Development Of Quantum Dots-Based Fluorescent Bio/Chem Sensors And Their Applications In Rapid Detection For Food Safety

Food safety concerns everyone in daily life. In recent years, due to the frequent outbreaks of food safety, it has increasingly become a major public health issue. Diseases result from food insecurity have not only caused serious economic losses, but also greatly threaten people’s health. Therefore, rapid, simple, and accurate detection methods are urgently needed for food safety.Biosensors and chemical sensors (bio/chem sensors) are highlighted interdisciplinary areas in both scientific research and engineering technology. Integrated immunology, biology, chemistry with physics, micro-electro-mechanical system (MEMS), electronics, and materials science, they are widely researched and applied in various fields. Bio/chem sensors show many great advantages, such as great selectivity, high accuracy, fast analysis, and simple operation. They are recognized as advanced detection methods and promising diagnostic tools. Among them, fluorescence detection is one type of sensitive and rapid optical techniques. It has been widely used in the development of most optical sensors and instruments.Nanotechnology, originated in 1980s, shows bright and broad application prospects due to its unique physical and chemical properties. The introduction of nanotechnology into the development of biosensors will bring new opportunities and expend bright innovate space. Quantum dots (QDs) as a special luminescent nanomaterial, has been widely used for fluorescent biosensing and cell imaging. It is a semiconductor nanocrystals with unique luminescence properties such as great stability, long fluorescence lifetime, narrow emission spectra, tunable size, and so on.This research work combined nanotechnology with optical sensing technology to develop new bio/chemical sensors for rapid detection in food safety by taking comprehensive advantages of various disciplines to enhance the performance of bio/chem sensors.The main research contents, results and conclusions are as follows:(1) Rapid detection of vegetable cooking oils adulterated with inedible used oil using fluorescence quenching method with aqueous CTAB-coated quantum dotsVegetable cooking oils adulterated with inedible used oils or impure oils have posed a severe food safety problem. Current detection methods cannot meet the needs for rapid detection of adulterated oils on line or in field. Therefore, the objective of this research was to develop a rapid optical sensing method based on fluorescence quenching of CdSe/ZnS quantum dots (QDs) for identification of adulterated vegetable cooking oils. High quality hydrophilic photoluminescent nanoparticles were synthesized by encapsulating hydrophobic QDs into the micellar structure of an amphiphilic surfactant cetyltrimethyl ammonium bromide (CTAB) via phase transfer method. TEM, AFM, and fluorescence spectroscopy were used to characterize the prepared CTAB-coated QDs. Oil samples were first captured into the core of the two-layer-structural micelle and then the fluorescence of CTAB-coated QDs, working as fluorescence probes, was selectively quenched by components of the adulterated oils. Heavy metal ions and free radicals were presumed to be main quenchers. After quenching for 1 min, fluorescence intensity was measured and converted to quenching percentage to determine the adulteration concentration. The results showed that in comparison with oil-soluble QDs, water-soluble CTAB-coated QDs had a greater ability to identify oil adulteration. A good quantitative relationship between quenching percentage and adulteration concentration (y=5.96x+14.99; R2=0.94) was obtained. The sensitive, simple and low-cost sensing method did not require sample pretreatment and could detect refined used oils at 0.4% or higher concentrations in soybean oil within 2 min, showing great potentials for rapid screening of used oils and quantitative analysis of used oil adulteration in field.