| As an important biomarker,micro RNAs(miRNAs)are of great significance for the diagnosis,prediction and treatment of tumors and other related diseases to achieve their high sensitivity and selectivity.However,due to the characteristics of small size,easy degradation,and sequence similarity among multiple miRNAs family members,higher requirements are put forward for their specific detection and high-sensitivity detection.In recent years,nanomaterials have attracted the attention of more and more researchers due to their fascinating properties such as surface and interface effects,quantum size effects,and macroscopic quantum tunneling effects.DNA-templated silver nanoclusters(DNA-AgNCs),as an emerging fluorescent nanomaterial,overcome a series of problems such as photobleaching,poor solubility,poor stability,and large cytotoxic side effects of traditional organic fluorescent dyes.The advantages of stability and biocompatibility,sub-nanometer size or DNA sequence-dependent fluorescence tunability have very broad application prospects in the fields of biosensing and imaging.In order to further improve the detection sensitivity,this paper combined a target-triggered entropy-driven cyclic amplification system with DNA-AgNCs to construct an enzyme-free,label-free fluorescent biosensor for the detection of tumor-related miRNAs.This research mainly includes the following two parts:(1)In the first part of the work,we designed the DNA signaling probe as a Y-shaped structure containing three DNA strands,one of which was used to hybridize to the Trigger strand released in the target-triggered entropy-driven amplification system,and the other Y2,Y3 The strand serves as the DNA template strand for the synthesis of AgNCs.In the absence of the target miRNA,the two DNA template strands are very close due to the formation of a Y-shaped structure,causing the AgNCs to emit red fluorescence;when the target miRNA is present,the strand displacement of the complex in the entropy drive is triggered to release the Trigger strand,which is combined with The Y1 chain in the Y-shaped structure is hybridized,the Y-shaped structure is opened,the Y2 and Y3 chains are separated,and the controllable transition from red fluorescence to yellow fluorescence is realized.Among them,the optimal excitation wavelength of yellow fluorescent AgNCs is 500 nm and the optimal emission wavelength is 570 nm;the optimal excitation wavelength of red fluorescent AgNCs is 565 nm and the optimal emission wavelength is 630 nm.Through the change of the ratio of yellow fluorescence to red fluorescence intensity,high-sensitivity detection of miRNA is realized.The target cyclic amplification and signal generation in this part of the work are two independent systems,which have the characteristics of universality,low background and large signal variation,and have obvious advantages compared with traditional AgNCs sensors.The results showed that the ratio of the two fluorescence intensities and the logarithm of the miRNA-21 concentration had a good linear relationship in the range of 0-100 n M,and the detection limit was 10.24 p M;it had good selectivity,and also realized the miRNA in human serum samples.-21 detection.(2)In the second part of the work,we designed the target cyclic amplification and signal generation in the same system,based on the fluorescence enhancement of guanine-rich(G-rich)AgNCs and the combination of two non-luminescent AgNCs to form a strong fluorescent nanometer Two kinds of AgNCs fluorescence enhancement phenomena of cluster dimer(dimer)were integrated into the fuel chain sequence in the entropy-driven amplification system,and the two entropy-driven sequence designs were integrated into a composite structure to construct a dual-entropy drive.The composite substrate finally utilizes each target to catalyze its respective entropy-driven process to achieve a significant enhancement of its corresponding AgNCs fluorescence signal,thereby realizing the separate and simultaneous detection and analysis of the two target miRNAs.Among them,the G-rich-enhanced AgNCs probe was used to detect miRNA-141,the optimal excitation wavelength was 515 nm,and the optimal emission wavelength was 585 nm.In the absence of the target,AgNCs and G-rich are in a state of separation,and no fluorescence appears;when the target exists,the entropy-driven strand displacement reaction is triggered,and the AgNCs are close to G-rich,and the orange fluorescence is greatly enhanced.The formation of nanocluster dimers was used as a signal probe for the detection of miRNA-21,and the optimal excitation wavelength was 565 nm and the optimal emission wavelength was 630 nm.In the absence of the target,the two AgNCs are separated and show no fluorescence;when the target is present,an entropy-driven strand displacement reaction is triggered,and the two AgNCs are approached,resulting in red fluorescent nanocluster dimers.This part of the work combines multicolor fluorescent AgNCs with multiple targets to screen out multiple analytes in one assay,with obvious advantages of simplicity,rapidity,and low sample and reagent consumption.The results showed that the G-rich-enhanced orange fluorescence at 585 nm gradually increased with increasing concentration of miRNA-141,and there was a linear relationship at lower concentrations of 0-0.5 n M,with a detection limit of10.51 p M.As the concentration of miRNA-21 increased,the red fluorescence of the dimer at 630 nm also gradually increased,and there was also a linear relationship at lower concentrations of 0-0.5 n M,with a detection limit of10.46 p M.The biosensor has good selectivity,and also realizes the detection analysis in human serum samples. |
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