High-efficiency Detection Of Endotoxin Based On Composite Nano-fluorescence Probes

Endotoxin,present in the outer membrane of all gram-negative bacteria,as a main reason for endotxemia and organs failure,which are severely harmful to human health.Traditional detection methods for endotoxin such as limulus reagent-based detection methods have long detection time,harsh detection conditions,and poor anti-interference ability,which have certain limitations for the rapid and accurate detection of endotoxin.High-sensitivity and rapid-detection fluorescence detection methods have shown promising applications in endotoxin detection.The rise of various nanomaterials and preparation techniques,especially the rapid development of fluorescence resonance energy transfer systems based on quantum dots and graphene oxide,provide powerful technical support for the development of new methods for endotoxin detection.At the same time,fluorescence polarization immunoassay technology has advantages such as rapid detection,simple operation,and quantitative measurement,which has a rapid progress in the detection of mycotoxins.This paper proposes to design and prepare nano-fluorescent probes with high-fluorescence intensity,low background interference and highly selective for fluorescence detection and fluorescence polarization detection of endotoxin in injections and blood.Due to the endotoxin of biochemical specimens with complex background and low levels,the rapid detection provides new methods and new approaches.The main research work and results are as follows:（1）Design and preparation of a GO-Apt-QD fluorescent probe based on fluorescence resonance energy transfer for the efficient and quantitative detection of endotoxin.First,quantum dots with high quantum yield and good photo stability are bound to Apt surface via EDC/NHS covalently,and then QDs-Apt fluorescent probes are prepared.The superior fluorescence quenching ability and adsorption of graphene oxide（GO）are used to further prepare GO-QDs-Apt fluorescent probe.Adding 200ng·mL^-1 endotoxin solution to this probe to investigate its fluorescence properties.Quantitative determination of endotoxin has a detection range from 10 ng·mL^-11 to 500ng·mL^-1 and the detection limit was 8.5 ng·mL^-1 under the condition of the quenching concentration of GO was 100?g·mL^-1and the quenching time of 30 minutes.This fluorescent probe is also suitable for the detection of endotoxin in the actual sample.In the vitamin B1 solution,the measured recoveries of the endotoxin were between 92.2%to 102.1%,and the RSD was less than 5%（n=6）.The results of this method agree well with the results of the traditional limulus reagent method.The GO-QDs-Apt fluorescent probes were prepared with 10?g·mL^-1 of?-D-glucose,Cu²⁺,Zn²⁺,Mg²⁺,Ca²⁺and aflatoxin,respectively.The fluorescent probes reacted with the endotoxin solution at100 ng·mL^-1,and then only the endotoxin solution had a fluorescence signal change,indicating that the method has good specificity.（2）LPS-FITC fluorescent probe was synthesized as a tracer.A fluorescence polarization detection method for endotoxin based on LPS-FITC and aptamer was established,which has the advantages of high sensitivity,simple operation and rapid detection.Under the reaction of EDC/NHS,aptamers that modified amino and carboxyl groups at 5 side were combined to form an aptamer complex,which increased the molecular volume,enhanced the change in fluorescence polarization values,and further improved detection sensitivity.When the aptamer complex was diluted 200-fold with PBS,the dilution of the fluorescent probe was 200-fold,and the competition binding time was 40 min,this method was established to achieve highly efficient endotoxin detection.The results showed that there was a good linear relationship between the detection concentration of 1 ng·mL^-1-10000 ng·mL^-1 and the detection limit was 1ng·mL^-1.In order to investigate the application of this method in actual samples,a standard method for fluorescence polarization was used to test the endotoxin in the blood of 5 clinically burned patients.The fluorescence polarization was measured within the range of less than 10 ng·mL^-1and then getting a good linear relationship between the change value of the endotoxin concentration.The linear equation was mp-mp₀=-0.539C（34）₁-0.678（R²=0.981）,which has a detection limit of 1.1 ng·mL^-1.Comparisons this test results with the turbidimetric kinetic method,this results were consistent with the previous.The LPS-FITC probe and aptamer complexes were treated with 10μg·mL^-1 ofβ-D-glucose,Cu²⁺,Zn²⁺,Mg²⁺,aflatoxin B1 solution and 100ng·mL^-1 endotoxin,respectively.The solution reaction only signaled to endotoxin,indicating that the established method has good specificity.The entire detection process can be quickly completed within 50 minutes,and then achieves a rapid detection of endotoxin with high sensitivity and high selectivity.