Application Of Carbon Quantum Dots And Silver Nano-based Biosensors For E.coli Detection

Food products are susceptible to contamination by a variety of bacteria,including pathogenic bacteria,during production,processing,transportation or storage.Escherichia coli（E.coli）is considered one of the most dangerous foodborne pathogens.Even at very low concentrations it can cause a variety of diseases（e.g.bloody diarrhea,kidney failure and even death）,and accurate detection of E.coli in food is needed to ensure food quality and safety.The development of a rapid,sensitive and selective method is still a hot issue for E.coli detection.Carbon quantum dots（CQDs）are one of the fluorescent nanoparticles that have been widely used in biosensors in recent years.This is due to the excellent advantages of CQDs,including biocompatibility,low toxicity,easy synthesis,good stability in aqueous solution,and small particle size.CQDs have many hydroxyl and carboxyl groups on their surface and use these functions to achieve binding to other biomolecules.The optical properties of CQDs lead to a wide range of applications of these particles in different fields,such as bioimaging,sensing,drug and gene delivery.This thesis focuses on the preparation of several carbon quantum dots and silver nanoparticles for the specific detection of E.coli using fluorescence resonance energy transfer（FRET）,internal filtration effect（IFE）and aptamer fluorescence,the most popular and facile of fluorescent biosensors.The final results show that the designed sensor has a wide linear range and a low detection limit.The cost reduction and the low detection limit are compared with the previous published papers.The main elements and innovations include the following:（1）In this chapter,carbon quantum dots were prepared from grapefruit peel as carbon source by microwave heating method.The prepared carbon quantum dots CQDs were analyzed by fluorescence spectroscopy,TEM analysis,XPS analysis,etc.,as well as their optical properties were also investigated.The prepared CQDs have high green fluorescence with excitation wavelength of 320 nm and maximum emission wavelength of 415nm.the average particle size is about 7.4 nm,uniform dispersion and good stability.And a FRET（fluorescence resonance energy transfer）based fluorescence method was constructed by combining carbon quantum dots with silver nanoparticles（AgNPs）for the rapid detection of E.coli.Its fluorescence transduction is based on the spectral overlap between the donor（CQDs）emission and the acceptor（nanoparticles）absorbance.The fluorescence of the aptamer-attached CQDs is burst in the presence of and silver nanoparticles.Upon addition of the specific E.coli solution,an aptamer-target complex is formed and the preferential interaction of the aptamer with the specific bacteria leads to the release of CQDs and AgNPs.After a period of incubation,the bacteria were centrifuged,resulting in the precipitation of E.coli aptamer couplers and AgNPs,hence the recovery of CQDs fluorescence.This method allows specific detection of E.coli in a wide range of pathogenic bacteria.The final results showed that the linear range of the sensor was 2×10³～2×10⁸CFU·m L^-1and the detection limit for E.coli was as low as 77 CFU·m L^-1.（2）To detect E.coli in food,a simple fluorescent biosensor based on single-stranded DNA（ss DNA）and carbon quantum dots（CQDs）was developed.The carbon quantum dots were prepared by superhydrothermal method using carrot juice as carbon source and analyzed by fluorescence spectroscopy,X-ray diffraction,infrared spectroscopy,and TEM analysis.The prepared carbon quantum dots have good water dispersion and photostability.ss DNA attachment induced a decrease in fluorescence intensity of CQDs at 445 nm（excitation at 347 nm）.In the presence of E.coli,ss DNA preferentially binds to E.coli through hydrogen bonding and its fluorescence is greater than that in the absence of E.coli.Under the optimized conditions,the results showed that the linear range of the sensor was 1×10²～1×10⁸CFU·m L^-1.The detection limit for E.coli was as low as 60 CFU·m L^-1.（3）Carbon quantum dots were prepared by hydrothermal method using celery juice as carbon source.A series of chemical and optical property analyses were performed on the prepared carbon quantum dots.And the fluorescence of carbon quantum dots（CQDs）was burst by internal filtration effect（IFE）using silver nanoparticles（AgNPs）as absorbers to construct label-free adapters.This strategy relies mainly on the absorption spectra of AgNPs overlapping with the fluorescence excitation or emission of fluorophores and the specific binding ability of E.coli aptamers.Upon addition of E.coli,the free aptamer sequence is first depleted to form some complexes,leading to AgNPs aggregation at high salt concentrations.At this point the absorption spectrum of AgNPs changes and no longer overlaps with the fluorescence emission spectrum of CQDs,leading to a significant fluorescence recovery of the adapted sensor.The excitation wavelength of the prepared carbon quantum dots was 348 nm and the maximum emission wavelength was 514nm.the average particle size was about 8.84 nm with uniform dispersion.the linear range of this sensor was 2×10²～2×10⁷CFU·m L^-1,and the detection limit for E.coli was as low as 185 CFU·m L^-1.