The Development Of Rapid And Micro-Volume Detection Of Exosomes From Pancreatic Cancer Based On Innovated Raman Probes And Advanced Dopamine Chip

Part 1 Isolation and identification of exosomes from cell lines PANC-1 and HPDE6-C7 and the examination of their typical moleculesIn this experimental part,we cultured the pancreatic cancer cell line PANC-1 and the normal cell line HPDE6-C7 according to the routine methods.The exosomes were isolated by using density gradient ultracentrifugation method.Obtained exosomes were scanned under the transmission electron microscope?TEM?to observe their diameters and sizes.Nanosight tracking analysis?NTA?technique was used to calculate the exosomes,the final number was the average of results of five experiments.Magnetic beads combined with flow cytometry analysis system and the nanoflow analysis method were used to identify the surface molecules on exosomes.BCA method was used to quantify the protein,ZETA distribution was obtained from instrument of Zetasizer Nano.From the experiment,images of TEM showed that the diameters of exosomes from PANC-1 and HPDE6-C7 cell lines?hereinafter referred to as EXO-PANC1,EXO-HPDE6-C7?were between 30nm and 180nm;the results of NTA showed that the concentration of EXO-PANC1 solution was2.72*10¹⁰±2.05*10⁹ particles/ml,while EXO-HPDE6-C7 solution was 2.43*10¹⁰±2.21*10⁹particles/ml;the protein concentration of EXO-PANC1 solution was 111.31?g/ml,EXO-HPDE6-C7 solution was 109.89?g/ml by BCA method;Zeta distribution of EXO-HPDE6-C7 was-25.4mV,EXO-PANC1 was-10.1mV.The results of magnetic bead combined with flow cytometry method showed that CD9,CD63 could not significantly distinguish exosomes of pancreatic cancer cell line PANC-1 from those of normal cell line HPDE6-C7,while Glypican-1?GPC1?and macrophage migration inhibitory factor?MIF?could do,the results of nanoflow analysis showed that there was high expression of MIF and GPC1antigens in EXO-PANC1.The results of this part demonstrated that the characters of exosomes from PANC-1 and HPDE6-C7 were consistent with what had been reported in current literatures?diameters,the expression of CD9,CD63 molecules?,GPC1 and MIF that was expressed highly in exosomes from pancreatic cancer cell line could be the targeted molecules in the examinations of subsequent experiments.Part 2 The exploration of related experimental conditions of dopamine chip packed by antibodies and SERS tagsIn this part we primarily designed the experimental steps for forming the dopamine chip,further we discussed the influence of the concentration of dopamine solution on the formation of chip.For the synthesis of nanotags that were detected by surface-enhanced Raman scattering technology,we firstly synthetized 18nm AuNPs as the core in the classical method,then the material structure-Au@Ag@pATP was formed step by step.To protect the fragile Ag layer,we added a layer of protective albumin from bovine serum?BSA?on the surface of Ag.To connect the antibodies to the surface of nanoparticles and not affect their stability and biocompatibility,we adhered the antibodies to the edges of nanoparticles through the polymerization of dopamine,therefore,forming the nanosphere structure of SERS tags which were packaged by multiple layers.We also identified the characters of SERS tags such as their images under TEM,ultraviolet spectrums,or SERS spectrums,we also explored the concentrations of AgNO3 or pATP solution which were the key impacted factors in the synthesis of SERS tags.Through the experiment,we knew that when the concentration of dopamine solution was 33mM,the polymerization on the chip was perfect.When the concentration of AgNO3 solution was 0.096mM,pATP solution was 9.6mM,the formation of SERS tags were with best presentations.Under TEM,we observed that the thickness of Ag was approximately 1nm,dopamine was about 3nm,the ultraviolet spectrum of Au nanoparticle showed that there was only one absorption peak?521nm?,while that of SERS tags showed that there were two absorption peaks?400nm and 500nm?which were derived from Ag and Au nanoparticles.The results of Raman spectrum showed that for SERS tags there was a correspondingly stronger Raman intensity peak(1072-1076cm^-1).The Raman signals still could be obtained on conditions of low power?0.05W?and short scanning