Design, Construction And Application Of Fluorescent Probe For Novel Biomarkers Of Liver Tumor

Liver cancer is a lethal malignancy with low survival rate.Early detection and intervention of liver cancer is one of the effective ways to improve the survival rate of patients.The gold standard for early diagnosis of liver cancer is alpha-fetoprotein(AFP)in serum,but it has been reported that AFP levels in early blood samples of some patients with liver cancer did not increase significantly.Based on this,researchers are working to expand the list of potential markers for liver cancer.For example,the liver’s high expression of Glucose-regulated protein 78(GRP78,an endoplasmic reticulum companion protein)and carboxylesterase(CE,a class of serine proteases)are two potential markers for liver cancer that have been identified in recent years.The establishment of sensitive,convenient and specific detection methods for these new markers has become one of the researches focuses in the field of chemical biology.Among the many detection methods,fluorescent labeling technology has attracted much attention because of its advantages of in situ,multi-level and high sensitivity.The core of fluorescence labeling technology is fluorescent probe.Therefore,this paper designed and constructed fluorescent probes targeting GRP78 and CE,two potential markers of liver cancer respectively,and applied the constructed probes to the specific imaging of liver cancer cells,liver tumor model mice,and human liver cancer tissues,specifically:First,fluorescent probe for GRP78: The existing detection of GRP78 mainly relies on traditional western blot analysis,which is unable to achieve real-time fluorescence imaging analysis of cells and vivo.To solve this problem,a "hairpin" peptide-based probe(pep FAM)was designed and constructed based on the fluorescence resonance energy transfer mechanism.The probe contained WIFPWIQL,a polypeptide sequence that was specifically bound to GRP78.Both ends of the peptide chain were sealed with 5-FAM fluorophore and dabcyl quench agent,respectively.Normally,the "hairpin" of the probe is in the closed state,energy transfer occurs,and fluorescence quenching occurs.When GRP78 is added,the "hairpin" of the probe is open,energy transfer is inhibited,and fluorescence is lit up due to the specific binding to the polypeptide sequence on the probe.This "hairpin" peptide-based probe successfully recognized GRP78 specifically and achieved specific imaging of liver cancer cells and mouse tumor models.Second,for CE fluorescent probes: the existing CE fluorescent probes have slow response speed and poor selectivity.To solve this problem,a series of amide structural units with different leaving groups were designed and constructed on alkaline blue 3 dye based on the method of "substrate hydrolytic enzymatic reaction" as CE response sites.The JFast selected from many probes shows the best comprehensive performance,that is,both ultra-fast response speed and high selectivity.In the presence of CE,JFast can maximize the fluorescence intensity of 676 nm at the main peak position in just 150 seconds without interference from other esterases.The molecular docking simulation results show that the distance between CE active cavity and JFast response site is the shortest.Imaging of cells and mice showed that the probe could light up areas of liver cancer cells and primary liver tumors.JFast was also used to distinguish between imaging human primary liver cancer tissue and adjacent tissue.In conclusion,we designed and constructed fluorescent probes with strong specificity and fast response for GRP78 and CE,two potential markers of liver cancer.The results of this study are expected to promote the early detection and diagnosis of liver cancer.