Ultrasensitive And Quantitative Stroke Biomarkers Detection MMP-9、S100-Β And NSE By Lateral Flow Controlled Surface Enhanced Raman Scattering

Research purpose:Cerebral vascular accident is an acute cerebrovascular disease.It is a group of diseases,including hemorrhagic and ischemic stroke,which are characterized by high morbidity,disability and mortality,caused by sudden rupture of cerebrovascular or obstruction of blood supply.For most stroke patients,imaging can clearly identify the lesions,judge the degree of brain injury,and objectively evaluate the prognosis.But some imaging examinations are often difficult to accurately judge the degree of injury,and some patients can not carry out dynamic imaging observation because of their critical condition.Recent clinical studies have found that neuron-specific enolase(NSE),serum matrix metalloproteinase-9(MMP-9),and serum biomarkers of S100-β protein are helpful in predicting the severity and clinical prognosis of stroke.Currently,enzyme-linked immunosorption test(ELISA)and Western blot test(Western blot)and other methods are used to detect biomarkers,which are usually time-consuming to operate,have large sample size and expensive detection instruments,and cannot be used as fast,low-cost and easy to operate clinical diagnostic analysis tools.Compared with traditional immunoassay methods,surface enhanced Raman scattering(SERS)technology has better anti-interference ability in complex sample systems.SERS technology also has good stability,convenience and reproducibility,and has become a powerful and sensitive analytical technology in chemical and biomedical analysis.Therefore,a laterally flow-controlled SERS immunoassay strip has been developed,which can be used to quantitatively analyze the biomarkers MMP-9,S100-β and NSE of stroke in low concentration range.This method is simple,rapid and sensitive.It provides a possibility for detecting the biomarkers of stroke in time and helps to evaluate the condition and prognosis of stroke patients.Research methods:A multiple SERS immunoassay strip for rapid detection of stroke was developed.Firstly,hollow Au-Ag bimetallic nanospheres were prepared.Secondly,5,5’-dithiobis(2-nitrobenzoic acid)modified HGNs@Ag to prepare nanoprobes with SERS properties.Then the prepared SERS probes were linked with the labeled antibodies and the immunolabeled SERS nanoprobes were prepared: HGNs@Ag@DTNB@MMP-9,HGNs@Ag@DTNB@S100-β and HGNs@Ag@DTNB@NSE.Finally,the paper strips fixed with basal probe for stroke biomarkers were prepared.Three detection lines were used to detect the biomarkers of mmp-9,S100-β and NSE respectively,and the changes of Raman intensity were recorded on the test line for ultra-sensitive quantitative detection.By optimizing the experimental conditions,including the type of running buffer,the amount of BSA in running buffer and the amount of S100-β-conjugated HGNs@Ag loaded on conjugate pad,high sensitivity detection of three stroke biomarkers was achieved.The results were compared with those of ELISA to verify the feasibility of this method in the clinical application of detecting biomarkers of stroke.The cross-reaction among the three types of stroke biomarkers in SERS immune test strips was evaluated.Blood samples of stroke patients were collected for ELISA and SERS immune test strips respectively,and the linear correlation of the two results was compared.Result:The limit of detection MMP-9,S100-β and NSE were 0.84 pg/ml,0.63 pg/ml and0.55 pg/ml respectively,while the clinical detection ranges of MMP-9,S100-β and NSE were 0.01-400 ng/ml,0.01-40 ng/ml and 0.02-80 ng/ml,respectively.Using different batches of SERS immunotest strips,mmp-9,s100-beta and NSE were detected respectively,and the results were in good agreement with each other.The cross reaction of multiple SERS immunoassay strips can be neglected.The smaller the cross reaction,the better the specificity of SERS immunoassay strips.Blood samples from stroke patients were collected for ELISA and SERS immunoassay strip detection,and the results of the two tests had good linear correlation.Conclusion:(1)Different batches of SERS FLA were used to test MMP-9,S100-β and NSE respectively.The results of MMP-9,S100-β and NSE obtained from three independent tests were in good agreement with each other,which shows that SERS immunoassay strips had good reliability and reproducibility.(2)The cross reactions between the SERS immunoassay strips with multiple SERS were negligible,which indicated that the strips had good specificity.(3)The detection of MMP-9,S100-beta and NSE by SERS immunoassay strip has a good linear correlation with the results of ELISA,which ensures the reliability and stability of clinical application.Compared with ELISA,the quantitative SERS strip has the advantages of ultra-sensitivity,fast,low cost,easy to use,no sample pretreatment and professional detection.