| Accurate and rapid detection of cancer-related low-abundance tumor markers can reflect the development of cancer in time,which is of great significance in the early diagnosis and treatment of cancer.Traditional fluorescent probes have two limitations:1)Non-specific stimuli in vivo will make the traditional "always on" mode of probe produce signal output once it encounters the target molecule,which will inevitably produce false positive signals;2)The fluorescent dye molecules of labeled probes are easy to be photobleached,resulting in insufficient detection sensitivity,which makes it impossible to achieve highly sensitive imaging detection of disease molecules.Therefore,the construction of stimulus-responsive plasma enhanced fluorescence detection system to achieve high sensitivity detection of low abundance target biomolecules is an effective way to solve the above scientific problems.Based on the above problems,in this paper,plasmon-enhanced fluorescence(PEF)was used as a signal amplification strategy to construct different signal-enhanced fluorescence nanoprobes,which were supplemented by DNA logic gate biological algorithm to achieve highly sensitive detection and controlled imaging analysis of different tumor markers.The specific research contents are as follows:(1)Gold nanopyramid(Au NBPs)was used as plasmonic substrate material.A plasmonic nanogap antenna was assembled by DNA strand replacement reaction mediated by Au NBPS,and a novel stimulation-responsive plasma nanocomposite was constructed by AND gate logic.This sensing mechanism was used for “double-lockdouble-key” activated highly sensitive detection and imaging analysis of two intracellular biomarkers(APE1 and mi RNA-21).The detection limit of APE1 was as low as 1.8m U/m L and that of mi RNA-21 was as low as 0.34 p M under the double amplification of PEF and strand displacement.In addition,the specific recognition of tumor cells can be realized based on the difference in the expression of APE1 and mi RNA-21 between tumor cells and normal cells.This work combines endogenous stimulation-response logic gate biological arithmetic with plasmonic nanogap antenna,and provides a new idea for ultrasensitive imaging of multiple biomolecules in cells.(2)Gold nanostars(Au NSTs)containing 20 hot spots with high symmetry were synthesized as plasmonic substrate material with stronger PEF effect,which had stronger fluorescence enhancement effect on dye molecules due to their excellent structural properties.Based on this,a DNA walking nanomachine based on Au NSTs was designed and constructed.Through the walking of the DNA walking nanomachine and the PEF effect of Au NSTs,the ultra-sensitive detection of mi RNA-373 in the cell was realized.At the same time,the microenvironment of tumor cells is weakly acidic and mi RNA-373 was used as the two input elements of the AND gate logic operation.At low p H,L0-LIP will anchor on the surface of cell membrane,while in the neutral environment of normal cells,p H insertion peptide(p HLIP)does not have this activity,which ensures the accuracy of signal detection.Compared with the uneven distribution of membrane proteins on the surface of the cell membrane,p HLIP can be uniformly anchored on the cell membrane,preventing the probe from "slipping away",thus improving the utilization rate of the probe and enhancing the intensity of the fluorescence signal.Furthermore,the optimal fluorescence enhancement effect was obtained by modulating the thickness of the silica shell coated on the surface of the Au NSTs,and the detection limit was reduced to 0.18 p M.This work has broad application prospects in the accurate imaging of intracellular tumor markers and the accurate differentiation of tumor cells from normal cells. |
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