DNA Functionalized Silicon Nanodots And Its Application In Fluorescence Detection And Cell Imaging

The spatial distribution of cellular molecules can be obtained via cell imaging,which plays a key role in understanding the psychology process of cells.More information is achieved by combination of cell imaging and fluorescence detection.DNA biomolecular ligands have been widely applied to the development of fluorescent probes due to their biocompatibility,water-solubility,tunability,and target recognition ability.DNA functionalized nanomaterial integrates the excellent properties of nanomaterial and DNA into one structure,which proves to be a powerful tool in fluorescence detection and imaging analysis.However,most of them with a sole emission signal are easily affected by the factors such as cellular uptake,probe concentration,and optical path length.The ratiometric fluorescent probes with dual-emission signals can conquer these shortcomings.Therefore,the development of DNA functionalized nano ratiometric fluorescent probes is crucial for the bioanalysis and imaging system.In this work,DNA functionalized fluorescent silicon nanodots(SiND)have been prepared and applied in the detection of nucleic acid,protein,metal ion and hydrogen ion.Moreover,the constructed ratiometric fluorescent probes have been subsequently utilized to dual-color cell imaging,cell recognition,and subcellular lysosome imaging.The main contents of this work are as follows:(1)A new method has been developed for the fluorescence detection of disease-associated miRNA-27a.The Cy5-DNA/SiND probe has been prepared via electrostatic interaction based on the dynamic quenching of SiND toward Cy5-tagged DNA probe.The fluorescence of Cy5-tagged DNA can be restored after hybridization with the synthetic target miRNA-27a(S-miRNA-27a).There is no involvement of the suspected carcinogen of diethylpyrocarbonate(DEPC)reagent in this enzyme-free method via replacement of U bases with T bases in the analyte.The stability of the system therefore has been improved.Under the optimal conditions,the method achieves a linear range of 0.5-20 nM with the detection limit of 0.16 nM,and the relative standard deviation of this method is 9%(c = 1 nM,n = 5).The established method has been successfully applied for the analysis of S-miRNA-27a in human urine and serum samples with satisfied recoveries of 90%-122%.(2)A ratiometric fluorescent probe has been constructed via covalent functionalization of SiND with Hg2+-specific 6-carboxy-X-rhodamine(Rox)-tagged DNA.A method for the detection of Hg2+ in human cervical cancer HeLa cells has been established with the ratiometric fluorescent probe on account of T-Hg2+-T interaction.For the Rox-DNA functionalized SiND,in the presence of Hg2+,the fluorescence of Rox can be quenched by the SiND due to the formation of stem-loop DNA relocating Rox close into SiND.The results of UV-vis absorption spectra and fluorescence lifetime measurements further indicate that the signal change is related to Hg2+ only.The fluorescence intensity change of Rox serves as the response signal,and the fluorescence of SiND insensitive to Hg2+ acts as the reference signal.Accordingly,a ratiometric method for the detection of Hg2+ is proposed.Under the optimum conditions,the linear range for Hg2+ is 10-1500 nM.The limit of detection and precision of this method for Hg2+ is 9.2 nM and 8.8%(c = 50 nM,n = 7),respectively.As for practical application,the recoveries in spiked human urine and serum samples are in the range of 81-107%.Moreover,the biofunctionalization of SiND improves the acid-base stability of SiND significantly,which is favorable for its application in the intracellular environment.This ratiometric fluorescent probe has been successfully used for dual-color visualization detection of Hg2+ in HeLa cells.(3)A method for detection of tumor marker mucin 1(MUC1)and dual-color imaging of cancer cells has been developed.The ratiometric fluorescent aptasensor(SiND-S2.2-Cy5)for selectively sensing MUC1 has been prepared by covalent attachment of the Cy5-tagged aptamer S2.2 to SiND.In the absence of MUC1,the fluorescence of Cy5 can be quenched by the SiND.In the presence of MUC1,the fluorescence of Cy5 can be restored due to structure switching of S2.2 via the interaction between MUC1 and S2.2.Under the optimum conditions,the linear range is 3.33-250 nM,and the limit of detection and precision of this method for MUC1 is 1.52 nM and 3.6%(c = 10 nM,n = 7),respectively.This method has been applied for the detection of MUC1 in human serum with recoveries of 87-108%.This ratiometric fluorescent aptasensor exhibits specificity for MUC1-possitive human breast cancer MCF-7 cells rather than MUC1-negative human normal breast MCF-10A cells and African green monkey kidney Vero cells,which can well achieve cell recognition between cancer cells and normal cells.(4)A ratiometric fluorescent probe has been developed for the detection of hydrogen ions in the subcellular organelle of MCF-7 cells.The pH-sensitive fluorescein(FAM)tagged aptamer AS 1411 targeted for nucleolin has been used to covalently modify the SiND.The optimal ratiometric pH biosensor(SiND-Apt-FAM)has been constructed by optimization of the amounts of the nanomaterial and the linker length of the aptamer.The application of the SiND-Apt-FAM in cellular lysosome has been investigated via the combination of synchronous fluorescence spectroscopy,fluorescence imaging and flow cytometry.This aptasensor shows cell specificity for nucleolin-overexpressed MCF-7 cells and HeLa cells rather than MCF-1OA normal cells,and it displays a wide pH response range of 4.5-8.0 in living cells.In addition,dual-color lysosome imaging and accurate measurement of pH in MCF-7 cells(pH 5.1)have been successfully conducted with this ratiometric fluorescent probe.