Dark-field Imaging Detection Of Biological Small Molecules By Gold Nanobipyramids@Metal Organic Frameworks Nanoprobe

Dark-field microscope（DFM）is an optical instrument that can observe and image samples by collecting scattering light from them.The key to dark-field microscopic imaging technology is avoiding the incident light from entering the field of view,which is mainly achieved by means of a condensor.On one hand,the light baffle above the light source prevents the light emitted from the center of the light source from entering the focal plane.On the other hand,the annular light is tilted away from the imaging area after irradiating the sample at an angle by the condensor,which thus allowing the observation of nanoparticles with high signal-to-noise ratio with the help of scattering light,and dark background imaging is achieved.Therefore,the dark-field microscopic imaging technique has high sensitivity and visibility,and can observe some small size particles easily.Virtually any matter can scatter light,but it usually appears as a broad band of non-characteristic mixed color scattering,so dark-field microscopic imaging is not suitable for highly sensitive spectral analysis.Localized surface plasmon resonance（LSPR）is the collective oscillation of free electrons on the surface of metal nanoparticles such as gold and silver under light excitation,which is manifested by the very strong absorption and scattering of light at specific wavelengths,so that LSPR nanoprobes are able to show vivid color under DFM.The combination of the unique LSPR properties of noble metal nanoparticles with the high spatial and temporal resolution imaging features of DFM greatly broaden the application scope of dark-field microscopic imaging technology.DFM can both image the noble metal nanoparticles through LSPR scattering light and capture their scattering spectra at the single particle level by coupled spectroscopy.The LSPR signal of plasmon nanoparticles are very sensitive to small changes in their own shape,composition,and surroundings,and is highly susceptible to changes in their scattering signal.Chemical measurements performed at the single-particle,single-site,or single-molecule level can substantially reduce the errors and information loss associated with averaging measurements.Thus,DFM-based measurements at the single-particle level can more precisely elucidate how nanostructure and environment affect chemical reaction processes and reveal the relationship between chemical kinetics and structure and activity.Currently,noble metal nanoparticles are often used as dark-field light scattering probes for reaction monitoring,analytical sensing,and cell imaging.However,in the field of analytical sensing,because of the single composition of precious metal nanoparticles,the variety of targets that can be detected is often limited,and the response mode is relatively single.Therefore,core-shell light scattering nanoprobes constructed with a precious metal"core"are gaining attention because of the diversity of functions brought about by the structural diversity of nanoprobes.Usually,the core-shell light scattering nanoprobes are prepared by using gold nanoparticles as the"core"and monomers or compounds that can respond to the target as the"shell",which are modified on the surface of gold nanoparticles by specific methods to form a core-shell structure.Under DFM,when the target is present,it can respond to the"shell"and cause the light scattering signal to change,thus realizing the single particle spectral measurement of the target.Metal organic frameworks（MOFs）are widely used in biomedical analysis because of their diverse structures,tunable pore sizes and biocompatibility,and the ability of MOFs with different structures to respond specifically to specific targets.Based on this,this paper innovatively combines the unique LSPR property of noble metals with the property of MOFs to respond to specific molecules,and prepares two novel core-shell light scattering nanoprobes and applies them to the dark-field imaging detection of small intracellular biomolecules.Details of the study are as follows:1.Dark-field imaging detection of glutathione in human serum by Au NBPs@ZIF-67nanoprobeA simple and rapid in situ synthesis strategy was proposed to use Au NBPs,Co（NO₃）₂·6H₂O and 2-Methylimidazole（2-MIM）as raw materials,and Hexadecyl trimethyl ammonium bromide（CTAB）was used as a linker to connect Au NBPs and ZIF-67 core,ZIF-67 was successfully grown around the Au NBPs,and the nanoscale spherical Au NBPs@ZIF-67 core-shell nanoprobes were finally formed by controlling the growth time.In the study,it was found that in the presence of reduced glutathione（GSH）molecules,it could bind to the central ion Co²⁺in ZIF-67 and undergo a redox reaction,and the Co²⁺was reduced by GSH thus leading to the disruption of the ZIF-67 framework and the Au NBPs were exposed.Monitoring the whole reaction process under DFM,we observed that the scattering spot of the core-shell nanoprobe gradually changed from green to red,and the single-particle scattering spectrum also shifted in intensity and peak.The results showed that the R/G value of the nanoprobe was linearly correlated with the GSH concentration in the range of 0.01–1μmol/L.The single particle dark-field imaging of GSH was highly sensitive and the detection limit was as low as 5.7 nmol/L.The assay was successfully applied to the determination of GSH in human serum samples with short time and good selectivity.2.Dark-field imaging monitoring of adenosine triphosphate level fluctuation in live cells by Au NBPs@ZIF-8 nanoprobeWe modified and simplified the synthesis method in Scheme 1,adjusted the amount of CTAB as a bridging agent,and successfully grew ZIF-8 core around Au NBPs,Zn（NO₃）₂·6H₂O and 2-MIM as reactants in the presence of CTAB,and finally obtained about 100 nm Au NBPs@by controlling the growth time,named ZIF-8 core-shell nanoprobe.Among them,Au NBPs was chosen as core because its scattering spot is red,which can clearly be distinguished from the background scattering of the cells.This is because the coordination of adenosine triphosphate（ATP）with the central ion Zn²⁺in ZIF-8 is much greater than that of the ligand 2-MIM and Zn²⁺in ZIF-8.Therefore,in the presence of ATP,the structure of ZIF-8 was disrupted due to the competitive coordination effect,and therefore the Au NBPs were gradually and completely exposed.The whole process of the reaction of Au NBPs@ZIF-8 with ATP was monitored in real time under DFM,and we observed that the scattering spot of the core-shell nanoprobe gradually changed from cyan to red,and the scattering spectral peaks also showed a blue shift and intensity We also observed a change in the blue shift and intensity of the scattering peaks.The results showed that the R/G value of the nanoprobe was linearly correlated with the ATP concentration in the range of 0.01–4 mmol/L,realizing the dark-field imaging detection of ATP with a detection limit as low as 5.28μmol/L.To demonstrate the potential of our synthesized core-shell nanoprobes to image intracellular biomolecules,we incubated Au NBPs@ZIF-8 with He La and observed the successful entry of the nanoprobes into the cells under DFM.We then applied an additional stimulus to modulate the expression level of ATP in the cell,and DFM successfully observed that the scattering properties of Au NBPs@ZIF-8 changed with the fluctuation of intracellular ATP level,enabling dark-field imaging monitoring of the fluctuation of intracellular ATP level.In conclusion,we innovatively combined Au NBPs and MOFs to synthesize two novel core-shell light scattering nanoprobes for dark-field imaging detection and cellular imaging of biological small molecules.Meanwhile,this in situ synthesis strategy we proposed has the advantages of simplicity,rapidity and economy,which provides a new idea for the construction of novel core-shell light scattering nanoprobes and their applications in biosensing.