Research On Lateral Flow Fluorescent Immunoassay Test Paper And Method With Up-conversion Material As Signal Material

Point of care testing(POCT)refers to medical tests carried out in or near the patient care site,and is one of the fastest growing areas of in the field of in vitro diagnostics.POCT provides a good means for disease diagnosis since it has many advantages,such as low cost,convenient use,saving on time,and simplicity,and thus plays a significant role in improving the quality of medical services and can fit the medical needs of remote districts.Upconversion nanoparticles(UNCPs,NaYF4:Yb/Er)are lanthanide-doped nanocrystals,can be excited by longer wavelength light and then emit shorter wavelength light.The low-energy photons are excited to emit high-energy photons,which violates the Stoke(anti-Stokes emission).This unique optical characteristic it has advantages such as high optical stability,large anti-Stokes shifts,sensitive to complex biological samples and multiple and narrow emission bands.So upconversion nanoparticles have been used as a new generation of fluorescence labels for immunoassays.In recent years,the study of lateral flow immunostrips based on upconversion nanoparticles have attracted more and more attention.Therefore,this dissertation designed and developed two newly lateral flow immunostrips for sensitive and rapid determination of oxidative stress markers 8-hydro-2'-deoxyguanosine(8-OHdG)and heat shock proteins70(Hsp70).In this dissertation,upconversion nanoparticles were used as fluorescent markers in combination with competitive and sandwich immunoassay,using 8-hydro-2'-deoxyguanosine(8-OHdG)and heat shock proteins70(Hsp70)as the model target analytes.The new methods using newly lateral flow immunostrips prepared for the rapid and sensitive determination of these two oxidative stress markers have been established and applied in the analysis of human urine and human serum samples.The research work provides new devices and new methods for the determination of 8-hydro-2'-deoxyguanosine(8-OHdG)and heat shock proteins70(Hsp70),and promotes the development of POCT methods,and have important scientific significance and application value.The research work was finally supported by.National Natural Science Foundation of China,"development of smart sensors based on mobile phone blood glucose meter and its application in quantitative assessment of DNA peroxidation damage"(21729501,January 2018--December 2021).This dissertation consists of introduction,research report,summary and outlook.The first chapter as the introduction mainly introduces the principle and classification of real-time determination method,real-time determination method,the luminescence mechanism of rare earth upconversion nanoparticles and the research progress of lateral flow fluorescence immunoassay.The second part is the research report part,and the third part is the summary and outlook and the research report consists of he following two parts:First,study on a new method for the detection of 8-hydro-2'-deoxyguanosine lateral flow immunostrips.Lateral flow immunostrips have received much attention as important tools for point of care testing(POCT).However,they are only few of research works for small biomarker molecules.In this work,a newly lateral flow immunostrips were designed and developed for the detection of 8-hydroxy-2'-deoxyguanosine(8-OHdG)as a model target of small biomarker molecules by integrating upconversion nanoparticles(UCNP)as fluorescence signal materials with a competitive immunoassay.The 8-OHdG antibody(Ab)was taken as the recognition element while UCNP-labeled Ab(UCNP-Ab)was taken as the signal probe.BSA was designed as carrier protein for 8-OHdG to form 8-OHdG-BSA conjugate.The conjugate of 8-OHdG-BSA was taken as the capture probe immobilized on the test line while an IgG antibody was taken as the capture probe immobilized on the control line.When the sample and the signal probe are loaded on a sample pad,8-OHdG analyte binds with the signal probe to form the 8-OHdG·UCNP-Ab bioconjugates.Both the formed bioconjugates and excess of the signal probe move along the strip,excess of the UCNP-Ab rather than the bioconjugates is collected by 8-OHdG-BSA immobilized on the test line while the signal probe is collected by IgG antibody immobilized on the control line.The fluorescence intensity on the test line was inversely proportional to the concentration of 8-OHdG from 0.1 to 10 nM with the limit of detection of 0.05 nM.The work demonstrates that competitive immunoassay using a carrier protein for small biomarker is a promising approach to design lateral flow immunostrips for POCT.Second,study on a new method for the detection of heat shock proteins70 lateral flow immunostrips.Heat shock protein 70(Hsp70)is a macromolecular biomarker.In this work,the heat shock protein 70(Hsp70)was used as the determination substance,and the heat shock protein 70 antibody(Ab1,orb79478)was used as the molecular recognition substance while the UCNP-labeled Ab1(UCNP-Ab1)was taken as the signal probe.The Hsp70 antibody(Ab2,orb24921)was taken as the capture probe immobilized on the test line while an IgG antibody was taken as the capture probe immobilized on the control line.When the sample and the signal probe are loaded on a sample pad,Hsp70 analyte binds with the signal probe to form the Hsp70-UCNP-Abl bioconjugates.Both the formed bioconjugates and excess of the signal probe move along the strip,the bioconjugates is collected by Hsp70 antibody(Ab2,orb24921)immobilized on the test line while the excess of signal probe is collected by IgG antibody immobilized on the control line.The fluorescence intensity on the test line was proportional to the concentration of Hsp70 in the sample.Based on this,a new method for real-time detection of Hsp70 by lateral flow immunostrips was established.The developed method for the determination of Hsp70 exhibited a linear range from 0 to 12 ng/mL with the limit of detection of 0.54 ng/mL while the relative standard deviation for 6 nM Hsp70 is 4.2%(n=7),the average relative standard deviation Hsp70 is 4.1%.The research results show that the designed real-time detection lateral flow immunostrips can be applied to the determination of biological macromolecules.