Impedance Biosensors for the Rapid Detection of Viral and Bacterial Pathogens Using Avian Influenza Virus Subtypes H5N1 and H7N2 and Escherichia coli O157:H7 as Model Targets

This research investigated impedance biosensors for the rapid detection of viral and bacterial pathogens using avian influenza virus (AIV) subtypes H5N1 and H7N2 and Escherichia coli O157:H7 as the model targets, which were chosen due to their impact on the agricultural and food industries. For the detection of AIV H7N2, a single stranded DNA aptamer was selected using systematic evolution of ligands by exponential enrichment (SELEX). The selected aptamer and a previously selected aptamer against AIV H5N1 were used in a microfluidics chip with an embedded interdigitated array microelectrode to fabricate an impedance biosensor for specific detection of AIV H7N2 and H5N1. The developed label-free biosensor was capable of detecting AIV H7N2 and H5N1 at a concentration down to 27×10-4 hemagglutinination units (HAU) in 30 min without sample pre-treatment, comparable to previously designed biosensors though with the advantage of DNA aptamers. Two impedance biosensors based on the use of screen-printed interdigitated electrodes were developed for the detection of E. coli O157:H7. The first was a label-free biosensor based on magnetic separation and concentration of target bacteria using antibody-labelled magnetic nanobeads and Faradic impedance measurement. It was capable of detecting 1400 cells or more of E. coli O157:H7 in a total detection time of 1 h. COMSOL Multiphysics software was used to analyze the biosensor using a simplified model and determine the role of the magnetic nanobeads in the impedance measurement. The second biosensor for detection of E. coli O157:H7 was based on aptamer-labeled magnetic nanobeads and glucose oxidase/Concanavalin A-coated gold nanoparticle labels. This biosensor was capable of detecting 8 cells or more of E. coli O157:H7 in 1.5 h. The lower detection limit of the developed impedance biosensor was comparable to the most sensitive biosensors published for the detection of E. coli O157:H7 and was also more rapid and more practical for in-field tests. Multiple impedance biosensor designs were developed in this research. The developed biosensor for AIV could conceivably be adapted for detection of other AIV subtypes and the developed E. coli O157:H7 biosensors could easily be adapted to detect different bacterial pathogens

A Bifunctional Nanocomposites Based Electrochemical Biosensor for In-field Detection of Pathogenic Bacteria in Food

This research focused on the application of electrochemical biosensors for the rapid detection of pathogenic bacteria, Escherichia coli O157:H7 and Salmonella Typhimurium, in foods. The possible presence of pathogenic bacteria in foods has always been a great threat to the wellbeing of people and the revenue of food companies. Therefore, the demand for rapid and sensitive methods to detect foodborne pathogens is growing. In this research, an impedimetric immunosensor was first developed for the rapid detection of E. coli O157:H7 and S. Typhimurium in foods. It was based on the techniques of immunomagnetic separation, enzyme labelling, and electrochemical impedance spectroscopy (EIS). This impedimetric immunosensor was capable of specifically detecting E. coli O157:H7 and S. Typhimurium within the range of 102 to 106 colony forming unit (cfu)/ml in the pure culture. The limits of detection (LODs) of E. coli O157:H7 in ground beef and S. Typhimurium in chicken carcass rinse water were 2.05 x 103 cfu/g and 1.04 x 103 cfu/ml, respectively. The second electrochemical biosensor was designed for rapid detection of E. coli O157:H7. This biosensor integrated magnetic GOx-polydopamine (PDA) based polymeric nanocomposites (PMNCs) which served dual functions as both the carrier and the label, and Prussian blue (PB) modified SP-IDMEs for measurement. The core-shell Abs/GOxext/gold nanoparticles (AuNPs)/magnetic beads (MBs)-GOx@PDA PMNCs acted efficiently to get a high load of enzyme onto the surface of bacterial cells. A filtration step separated the free PMNCs from the bonded ones and reduce the background noise to achieve better sensitivity. The constructed biosensor had been proved to be able to detect E. coli O157:H7 with the LOD of 52 cfu/ml in the pure culture. The third electrochemical aptasensor was developed to detect S. Typhimurium based on the concept of the bifunctional nanocomposites. The ssDNA aptamers were used as the biorecognition element. The achieved LOD in the pure culture was 96 cfu/ml. The biosensors developed in this research exhibited good specificity, reproducibility, and easy-to-operate, and are expected to find broad applications in the detection, especially in-field detection, of foodborne pathogens

An Impedimetric Aptasensing System for the Rapid Detection of Salmonella Typhimurium

Salmonella Typhimurium is a foodborne pathogen associated with raw and undercooked eggs, poultry, beef, fruits, and vegetables. In the United States, Salmonella is responsible for approximately 1.2 million illnesses, 23,000 hospitalizations, and 450 deaths annually. For many years, conventional detection methods such as culture-dependent and PCR-based methods have been the “golden standards” for the detection of this pathogen due to their high sensitivity and reliability. However, they still have some disadvantages such as long enrichment steps and high costs that need to be overcome. The development of a rapid and reliable method for the detection of S. Typhimurium is needed due to the significant threat S. Typhimurium poses to public health. The goal of this study was to develop an impedimetric aptasensor for the rapid detection of Salmonella Typhimurium using a system setup from our previous study. In this study, gold interdigitated array microelectrodes were immobilized with NH2-Salmonella Typhimurium aptamers to capture S. Typhimurium cells in pure culture samples. The impedance change caused by the capture of S. Typhimurium cells by the aptamers at the sensor-sample interface was measured in the presence of a redox probe and recorded using a laptop with LabVIEW software. The results showed that there was a linear relationship with a correlation coefficient of 0.93 between the impedance change and the log value of S. Typhimurium in a range of concentrations from 101 to 105 CFU/50 μL in pure culture samples. The total detection time from sampling to results was less than one hour. The developed impedance aptasensor was highly specific to S. Typhimurium. The aptasensor has the potential to be used as a preliminary and rapid preventive stage to isolate samples that may contain S. Typhimurium before being sent for further validation with other conventional methods like microbial plating

