Preparation And Molecular Dynamics Simulation Of A Novel Fluorescent Microspheres And Its Application In The Detection Of Myocardial Infarction Marker By Immunochromatography

Cardiovascular diseases(CVDs)are the number one cause of death worldwide.Myocardial infarction(MI)is a common manifestation of ischemic heart disease.According to the "Chinese Cardiovascular Report 2017",the overall mortality of myocardial infarction in China has shown an upward trend in the past 15 years,of which the incidence of individual morbidity is become younger.Therefore,effectively detect the MI markers in advance and predict the occurrence and development of MI,has very important significance for the prevention and treatment of potential patients.In this paper,a rapid detection technique was developed for the early detection of C-reactive protein(CRP)and cardiac troponin I(cTnI)MI markers in clinical detection by lateral flow immunochromatographic analysis.The one-step synthesis of core-shell nanospheres with distinct functional areas was used to solve the key technical problems in the detection platform,effectively improving the sensitivity and stability of the detection,thus has important theoretical significance and practical value.This paper combines the immunology,polymer chemistry,and materials science as well as computer simulation techniques to systematically study antibody synthesis,synthesis and preparation of fluorescent immuno-microspheres,assembly of the test strip,instrument commissioning,and the detection of clinically samples during the development of the lateral flow immunochromatographic analysis.One-step synthesis of core-shell nanospheres with distinct functional areas was studied.The synthesis mechanism of microspheres was studied through the combination of experimental parameters and dissipative kinetics simulations.The effect of fluorescent dyes on the secondary structure of antibody proteins was investigated to study the effect of dye on the stability of the methodological.The red and green fluorescent detection systems were constructed for rapid clinical detection of cTnI and CRP.The main research results are as follows:(1)3 positive hybridoma cells stably secreting anti-cTnI monoclonal antibodies were obtained,and the subtypes of the 3 antibodies were identified as IgG1 by the mouse monoclonal antibody IgG/subclass detection kit.The antibody remained stable after 6 months of storage at 4°C and-20°C.(2)By adjusting the experimental parameters(KPS,functional monomer,reaction temperature and stirring rate),carboxyl functionalized polystyrene microspheres with a uniform particle size and good dispersibility were prepared from 200 nm to 600 nm.The microspheres have a core-shell structure.The interior of the sphere is a polystyrene core,which can effectively envelop fluorescent dyes.The outer layer is a carboxyl-functionalized polyacrylic acid shell,which is used to connect antibody proteins.The well-defined functional areas are conducive to the loading of fluorescent dyes and the binding to antibodies.(3)Fluorescent nanoparticles coated with NR and FITC were prepared by using swelling and dispersing technique.The maximum fluorescence emission wavelengths were 606 nm and 502 nm,respectively.The blank CPNs were characterized using Fourier transform infrared spectroscopy and thermogravimetric analysis,to study the changes of functional groups and thermal information of R-CPNs and G-CPNs which further confirmed the well combination of fluorescent dyes and polystyrene microspheres;the relationship between NR/FITC and the secondary structure of the antibody protein was investigated using circular dichroism chromatography which providing the theoretical support for the study of the stability and reproducibility of lateral flow immunoassay methods.(4)Using computer simulations to construct the stable conformations of polystyrene,acrylic acid,NR and FITC molecules.Their solubility parameters and the interaction parameters between the molecules were calculated,and the microspheres were deduced by means of mesoscopic kinetics.The synthesis process mainly includes three stages: the initial synthesis material is uniformly distributed,the mid-stage formation of small aggregates,the formation of large aggregates at the end of the process to form a stable microsphere structure.Besides,the density of the fluorescent dyes in the microspheres was studied at the same time.The distribution provides theoretical support for the experimental results of microspheres.At the same time,it provides new ideas and new methods for constructing new functional nanospheres.(5)The application of R-CPNs and G-CPNs in the detection of cTnI was studied.After the coupling reaction between R-CPNs/G-CPNs and cTnI-McAb1,the morphology remained uniform.The PDI was 0.057 and 0.062,respectively,which were less than 0.1,showing a good monodispersed;The optimal coupling time and antibody concentration during the coupling reaction between the microspheres and cTnI-McAb1 was 120 min and 500 ?g/mL,respectively.The optimized immunoreaction time was 15 min during sample detection,and the optimized NR-LFIA which use to cTnI detection were within the LoD of 0.016 ng/mL and the detection range is 0.016-80 ng/mL.The LOD of the FITC-LFIA for cTnI is 0.18 ng/mL,and the detection range is 0.18-80 ng/mL.The LFIA has good specificity,repeatability and stability and can be applied to the detection of clinical samples.(6)The application of R-CPNs and G-CPNs in the detection of CRP was studied.After the coupling reaction with CRP-McAb1,the morphology of Im-R-CPNs/Im-G-CPNs remained homogeneous,PDI was 0.048 and 0.059,respectively,showing a good monodispersed;The optimal coupling time and antibody concentration during the coupling reaction between the microspheres and cTnI-McAb1 was 75 min and 300 ?g/mL,respectively.The optimized immunoreaction time was 3 min;MES with pH 6.5 was used as a buffer during the coupling reaction,the buffer can effectively maintain the stability of the conjugated system.The optimized NR-LFIA which use to CRP detection were within the LoD of 0.091 mg/L and the detection range is 0.1 to 160 mg/L.The LoD of the FITC-LFIA for CRP is 0.37 mg/L,and the detection range is 0.4-200 mg/L.The LFIA has good specificity,repeatability and stability and can be applied to the detection of clinical samples.