Mechanism Research Of Sandwich Biosensor For Two Foodborne Pathogens Based On Carbon Quantum Dots And DNA Tetrahedron Signal Amplification

Foodborne pathogens cause a variety of diseases and serious economic risks,which is a public problem concerned.Food,animals,drinking water,and even equipment used to process or transport food and water may all be hosts for foodborne pathogens.Most foodborne pathogens proliferate rapidly,reaching disease-causing levels in a matter of hours.The existing commercial detection methods of foodborne pathogens include culture,immunological,and nucleic acid-based techniques,which have shortcomings such as poor specificity,time-consuming,laborious,and high requirements for professional operators.Therefore,sensitive and rapid detection of foodborne pathogens is needed to ensure food safety and address the global challenges posed by foodborne pathogens.Carbon quantum dots(CDs)have attracted the attention of researchers because of their strengths of simple preparation,favorable fluorescence performance,low toxicity,and good biocompatibility.Aptamers are gradually replacing antibodies as molecular recognition materials in the detection field,due to their more tolerant working,storage,and transportation requirements.However,aptamers may intertwine with each other,affecting their recognition of the target.DNA tetrahedron(Td)formed by DNA self-assembly through base complementary pairing is a candidate The freely editable Td structure has four vertices,which can not only couple the aptamer recognition target,but also couple the CDs,so as to realize the amplification of fluorescence signal.The purpose of this study is to use CDs and combination of Td to build two kinds of signal amplification method on the basis of the existing magnetic separation layer testing design,further improve the detection sensitivity,achieve sensitive detection of foodborne pathogenic bacteria,and applied to the practical analysis of food-borne pathogens in food samples,the selectivity,sensitivity and accuracy of analysis method to evaluate.In this study,a variety of CDs were prepared,a stable Td structure was constructed,and two signal amplification methods were constructed.The main research results are as follows:1.Preparation and characterization of N-CDsIn order to explore the effects of different precursor substances and reaction conditions on nitrogen-doped carbon quantum dots(N-CDs),three N-CDs with different solvents,five N-CDs with different proportions of water and ethanol as solvents,three N-CDs with different p H(p H=2,7,and 12),three N-CDs with different preparation temperatures(120?,160?,and 200?),and four N-CDs with different lengths of time(4-10h)and four N-CDs with different precursors(?-cyclodextrin,p-phenylenediamine(PPDA),citric acid and urea or ammonium acetate)were prepared.Atomic force microscopy(AFM),Fourier transform near-infrared spectroscopy(FT-IR),fluorescence spectrophotometry and ultraviolet spectrum scanning were used to characterize,then the morphological characteristics,structural functions and fluorescence characteristics were analyzed.It was found that the polarity of solvent was related to the formation of surface groups.The N-CD prepared by solvent with large polarity was more likely to generate oxygen-related groups on the surface,which adjusts the band gap width and leads to the red shift of fluorescence wavelength.The increase of p H promoted the aggregation between N-CD and the increase of N-CD particle size,but N-CD did not show size-dependent fluorescence characteristics.When the preparation temperature was too high,the non-radiation trap on N-CD surface led to strong thermal quenching and reduced the fluorescence intensity of N-CD.The introduction of N source greatly improved the fluorescence performance of N-CD.In addition,?-cyclodextrin,PPDA,citric acid and urea or acetate N-CD prepared in the experiment had high biosafety,which lays a theoretical foundation for the future research of non-toxic N-CD in the detection of foodborne pathogens.2.Design,preparation,and optimization of TdIn this work,several Td structures were designed,and their morphology in different preparation conditions and ion concentration may form were predicted through Nupack.Four of the sequence designs with the highest yield in theory were selected and self-assembled,the synthesis Td were verified through the gel electrophoresis,and Td with the highest yield and the most stable structure was obtained by optimizing the synthesis method and buffer solution.The Td structure was successfully designed and constructed to overcome the disordered fixation,entanglement and steric hindrance of the aptamers on the substrate surface.This rigid structure is an ideal identification element for pathogenic microorganisms,which is easier to capture pathogenic microorganisms with biological activity.3.Amplification signal based on single CD and Td was used to sandwich detect Staphylococcus aureusTimely detection of Staphylococcus aureus(St.aureus)is critical because it can multiply to disease-causing levels in a matter of hours.Herein,a simply and sensitive Td fluorescence signal amplifier with nitrogen-doped blue CDs(b CDs)was prepared for sandwich detection of St.aureus.b CD was modified at the apex of Td,and aptamer on Td was used to accurately identify and"adsorb"the amplifier to the surface of St.aureus.The stable triangular structure can not only anchor the aptamers firmly but also distance the aptamers from each other,preventing them from being too close.AFM demonstrated the successful preparation of this signal amplifier.Cytotoxicity test results showed that the cell survival rate increased slightly four days after adding the fluorescent probe into cell culture medium,indicating that the probe has a high biosafety.The probe with rigid pedestal can easily capture the target and was an ideal identification element for pathogenic microorganisms.The fluorescence signal intensity of the target was amplified by 4.72 times with this probe.The strategy showed a stronger fluorescence intensity change,sensitivity(linear range of 7.22×10~0-1.44×10~9CFU·m L^-1 with a LOD of 4CFU·m L^-1),and selectivity.The recovery rate in qualified pasteurized milk and drinking water samples was 96.54%to 104.72%.Compared with simple aptamer sandwich detection,these fluorescence signal amplifiers have improved fluorescence detection of St.aureus.Additionally,this fluorescent signal amplification strategy may be applied to the detection of other food pathogens or environmental microorganisms in the future.4.Amplification signal of Td-CD fluorescent microspheres prepared based on multiple CDs and Tds was used for sandwich detection of Salmonella typhimuriumBased on the fluorescence signal amplifier monomer constructed in the previous experiment,CD₄₂₁ with high fluorescence quantum yield was prepared in this experiment,streptavidin(SA)was modified on its surface,multiple Td and CD₄₂₁ were assembled by specific binding between streptavidin biotin,and Td-CD₄₂₁ fluorescent microspheres with uniform size and good dispersion were obtained to further amplify signals.Sensitive detection of Salmonella typhimurium(Sa.Typhimurium)by magnetic separation.These Td-skeleton fluorescent microspheres can actively recognize Sa.Typhimurium and fluorescently bind to the target.The results showed that the fluorescence intensity of microspheres increased by3.05 times,and the linear detection of Sa.Typhimurium in the concentration range of 10-1.0×10~8CFU·m L^-1 was successfully achieved,and the detection sensitivity reached9CFU·m L^-1.The recovery rate in real milk and drinking water samples is 95.35%-103.01%.In addition,this fluorescence signal amplification strategy provides new insights into the analysis of various food threat factors and other fluorescence imaging applications.