Research On Detection Method Of Pathogenic Bacteria And Antibiotics In Milk Based On Upconversion Fluorescence Biosensing

Milk contains essential nutrients for human growth and development,and it is widely accepted as part of the human diet worldwide.The rise in milk consumption has prompted a lot of attention to quality and safety issues.Traditional milk quality and safety testing procedures in practical applications suffer from the limitations of being cumbersome,costly,requiring elongated operational time.Rapid,low-cost,accurate,and sensitive technologies for testing milk quality and safety are critical for the nation’s dairy industry’s development.Upconversion fluorescence detection technology has been used in the field of food safety detection owing to its strong stability,high sensitivity,reliability,and safety features.Therefore,this study took milk as the research object and integrated upconversion fluorescence biosensing technology with the existing methods,and the study aimed to provide new ideas for milk safety testing by designing novel methods for detecting harmful pollutants in milk.The main research contents are as follows:1.Detection of pathogenic bacteria in milk based on black hole quencher-assisted upconversion fluorescence biosensing.To address the issues of long detection time,complicated operation steps,and high costs associated with conventional detection methods,this study proposed the development of a sensing detection platform based on fluorescence resonance energy transfer（FRET）between the black hole quencher-1（BHQ-1）and the upconversion nanoparticles（UCNPs）.Using the typical Gram-positive Coccus-Staphylococcus aureus（S.aureus）as the detection object,a mixed system of UCNPs-aptamer and complementary deoxyribonucleic acid（c DNA）-BHQ-1 was constructed.In the presence of the target pathogenic bacteria,UCNPs-aptamer preferentially recognized and captured the target bacteria,causing the UCNPs-aptamer-c DNA-BHQ-1 complex to dissociate,resulting in enhanced upconversion fluorescence.Under optimal experimental conditions,the relative fluorescence intensity of the sensing system at 540 nm was correlated with the logarithmic concentration of S.aureus ATCC^?29213^TM.The obtained concentration range was 36-3.6×10⁷cfu/m L and the limit of detection was 6 cfu/m L.The specificity of the sensing platform was monitored in the presence of other commonly occurring pathogenic bacteria,and the results indicated strong affinity for the target bacteria.The method was applied to the detection of S.aureus in milk samples,and further validated by traditional plate counting method with no significant difference.The above research showed the created simple,specific,and sensitive method provided a new idea for the detection of pathogenic bacteria in milk.2.Detection of pathogenic bacteria in milk based on graphene oxide quantum dots-assisted upconversion fluorescence biosensing.Given the inability to fully comprehend the detailed mechanism of recognition and capture between aptamers and pathogenic bacteria,this study proposed a new idea to establish a sensing detection platform based on FRET between UCNPs and graphene oxide quantum dots（GOQDs）from the standpoint of pathogen-specific gene.Using the nuc gene of S.aureus as the detection object,a mixed system of UCNPs-ss DNA and GOQDs was constructed.In the presence of the S.aureus nuc target strand,it hybridized to ss DNA on the fluorescent probe,resulting in higher upconversion fluorescence with increased concentrations of nuc target strand.Under optimal experimental conditions,an excellent linear relationship was observed between the fluorescence intensity of the sensing system at 545 nm and the logarithm value of the nuc target strand concentration.The detected concentration range was 1×10^-17-1×10^-11 mol L^-1with an obtained limit of detection of 0.98×10^-17mol L^-1.Furthermore,the detection method was highly specific,capable of discriminating single base difference,and had good recovery rates when applied to the analysis of milk samples for S.aureus.The developed sensing platform provided a new idea for the detection of pathogenic bacteria in milk.Compared with the previous research,the detection accuracy was improved.3.Detection of antibiotics in milk based on Au NPs-assisted upconversion fluorescence biosensing.A label-free detection method based on the FRET between UCNPs and Au NPs was proposed in this study to circumvent the shortcomings linked with the conventional antibiotic detection methods,which were time-consuming,labor-intensive,and costly.A mixed system of aptamers,Au NPs,Na Cl,and UCNPs was built for the target tetracycline（TC）detection.Despite being exposed to high concentrations of Na Cl in the absence of TC,aptamer-adsorbed Au NPs remained dispersed in such high Na Cl environment,thereby quenching the upconversion fluorescence.The specific binding of TC to aptamer increased with its rising concentration,therefore,the Au NPs unprotected by aptamer aggregated to varying degrees,resulting in the increase in upconversion fluorescence.The relative fluorescence intensity at 545 nm of the sensing system and the logarithm of the TC concentration was linearly related at the optimal experimental conditions.Furthermore,the sensor was highly specific for TC.The method’s accuracy and reliability were established by comparing it with the detection findings of the enzyme linked immunosorbent assay（ELISA）method for the detection of TC in milk samples.In addition to having the ability to perform rapid and accurate analysis,this method offered a new insight into the detection of antibiotics in milk.4.Detection of antibiotics in milk based on Mn O2 nanosheets-assisted upconversion fluorescence biosensing.Considering the low sensitivity of antibiotic detection methods,this study explored a detection method based on the FRET between UCNPs and Mn O₂nanosheets.Incorporating Mn O₂ nanosheets and UCNPs-aptamer for the detection of TC was constructed.In the presence of TC,the UCNPs-aptamer adsorbed on the surface of Mn O₂nanosheets preferentially bound to TC,triggering the upconversion fluorescence enhancement.A good linear relationship was established between the relative fluorescence intensity at 450 nm of the sensing system and the logarithm of the TC concentration under optimal conditions.The detection range was found to be 0.01-100 ng/m L with a detection limit of 0.0085 ng/m L.Furthermore,the method had high selectivity and anti-interference ability.The proposed method for the detection of TC in milk samples was validated by the ELISA method with no significant difference,indicating the method was accurate and reliable.The developed method was simple and sensitive,and it provided a novel approach to detecting antibiotics in milk.Compared with the previous research,the detection sensitivity was improved.5.Detection of antibiotics in milk based on MXene nanosheets-assisted upconversion fluorescence biosensing.In the previous research,aptamer-modified UCNPs were employed as the signal probes for antibiotic detection.However,the high density of aptamers directly modified on UCNPs invariably resulted in crowding and tangling,which affected the detection sensitivity.In this study,DNA tetrahedral nanoprobes with excellent mechanical rigidity,structural stability,and good density and orientation control were introduced,and a new idea of FRET sensing detection based on UCNPs and MXene nanosheets was proposed.A mixed system of MXene-Ti₃C₂ nanosheets and DNA tetrahedral fluorescent nanoprobes was built utilizing TC as the detection target.In the presence of TC,the DNA tetrahedral fluorescent nanoprobes preferentially bound to the TC,which dissociated from the MXene nanosheets,resulting in enhanced upconversion fluorescence.Under the optimal experimental conditions,a good linear relationship was established between the fluorescence intensity at 540 nm of the sensing system and the logarithm of TC concentration.The detection range was 0.01-100 ng/m L with a limit of detection of 0.0057ng/m L.The specificity of the proposed approach was examined revealing high selectivity for TC in the presence of other potential antibiotics.Consequently,the results obtained using this approach and ELISA method for monitoring TC in milk samples did not differ significantly.The developed method was consistent and sensitive,offering a novel approach to detecting antibiotics in milk.Compared with the previous two studies,the detection sensitivity was further improved.