| Micro RNAs(miRNAs)are a group of endogenous RNA molecules that perform important roles in gene modulation.Increasing evidence suggests that changes in miRNA expression levels are closely associated with the progression and prognosis of various diseases and have been recognized as biomarkers for the diagnosis of cancer.Exploring effective signal amplification strategies and selecting suitable DNA probe carriers are urgently needed to prepare ultrasensitive detection of miRNA21 biosensors and to achieve in vivo miRNA21 in situ imaging and therapy.DNA walker is a dynamic DNA nanodevice that provides the possibility of signal amplification by driving the cyclic and progressive movement of DNA walker under the action of a driving force.At the same time,the spontaneous motion of DNA walker meets the requirements of real-time imaging for intracellular detection,and also stimulates various biological applications including drug delivery and diagnosis.This thesis focuses on exploring multiple DNA walker models to select the optimal model,and from the optimal model as the basic system,it can be expanded into an ex vivo sensitive detection of miRNA21 synergistic gene therapy biosensor and multiple amplification means combined with ultrasensitive detection of miRNA21 electrochemical sensor.Based on the fact that the basic system of DNA walker needs to meet the requirements of multienvironment use,the selection of the driver considers the introduction of endogenous substances to provide a new design strategy for target detection and imaging.walker driver can achieve the overall system for accurate differentiation of tumor cells from normal cells and achieve the experimentally set goal of sensitive detection and in situ imaging for different intracellular miRNA21 in vitro and in vivo.There are two specific works in this paper:(1)3D DNA walker responds to dual intracellular switches to build fluorescent analysis and cell therapy biosensorsIn the second chapter of the work,this paper hopes to develop simple and versatile DNA nanostructures to realize biosensors with sensitive detection of miRNA21 in vivo and ex vivo with a gene therapy effect.Firstly,a 3D DNA walker is constructed,bio affinity gold nanoparticle(Au NP)is selected as the carrier,and the walker-block is modified with sub-stable hairpin H1 on Au NP,which contains AP sites on the hairpin H1 loop and the antisense sequence of survivin m RNA is hidden in the stem,which is used to regulate the content of survivin protein in different cells and achieve the effect of gene therapy in tumor cells.gene therapy effect.The initial construction of 3D DNA walker was designed with nine models for oxDNA coarse-grained simulation,and the optimal model was selected to ensure the versatility and signal excellence of 3D DNA walker.The experimental design principle is that the biosensor is activated in the presence of dual-switch miRNA21 and APE1 enzymes,and the miRNA21 is detected efficiently and conveniently in vitro using fluorescence real-time monitoring,and the biosensor is transported to the corresponding location in vivo by endocytosis to turn on the in situ imaging fluorescence signal while releasing the antisense sequence of survivin m RNA to target and regulate survivin protein synthesis and apoptosis in tumor cells.This chapter has achieved the goal of gene therapy for tumor cells.The experiments in this chapter achieved the in vitro detection of miRNA21 at different concentrations with a detection limit of 3.868 p M and good linearity of miRNA21 in the range of 100 p M-100 n M.The detection process was simple and easy,with rapid response,and the sensitive in vitro detection was successfully achieved.Meanwhile,precise imaging of miRNA21 in different cells and accurate regulation of survivin protein content were achieved,and the reduction of survivin protein in tumor cells to 50% of the original level was realized.Meanwhile,it successfully caused apoptosis of tumor cells,and the survival rate of both MCF-7 and Hela cells was reduced to 56%,realizing the integration of tumor cell detection and treatment.(2)Construction of a multi-amplified dual-mode biosensor for sensitive detection of micro RNA21 based on APE1 enzyme-driven 3D DNA walkerBased on the optimal model of 3D DNA walker in Chapter 2,the experiments in Chapter 3 superimposed multiple signal amplification strategies to achieve in vitro fluorescence and electrochemical bimodal detection of miRNA21 to reduce the detection limit and broaden the linear range of miRNA21.The experiments combined the advantages of the high flexibility of the single-legged DNA walker,high stability of multi-legged DNA walker hybridization,and high efficiency of non-linear HCR amplification,which caused significant enhancement of the electrochemical signal.The main principles of the experiment are: in the presence of target miRNA21 and driver APE1 enzyme,the fluorescent biosensor is activated for fluorescence real-time quantitative monitoring of miRNA21;the gold nanoparticles and the remaining DNA strands connected by sulfhydryl groups on the gold nanoparticles after enzymatic cleavage using the fluorescent sensor can be used as multi-legged DNA walker to walk and cleave on the electrochemical sensor,and the cleavage generates HCR trigger strand,which triggers nonlinear HCR,and the addition of signal molecules triggers strong amplification of the electrical signal to achieve a bimodal detection mode.The experimental data showed that the detection limit of miRNA21 the fluorescent was 6.65 p M and the linear range was 50 p M-100 n M;the detection limit of miRNA21 of the electrochemical sensor was reduced to 1.6 p M and the linear range was 5 p M-10 n M,which successfully realized the bimodal detection of miRNA21 in vitro and the bimodal sensor had excellent selectivity,stability,and reproducibility. |
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