| Liquid biopsy a branch of in vitro diagnosis,which mainly evaluate the diseases by blood detection.With the advent of the era of precision medicine,liquid biopsy has been widely used in tumor diagnosis,pregnancy screening and other fields due to its non-invasive,sensitive and dynamic characteristics.Especially in the early screening,mutation typing,medication guidance and relapse monitoring of tumors,liquid biopsy has an immeasurable development prospect,and was selected as "2015 top ten breakthrough technologies" by MIT science and technology review.It is estimated that the market size of liquid biopsy will reach US $2.05 billion in 2022,with a compound annual growth rate of 23.4%.Circulating tumor cells(CTC),circulating DNA(ct DNA)and exosomes are three major detection objects of liquid biopsy.Exosomes are small vesicles with a diameter of 40-150 nm secreted by cells.They circulate freely in body fluid,transfer information to adjacent or distant cells,and participate in many physiological and pathological processes such as immune regulation and tumor metastasis.Tumor derived exosomes carry some cytoplasm and membrane components of tumor cells.Therefore,compared with CTC or ct DNA,exosomes not only have more quantity than CTC,but also carry many kinds of tumor related molecules such as nucleic acid,protein,lipid,polysaccharide and so on.It has been known that some important molecules,such as HER2 and PD-L1,will enter the blood with exosomes from tumor tissue.However,the amount of tumor derived exosomes in the blood is very small.Most of the existing detection methods are based on the horseradish peroxidase(HRP)catalyzed ELISA system,which often brings false negative results due to the lack of sensitivity.Therefore,it is urgent to develop new high sensitive detection methods for exosomes.In recent years,more and more attention has been paid to Gaussia luciferase(Gluc).Compared with traditional luciferase,it has the advantages of high luminous efficiency,small molecular weight and simple reaction system.The purpose of this study is to develop a novel exosomes detection method based on the Gluc catalytic luminescence system,and to detect the surface marker protein of exosomes from patients’ blood,so as to provide a basis for the diagnosis and treatment of tumors.For this goal,the following research contents were done in this paper.1.The fusion protein of Gluc and streptavidin(SA)was designed and purified in eukaryotic expression system,and identified by Western blot.Verify the luminous efficiency of Gluc-SA and the linear relationship between luminous value and its concentration.To find the best concentration of substrate,optimize the detection parameters,and keep the same in the following experiments.To verify the recognition ability of Gluc-SA to biotin,the linear relationship between luminous intensity and biotin concentration was evaluated,and the minimum detection limit(LOD)was calculated.At the same time,it was compared with HRP-SA in detecting biotin.The results show that the designed gluc-sa has the advantages of correct expression,high efficiency of catalytic luminescence,good linearity,and can combine with biotin,and realize the quantitative detection of biotin in a wide range,especially the linear relationship in the low concentration range is better than the conventional HRP catalytic color system,and has a lower LOD than the HRP system.2.In order to find a method to capture exosomes from the samples,Gluc was expressed on the exosomes secreted by HEK293 T cells,and an accurate and quantitative exosomes standard was prepared.It was found that a commonly used Maxi Sorp surface could capture exosomes from samples,and the efficiency was higher than that of CD63 or CD9 antibody.When the exosomes were mixed with 10% plasma,Maxi Sorp could still capture the exosomes,and the amount of capture was linear with the amount of exosomes in the sample.The results show that Maxi Sorp surface is very suitable for the detection of plasma exosomes.3.Combined with the Gluc-SA bioluminescent reporter molecule and maxisorb surface capture,a new method for the detection of exosomes surface markers was constructed,including the steps of sample pretreatment,sealing,incubation,biotinylated antibody recognition,gluc-sa binding and luminescent detection.Using this method,the exocrine HER2 was successfully detected in the culture medium of BT474 cells(HER2+),while the luminescent value of the culture medium of MB-MDA-231 cells(HER2-)was very low.Exosomes expressing PD-L1 were prepared as standard for quantitative detection.Then the standard curve of luminescent value exosomes PD-L1 content was drawn and LOD was calculated.PD-L1 in the plasma of patients with head and neck cancer was measured quantitatively and compared with that of healthy people.The results showed that the detection results of this method were highly consistent with the clinical situation and the results of the traditional methods reported in the literature.In conclusion,we developed a novel method for the detection of exosomes surface markers based on Gluc luminescence reporting system.Compared with the conventional products on the market,this method is simpler,cheaper and more sensitive.This study is helpful to the clinical translation of exosomes detection and promote the application of liquid biopsy. |
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