| Lung cancer is one of the cancer types with the highest morbidity and mortality in the world.In order to reduce mortality,early detection and early treatment are still effective ways.Therefore,the early diagnosis of lung cancer has become extremely important.Clinically,the diagnosis of lung cancer is based on tissue imaging and tissue biopsy.However,these methods still have many drawbacks,including radiation caused by imaging techniques,increased risk of cancer,and pain caused by tissue puncture.Liquid biopsies provide a new diagnostic dimension for cancer based on molecular information obtained from fluid samples such as peripheral blood and urine.Liquid biopsy is non-invasive,safe,and easy to repeat compared to traditional tissue sampling,allowing physicians to discover a range of information about a disease or tumor through a simple blood sample.Many biomolecules have been extensively studied as cancer markers.Among them,only protein molecules in body fluids have been approved as cancer biomarkers by various national food and drug administrations(FDA).The detection of serum protein markers also plays an important role in the diagnosis and prognosis of lung cancer.However,the protein markers currently used are broadspectrum type,and the sensitivity and specificity for the diagnosis of lung cancer still appear to be weak.Therefore,more protein markers that can be used for the diagnosis and prognosis of lung cancer need to be discovered and evaluated.In addition,in order to improve the diagnostic effect and efficiency,simultaneous detection tests of various protein markers also need to be developed.Single-molecule array technology(Simoa)is a digital form of enzyme-linked immunosorbent assay(ELISA)used for immunoassay,which captures and individually seals magnetic beads immunocomplexes in femtoliter microwells on the Simoa disc.It achieves high sensitivity detection of protein markers.The average sensitivity of its detection is 1000 times that of traditional technology,and it has broad application prospects in life sciences,in vitro diagnosis,concomitant diagnosis,blood screening,drug research and development,etc.This project combines single-molecule array technology(Simoa)to develop a series of highly sensitive protein detection assays.The developed assay screened several new protein markers of potential value for the diagnosis of lung cancer.The main contents include the following several aspects:(1)Highly sensitive immunoassay was developed for the detection of EphA2 and application evaluate in lung cancer based on Simoa.EphA2 was demostrated to be overexpressed in 90% of lung cancer tissue samples.The expression of EphA2 was positively correlated with smoking history,and its high expression predicted poor survival.Tissue biopsy demonstrates that overexpression of EphA2 has a certain diagnostic and prognostic value for the detection of lung cancer.Whether serum EphA2 has the same value in lung cancer testing requires further evaluation.This project develops an ultrasensitive detection serum EphA2 test based on Simoa technology.The developed EphA2 Simoa assay is characterized by fast,high sensitivity and wide detection range(0.5-1024 pg/m L).The detection limit of EphA2 in this test reached0.102 pg/m L.At the same time,selective experiments demonstrated that the developed Simoa assay is highly specific for EphA2 detection.The validation test and the spike recovery test also demonstrate that the assay can be used for the detection of EphA2 in human serum.Serum EphA2 protein concentrations were measured in 26 healthy people,20 benign lung disease patients,and 39 lung cancer patients using the developed method.The results suggest that high expression of serum EphA2 may be associated with lung cancer and may be an early biomarker of lung cancer development.(2)Ultrasensitive immunoassay was developed for the detection of b FGF and application evaluate in lung cancer based on Simoa.b FGF has a very low concentration in human serum and is in the sub-picogram level,requiring highly sensitive detection techniques to accurately assess its diagnostic value.This project developed a b FGF ultrasensitive detection assay based on Simoa technology.It has a wide detection range(0.125-128 pg/m L)and a low detection limit for b FGF,reaching 0.046 pg/m L.At the same time,selective experiments have shown that the developed Simoa assay is highly selective for b FGF.The validation test and the spike recovery test also prove that this assay can be applied for the detection of b FGF in human serum.In addition,this project used the developed b FGF Simoa assay carry out the preliminary evaluation of b FGF in clinical lung cancer serum samples,and obtained good results.When distinguishing between healthy and lung cancer groups,the sensitivity and the specificity of b FGF was90% and 92.6%,and the area under the ROC curve(AUC)was 0.929.Therefore,the developed b FGF Simoa assay in this project has shown potential application prospects for the early diagnosis and related research of lung cancer.