| In recent years,cancer has become the leading cause of human death.The key to improving the survival rate of cancer patients lies in early detection and early treatment.However,traditional detection methods including magnetic resonance imaging,ultrasound and biopsy are not efficient for early cancer detection because the results of these methods lied on the phenotypic characteristics of tumors.The discovery of cancer biomarkers provides a new strategy for accurate diagnosis of early cancer.A large number of studies have shown that accurate and sensitive detection of cancer biomarkers can indirectly achieve accurate diagnosis of early cancer.DNA nanomachines based on catalytic hairpin assembly reactions are an enthalpy-driven,enzyme-free amplification strategy proposed in recent years.It has been widely used in the detection of cancer biomarkers.However,there are two problems in its application in the early diagnosis of cancer,which affect diagnostic accuracy.On the one hand,it has low signal amplification efficiency,background interference and slow response speed for the detection of intracellular targets.On the other hand,it cannot detect two different types of cancer markers.To solve these two problems,this article has carried out the following work:(1)An efficient and reliable localized catalytic hairpin assembly-based DNA nanomachine for miRNA-21 imaging in living cellsA localized catalytic hairpin assembly-based DNA nanomachine(LCHA nanomachine)was developed for rapid,efficient and reliable imaging of miRNA-21 in living cells.The nanomachine was simply constructed by one-step self-assembly process of a stator strand,a pair of hairpin probes from CHA and an AS1411 aptamer.Benefiting with spatialconfinement effect,a pair of hairpin probes with high collision frequency was rapidly and efficiently assembled through catalyst of miRNA-21 on a stator strand in every nanomachine.Comparing with the free-CHA nanomachine,LCHA nanomachine shortened 4.5-fold reaction time for reaching a plateau and significant improved sensitivity for miRNA-21 detection in vitro.Importantly,the nanomachine was successfully applied for miRNA-21 imaging in living cells.With the assistance of an AS1411 aptamer and stator strand,the pair of hairpin probes with the ratio of 1:1 synchronously transported into a co-site of cytoplasm,which ensures efficient imaging for trace miRNA-21.Signal output of ratio of 6-carboxyfluorescein(FAM)to tetramethyl rhodamine(TAMRA)intensities guaranteed reliability through avoiding the interference from different amount of the nanomachine that enters into cells.Notably,the nanomachine can distinguish the miRNA-21 expression level in different kinds of cancer cells.This method can realize the accurate detection of cancer biomarkers and provides a powerful method for the accurate diagnosis of early cancer.(2)A localized catalytic hairpin assembly and enzyme-based AND logic gate for the simultaneous detection of miRNA-21 and APE1Although the LCHA nanomachine proposed in the previous work can achieve efficient and accurate detection of a single cancer marker,it cannot achieve simultaneous detection of multiple types of targets.However,cancer is a multi-stage disease.The detection of a single type of cancer marker cannot accurately reflect the type and stage of the disease.It is easy to produce false negative or false positive signals in the diagnosis.Based on this fact,taking advantage of the programmability and predictability of DNA nanomachines,we constructed an AND logic gates combined the localized catalytic hairpin assembly reaction and the enzyme cleavage reaction to realizing different types of cancer markers(miRNA-21 and APE1)detection.In the simultaneous presence of miRNA-21 and APE1,miRNA-21 will trigger the LCHA reaction,the hairpin structure of H1 is opened,the FAM on H1 is far away from the quenching group BHQ,and the fluorescence of FAM is recovered,realizing the detection of miRNA-21.At the same time,the 3’ end of H2 will hybridize with the 5’ end of the S containing the restriction site to form a double-stranded structure at the restriction site.APE1 enzyme cleavage reaction occurs at this site,the fragment with TAMRA is cleaved and released into the system,the fluorescence signal is restored,realize the detection of APE1.The FAM fluorescent signal in the AND logic gate responds to miRNA,and the TAMRA fluorescent signal responds to APE1,thereby realizing the simultaneous detection of miRNA-21 and APE1. |
