Development Of Metal Composite Nanoenzyme-labeled Lateral Flow Immunoassay For Rapid Detection Of Cardiac Biomarkers

Acute myocardial infarction（AMI）is a common coronary artery disease with high mortality.As a key diagnostic link for AMI,cardic biomarkers are mainly tested by central laboratories in clinical medical institutions.But its operation steps are cumbersome,requiring regular equipment maintenance and professional operators,which greatly limits the ability of suburban/rural areas to detect cardic biomarkers,prolongs the diagnostic time of AMI,and is not conducive to the development of graded diagnosis and treatment.Therefore,the development of point of care testing（POCT）for cardiac biomarkers is a research focus of domestic and foreign researchers in the field of rapid diagnosis of AMI.Lateral flow immunoassay（LFIA）is a solid-phase immunochromatographic method based on paper chromatography analysis combined with antigen antibody immune response,and is an ideal POCT platform.The colorimetric LFIA is well suited for the rapid in vitro diagnosis needs of AMI patients during transportation outside the chest pain center（at home,primary medical institutions,emergency vehicles,etc.）.However,the sensitivity of the classic colorimetric LFIA test strip cannot meet the needs of detecting low abundance cardiac biomarkers（such as cardiac troponin I（cTnⅠ）and Heart-type fatty acid-binding protein（H-FABP））in the early blood of AMI patients.With the development of nanoenzyme materials,the sensitivity of enzyme mediated colorimetric enhanced LFIA has been greatly improved.However,nanoenzyme materials have drawbacks such as complex synthesis processes,harsh reaction conditions,difficulty in surface functionalization,and high raw material costs,making it extremely difficult to meet industrial production needs.Therefore,it is of great practical significance to seek suitable nanoenzyme synthesis strategies for the industrialization of enzyme mediated colorimetric enhanced LFIA for detecting cardiac biomarkers.This paper adopts two technical routes to solve the above problems:one pot synthesizes a new trimetallic nanoenzyme:AuPt@Fe_xO_y NPs;One step large-scale implementation of gold nanoparticles（Au NPs）coated with CeO₂ shell with peroxidase-like（POD-like）activity:Au@CeO₂ NPs.Both methods have the characteristics of mild reaction conditions,simple operation,and low cost,and are respectively used as labeling probes of enzyme mediated colorimetric enhanced LFIA to detect early cardiac biomarkers in AMI:cTnⅠ and H-FABP.The constructed LFIA has excellent sensitivity,specificity,and stability,and shows good applicability for the detection of serum samples from clinical AMI patients.And as a universal POCT platform,the above two LFIA can be used for the detection of other biomarkers by replacing relevant antibodies.The main research work is as follows:1.A novel trimetallic nanoenzyme AuPt@Fe_xO_y NPs with excellent localized surface plasmon resonance（LSPR）effect and POD-like catalytic activity was synthesized through redox interaction-engaged strategies.This nanoparticle has the characteristics of simple synthesis method,mild reaction conditions,and low cost.And we have preliminarily explored the regulatory mechanisms of LSPR effect and POD like activity by changing the proportion of precious metals in AuPt@Fe_xO_y NPs.AuPt@Fe_xO_y-LFIA for cTnⅠ detection was prepared using an immunosandwich method using cTnⅠ monoclonal antibody coupled AuPt@Fe_xO_y NPs.Based on the excellent optical properties of Au and the POD-like activity of Pt and Fe_xO_y,AuPt@Fe_xO_y-LFIA has two colorimetric modes:conventional colorimetric mode based on LSPR effect and enzyme mediated colorimetric enhancement mode.By optimizing the reaction conditions,the Limit of detection（LOD）of cTnⅠ using AuPt@Fe_xO_y-LFIA is 0.48 ng m L^-1（conventional colorimetric mode）and 0.026 ng m L^-1（enzyme mediated colorimetric enhancement mode）.The experimental results show that the AuPt@Fe_xO_y-LFIA has the characteristics of high sensitivity,time-saving,economy,convenience,quantifiability,and strong stability.Meanwhile,AuPt@Fe_xO_y-LFIA has high specificity and detection repeatability for cTnⅠ detection.2.Au nanoparticles with CeO₂ catalytic shell,namely Au@CeO₂ NPs,were one step synthesized in large quantities by redox interaction-engaged strategies.As a new strategy to improve the detection sensitivity of Au NP-LFIA,Au@CeO₂ NPs have overcome a series of pain points and difficulties that hinder the large-scale industrialization of LFIA,including the cumbersome operation,poor stability,and high cost of previous methods such as aggregation chromogenic probe method,noble metal enhancement method,HRP mediated colorimetric enhancement method,and other catalytic shell coating Au NPs.Au@CeO₂-LFIA for H-FABP detection was prepared using an immunosandwich method using H-FABP monoclonal antibody coupled Au@CeO₂ NPs.Because of suitable oxidation vacancies based on CeO₂ and the ability to quickly convert between Ce³⁺and Ce⁴⁺,Au@CeO₂ NPs exhibit excellent POD-like activity.Combined with the inherent LSPR effect in the nucleus of Au NPs,Au@CeO₂-LFIA also has both conventional colorimetric mode and enzyme mediated colorimetric enhancement mode.After optimizing the experimental conditions,the LOD of Au@CeO₂-LFIA for H-FABP detection is 0.35 ng m L^-1.The experimental results indicate that,Au@CeO₂ NPs can be synthesized on a large scale in one step at room temperature,and the detection platform has strong specificity,high sensitivity,convenience,and can be stored for a long time.3.cTnⅠ and H-FABP have strong complementarity in hemodynamics,sensitivity and specificity of AMI diagnosis.In order to make early diagnosis of suspected AMI patients more quickly,efficiently,and accurately,we use AuPt@Fe_xO_y-LFIA and Au@CeO₂-LFIA to jointly detect cTnⅠ and H-FABP in serum samples of AMI clinical patients.Both LFIA showed excellent applicability and good consistency with the detection results of commercial ELISA kits,with correlation coefficients R² of 0.97 and0.98,respectively.When using AuPt@Fe_xO_y-LFIA and Au@CeO₂-LFIA for dynamic detection of serum samples from AMI patients,the dynamic changes of the two biomarkers confirm the important significance of cTnⅠ and H-FABP combined detection in the early onset of AMI.Moreover,the two LFIAs demonstrate lower usage thresholds and lower single testing costs for clinical samples compared to central laboratory methods.In summary,the metal composite nanoenzyme labeled side LFIA we prepared not only solve the problem of the previous conventional colorimetric LFIA being unable to meet the detection sensitivity of cardic biomarkers,but also meet the industrial production needs of nanoenzyme labeled probes.The prepared LFIA achieve rapid,accurate,and real-time detection of cardic biomarkers represented by cTnⅠ and H-FABP,without relying on professional equipment and personnel.The combination of AuPt@Fe_xO_y-LFIA-based detection of cTnⅠ,supplemented by Au@CeO₂-LFIA-based detection of H-FABP,not only helps promote the grading diagnosis and treatment of AMI patients,but also provides a new option for early diagnosis and disease monitoring of AMI outside hospitals/underdeveloped areas.