Development of biosensors for detection of bacterial toxins and contaminants in complex food matrices

• Main Author: Klisara, Nevena
• Other Authors: Bo Liedberg
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2020
• Subjects: Engineering::Materials
• Online Access: https://hdl.handle.net/10356/137293
• Institution: Nanyang Technological University

This study attempts to merge novel sampling strategies and biomolecular sensing tools for the development of next-generation of rapid and sensitive bioassays for the detection of bacterial toxins and contaminants in complex food matrices. State of the art sampling, extraction and biosensor technologies are combined to develop prototype assays for onsite detection of toxins/proteases in contaminated food products. Considering the vast number of foodborne diseases and caused deaths, the assay for the fast and reliable analysis could assist in the early detection and subsequent prevention of foodborne illnesses. The conventional methodologies are tedious and cumbersome with requirements for trained personnel and expensive instruments. Thus, there is an urgent need to develop an on-site assay to meet requirements such as low cost, fast response, selective and sensitive with no need for sophisticated instrumentations. In this study, emphasis will be on botulinum neurotoxin (BoNT) as a model target for the development of assays, one of the most toxic substances known to man (lethal dose for humans 3-10 ng/kg). Several outbreaks of infant botulism in Australia, Italy, UK and Ireland due to contaminated dairy products and other food-botulism outbreaks have raised demands for efficient diagnostic tools to detect the toxin or the toxin-producing clostridia spores. The focus of this thesis was to develop assays where efforts emphasized on the use of synthetic peptide as recognition molecule which can achieve sensing performance sufficient enough to be utilized in the food safety monitoring and overcome limitations of the gold standard assay – the mouse bioassay. On the other hand, utilizing the peptide as a substrate provides easy and more precise detection, a higher degree of robustness and stability compared to conventional approaches using antibodies. The sensing strategy is based on observing the change in fluorescent/colorimetric response due to the reduction in the number of reporter molecules (in solution or on the membrane) upon the cleavage of peptide by targeted protease. As for assaying in foods, the sample preparation plays a crucial role in successful target detection, especially at low concentration levels, therefore the magnetic beads were used as facilitators in the sample clean-up process due to their easy manipulation by the external magnetic field. Utilizing the peptide and magnetic beads, two assays were developed. In the first assay – the solution-based assay – the reduction in the fluorescence intensity upon cleavage was observed depicting the LOD of 0.5 - 1 nM (25-50 ng/mL) in ~6h of total assay time. In order to simplify the assay by excluding the instrument for the signal read-out and unstable fluorescence dye, the solution-based assay was translated to dipstick assay with naked-eye detection. In this assay, magnetic beads served as mediators in the clean-up process as well as reporter molecules simplifying the overall assay performance. The dipstick assay yielded LOD of 1 nM (50 ng/mL). Further improvements in assay sensitivity, a reduction of overall assay time with a more simple testing approach was accomplished with the development of sorbent incorporated membrane-based and bulb/tip clean-up integrated within dipstick assay. In this alternative approach, the dipstick assay offered clean-up and detection as one step, and better sensitivity (0.1 nM; 5 ng/mL) thanks to the stable fluorescence nanobeads. A potential application of sorbents integrated dipstick test across a wide range of foods was proven by testing liquid, solid and canned food in this study. Assays responses for BoNT/A protease met requirements for analytically relevant levels (below that of oral toxicity; 1 µg/kg) in food matrices. Developed assays offer the avenue for excluding conventional long and tedious sample pre-treatments with utilization of magnetic beads and/or sorbents. The proposed assays deliver new approaches for on-site food safety monitoring capable of overcoming threats related to accidental contamination in the food supply chain due to improper handling of food products as well as from deliberate threats as terrorist attacks.