Electrical monitoring of infection biomarkers in chronic wounds using nanochannels.

Chronic wounds represent an important healthcare challenge in developed countries, being wound infection a serious complication with significant impact on patients' life conditions. However, there is a lack of methods allowing an early diagnosis of infection and a right decision making for a correct treatment. In this context, we propose a novel methodology for the electrical monitoring of infection biomarkers in chronic wound exudates, using nanoporous alumina membranes. Lysozyme, an enzyme produced by the human immune system indicating wound infection, is selected as a model compound to prove the concept. Peptidoglycan, a component of the bacterial layer and the native substrate of lysozyme, is immobilized on the inner walls of the nanochannels, blocking them both sterically and electrostatically. The steric blocking is dependent on the pore size (20-100 nm) and the peptidoglycan concentration, whereas the electrostatic blocking depends on the pH. The proposed analytical method is based on the electrical monitoring of the steric/electrostatic nanochannels unblocking upon the specific degradation of peptidoglycan by lysozyme, allowing to detect the infection biomarker at 280 ng/mL levels, which are below those expected in wounds. The low protein adsorption rate and thus outstanding filtering properties of the nanoporous alumina membranes allowed us to discriminate wound exudates from patients with both sterile and infected ulcers without any sample pre-treatment usually indispensable in most diagnostic devices for analysis of physiological fluids. Although size and charge effects in nanochannels have been previously approached for biosensing purposes, as far as we know, the use of nanoporous membranes for monitoring enzymatic cleavage processes, leading to analytical systems for the specific detection of the enzymes has not been deeply explored so far. Compared with previously reported methods, our methodology presents the advantages of no need of neither bioreceptors (antibodies or aptamers) nor competitive assays, low matrix effects and quantitative and rapid analysis at the point-of-care, being also of potential application for the determination of other protease biomarkers.

Direct competitive immunosensor for Imidacloprid pesticide detection on gold nanoparticle-modified electrodes.

A direct competitive immunosensor for the electrochemical determination of Imidacloprid (IMD) pesticide on gold nanoparticle-modified screen-printed carbon electrodes (AuNP-SPCE) is here reported for the first time. Self-obtained specific monoclonal antibodies are immobilized on the AuNP-SPCE taking advantage of the AuNPs biofunctionalization abilities. In our biosensor design, free IMD in the sample competes with IMD conjugated with horseradish peroxidase (IMD-HRP) for the recognition by the antibodies. After that, 3,3',5,5'-Tetramethylbenzidine (TMB) is enzymatically oxidized by HRP, followed by the oxidized TMB reduction back at the surface of the SPCE. This process gives an associated catalytic current (analytical signal) that is inversely proportional to the IMD amount. The main parameters affecting the analytical signal have been optimized, reaching a good precision (repeatability with a RSD of 6%), accuracy (relative error of 6%), stability (up to one month), selectivity and an excellent limit of detection (LOD of 22 pmol L-1), below the maximum levels allowed by the legislation, with a wide response range (50-10000 pmol L-1). The detection through antibodies also allows to have an excellent selectivity against other pesticides potentially present in real samples. Low matrix effects were found when analysing IMD in tap water and watermelon samples. The electrochemical immunosensor was also validated with HPLC-MS/MS, the reference method used in official laboratories for IMD analysis, through statistical tests. Our findings make the electrochemical immunosensor as an outstanding method for the rapid and sensitive determination of IMD at the point-of-use.

Electrical Evaluation of Bacterial Virulence Factors Using Nanopores.

In this work, we propose a novel methodology for electrical monitoring using nanoporous alumina membranes of virulence factors secreted by bacterial pathogens. Bacterial hyaluronidase (HYAL), which is produced by a number of invasive Gram-positive bacteria, is selected as a model compound to prove the concept. Our electrochemical setup takes advantage of the flat surface of indium tin oxide/poly(ethylene terephthalate) (ITO/PET) electrodes for their assembly with the nanoporous membrane. The proposed analytical method, based on the electrical monitoring of the steric/electrostatic nanochannels blocked upon formation of an antibody-HYAL immunocomplex, reached detection limits as low as 64 UI/mL (17.3 U/mg) HYAL. The inert surface of the ITO/PET electrodes together with the anti-biofilm properties of the 20 nm pore-sized alumina membranes allows for culturing the bacteria, capturing the secreted enzymes inside the nanochannels, and removing the cells before the electrochemical measurement. Secreted HYAL at levels of 1000 UI/mL (270 U/mg) are estimated in Gram-positive Staphylococcus aureus cultures, whereas low levels are detected for Gram-negative Pseudomonas aeruginosa (used as a negative control). Finally, HYAL secretion inhibition by RNAIII-inhibiting peptide (YSPWTNF-NH2) is also monitored, opening the way for further applications of the developed monitoring system for evaluation of the antivirulence potential of different compounds. This label-free method is rapid and cheap, avoiding the use of the time-consuming sandwich assays. We envisage future applications for monitoring of bacterial virulence/invasion as well as for testing of novel antimicrobial/antivirulence agents.

In Situ Plant Virus Nucleic Acid Isothermal Amplification Detection on Gold Nanoparticle-Modified Electrodes.

Solid-phase isothermal recombinase polymerase amplification (RPA) offers many benefits over the standard RPA in homogeneous phase in terms of sensitivity, portability, and versatility. However, RPA devices reported to date are limited by the need for heating sources to reach sensitive detection. With the aim of overcoming such limitation, we propose here a label-free highly integrated in situ RPA amplification/detection approach at room temperature that takes advantage of the high sensitivity offered by gold nanoparticle (AuNP)-modified sensing substrates and electrochemical impedance spectroscopic (EIS) detection. Plant disease ( Citrus tristeza virus (CTV)) diagnostics was selected as a relevant target for demonstration of the proof-of-concept. RPA assay for amplification of the P20 gene (387-bp) characteristic of CTV was first designed/optimized and tested by standard gel electrophoresis analysis. The optimized RPA conditions were then transferred to the AuNP-modified electrode surface, previously modified with a thiolated forward primer. The in situ-amplified CTV target was investigated by EIS in a Fe(CN6)4-/Fe(CN6)3- red-ox system, being able to quantitatively detect 1000 fg µL-1 of nucleic acid. High selectivity against nonspecific gene sequences characteristic of potential interfering species such as Citrus psorosis virus (CPsV) and Citrus caxicia viroid (CCaV) was demonstrated. Good reproducibility (RSD of 8%) and long-term stability (up to 3 weeks) of the system were also obtained. Overall, with regard to sensitivity, cost, and portability, our approach exhibits better performance than RPA in homogeneous phase, also without the need of heating sources required in other solid-phase approaches.