Development Of Novel Immunosensors And Its Application In Secretory Autophagosomes Detection

Our previous studies have found that tumor cell-released autophagosomes(TRAP)induced the differentiation of B cells into immunosuppressive IL-10⁺B cells via TLR2-My D88-NF-?B signaling pathway,which inhibited T-cell proliferation and anti-tumor immune response.In addition,TRAP promted neutrophils to produce ROS and activate caspase-3,which contributed to the apoptosis of neutrophils and inhibition of T-cell proliferation.Moreover,TRAP induced polarization of M2-like macrophages through TLR4-My D88-p38-STAT3 signaling pathway,which promoted high expression of PD-L1 and IL-10 and inhibited T-cell proliferation.It can be seen that TRAP is closely related to the occurrence and development of tumors,and whether the TRAP content in peripheral blood reflects the tumor progression is worthy of further study.Because of the lack of direct method of detecting TRAP and the low concentration of TRAP in peripheral blood,highly sensitive detection method is needed.Therefore,the novel method of quantitative detecting TRAP and the relationship between TRAP content of peripheral blood and tumor are worthy of further studies.ObjectivesTo investigate novel methods of sensitive detection of TRAP.In other words,to develop novel immunosensors utilizing the high sensitivity and high specificity of immunosensors and the excellent properties of carbon nanomaterials,in order to detect the content of TRAP in peripheral blood of human or mouse and explore the relationship between the content of TRAP in peripheral blood and cancer.Methods1.Design,development and application of the novel enzyme cascade immunosensorCarbon nanocomposites(MWCNTs-PDA-Au NPs)prepared using multi-walled carbon nanotubes(MWCNTs),dopamine hydrochloride(DA)and chloroauric acid(HAu Cl₄)were used as substrate materials on the electrode surface of the immunosensor.An enzyme cascade system of?-galactosidase(Gal)and tyrosinase(Tyr)was applied to the immunosensor.The carbon nanocomposites were observed and analyzed by transmission electron microscopy and UV-visible absorption spectroscopy respectively to verify whether they were successfully synthesized.The performance of carbon nanocomposites was verified by cyclic voltammetry in hexaammine ruthenium chloride solution.The feasibility of the enzyme cascade reaction was verified in the solution phase.The construction of the immunosensor was monitored by cyclic voltammetry in potassium ferricyanide solution.The model antigen CEA was quantitatively detected by the immunosensor and the methodological evaluation and preliminary application of the immunosensor were carried out.2.Design and development of specific nanomagnetic beads for enrichment and purification of TRAPThe Fe₃O₄-Au composites were synthesized by using ferroferric oxide(Fe₃O₄)magnetic nanoparticles and gold(Au)nanoparticles,and the LC3B antibody was then coupled to prepare specific nanomagnetic beads for enrichment and purification of TRAP.Scanning electron microscopy and UV-visible absorption spectroscopy were used to verify the successful synthesis of Fe₃O₄-Au composites.The specificity of LC3B antibody was analyzed by Western blot.The preparation and performance of the specific nanomagnetic beads were analyzed by laser confocal microscopy.The performance of the nanomagnetic beads was analyzed by Western blot.3.Design and development of the novel immunosensor based on graphene oxide hydrogel compositesThe graphene oxide hydrogel(GH)was preparaed with graphene oxide(GO)and then mixed with methylene blue(MB)to form graphene oxide hydrogel-methylene blue(GH-MB)complex,which was modified on the surface of the electrode as base material.The sandwich type immunosensor was constituted by GH-MB together with the specific nanomagnetic beads.Scanning electron microscopy and UV-Vis absorption spectroscopy were used to verify the successful synthesis of GH-MB composites,and the performance of GH-MB was verified by cyclic voltammetry in potassium ferricyanide solution.Western blot and flow cytometry analysis were used to select appropriate LC3B antibody for the immunosensor.The construction of the