| Cancers are becoming the leading causes of death,which are seriously threatening human health and life.Nearly 8.2 million cancers related deaths,32.6 million people living with cancer and 14 million new cases happened every year.In many cases,cancers are not diagnosed and treated until cancer cells have already invaded surrounding tissues and metastasized throughout the body.The identification of low abundance disease-specific protein is challenging due to the presence of other proteins in a wide dynamic range.The quantified detection of proteins in clinical fluid is believed to be especially important in the disease diagnosis and subsequently observing disease progression in response to therapy.This accelerates the need of quick detection of proteins such as C-X-C chemokine receptor type 4(CXCR4),C-C chemokine receptor type 3(CCR3),alpha-fetoprotein,carcinoembryonic antigen for diagnosis purposes and monitoring tumor progression.Conventional tools for protein analysis,such as western blot and enzyme-linked immunosorbent assays,are limited by their low sensitivity,lengthy processes,and high sample demand.To overcome these limitations,this thesis proposes the advancement of photonic crystals and plasmonic silver film to be a compelling platform for improving the sensitivity of surface-based fluorescent assays used in clinical diagnostic applications and life science research.The main contributions to this thesis are as follows.1.Revealing chemokine receptor CXCR4 expression,distribution and internalization levels in different cancers helps to evaluate cancer progression or prognosis and to set personalized treatment strategy.This thesis describes a sensitive and high-throughput immunoassay for determining CXCR4 expression and distribution in cancer cells.The assay is accessible to a wide range of users in an ordinary lab only by dip-coating poly(styrene-co-N-isopropylacrylamide)spheres on the glass substrate.The self-assembled spheres form three-dimensional photonic colloidal crystals which enhance the fluorescence of CF647 and Alexa Fluor 647 molecules by a factor of up to 1000.CXCR4 in cells is detected by using the sandwich immunoassay,where the primary antibody recognize CXCR4 and the secondary antibody is labeled with CF647 molecule.With the newly established assay,I quantified the total expression of CXCR4,its distribution on the cell membrane and cytoplasm,and revealed their internalization level upon SDF-1?activation in various cancer cells,even for those with extremely low expression level.2.A highly sensitive microarray assay that can detect different types of chemokine receptors simultaneously in a single cell sample is reported in this thesis.This assay is constructed in an immuno-dot blot format on colloidal photonic crystal film where fluorescent dyes,AF647 and FITC,can enjoy dramatic signal enhancement up to 820×and 550×folds,respectively.The high enhancement for two-color fluorescence allows highly sensitive detection of co-expression of chemokine receptors and their ratio changes after binding with the respective ligands and drugs in an identical cell sample.This will help to achieve chemokine receptor-related drug screening,therapeutic intervention or prognosis evaluation.This method can also be used as a valuable and universal tool to analyze other proteins involved in cells and their interoperable changes after physiological or therapeutic responses.3.Finally,a novel low-cost plasmonic silver needle(PSN)for immunofluorescence detection of tumor biomarkers is reported in this thesis.The fluorescence signal is enhanced on the needle by up to 220-fold,allowing high-performance detection of tumor biomarkers down to 0.08 ng m L-1.To assess the clinical potential of the proposed assay technique,PSN-based sandwich immunoassay for the detection of alpha-fetoprotein and carcinoembryonic antigen were performed for blood as well as for serum sample.The results from serum sample have an excellent agreement with an electrochemiluminescence assay.The small relative error and a good linear correlation suggest that the accuracy and precision of this analytical technology is satisfactory.This assay technique with lower cost,use of less sample,higher sensitivity and easier procedure shows great promise for the facile and early cancer diagnosis. |
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