Functional Nanomaterials Combined With Isothermal Nucleic Acid Amplification Technology For Detection Of Tumor Biomarkers

Malignant tumor has become an important disease threatening human life and health.To achieve rapid,sensitive and efficient analysis and detection of tumor markers is of great significance for early screening,detection,diagnosis,treatment effect and prognosis of cancer.It has become hot research issue,so the research objects of our experiments are the tumor marker miRNA-155 and circulating tumor cells（CTCs）.However,the content of tumor markers is extremely low and difficult to detect.DNA amplification was introducing to improve sensitivity.A series of in vitro detection methods with high selectivity and sensitivity for the detection of miRNA-155 and CTCs were constructed based on isothermal amplification of nucleic acids,such as DNA walker,hybrid chain reaction（HCR）,combined with functional nanomaterials AuNPs,Au@Fe₃O₄ and Au@luminol.The specific content is divided as followed three parts:（1）A colorimetry nucleic acid sensor was designed to visually and supersensitively detect miRNA-155,based on gold nanoparticles（AuNPs）,Au@Fe₃O₄ composite nanomaterials and DNA walker.The DNA walker（including walking chain and orbital chain）was modified on the surface of Au@Fe₃O₄ composite nanomaterial.In the presence of the target,the walking chain walked along the orbital chain and cut the orbital chain to obtain the short single-stranded DNA（product chain DNA）.The product chain DNA supernatant was obtained by magnetic separation,and the product chain DNA was added to the AuNPs solution.The experimental principle was single DNA make gold nanoparticles in high salt solution keep stable.When the concentration of product chain varying from low concentration to high concentration,the color of AuNPs in NaCl solution gradually changes from blue to red wine,so as to realize quantitative detection of miRNA-155.This method possessed no large instruments and equipment,good visualization,great stability,strong real-time detection,high sensitivity,specificity,and had potential application prospect in the field of clinical diagnosis and related biochemical analysis.（2）A target-driven dual DNA walker signal amplification electrochemiluminescence（ECL）nucleic acid sensor was constructed,which was used for the efficient and ultra-sensitive detection of miRNA-155.The first DNAzyme-driven DNA walker was fixed on the AuNPs surface.In the presence of the target,the walker started work,the walking chain walked along the orbital chain,and the second DNA walker was cut.The second walker was propelled by toehold mediated strand displacement reaction（TMSDR）and“walking”efficiently along DNA“track”,introducing considerable Au@luminol signal molecules attached on the electrode surface and achieving ECL signal.Due to the amplification of the two DNA walkers,this method had an extremely low detection limit（3.3 aM miRNA-155）compared with the traditional nucleic acid detection method.This method required no cleaning steps and simpler operation,providing a possible method for early clinical and immediate detection of miRNAs and cancers.（3）An ultra-sensitive detection method for circulating tumor cells（CTCs）based on dual-aptamer recognition,enzyme-free chemiluminescence detection,hybridization chain reaction（HCR）signal amplification and magnetic separation was proposed.In this experiment,magnetic Fe3O4 nanoparticles modified with AS1411 aptamer（MNs-AS1411）were prepared to capture and separate CTCs in whole blood samples.Through HCR self-assembly of hairpin nucleic acid,HCR aggregates containing multiple（MUC1）aptamer and-SH.The HCR aggregates（ALHA）modified by Au@luminol were formed by labeling Au@luminol nanoparticles at the thiol end,which could recognize CTCs captured by the magnetic sphere and produce chemiluminescence in the K2S2O8 solution.Nucleic acid dual-aptamer as the capture probe and the recognition probe overcome the difficulty of antibody inactivation,low capture efficiency and high cost.Enzyme-free chemiluminescence detection possess no background light source,high sensitivity and easy control.HCR signal amplification combined with magnetic separation can further improve sensitivity and selectivity.This method can realize the highly sensitive with detection limit of 3 CTCs cells/mL,providing a possible detection approach for the early diagnosis and warning of cancer spread,as well as the evaluation of treatment effect.