immunosensor was monitored by cyclic voltammetry in potassium ferricyanide solution.The model antigen LC3B was quantitatively detected by the immunosensor and the methodological evaluation of the immunosensor was carried out.The immunosensor could be used to detect TRAP.4.Investigation of the novel immunosensor based on graphene oxide hydrogel composites for TRAP detection and its application4.1 Detection of TRAPs derived from tumor cell lines by the novel immunosensorThe B16F10 cells were incubated under hypoxia in 24-well plate for 24 h with 3×10²?1×10³?3×10³?1×10⁴?3×10⁴?1×10⁵?3×10⁵cells/well,respectively,and the concentrations of TRAPs in culture supernatant were detected with the immunosensor.The B16F10 cells were incubated under hypoxia in 24-well plate with 1×10⁵cells/well for 12 h,24 h,48 h,72 h,respectively,and the concentrations of TRAPs in culture supernatant were detected with the immunosensor.The Hep G2 cells,EL4 cells,Hepa 1-6 cells,B16F10 cells and MDA-MB-231were incubated under hypoxia in 24-well plate with 1×10⁵cells/well for 24 h,and the concentrations of TRAPs in culture supernatant were detected with the immunosensor.4.2 Detection of TRAPs derived from mouse peripheral blood by the novel immunosensorC57BL/6 mice were injected with 10 ng of TRAPs derived from B16F10 cells.The blood was collected from mice eyes after 10 min,20 min,and 30 min and the concentrations of TRAPs in the plasma were measured with the immunosensor.The C57BL/6 mice were injected with10 ng,30 ng,100 ng of TRAPs derived from B16F10 cells.The blood was collected from mice eyes after 10 min and the concentrations of TRAPs in the plasma were measured with the immunosensor.The tumor vaccine developed by our group was used to treat 4T1 tumor-bearing mice and the normal saline group was as control.The blood was collected from mice eye at 30days and the concentrations of TRAPs in the plasma were measured with the immunosensor.The TRAPs from peripheral blood of 4T1 tumor-bearing mice enriched by the nanomagnetic beads were identified by transmission electron microscopy.The TRAPs enriched by the nanomagnetic beads were identified by Western blot.4.3 Detection of TRAPs derived from human peripheral blood by the novel immunosensorThe concentrations of TRAPs in plasma from 12 cancer patients and 12 healthy people were measured with the immunosensor.The concentrations of IL-1?,IL-6 and IL-10 in peripheral blood from the 12 cancer patients and 12 healthy people were measured using ELISA kits and the correlation between IL-1?,IL-6 and IL-10 with TRAP content was analyzed by Spearman method.Results1.Design,development and application of the novel enzyme cascade immunosensorTransmission electron microscopy showed that carbon nanotubes(MWCNTs)were successfully loaded with gold nanoparticles(Au NPs)after mixed with polydopamine(PDA).The UV-visible absorption spectroscopy showed that the carbon nanocomposites had characteristic absorption peak of gold nanoparticles,which indicated that the composites were successfully synthesized.Cyclic voltammetry showed that the carbon nanocomposites could amplify electrical signals and enhance conductivity.The enzyme cascade reaction was feasible in solution phase.Phenyl-?-D-galactoside(P-GP)could be hydrolyzed by Gal followed by oxidation with Tyr.Cyclic voltammetry showed that the immunosensor was successfully constructed.The linear range of CEA was from 10 pg/m L to 10 ng/m L,and LOD was 8.39pg/m L(S/N=3).The immunosensor was confirmed with good properties.2.Design and development of specific nanomagnetic beads for enrichment and purification of TRAPScanning electron microscopy showed that Fe₃O₄ was successfully synthesized and loaded with gold nanoparticles.The UV-visible absorption spectroscopy showed that the Fe₃O₄-Au composites had characteristic absorption peak of gold nanoparticles.Western blot result showed LC3B Ab₁ had good specificity,so it was used to prepare nanomagnetic beads.Laser confocal microscopy showed that nanomagnetic beads with specific enrichment and purification of TRAP had been successfully prepared and had good specificity.Western blot results showed that the nanomagnetic beads had highly