Ultra-sensitive Detection Of Fn14,P-cadherin And N-Cadherin Based On Single Molecule Array Technology

Early detection is important for the diagnosis and treatment of diseases.Earl y prevention,early detection and early treatment are the key to reducing the incidence of disease,improving the prognosis and improving the survival rate of patients.Compared with tissue biopsies currently widely used in clinical practice,liquid biopsy has received more and more attention due to its advantages such as non-invasiveness,high accuracy and real-time monitoring.Protein biomarkers are the most commonly used detection methods in liquid biopsy,but traditional protein detection methods are not sensitive enough to limit the detection of some low-abundance proteins.Single Molecule Array technology(Simoa)is an ultrasensitive bead-based digital enzyme-linked immunosorbent technology.Beads containing a single immunocomplex are loaded into microwells(~46 fL)and produce fluorescence through enzyme-catalyzed reactions in extremely small volumes.By measuring the number of fluorescent microwells in arrays arranged on a circular Disc,the concentration of target molecule was determined.This paper developed ultra-sensitive detection of three biomarkers for Fn14,P-cadherin and N-Cadherin which based on single-molecule array technology,as follows:1.Fibroblast growth factor inducible-14(Fn14)is a receptor protein that plays an important role in the progression of cancer and some other diseases.Here,an ultrasensitive assay was developed for the detection of Fn14 based on Simoa.To obtain better performance for Fn14 detection,assay conditions including reagent concentrations and measurement parameters were optimized and 44 different antibody pairs were screened.The detection range of Fn14 is 1.26 pg/mL to 3,683 pg/mL with a lower LOD of 0.32 pg/mL,which is much lower than that of conventional ELISAs.2.Overexpression of P-Cadherin is associated with the development of some malignant diseases.In this paper,the Simoa assay we developed for P-Cadherin showed good linearity in the dynamic range of 1.38 pg/mL to 1,886 pg/mL with a LOD as low as 0.65 pg/mL.3.N-Cadherin,a member of the cadherin family,is a key transmembrane cell adhesion molecule,and its overexpression is also associated with the development of malignant diseases such as cancer.In this work,we have developed a new N-Cadherin ultra-sensitivity detection method.The method has a linear range of 11.40 pg/mL to 10,500 pg/mL and a detection limit of 2.85 pg/mL,which is much lower than the LOD of conventional immunoassays.It allows us to accurately quantify N-Cadherin protein in serum even at very low concentrations.In addition,the total operations of these assays are automated and only take approximately an hour to accomplish.Furthermore,these assays were successfully applied to the determination of spiked target proteins in serum samples with satisfactory performance.