Magnetic electrochemical and colorimetric immunoassays for biological and chemical contaminants

The research activity, carried out during my PhD, is focused on the development of magnetic electrochemical and colorimetric immunoassays for biological and chemical contaminants. The common theme of my PhD thesis, devoted to the development and application of sandwich/competitive immunoassays, is the use of magnetic beads (MBs) as a support of an immunological complex. The MBs are micron scale spheres constituted of a dispersion magnetic material (iron oxides) clad with a thin polymer shell which defines their surface area and shields them from iron exposure. Depending on the functional groups, present on their surface (tosyl, amino, carboxyl), the beads can be used to covalently and stably bind a great variety of biological elements (i.e antibody, DNA, etc.). They allow an efficient separation of the target analyte from the sample thanks to the use of simple magnets, giving highly reproducible results. Their spherical shape ensures a larger surface/volume ratio compared to the wells of the ELISA plate. This allows to immobilize a large number of biomolecules on each MB surface, improving the sensitivity of the assay and reducing the reaction times. The first year of my PhD was devoted to demonstrate the applicability of an ELIME (Enzyme-Linked-Immuno-Magnetic-Electrochemical) assay to reveal Salmonella (the major foodborne pathogenic bacterium) in green vegetables. In my previous work, a rapid, low-cost and easy to-use ELIME assay for Salmonella, has been developed and successfully applied to irrigation water, the primary source of vegetable contamination. Outbreaks of salmonellosis have traditionally been linked to the consumption of food products of animal origin (like meat and eggs) but, more recently, an increasing number of outbreaks have been associated with contaminated fruits and vegetables, especially raw leafy green vegetables. To protect consumer’s health, food safety regulations have established a zero tolerance for Salmonella in a defined amount of a given food product (25g for vegetables). The routine method for detecting Salmonella is the standard cultural method (EN/ISO 6579) which is very sensitive and inexpensive, but labor-intensive, extremely time consuming (up to 5 days to obtain results), not suitable for testing a large number of samples and not compatible with the shelf life of vegetables. For these reasons, simple and rapid tests, able to perform multi-sample analyses, are needed. Biosensors potentially provide a powerful means to detect pathogenic bacteria with the advantages of rapidity, sensitivity and specificity. In this contest, my attention has been focused on the developed of an ELIME assay which employs magnetic beads as support of a sandwich immunological chain, coupled with a strip of eight-magnetized screen-printed electrodes localized at the bottom of eight wells (8well/SPE strip), connected with a portable electrochemical detector. The applicability of the ELIME assay to detect Salmonella in vegetables was demonstrated by analyzing 13 uncontaminated Salmonella leafy green vegetables, available on Italian markets, experimentally inoculated with the target bacterium (1-10 CFU/25g), and pre-enriched in two different broths. In parallel, the same samples were analyzed by RTi-PCR method. During my experimental activity great attention was paid to the evaluation of the sample matrix effect and to the selection of the medium broth to be used during the pre-enrichment phase. This phase is necessary to allow the target Salmonella to multiply until reaching a detectable concentration by ELIME and RTi-PCR assays and also to overcome the inability of both techniques to distinguish between living and death cells. A confirmation of the ability of both methods to detect such a low Salmonella concentration, after 20 h (ELIME) and 8 h (RTi-PCR) of pre-enrichment, was performed with the ISO method. This research, made in collaboration with the Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, was published in: Talanta 166 (2017) 321– 327. During the second and third year of my PhD, I have carried out two works at the same time. The first one concerned the development of an ELIME assay for detecting Campylobacter jejuni and Campylobacter coli. Campylobacter genus is the most important cause of acute bacterial diseases in humans worldwide after Salmonella. Many bacterial species in the genus of Campylobacter are considered harmful and may cause several infectious diseases, but the thermophilic species C. jejuni and C. coli are responsible for over 95% of the infections (80-85% C. jejuni and 10-15% C. coli). Conventional bacterial testing methods (ISO 10272-1) rely on specific microbiological media to isolate and enumerate viable bacterial cells in food. These traditional methods are very sensitive and inexpensive, but require