Aptamer-based SPR Biosensor for Detection of Avian Influenza Virus

Rapid and specific detection of avian influenza (AI) virus is urgently needed with the concerns over the outbreaks of highly pathogenic H5N1 avian influenza in animal and human infection. Aptamers are artificial oligonucleic acid that can specifically bind to target molecules. They show comparable affinity for target virus and better thermal stability than monoclonal antibodies. Those advantages make aptamers promising candidates in diagnostic and detection applications. The goal of this research was to use DNA&ndashaptamer as the specific recognition element in a portable surface plasmon resonance (SPR) biosensor for detection of AI H5N1 virus in poultry. A SPR biosensor was fabricated using the selected aptamers based on streptavidin&ndashbiotin method. Streptavidin was directly adsorbed onto the surface of a gold waveguide in the SPR biosensor. Then, biotinylated aptamers were immobilized on the sensor surface via streptavidin&ndashbiotin binding. The immobilized aptamers captured AI H5N1 virus in a sample solution, causing an increase in refraction index (RI). Performances of the aptamer&ndashbased SPR biosensor were studied in streptavidin modification, aptamer immobilization and virus detection. The optimal concentrations of streptavidin and aptamers were determined to improve the sensitivity of the biosensor. The response of the aptamer&ndashvirus interaction was shown to be virus titer&ndashdependent, and a linear range for the titers of AI H5N1 was found between 0.128 and 1.28 HA unit. The aptamer&ndashbased SPR biosensor could detect the H5N1 virus at a titer greater than 0.128 HA unit within 1.5 h. No significant interference was observed from non&ndashtarget subtypes such as AI H7N2, H9N2, H2N2, H1N1 and H5N2. The aptamer&ndashbased SPR biosensor was further evaluated for detection of AI virus in poultry swab samples. All of the AI viruses used in this study were killed ones to ensure biological safety

Emerging (Bio)Sensing Technology for Assessing and Monitoring Freshwater Contamination - Methods and Applications

Ecological Water Quality - Water Treatment and ReuseWater is life and its preservation is not only a moral obligation but also a legal requirement. By 2030, global demands will exceed more than 40 % the existing resources and more than a third of the world's population will have to deal with water shortages (European Environmental Agency [EEA], 2010). Climate change effects on water resources will not help. Efforts are being made throughout Europe towards a reduced and efficient water use and prevention of any further deterioration of the quality of water (Eurostat, European Comission [EC], 2010). The Water Framework Directive (EC, 2000) lays down provisions for monitoring, assessing and classifying water quality. Supporting this, the Drinking Water sets standards for 48 microbiological and chemical parameters that must be monitored and tested regularly (EC, 1998). The Bathing Water Directive also sets concentration limits for microbiological pollutants in inland and coastal bathing waters (EC, 2006), addressing risks from algae and cyanobacteria contamination and faecal contamination, requiring immediate action, including the provision of information to the public, to prevent exposure. With these directives, among others, the European Union [EU] expects to offer its citizens, by 2015, fresh and coastal waters of good quality

Development of a low cost biosensing platform for highly sensitive and specific on-site detection of pathogens and infections

A highly sensitive, specific, real time, and field-deployable surveillance tool is critical to the control of pathogens and infections, as well as ecological impact of chemicals exposure. This work investigates the development of a low cost biosensing platform that can be used for viral disease diagnosis and chemical detection. The sensing mechanism is known as AC electrokinetics (ACEK) capacitive sensing. By applying an inhomogeneous AC electric field on sensor electrodes, positive dielectrophoresis is induced to accelerate the travel of analytes. The same applied AC signal also directly measures the capture of target by the probe on sensor surface. The realized sensing platform is not only rapid but also highly sensitive and specific. Built on our initial proof-of-concept of ACEK capacitive sensing, this work studies in details the immobilization of probes on electrode surface, electrode design, the interactions between biomolecules such as nucleic acids and testing buffers, and the effect of dielectrophoresis and accompanying ACEK phenomena. Experimental comparisons are made between sensors with various probe immobilization, different electrode designs, testing buffer and detection protocols. As a result, much higher sensitivity and selectivity have been achieved. We are able to successfully detect virus particles in nasal swab samples, specific antibody in serum and whole genome nuclei acids in serum. To extend the application of this sensing method on other electrode platform, polyimide-based laser printed electrodes are also investigated and successfully demonstrated for small molecule detection. However, this type of sensor exhibits high internal resistance, making it only suitable for chemical or particle detection in highly resistive electrolyte, such as de-ionized water. With procedural and design improvements discussed in this work, it is expected that ACEK capacitive sensing will become a disruptive technology in on-site biochemical detection