Preliminary Study On Tumor Biomarkers Enhanced Detection By Protein And Nucleic Acid Micro/nano Biosensing System

Cancer seriously endangers human life.The early and non-invasive tumor biomarkers detection will facilitate cancer screening,diagnosis,and treatment.We focus on prostate cancer and breast cancer,take traditional and novel biomarkers as targets,and aim for accurate and safe cancer management.Based on micro-nano materials,immunoassay,and isothermal amplification,we constructed novel biosensing to achieve simple,fast,sensitive,and high-throughput biomarkers in vitro detection.Meanwhile,we also designed a versatile system to realize multi-targets intracellular imaging and cancer therapy.Specific research as follows:1.A novel enzyme-free fluorescent cascaded immunosensor for protein tumor markers detection:we designed a novel enzyme-free fluorescent immunosensor based on etching sensitization and aggregation-induced luminescence.The antibody-labeled immune probes（AgNPs-Ab2,MBs-Ab1）were prepared by silver nanoparticles（AgNPs）and magnetic spheres（MBs）.AgNPs-Ab2 was captured by MBs-Ab1 in the presence of prostate-specific antigen（PSA）.After magnetic separation,hydrogen peroxide（H₂O₂）etched AgNPs to lots of Ag⁺.Ag⁺specific complexed with aggregation-induced luminescent material（TPE-4TA）to generate fluorescent aggregates（Ag⁺-TPE-TA）,effectively reducing background and improving sensitivity with LOD of 15.66 pg/m L for PSA.The portable sensing was suitable for serum sample detection with high accuracy.2.A highly sensitive dual-enzyme cascade signal amplification biosensor for miRNA detection:the dual-enzyme cascade signal amplification biosensor was designed by DSN-assisted target recycling and HRP catalytic system.We prepared high-performance PS-ssDNA-HRP probes by polystyrene microspheres（PS）.ssDNA could specifically capture miRNA,and then DSN specifically cleaved ssDNA in ssDNA/miRNA,releasing miRNA and HRP.Target recycling and enzymatic catalysis enabled dual signal amplification.Finally,change of color and absorbance enabled visual qualitative and microplate quantitative.This simple and easy-operation biosensor first combined DSN with HRP,enabling an ultra-low LOD of fM without PCR amplification in 40 min.In addition,this platform achieved better performance for miRNA-21 detection in clinical breast cancer samples,which was highly consistent with qRT-PCR results.3.A“dual-key specific unlocking”high-throughput biosensor for miRNAs simultaneous detection:based on target recycling,signal enrichment,and liquid-phase chip technology,we designed a“dual-key specific unlocking”high-throughput biosensor.The blocked probes（FS-HDs）were designed via hairpin DNA（HDs）labeled with fluorescent encoded nanospheres（FS）.The miRNA/DSN could specifically unlock FS-HDs to FS-ssDNA and then enriched the FS on PS.Finally,it could achieve miRNA quantification by flow cytometry.This dual-unlocking strategy improved the specificity.The target recycling and signal enrichment were both beneficial for higher sensitivity with LOD of 3.39 fM,which was 3 orders higher than traditional methods.Furthermore,this sensor enabled miRNA-21 and miRNA-141 simultaneous detection based on color-coded FS-HDs.This sensor was performed in one-pot without complex purification,and allowed for miRNA-21 detection in breast cancer blood samples with higher consistency of qRT-PCR,demonstrating a new avenue for multiple miRNAs specific testing.4.A dual-modality imaging and dual-gene silencing versatile theranostics system:based on MnO₂ nanosheets and DNAzyme,we constructed a versatile theranostics system with dual-modality imaging and dual-gene silencing.Considering the lower cellular delivery,insufficient cofactors,and inability degradability of traditional probes,polyethyleneimine（PEI）was modified on MnO₂ to facilitate the loading of nucleotide probes and construct multifunctional probes（MnPDs）.MnPDs were dissolved into Mn²⁺instantaneous by overexpressing GSH in the tumor.Mn²⁺could be used for tumor-enhancing MR imaging and provide cofactors for DNAzyme.More importantly,the released probes enabled simultaneous imaging of miRNA and telomerase,as well as EGR1 and SUR m RNAs silencing with potent antitumor effect,providing new insights for accurate and efficient breast cancer theranostics.