(3)Ultrasensitive immunoassay was developed for the detection of Pro GRP and application evaluate in lung cancer based on Simoa.Pro GRP is a highly specific small cell lung cancer biomarker with low serum concentration and poor stability.This project develops the corresponding Pro GRP Simoa ultrasensitive detection assay based on single molecule array technology.Compared with the previously reported methods,the test has the advantages of fastness,high sensitivity and wide detection range(0.03125-512 pg/m L).The detection limit is as low as 24 fg/m L.The validation test and the spiked recovery test proved that the developed test also achieved good detection results.This assay did not achieve the desired results in the selected clinical samples and may be related to the poor stability of Pro GRP in serum.In addition,this assay has potential applications for studying the degradation of Pro GRP in serum and related enzyme kinetics because of the very low detection limit.(4)For the first time,this project developed a highly sensitive assay for the detection of human AEG-1 autoantibodies using Simoa technology.To a certain extent,the application range of this technology in the field of biomarker detection has been expanded.The developed human AEG-1 autoantibody Simoa assay has the characteristics of rapidity,high sensitivity and wide detection range,and provides technical support for research in related fields and discovery of autoimmune antibodies.In addition,the human AEG-1 autoantibody Simoa assay developed in this project presents potential applications for early diagnosis and related research on lung cancer and other diseases.(5)Simultaneous evaluation of the application value of FORL1 and FGF21 in the diagnosis of lung cancer based on dual Simoa assay.FOLR1 is usually used as a target protein for lung cancer targeted therapy.Serum FOLR1 has rarely been studied for the diagnosis and prognosis of lung cancer.It has been reported that FGF21 has a certain anti-cancer effect,and whether its serum level has changed with the development of lung cancer has not been reported.In this project,a single molecule array technique was used to develop a duplex assay for simultaneous detection of serum FOLR1 and FGF21 and to evaluate the value of both in lung cancer.The duplex Simoa assay developed is fast,highly automated and highly sensitive.The detection limits of FOLR1 and FGF21 were 3.8 pg/m L and 1.6 pg/m L,respectively.Then,the serum validation test and the spike recovery test also proved that this assay can be used for simultaneous detection of FOLR1 and FGF21 in human serum.In addition,this chapter provides a preliminary assessment of the concentrations of FOLR1 and FGF21 in clinical lung cancer serum samples based on the developed duplex Simoa assay.By comparing the concentrations of FOLR1 and FGF21 in the serum of healthy,lung cancer and lung disease patients,the results showed that the concentration of FOLR1 was significantly different,which has certain diagnostic value in the diagnosis of lung cancer.(6)An immunoassay for the quadruplex detection of lung cancer markers based on Simoa technology.This project developed a corresponding quadruplex Simoa assay for four classical lung cancer markers,which has the advantages of high efficiency,fastness,high sensitivity and small sample size.Serum validation tests have shown that the quadruplex Simoa assay can be used for simultaneous detection of CEA,CA125,Cyfra21-1 and HE4 in human serum.In addition,a preliminary assessment of the concentration of four protein markers in clinical lung cancer serum samples was performed based on the developed quadruplex Simoa assay.The results show that this quadruplex Simoa assay meets the needs of clinical testing and has broad application prospects in lung cancer detection and related fields.(7)Glucose was detected based on blue-green fluorescent silicon nanodots synthesized in situ.Glucose is not only an important energy source for the human body,but also a biomarker for many diseases.This project proposes a new method to directly monitor glucose levels by one-step synthesis of water-soluble fluorescent silicon nanodots(SND).SND was synthesized by a mild redox reaction between glucose and aminopropyltriethoxysilane(APTES).SND exhibits good dispersibility in water,high blue fluorescence,strong light stability and wide p H tolerance,with excitation /emission peak wavelength of 410/475 nm.By studying the emission characteristics of SND,this method can detect low concentrations of glucose in a wide detection range based on monitoring the visible light emission of in situ synthesized SND. |
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