Preparation Of Quantum Dot Fluorescent Probes And Their Application In The Detection Of Gram-negative Bacteria

Bacterial infections have become a serious threat to human health due to their high morbidity and mortality.The development of accurate,rapid and effective bacterial detection methods is important for the prevention and control of bacterial infections.Fluorescent probes based on quantum dots（QDs）have broad application prospects in bacterial detection.Typically,these probes use antibodies or aptamers as their recognition elements（receptors）.Such probes are expensive and require harsh detection and storage conditions.This is not suitable for rapid diagnosis bacterial infections at early stage.This work is based on the modification of QDs combined with fluorescence detection technology to construct fluorescent probes for rapid and sensitive detection of Gram-negative bacteria.These probes can bind to different sites on lipopolysaccharide by the structural design and optimization of QDs ligands.The fluorescence properties and detection performance of these fluorescent probes were further studied in detail.The main research contents are as follows:（1）The fluorescent probe QDs/MPA-APBA were constructed by using 3-mercaptopropionic acid（MPA）and 4-aminophenylboronic acid（APBA）.The probe QDs/MPA-APBA can bind to the cis-diol structure on the lipopolysaccharide on the surface of Gram-negative bacteria.Compared with traditional methods,it can distinguish Gram-negative bacteria and Gram-positive bacteria in 30 min.It’s quenching of fluorescent（1-I/I₀）to Escherichia coli is 0.22 and 0.50 at pH=7.4 and 9.0,respectively,indicating that the probes are better suited for detection under alkaline condition.（2）L-cysteine（Cys）and APBA were used to introduce a Wulff-type boronic acid structure to construct a fluorescent probe QDs/Cys-APBA.This structure can improve the probes’stability of the detection performance under different pH conditions.The probe can complete qualitative analysis of Escherichia coli and Pseudomonas aeruginosa within 30 minutes,with the limit of detection of 58 CFU/m L and 97CFU/m L,respectively.The QDs/Cys-APBA retains stable detection performance to Escherichia coli under different pH conditions（5.0-9.0）,showing the high selectivity to Gram-negative bacteria.In addition,QDs/Cys-APBA can detect Escherichia coli in milk to accurately assess the bacterial contamination levels.（3）Cys and polymyxin B（polymyxin B）were used to modify QDs to construct a narrow-spectrum antibiotic-modified fluorescent probe QDs/polymyxin B.The probe can specifically bind to the lipid A on lipopolysaccharide.QDs/polymyxin B can detect Escherichia coli and Pseudomonas aeruginosa within 20 minutes with lower limit of detection 15 CFU/m L and 4 CFU/m L,respectively,and the probe shows excellent antibacterial activities against Escherichia coli.In addition,QDs/polymyxin B can detect Escherichia coli in artificial urine to achieve the rapid diagnosis of urinary tract infections.In this paper,the fast and sensitive detection of Gram-negative bacteria was realized by functional design of the ligands of QDs in fluorescent probes.A fluorescent probe with better anti-interference ability was constructed by introducing a ligand with a"Wulff-type"structure.The introduction of antibiotic ligands further improves the sensitivity of the probe,while endowing it with excellent bacteriostatic properties.