Development of biosensors for the detection and quantification of small molecules and micro-organisms

This Ph.D. research includes two tightly-connected projects that describe biosensor developments for food safety applications: - Selection of aptamers for the detection of biogenic amines in seafood. - Development of a biomimetic sensor for the detection of Campylobacter in poultry cecal samples. Selection and characterization of histamine-binding aptamers. Histamine is an indicator of seafood freshness and the causative agent of scombroid food poisoning. Outbreaks of histamine poisoning are associated with the consumption of spoiled fish products that contain high amounts of histamine. Individuals with low activity of histamine-metabolizing enzymes are more susceptible to scombroid food poisoning. Traditional analytical techniques for detecting histamine are based on accurate, but time-consuming and laborious, chromatographic techniques. Biosensors are a promising tool for routine seafood analysis since they can offer rapid on-site detection. The objective of this study is to develop aptamers as bio-recognition elements that can bind specifcally to histamine. For this purpose, we used a magnetic beads-based SELEX. In preparation for SELEX (Systematic evolution of ligands by exponential enrichment), the amino acid L-histidine (which is the precursor of histamine) was immobilized on NH2-terminated magnetic beads through EDC crosslinking. The immobilization was verifed using FT-IR ATR spectroscopy. To enhance the ability of the selected aptamers to operate in a complex environment, the selection process was also performed in the presence of a food matrix. Two SELEX types were performed: A SELEX was done in binding buffer (SELEX No. 1), incubating the random oligonucleotides with histamine-coated beads. Then, a matrix SELEX was performed containing tuna extract as food matrix (SELEX No. 2). SELEX No. 1 resulted in the selection of two aptamers (HAPT 1 and HAPT 2), whereas SELEX No. 2 resulted in the selection of 20 aptamers (HAPT 2 - HAPT 21). The aptamer (HAPT 2) that appeared in both SELEXs was chosen for further characterization. Post-SELEX characterization was carried out to determine the affinity and selectivity of HAPT 2. The selection of histamine-binding aptamers is the first step toward the fabrication of histamine-detecting biosensors. Development of a biomimetic sensor for the detection of Campylobacter in poultry cecal samples. Campylobacter is responsible for an enteric foodborne disease called campylobacteriosis that has complications, such as toxic megacolon, sepsis, or Guillain-Barré syndrome. Humans acquire the bacterium from animals that act as a reservoir. The primary sources of infection concern food products of animal origin, mainly poultry that is poorly handled or undercooked. Traditionally, the detection of Campylobacter is a 2 - 4 days cultivation process that necessitates to have an appropriate laboratory infrastructure. However, the development of biomimetic sensors is advancing fast, aiming to create sensors for rapid on-site identification and quantification of pathogens. In the absence of early detection, the presence of Campylobacter can lead to the rapid spread to the bacterium, posing a severe risk to public health. This project focuses on the development of a biomimetic sensor for the detection of Campylobacter coli and Campylobacter jejuni. The sensor combines surface imprinted polymers (SIPs) as biomimetic receptors with the heat-transfer method for the label-free detection of the bacteria. The SIPs were embossed with surface cavities complementary to the shape and size of Campylobacter. We performed dose-response measurements on Campylobacter-imprinted SIPs and we observed a concentration-dependent increase of the sensor response. The detection limit was calculated at 1×103 CFU/ml for C. coli and 6.6 × 103 CFU/ml for C. jejuni. The selectivity and inclusivity are two important sensor performance features. We assessed the inclusivity of Campylobacter-imprinted SIPs using different strains of the same Campylobacter species. The SIPs demonstrate remarkably high selectivity at species level. We tested the selectivity of Campylobacter-imprinted SIPs using different bacterial species that belong to the Order of Campylobacterales. The sensor response showed that the Campylobacter SIPs are inclusive, meaning that different strains of the same species can bind to them. Furthermore, we accomplished the detection of C. coli and C. jejuni in chicken cecal droppings suspended in phosphate-buffered saline (PBS). The limit of detection in those suspensions was 1.1 × 103 for C. coli and 26.5 × 103 CFU/ml for C. jejuni. The developed biomimetic sensor is applicable for the detection of pathogens in real samples.

Publication year:2022

Accessibility:Open