The Application Of Dark-field Light Scattering Properties Of Gold Nanorods On The Detection Of MicroRNA

Cancer is the main cause of morbidity and death in the world.Micro RNA is considered as a tumor biomarker,and its abnormal expression in the body often indicates the pathological changes of the organism.At present,there are many methods for detecting micro RNA,but some of them still have many limitations such as insufficient sensitivity,low specificity,and difficulty in achieving intracellular content analysis.Therefore,there is an urgent need to develop sensitive and accurate micro RNA analysis methods.Gold nanorods(Au NRs)are a kind of anisotropic noble metal nanomaterial with unique electron oscillation plasmon resonance properties in the transverse and longitudinal.The dark-field light scattering of Au NRs is size dependent,the scattering spectrum,scattering intensity,and scattering color can change along with the morphology of Au NRs.The assembly of two Au NRs also affects the dark-field light scattering properties.Under the dark field microscopy(DFM),Au NRs present stable and obvious light scattering signals,and are widely used in the analysis and detection of micro RNA.Therefore,in order to solve the above mentioned problems in micro RNAdection,this thesis takes Au NRs as photostability probes to develop more sensitive and accurate micro RNA sensors.The specific research contents are as follows:1.Size-dependent plasmon resonance scattering characteristics of gold nanorods for highly sensitive detection of micro RNA-27 a.Micro RNAs,as one kind of significant biomarker,play indispensable roles in diagnosis and treatment of cancers.Yet owing to low expression and high sequence homology,the sensitivity and specificity for micro RNAs detection is greatly challengeable.Herein,a sensitive sensing platform with high specificity was structured to detect micro RNA-27 a based on micro RNA-27 a triggered chemical etching of Au NRs to smaller size,which accompanied by the significant blue shift as well as the great intensity decrease in the localized surface plasmon resonance(LSPR)scattering and remarkable color variability from red to green.When combined with the strand displacement reactions as well as liposome signal amplification and transduction,the proposed bioassay presented high selectivity toward micro RNA-27 a with a dynamic range from 100 f M-3 p M and the limit of detection as low as 16.5 f M(3s/k)under dark-field microscopy.Additionally,the remarkable discrimination of single nucleotide difference suggested a superior selectivity,which was able to detect micro RNA-27 a extracted from breast cells.The strategy put forward in this work is universal,presenting the amusing application prospects in early diagnosis of various cancers by adapting the corresponding nucleotide sequences.2.Two-dimensional analysis method for highly sensitive dual-targeting detection with Au NRs dimer.It is a challenge to achieve simultaneous detection of multiple biomarkers with the same nanomaterial.In this work,the hairpin DNA selectively modified on the surface of Au NRs to construct two kinds of nanoprobes.When targets existed,they would assemble Au NRs to end-to-end and side-by-side Au NRs dimers(called as ARETE dimer and ARSBS dimer,respectively).Compared with single Au NR,their dark-field scattering intensity and R percentage variation was extremely obvious.Based on this,micro RNA-21 and micro RNA Let-7a in breast cancer cells was detected.First,different hairpins were modified on the ends or sides of the Au NRs.Catalyzed hairpin assembly(CHA)cycle was triggered in the presence of micro RNA-21 or micro RNA Let-7a,and Au NRs were self-assemblied to ARETE dimers or ARSBS dimers.The analysis methods,such as the R percentage or scattering intensity,were difficult to distinguish single Au NR,ARETE dimer and ARSBS dimer.Thus,the twodimensional analysis method was constructed to adress this issue: The three states of Au NRs were distinguished greatly by combining the R percentage and scattering intensity with the data processing method of principal component analysis and counted to achieve the purpose of detecting dual targets.The fraction of dimers presented a linear relationship with the amount of micro RNA in the living cells.The detection limit of ARSBS dimer and ARETE dimer were1.72 f M and 0.53 f M,respectively.The number of recognized hairpins on the ends of ARETE dimer was less,and the electric field strength was stronger,resulting in its sensitivity about 3.2times higher than that of ARSBS dimer.This method not only realized the sensitive detection of dual targets,but also realized the intracellular high-resolution imaging,which developed a new way for the oriented assembly of nanomaterials and biological detection in living cells.In summary,this dissertation used dark-field microscopy imaging technology as the platform and Au NRs as optical probes to construct a high-sensitivity and high-selectivity analysis method for micro RNA detection,which had been successfully applied to the analysis detection of micro RNA in cell extract and high-resolution imaging in living cells.The experiment involved signal amplification methods such as strand displacement reaction and CHA cycle to further improve detection sensitivity and selectivity,and our method has great application prospects in the early diagnosis of cancer.