time?10ms?.The above optimized experimental conditions for dopamine chip-SERS system that had been explored in this part of experiment would be further used in the subsequent establishing of the studying system.Part 3 Application of dopamine chip-SERS examined system on testing exosomes from the cell lines and optimization of this systemIn this part we detected the EXO-PANC1 and EXO-HPDE6-C7 by using the previously established dopamine-SERS systems with different targeted antigens individually.Results showed that dopamine chip-SERS examined system on targets of GPC1/MIF could distinguish EXO-PANC1 from EXO-HPDE6-C7,while when we regarded CD9,CD63 as examined targets,we could not distinguish the two types of exosomes efficiently.We further added the epidermal growth factor receptor?EGFR?,and evaluated the tested efficiencies of dopamine chip-SERS system for targets of MIF/GPC1/EGFR/CD63 antigens separately,we found that there was the same lowest detected limitation(9.0*10^-19mol//L)in MIF/GPC1/EGFR groups,while CD63 group could not detect exosomes in that lowest concentration.From the linear equations in different groups,we found that the slope of linear equation of MIF group was the highest,which demonstrated that its sensitivity was higher.Next,we examined EXO-PANC1 for the target of MIF and drew the standard curve,the linear fitting equation for exosomes'solution in low concentration was y=-2.4152+1.2392x.This system could detect EXO-PANC1 in 2?L exosome solution with concentration of5.44*10² particles/ml,and the sensitivity of it was increasing dramatically compared with commercial ELISA kit.Results of this part showed that dopamine chip-SERS detected system with target of MIF had higher diagnosis efficiency than other systems,which could be applied to the subsequent examination of clinical samples.Part 4 Examination of serous samples from patients with pancreatic cancer using dopamine chip-SERS detection system and the evaluation of its testing efficiencyIn this part of experiment,firstly we examined the serous samples from patients with pancreatic cancer and the healthy using dopamine chip-SERS system with the target of MIF/GPC1/EGFR,the results showed that only MIF-SERS system could distinguish:patients with pancreatic cancer from the healthy,pancreatic cancer patients in P1² from P3stages,pancreatic cancer patients with metastasis from non metastasis?P<0.05?.We further compared the examined results of MIF-SERS system with clinical monitors such as cancer antigen 19-9?CA19-9?,and carcinoembryonic antigen?CEA?or the tested results of MIF-ELISA commercial kit,and found CA199 could distinguish patients with pancreatic cancer from the healthy?P<0.05?,but couldn't distinguish:patients with pancreatic cancer in P1²stages from P3 stage,metastasis from non metastasis?P>0.05?,it had the similar sensitivity?68.2%vs 62.5%?and specificity?72.7%vs 76.2%?with MIF-SERS system.The analysis of results of CEA showed that this biomarker could not identify the three groups above?P>0.05?,its sensitivity reduced by 21.6%,specificity by 12.6%comparing with MIF-SERS system.The sensitivity and specificity of ELISA method decreased by 21.6%,16.2%,separately in comparison with MIF-SERS system.In addition,considered the above classical methods,the serous volume for MIF-SERS system was shrunk dramatically?1-2mL vs 2?L?.According to the tested values of the samples,we formulated the clinical referenced range of log?Raman intensity?primarily for the target of MIF,it was pancreatic cancer vs the normal:3.77±0.15 vs 2.67±0.80,cutoff value:3.55,the threshold of Raman signal intensities for the imaged dots was 20.Next,we performed the combined analysis for testing results from MIF,GPC1,EGFR groups by statistical method,and found that the sensitivity of combined diagnosis was elevated by 12.5%comparing with single MIF-SERS system,while its specificity was comparable with that of MIF-SERS system?75.0%vs 76.2%?,its cutoff value?pancreatic cancer patients vs the healthy?was 0.27 via calculating.Through this part of experiment,we established the dopamine-SERS detection system with target of MIF on the whole,this system in combination with other biomarkers such as GPC1/EGFR was hopeful to be applied to the clinical examination and could help for the diagnosis of pancreatic cancer.