Economic losses to agriculture due to pest and pathogen infections are estimated at $40 billion annually and the economic losses to the health care industry due to food borne illnesses are estimated at $15 billion in the U.S. alone. Early detection of pest or pathogen infection in agricultural crops and reliable detection of harmful pathogens in food are important to minimize agricultural productivity loss, ensure food safety, improve food quality and minimize food related public health issues. There is a pressing need to develop rapid, highly selective and sensitive detection technologies for early identification of plant and human pathogens. While a variety of molecular methods are currently being used for this purpose, an inexpensive, high selective, rapid method for the detection of pathogens is highly desired. Electrochemistry biosensors offer unique advantages to this application. Electrochemical sensors have been widely explored for medical and environmental sensing applications, but not as much for food and agricultural applications. An electrochemical biosensor uses a highly selective bio-recognition element such as enzymes, antibody, aptamer or virus and is capable of detecting biding events with ultra-low detection limits. This presentation will focus on some of the recent developments in our lab in the development of electrochemical biosensors for detection of crop diseases and fungal plant pathogens.

Rational design of synthetic peptides has become a critical step during development of functional (cleavage) assays for proteases. In this study we describe a high throughput proteomic methodology to screen for peptides that are effectively and specifically cleaved by the protease. It is based on rationally designed peptide libraries that are screened using Self Assembled Monolayers Desorption Ionization-Mass Spectrometry (SAMDI-MS)¹ to identify potential peptide hits. We start by describing experiments of matrix metalloproteinase-7 (MMP-7)² a potential cancer biomarker. However, the focus is on Omptin proteases present on the surface of E.coli and Salmonella. These membrane proteases, e.g. OmpT and PgtE, serve as a defense mechanism and contribute to the virulence of various pathogenic strains by inactivating/digesting host generated antimicrobial peptides. We demonstrate that the catalytic efficiency (k_cat/K _M) of OmpT targeting LL37, an anti-microbial peptide and a natural substrate to OmpT, can be improved ~400-fold by replacing natural cleavage sequence ARRA with and FRRV identified from a SAMDI-MS screening³. A portion of the FRRV-modified LL37 peptide was subsequently employed in a fluorescent quenching assay enabling detection of OmpT in detergent micelles down to about 2 nM. It is also shown that it is possible detect at least 10⁴ CFU/mL E.coli cells with over expressed levels of OmpT in the membrane as well as wild type E. coli K12 cells. This is fully comparable to the sensitivity levels obtained by commercial culture-independent test kits opening up for rapid detection of pathogenic strains of E.coli and other bacteria.

The quantification of nitrites (NO₂^-) has gained an increasing relevance in biomedicine due to its recognition as a central homeostatic species in the NO physiology, as well as an important signalling molecule. Though, the circulating levels of nitrites in body fluids have been difficult to measure due to sampling issues and the poor performance of common diagnosis methods. In clinical applications, the detection of nitrites in urine is widely used, as a marker of NO production under infection and/or inflammatory conditions. Thus, the implementation of an effective and easy-to-use point-of-care-test (POCT) for quick and effective NO₂^- readings would be surely welcome either in translational medicine or clinical diagnosis. Other markets for nitrites testing exist in the food industry, and pollution control (the main international authorities, for instance, have promulgated rules restricting nitrite concentrations in drinking water and foodstuff). Our research group has been focused on the construction of a POCT for nitrites quantification using enzyme-based electrochemical biosensors. In this communication, the progress made over the last years will be shortly reviewed. Several second generation biosensors using redox mediators were initially proposed. These were followed by several third generation configurations based on a direct electron transfer approach.  Recently, the electrodes system was fully miniaturized using disposable screen-printed electrodes and the proof-of-concept was established. The technology is now mature to start its transference into the market.