Novel Optical Sensors For Detecting Biomarkers Using Isothermal Nucleic Acid Amplification Technology

Cancer is a threat to human health,so the early diagnosis of cancer is of great significance for the detection and timely treatment of cancer.However,in the early stage of cancer,the concentration of biomarkers is low,and the traditional methods relying on the interaction of antigen and antibody in enzyme-linked immunosorbent assay(ELISA)have low detection sensitivity,which is difficult to meet the requirements of early cancer diagnosis.Therefore,it is of great significance to construct biosensor to detect cancer related biomarkers in the early diagnosis and treatment of cancer.Isothermal amplification of nucleic acids technology can realize the process of nucleic acid amplification under constant temperature,which can make up for the shortage of polymerase chain reaction(PCR).In this paper,based on the isothermal amplification techniques,three fast and ultra sensitive optical biosensors were constructed for the detection of exosome and glutathione,respectively.The contents of this paper mainly include the following three parts:(1)Cancer cells can secrete various types of humoral factors in their microenvironment.These humoral factors are biomarkers for disease diagnosis and prognosis.Early diagnosis is very important to improve the prognosis of cancer patients.This paper describe a simultaneous binding of double-positive exosomes proteins-based(called“dual-binding”)recognition mode(DRM)for accurate,non-purified,low-cost,and visual detection of exosomes surface proteins.This strategy relies on two aptamer probes to recognize the exosome surface intrinsic protein CD63 and high expression protein Ep CAM simultaneously.By dual binding-mediated toehold activation and a catalytic hairpin assembly(CHA)strategy is introduced to controlled form abundant of G-quadruplex DNAZyme that catalyze the etching of Au@Ag nanorods(Au@Ag NRs).The strategy eliminates the influence of interfering proteins by double binding recognition mode,and improves the specificity and accuracy of detection.This strategy requires neither time-consuming sample purification nor complex biomarkers,which is simple and sensitive.Therefore,this strategy has broad application prospects in the early diagnosis,treatment response and recurrence monitoring of cancer.(2)Exosomes as non-invasive biomarkers for cancer diagnosis have great prospects.However,the separation and purification of exosomes are very complex,so the detection of exosomes is still a challenge.Herein,We report a sensor based on the proximity ligation,non-purification strategy establishes a potential platform for the highly sensitive and specific detection of exosomes.The strategy relies on the adjacent connection of two probes to trigger the formation of primers,which can trigger branched hybridization chain reaction(BHCR)to achieve the amplification of the target.This strategy uses double binding recognition mode to improve the specificity and accuracy of detection.Based on the amplification strategy of b HCR,this method has high sensitivity for the detection of exosomes,and the detection limit is as low as 4.47×10~5particles/m L.Our strategy can monitor exosomes in serum without additional purification steps,and has the advantages of simple operation and short analysis time.Our results demonstrate that our biosensor has great potential in early diagnosis of cancer as a conventional biological detection method.(3)The abnormal expression of glutathione is closely related to the occurrence of some tumor diseases.It can be used as a tumor biomarker to participate in the diagnosis of tumor diseases.Therefore,monitoring the expression level of glutathione is of great significance in clinical diagnosis.In this work,a DNA nanomachine mediated HCR reaction based on tricyclic DNA probe was designed for ultra sensitive detection of glutathione.The strategy relies on the release of primers by S-S cleavage mediated by the target,which can initiate the hybridization chain reaction between DNA nanomachines.In this strategy,the Y-shaped design of DNA nanomachine significantly improves the detection performance.The three DNA functional modules of DNA nanomachine are used for signal recognition and signal amplification respectively.Based on the amplification strategy of this scheme,our biosensor has high sensitivity for the detection of glutathione,and the detection limit is 3.63 n M.Therefore,our biosensor has a broad application prospect in early disease diagnosis.