(2) A target-responsive and size-dependent hydrogel aptasensor embedded with QD fluorescent reporters for rapid detection of avian influenza virus H5N1A target-responsive hydrogel based fluorescent aptasensor was developed for rapid detection of avian influenza virus (AIV) H5N1. A specific aptamer against H5N1 was selected and two single-stranded DNAs (ssDNAi and ssDNA2) were designed to be partially complementary to two ends of the aptamer. Both aptamer and ssDNA1 were functionalized with acrydite at 5’-terminal for hydrogel synthesis by polymerization. Quantum dots (QDs), used as fluorescence reporters, were labeled at 5’-terminal of ssDNA2 and quenchers for QDs were conjugated at 3’-terminal of the aptamer. Hybridization between aptamer and ssDNAs formed the crosslinker to complete the QD-aptamer hydrogel. Initially, without targets, the prepared hydrogel remained in a shrunken state because of the crosslinking in the polymer network and the QD was quenched. With target AIVs, the crosslinking was dissociated due to the binding reaction between the aptamer and target, resulting in the abrupt swelling of the hydrogel and the release of aptamer-quencher and ssDNA2-QD. The response of aptamer upon target binding was demonstrated by quartz crystal microbalance (QCM) method and the microstructure of hydrogel was characterized by SEM. Spectrophotometric results indicated fluorescent changes at the emission peak were correlated to the titers of H5N1. The total detection time from sampling to results was 30 min. The lowest detection limit was 0.2 HAU with a detecting range of 2-2.3 to 26 HAU 20 μL-1. The size-dependent property of this aptasensor was examined quantitatively and qualitatively. This simple, low-cost, and specific aptasensor has great potential for the in-field rapid detection of AIV H5N1.(3) A fluorescent aptasensor coupled with nanobead-based immunomagnetic separation for simultaneous detection of four foodborne pathogenic bacteria in foods and its in-field applicationsThere is a growing need for rapid detection of multiple foodborne pathogens. The objective of this study was to develop an aptasensor for rapid, sensitive, specific, quantitative, and simultaneous detection of Escherichia coli (E. coli) O157:H7, Staphylococcus aureus (S. aureus), Listeria monocytogenes (L. monocytogenes), and Salmonella Typhimurium (S. Typhimurium) in food using magnetic nanobeads (MNBs) for separation and quantum dots (QDs) as fluorescence reporters. Streptavidin-coated 25 nm MNBs, conjugated with four corresponding biotin-labeled antibodies, respectively, were used to simultaneously capture and magnetically separate four bacterial pathogens from the food matrix in 45 min. Streptavidin-coated QDs with emission wavelengths of 528,572,621, and 668 nm, conjugated with four corresponding biotin-labeled aptamers, were used to label the separated MNB-cell complexes. The fluorescence intensities of all reporting QDs in the MNB-cell-QD complexes were measured simultaneously with a portable spectrometer for quantitation of four different types of bacterial cells. SEM and confocal microscopy were used for characterization of the binding between nanobeads, QDs, and bacterial cells and a simulation model was used to analyze the magnetic separation. Results showed for E. coli O157:H7, S. aureus, L. monocytogenes, and S. Typhimurium, the capture efficiencies of antibodies with 25 nm MNBs were 90.4%,87.5%,92.0%, and 92.0%, respectively. The limits of detection for E. coli O157:H7, S. aureus, L. monocytogenes, and S. Typhimurium were 80,100,47, and 160 CFU mL-1 in pure culture and 320,350,110, and 750 CFU mL-1 in ground beef, respectively. The developed aptasensor was capable of simultaneously detecting four bacteria within 2.5 h in a broad range of 101-104 CFU mL-1, showing great potential in multiplex detection of more other foodborne pathogens.In addition, multiple common pathogenic bacteria (E. coli O157:H7, L. monocytogenes, and S. Typhimurium) in foods were detected in field to verify the feasibility of a portable fluorescent biochemical rapid analyzer developed by the collaborators. Magnetic beads were conjugated with three types of specific antibodies for simultaneously capturing the targets in ground beef, lattice and shrimp in a magnetic field. QDs with three different emission wavelengths were conjugated with three different antibodies respectively as fluorescent labels to three different target pathogens which then can be simultaneously detected. Using this instrument, the total detection time from sampling to results was less than 60 min. Therefore, this technology could realize fast and in field detection of multiple foodborne pathogens in meat, vegetables, and aquatic products during logistics and sales, facilitating in-line monitoring and early warning of the outbreaks of foodborne diseases and effectively improve the management of food safety.