Study On The Methods For Detecting Huperzine A With Gold Nanoparticle Probes Labeled By Enzyme

Huperzine A(HupA)as an efficient and reversible acetylcholinesterase(AchE)inhibitor,is currently the most promising drug for Alzheimer’s disease(AD)treatment.With the acceleration of the aging of world population,Alzheimer’s disease increased year by year.Thus the demand for it is increasing.Currently,the medical profession hasn’t found a suitable succedaneum for huperzine A.Artificial-synthesized methods of huperzine A only stuck in the laboratory stage,Currently,Huperzine A is mainly extracted from plants such as the genus Lychee and the genus,but the wild resources of these plants are scarce and the content of Huperzine A is very low.Therefore,establishing a Huperzine A detection with high sensitivity will have important practical significance for the full use of such resources.Recently,nanotechnology develops rapidly,especially Nanogold labeling technology has been attracted intense attention in practice fields.The technology having the advantages of easy-to-operation,fast,accurate and non-toxic,provides a new idea for the detection of small molecule drugs.Gold nanoparticles(AuNPs)with a large specific surface area and good biocompatibility,can be combined with antibody,enzyme and other biological macromolecules through the electrostatic interaction,and can also be combined with DNA modified by thiol or PolyA.The conjugate’s property is stable and can hardly change the activity of biological molecules.These functional nanogold probe used in immunoassay,can significantly improve the detection sensitivity of the method.Magnetic nanoparticles(MMPs)are a kind of new functional materials,referring to the magnetic microspheres coupling with antigens or antibodies.Magnetic beads with high magnetic response,under the action of the external magnetic field,can quickly separate or concentrate the target,having the advantages of easy-to-operation,efficient and not influencing biological activity,etc..Magnetic beads have unique superiority in the field of immunoassay and cell separation.A novel enzyme immunosorbent assay with high sensitivity for detection of HupA was established by using AuNPs probes and MMPs probes based on the principle of competetive immunoassay.This method was applied to detect actual samples,which provides a new strate-gy for the analysis of the effective components of the medicine.The paper is divided into three chapters,the main contents are as follows:Chapter 1:The study of the method for detecting Huperzine A with gold nanoparticle probes.AuNPs were prepared by the classic trisodium citrate reduction,and modified with enzyme and antibody to prepare AuNPs probes.The preparation conditions of the probe were optimized,such as labeling pH,the optimal quantity of antibody,the ratio of enzyme and antibody.Then characterize the prepared AuNPs probes by UV spectrophotometry and transmission electron microscopy(TEM).A new method for detecting HupA was established by introduced AuNPs probes into the immunoassay based on competitive reaction.After optimizing the experimental conditions such as the dilution ratio of coating antigen and nano-gold probe and the optimal competitive reaction time,we can get a standard working curve for HupA.The linear equation was 1=0.2985 lgC + 0.1704(r=0.9960),the linear range was from 0.5 ng/mL to 150 ng/mL and the limit of detection was 602.56 pg/mL.By the established method we detect the content of HupA in Huperzia serrata and compared with the national standard with satisfactory results.This method with a short detection time,high sensitivity and good repeatability,is a simple,efficient and economical detection system,more conducive to generalize.Chapter 2:The study of the method for detecting Huperzine A based on magnetic beads and gold nanoparticle probes.Firstly,we labeled the coating antigen on magnetic beads.Based on magnetic separation and AuNPs probes,a new enzyme immunoassay technique for the detecting huperzine A was established,which was rapid,efficient and sensitive.After optimizing the experimental conditions such as the dilution ratio of the MMPs probes and the AuNPs probes and the optimal competitive reaction time,the detection system was finally established.Under the optimized experimental conditions,this method has a good linearity in the range of 0.075～180 ng/mL,the linear equation was 1=0.2813 lgC+0.3051(r=0.9950),the detection limit was 160.32 pg/mL.By this method we detect the content of HupA in tablets and compared with the national standard with satisfactory results.The method established in this study has higher sensitivity,wider linear range and shorter detection time.Chapter 3:The study of the method for detecting Huperzine A based on magnetic beads and DNA-AuNPs probes.On the basis of the first two methods,the DNA-nano gold probes detection method was established based on the magnetic beads,and the precision,repeatability and specificity of the method were optimized.The results were all within the allowable range,The linear range of this method was 0.008～16 ng/mL,the linear equation was I=0.3006 lgC+0.6142(r=0.9952)and the detection limit was 19.41 pg/mL.The recovery rate of endophytic fungus was in the range of 99.33～103.73%.The introduction of DNA further enhances the sensitivity of the method,which is more sensitive than the first two methods.