several days to generate results because they rely on the ability of microorganisms to multiply to produce visible colonies. Also in this case, rapid and reliable methods, able to perform multi-sample analyses, are required. As the key element of the ELIME assay is the antibody that is specific to antigens located on the surface of the Campylobacter cells, in a preliminary phase of my study, I examined the antigen-antibody interaction by testing with ELISA four commercial antibodies versus pure broth cultures of C. jejuni grown in two different media, evaluating their influence on the antigenic expression. The reactivity of the antibodies to the antigens was also estimated using whole cells C. jejuni and C. coli inactivated with NaN3 and heat killed cells. These ELISA experiments allowed me to establish the best conditions in terms of culture medium, cell treatment and antibodies able to recognize both C. jejuni and C. coli. After that, six combinations of the selected antibodies (unconjugated and conjugated with AP or HRP) were employed to set-up the ELIME assay in which tosylactivated magnetic beads (MBs), used as support of a sandwich immunological complex, were coupled to a strip of 8-magnetized screen-printed electrodes, that act as electrochemical transducers. Each antibody combination, except one (proved to be failed), was used to construct calibration curves for C. jejuni (ATCC 29428) and C. coli (ATCC 33559), previously grown in Bolton Selective Enrichment Broth and then heat treated (100 oC for 5 min). The antibody pair consisting of goat polyclonal antibody anticampylobacter, coated on the surface of MBs, and the same antibody conjugated with AP (to label the captured cells) was chosen for further experiments, given the best response for both Campylobacter species and the lowest detection limit. The selectivity of the ELIME assay, designed so that the two sequential incubations for the two immuno-recognition events occur in a single step of 1 h, was proved by inclusivity and exclusivity test performed analyzing other C. jejuni strains and non target microorganisms. Although promising results were attained, yet research has to demonstrate the applicably of the ELIME assay in different food categories. The paper concerning this work, in collaboration with the Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, is under preparation. The other work, carried out during the second and third year of my PhD, was focused on the development of rapid screening methods (based on the use of MBs) for algal toxin detection in sea water, to be integrated into an automated system. This activity is part of the SMS project (Sensing toxicants in Marine waters makes Sense using biosensors), approved in the EU call “OCEAN 2013”. The project promotes a multidisciplinary scientific approach, involving industrial partners, to develop a platform that will allow a real-time detection of various contaminants such as boat paints, pharmaceutical compounds, toxic algae and their toxins. In the last years, there is a growing worldwide phenomenon: the "Harmful Algal Blooms" (HAB). The main reason for the increased frequency of this phenomenon is constituted by the anthropogenic activity, including excessive nutrient loading from fertilizers or sewage waste, food web alterations, introduced species, water flow modifications, and climate changes. During algal blooms, toxic substances called algal biotoxins can be produced. Marine biotoxins represent a serious hazard, because they can accumulate in fish and mussels and enter the food chain. The detection of marine toxins released by algae has mostly been directed on the analysis of fish/shellfish homogenate rather than seawater samples. To fill this gap, my work was devoted to the re-modeling conventional ELISA kits to set-up non-automated direct competitive ELIMC (Enzyme-Linked Immuno-Magnetic Colorimetric) assays for the detection of Domoic Acid (DA), Okadaic Acid (OA) and Saxitoxin (STX) in seawater. For each toxin, linear working range, sensitivity and detection limit of the ELIMC assays were calculated. After evaluation of seawater matrix effect and recovery study, ELIMC assays were integrated within a novel automated networked system (ASMAT - Analytical System for Marine Algal Toxins) based on the micro Loop Flow Reactor (μLFR) technology, suitable for on-line monitoring of marine toxins. Once established the best operational conditions, ASMAT was calibrated towards DA, OA and STX verifying its capability to detect sub-ppb levels of the target toxins. Analysis of DA, OA and STX in real samples of marine water, sampled in Alonnisos (Greece) were carried out in laboratory using ELIMC assays, ASMAT and ELISA Kits, for a comparative evaluation Finally, on-line suitability of ASMAT as an early warning alarm system was demonstrated by a field installation within a floating platform in the port of La Spezia (Italy).