Research On A New Immunoassay Method Based On Time-resolved Fluorescent Microspheres

Optical biosensing technology has the benefits of high detection sensitivity and quick detection speed in the field of life science,and it can perform quantitative analysis of chemical and biological data.Due to its benefits of low background interference,simple operation,and high sensitivity,time-resolved fluorescence immunoassay technology has steadily developed into a mature and advanced detection method in optical biosensing technology.It can detect polypeptides,chemicals,pathogen antigens and antibodies,indicators,medicines,nucleic acids,and other compounds.Because of their excellent fluorescence labeling efficiency and detection sensitivity,time-resolved fluorescent microspheres are extremely useful in immunoassays.Current time-resolved fluorescent microspheres rely on rare earth ion complexes to absorb ultraviolet light and generate fluorescence,with an excitation wavelength of 360 nm.Large Stokes shifts are produced in the UV area of nm,which decreases background noise.The goal of this research is to create a new type of time-resolved fluorescent microspheres with increased sensitivity.Because of its quick detection time and numerous advantages for on-site screening,time-resolved fluorescence lateral flow chromatography has gotten a lot of interest.It does,however,have drawbacks,such as low reproducibility of experimental outcomes.Poor repeatability in lateral flow chromatography has a number of drawbacks.In response to the aforementioned issues,we have completed a number of tasks,the details of which are as follows:The second chapter using the swelling principle to convert the singlet oxygen energy,we design and construct a novel anti-Stokes fluorescent microsphere with high sensitivity,which is inspired by the luminous oxygen channel analysis technique.The experimental conditions were optimized to improve the efficiency of singlet oxygen,a rare earth fluorescent dye molecule Eu（TTA）₃（TPPO）₂ was prepared and combined with organic molecules that can transfer luminescence energy,and a rare earth fluorescent dye molecule Eu（TTA）₃（TPPO）₂ was prepared and combined with organic molecules that can transfer luminescence energy.The photosensitizer-coated polyphenylene was combined with 4-（2-phenyl-5,6-dihydro-1,4-oxthihexyl-3-yl）-N,N-tetracosylaniline.The singlet oxygen energy transfer occurred on the same microsphere during the reaction in the ethylene microsphere dispersion system,which raised the singlet oxygen level on the microsphere’s surface and considerably improved the microsphere’s sensitivity.It is also confirmed that the time-resolved fluorescent microspheres have the benefits of uniform particle size,good dispersion and stability,high fluorescence efficiency,and strong anti-interference capacity through a series of characterizations.The new time-resolved fluorescent microspheres were then tested in immunoassays to see if they could be used in immunoassays.In the second chapter,Because myoglobin（Myo）can be employed as a marker for the early identification of acute myocardial infarction,myoglobin-based detection approaches have been extensively researched.The fluidic chip addresses the issue of time-resolved fluorescence lateral flow chromatography’s poor repeatability,combines the advantages of microfluidic chip technology and time-resolved fluorescence immunoassay technology to perform convenient myoglobin detection,greatly improves analysis efficiency,and develops new immunoassay detection methods.We use glass slides to immobilize antibodies and pre-embed the glass slides in the microfluidic chip,which increases the automation of the analysis process by concentrating the reagent reaction,sample separation,product detection,and other operations on a micro-sized chip.The entire detection procedure takes only ten minutes,and the analytical efficiency is good,additionally,the microfluidic chip can achieve a tightly controlled reaction time,which improves detection reproducibility.This is a new immunological technology based on the immunosandwich method with two antibodies.On a glass slide,a composite structure of fluorescent microspheres may be built,the properties of time-resolved fluorescent microspheres mixed with myoglobin antibody can be formed.The fluorescence is bright and stable,and it may be detected immediately on the glass slide.This approach can be used as a foundation for quantitative myoglobin analysis,it has a high sensitivity and a low detection limit of 3.85 ng/m L,making it acceptable for clinical testing of myoglobin.