to produce sensitive and efficient electrochemical magneto-immunosensors. However, MB effective handling requires training, and MB washing after each incubation step is time consuming and contributes to raise result variability. Consequently, most of the electrochemical magneto-immunosensors reported to date, which entailed relatively long and complex multi-step procedures, would be difficult to carry out at point-of-care (POC) settings or by laypersons. For this reason, here we targeted the development of a simplified detection path, which is fast and simple enough to be operated at a POC setting, sufficiently efficient to provide analyte quantitation comparable to classical diagnostic methods, and dependent on minimal technical requirements to facilitate method global exploitation. As a proof-of-concept, we optimized an extremely simple, fast and efficient electrochemical magneto-immunosensor for detection of matrix metalloproteinase 9 (MMP-9). To accomplish this, we optimized MB immunomodification, produced an immunomodified Poly-HRP signal amplifier, developed a single-step magneto-immunoassay, and optimized electrochemical detection using a multiplexed magnetic holder and a ready-to-use commercial substrate solution. The sensor was finally calibrated by detecting MMP-9 in clinical samples. This electrochemical magneto-immunosensor detected MMP-9 in just 12-15u202fmin, displaying linear response between 0.03 and 2u202fngu202fmL-1 of MMP-9, limits of detection (LOD) and quantification (LOQ) of 13u202fpgu202fmL-1 and 70u202fpgu202fmL-1, respectively, %CV<u202f6%, and accurate quantification of MMP-9 in patient plasma samples. These results were comparable to those afforded by a 5-h reference ELISA that used the same antibodies, confirming the applicability of our simplified method.

NaNO3/NaCl Oxidant and Polyethylene Glycol (PEG) Capped Gold Nanoparticles (AuNPs) as a Novel Green Route for AuNPs Detection in Electrochemical Biosensors

Gold nanoparticles (AuNPs) have been exploited as signal-producing tags in electrochemical biosensors. However, the electrochemical detection of AuNPs is currently performed using corrosive acid solutions, which may raise health and environmental concerns. Here, oxidant salts, and specifically the environmentally friendly and occupational safe NaNO3/NaCl mixture, have been evaluated for the first time as potential alternatives to the acid solutions traditionally used for AuNPs electrooxidation. In addition, a new strategy to improve the sensitivity of the biosensor through PEG-based ligand exchange to produce less compact and easier to oxidize AuNPs immunoconjugates is presented too. As we show, the electrochemical immunosensor using NaNO3/NaCl measurement solution for AuNPs electrooxidation and detection, coupled to the employment of PEG-capped nanoimmunoconjugates, produced results comparable to classical HCl detection. The procedure developed was next tested for human matrix metallopeptidase-9 (hMMP9) analysis, exhibiting a 0.18-23 ng/mL linear range, a detection limit of 0.06 ng/mL, and recoveries between 95 and 105% in spiked human plasma. These results show that the procedure developed is applicable to the analysis of protein biomarkers in blood plasma and could contribute to the development of more environmentally friendly AuNP-based electrochemical biosensors.