The Preparation Of Bifunctional Hybrid Nano-flowers And Their Application In The Enzyme-linked Immunosorbent Assay For Helicobacter Pylori Detection

Helicobacter pylori(H.Pylori,HP)is a kind of gram-negative bacterium which requires very stringent growth conditions.It is the only known human pathogenic microorganism that can live in the human stomach and cause infection.As the infection of HP will be accompanied for a lifetime without treatment or intervention and may induce diseases such as gastric cancer.It is estimated that about 50%of mankind are infected,with infection rates in some developing countries reaching as high as 90%.The high threshold of detection is one of the important factors restricting the prevention and control of HP.However,the existing diagnostic detection methods can't achieve accurate quantitative analysis,and the invasive methods will cause trauma and pain to patients,which leads to the poor acceptance of these methods.Non-invasive methods,represented by ¹³C/¹⁴C isotope breath test,is limited in scope because of concerns with radioactivity.Therefore,it is necessary to develop a specific and sensitive in vitro non-invasive immunodiagnostic method.In this study,the New Zealand white rabbit was immunized wi th HP and purified serum antibodies were obtained subsequently by ammonium sulfate precipitation method.Then,a kind of organic-inorganic bifunctional hybrid nano-flower(R-HP-Ab-HRP@Cu²⁺NFs)including rabbit polyclonal antibody of HP(R-HP-Ab)labeled with horseradish catalase(HRP)was prepared by one pot method with copper ion as metal substrate.And,a new non-invasive indirect enzyme-linked immunosorbent assay(i ELISA)method based on NFs was established.As the NFs prepared in this paper is rich with R-HP-Ab,the function of HP specific capture can be well realized,and the abundant HRP in NFs can simultaneously meet the requirements of catalyzing3,3',5,5'-tetramethylbenzidine(TMB)color rendering in the presence of H₂O₂,so as to achieve the function of signal amplification.Otherwise,it could combine the steps of enzyme-labeled secondary antibody after the incubation of primary antibody in the traditional ELISA process into one,thus achieving the effect of saving detection time.Therefore,this method is very advantageous for sensitive non-invasive detection of HP in vitro.As shown in the results:Firstly,according to the SDS-PAGE results,the purity of the purified anti-Hp antibody reached more than 90%,and the titer of the purified antibody reached 1:128000.In addition,the NFs synthesized by R-HP-Ab-HRP,including HRP and R-HP-Ab at the ratio of 1:1 and doping copper ion showed the best performance.Then,the NFs was characterized by Transmission electron microscopy(TEM),Scanning electron microscopy(SEM),Energy dispersive spectrum(EDS)and Dynamic Light Scattering(DLS).The results showed that NFs had a stretched petal-like structure with a size of about 5?m under electron microscope,however,the equivalent diameter of NFs in DLS analysis was about 1500 nm that may due to the petal-like imperfect sphere structure.In addition,the EDS analysis showed that all elements in NFs were evenly distributed.In addition,the bifunctional activity of NFs was verified,which proved that NFs had good HP recognition specificity and catalytic TMB chromoenic activity.Further,the i ELISA based on NFs or R-HP-AB-HRP were respectively established,and the i ELISA process conditions were optimized and the two detection methods were compared.And the established i ELISA method based on R-HP-Ab-HRP@Cu²⁺NFs showed a prominent linearity with HP at a concentration of0-10⁵ CFU/m L(R²=0.9997).Moreover,the limit of detection(LOD)reached 50CFU/m L,and not only the detection sensitivity was 20 times higher th an that based on R-HP-Ab-HRP but also the stability of R-HP-Ab-HRP in NFs was improved.In addition,the OD450 nm value was still linearly related to the concentration of HP at a range of0-10⁵ CFU/m L(R²=0.9952)with a LOD of 50 CFU/m L in the system of artificial saliva.From all has been discussed above,this study provided a detection method with strong specificity,high sensitivity and simple operation for non-invasive in vitro diagnosis of HP,which is expected to reduce the threshold of HP detection a nd improve the popularity of HP detection.