efficient in enriching TRAP.3.Design and development of the novel immunosensor based on graphene oxide hydrogel compositesScanning electron microscopy showed that GH had characteristic porous structure.UV-visible absorption spectroscopy showed that the GH-MB composites had characteristic absorption peaks of GH and MB,suggesting successful synthesis of GH-MB composites.Cyclic voltammetry showed that GH-MB could amplify electrical signals.Western blot and flow cytometry analysis showed that LC3B Ab₂ had good specifity and had different antigenic epitopes from LC3B Ab₁.Cyclic voltammetry showed that the immunosensor was successfully constructed.The linear range of the LC3B was 10 pg/m L-100 ng/m L,and LOD was 2.696pg/m L(S/N=3).The immunosensor had good specificity and high stability and could be used for the detection of TRAP.TRAP was detected using the constructed immunosensor with linear range of 60 pg/m L-600 ng/m L and LOD of 15.739 pg/m L(S/N=3).4.Investigation of the novel immunosensor based on graphene oxide hydrogel composites for TRAP detection and its application4.1 Detection of TRAPs derived from tumor cell lines by the novel immunosensorThe concentrations of TRAPs increased with the increase of cell numbers until cell number was 1×10⁵.The concentrations of TRAPs released by tumor cells increased with the increase of culture time until 24 h.TRAPs were found in culture supernatant of different tumor cell lines instead of the cell-free medium and the concentrations were different.4.2 Detection of TRAPs derived from mouse peripheral blood by the novel immunosensorThe concentration of autologous TRAP was very low in mice.The concentration of TRAP was highest at 10 min after injection and then gradually decreased.With the increase of injection quantity of TRAPs,the concentration of TRAPs in peripheral blood was increased gradually.The concentrations of TRAPs in peripheral blood of tumor-bearing mice in normal saline group were significantly higher than normal mice,and concentrations of TRAPs were significantly reduced after tumor vaccine treatment.Transmission electron microscopy result showed that TRAPs from peripheral blood of 4T1 tumor-bearing mice were enriched by the nanomagnetic beads successfully.Western blot result showed that the enriched TRAPs had high purity.4.3 Detection of TRAPs derived from human peripheral blood by the novel immunosensorTRAPs existed in plasma of cancer patients and healthy people and the concentrations of TRAPs in plasma of cancer patients were significantly higher than that of healthy people.The levels of IL-6 and IL-10 in plasma of cancer patients were higher than those in healthy people and the levels of IL-6 and IL-10 were positively correlated with the concentrations of TRAPs.Conclusions1.The enzyme cascade immunosensor based on carbon nanotube composites has been successfully developed with high sensitivity and good specificity.The carbon nanotube composites with good conductivity provided many active sites for antibody conjugation,which was responsible for signal amplification.And the enzyme cascade system showed an enhanced selectivity compared with that based on a single enzyme.2.The specific nanomagnetic beads for enrichment and purification of TRAP have been successfully developed with strong anti-interference ability and good specificity.The efficiency and purity of TRAP enrichment were satisfied.3.The immunosensor based on graphene oxide hydrogel composites has been successfully developed with excellent performance.The graphene oxide hydrogel composites not only acted as a redox probe,but also coupled antibodies and amplified electrical signals.The immunosensor could be used for TRAP detection.4.Immunosensor can be applied to:detection of secreted TRAP in tumor cell culture;detection of TRAP content in peripheral blood of 4T1 tumor-bearing mice and evaluation of tumor vaccine treatment effect;content of TRAP in peripheral blood of cancer patients.The results of the test showed that the TRAP in the peripheral blood of cancer patients was significantly higher than that of healthy people,and the levels of IL-6 and IL-10 in peripheral blood of human were related to the content of TRAP.