Construction Of Exosome SORL1 Detection Platform Based On 3D Porous Microfluidic Chip And Its Application In Early Diagnosis Of Colorectal Cancer

In recent years,the incidence and mortality of Colorectal cancer(CRC)have shown a significant upward trend.It is one of the major diseases that seriously endanger human health,and the age of onset is earlier.Early symptoms of colorectal cancer are hidden and difficult to diagnose.At the same time,the sensitivity and specificity of the widely used tumor markers in clinical practice are not ideal,and accurate diagnosis cannot be achieved.Therefore,it is of great significance to screen new tumor markers and develop new detection technologies for promoting early diagnosis of colorectal cancer and improving survival rate of patients.With the development of liquid biopsy technology represented by exosomes and microfluidic chip technology,new exosome detection technology using microfluidic chip as carrier has emerged,which is expected to improve the detection efficiency and is a new trend in the development of early tumor diagnosis technology.In this study,an efficient exosome enrichment platform based on three-dimensional porous microfluidic chip was constructed,and Sortilin-related receptor 1(SORL1)was screened as a colorectal cancer-specific exosome membrane surface protein.Combined with quantum dot fluorescence imaging and artificial intelligence image analysis system,a rapid and sensitive detection system was constructed.Objective:A platform for efficient enrichment and rapid capture of exosomes based on three-dimensional porous microfluidic chip was constructed.The exosomes of colorectal cancer were analyzed by depth mass spectrometry,and the membrane surface proteins of colorectal cancer specific exosomes were screened by multi-level expression.Furthermore,the method of detecting differential exosomal membrane proteins based on high-specificity quantum dots labeling was designed,and the application value of the detection system in the specific diagnosis of early colorectal cancer was preliminarily evaluated by combining quantum dots fluorescence images and artificial intelligence image integrated analysis system.Methods:1.Based on the plasticity of Polydimethylsiloxane(PDMS)and the easy solubility of NaCl particles to construct a porous structure,a three-dimensional porous sponge structure chip was fabricated.SEM)for structural characterization.The functionalized 3D porous sponge structure of CD9 antibody can bind to the exosomes entering the microfluidic chip.The exosome capture efficiency of the chip was verified by Nanoparticle tracking analysis(NTA)and fluorescence analysis.2.Label-free proteomics quantitative analysis technology was used to quantitatively compare the proteome of the samples,and combined with the screening and location of the database,the range of differentially expressed proteins on the surface of exosomes between colorectal cancer and healthy controls was determined.Flow cytometry analysis using latex beads and Western blot were used to further verify the localization and expression of differential proteins in cell lines and clinical serum samples.3.Silicon quantum dots(Si-QD)-SORL1 antibody complexes(Si-QD-SORL1)were prepared and subjected to Transmission Electron Microscopy(TEM).TEM)and Ultraviolet-visible spectrum(UV-vis)scanning were used to characterize the Si-QD-SORL1 complex.4.Confocal laser scanning microscopy(CLSM)based quantum dots fluorescence imaging technology to obtain high-resolution fluorescence images,based on Artificial intelligence(Artificial intelligence,AI)image integrated analysis system was used to extract and analyze the features of the obtained fluorescence images to construct a rapid and sensitive colorectal cancer detection platform.The diagnostic efficacy of the detection system was evaluated by Receiver operator characteristic curve(ROC).Results:1.The novel 3D porous PDMS sponge structure chip was successfully designed and fabricated.The optimal pore structure was obtained when the mass ratio of NaCl to PDMS was 3.0:1.The CD9 antibody was efficiently immobilized in the porous PDMS structure at 37℃ for 2 hours.NTA and fluorescence analysis results showed that the exosome capture efficiency of the chip could reach up to 90%.2.Five protein molecules including SORL1 and PMSA3 were included in the candidate range of early diagnosis of colorectal cancer by combining labelless quantitative proteomics analysis,protein molecular localization in the database and comprehensive evaluation of antibody availability.Western blot results showed that SORL1 was highly expressed in colorectal cancer exosome samples,and flow cytometry results showed that SORL1 was located on the surface of the exosome membrane.Therefore,SORL1 was focused on as a CRC specific exosomal membrane protein for subsequent studies.3.TEM results showed that the Si-QD and SORL1 antibodies went from dispersed two separate state substances to successfully binding into an integral complex.UV-vis spectral scanning revealed a clear Si-QD absorption peak at 357 nm,which was significantly reduced by the addition of the SORL1 antibody to form the Si-QD-SORL1 complex.Both TEM and UV-vis spectroscopic scanning results showed that the Si-QD-SORL1 complex was successfully synthesized.4.The three-dimensional porous microfluidic chip-based SORL1 detection technology and AI-based image integrated analysis system were used to detect the serum samples of 106 colorectal cancer patients and 100 healthy controls in the sample bank.The results showed that the expression of SORL1 was significantly up-regulated in colorectal cancer patients.the Area under the curve(AUC)was 0.98.Meanwhile,this study focused on the diagnostic results of 81 patients with early colorectal cancer(stage I and II).The results showed that the serum samples of early colorectal cancer patients and healthy controls could be effectively separated,and the diagnostic sensitivity reached 97.53%,and the AUC was 0.99.The system shows good diagnostic efficacy in young patients with colorectal cancer,patients with small tumors and patients with negative CEA.In addition,the accuracy and specificity of the SORL1 detection platform based on 3D porous microfluidic chips in the diagnosis of CRC were significantly higher than those of the conventional marker CEA and pan-cancer marker EpCAM.Conclusions:1.In this study,a novel three-dimensional porous sponge structure microfluidic chip was successfully designed and prepared.SORL1 was identified as a specific exosomal membrane protein for the early diagnosis of colorectal cancer through multi-level expression screening,which has good diagnostic efficiency in the detection of colorectal cancer-related exosomes and provides new ideas for the early diagnosis of colorectal cancer.2.In this study,the SORL1 detection technology based on high-specificity quantum dots labeling technology and the image integrated recognition system based on artificial intelligence were established,which provided a new and efficient detection technology platform for the detection of clinical exosome markers and early diagnosis of colorectal cancer.