Single Cell Analysis Based On Dark Field Scattering Imaging

Recently,label-free localized surface plasmon resonance?LSPR?technique receives promoting interesting due to its wide applications in catalysis,phototherapy and biosensing.Au,Ag and Cu nanoparticles are ideal plasmonic materials,the LSPR signal depends on the morphology,sizes,composition,as well as surrounding dielectric environment of the nanoparticles.The scattering light of an individual plasmonic nanoparticle can be readily observed under a dark-field microscope?DFM?,providing a powerful means for the in-situ investigations of biological processes and localized chemical reactions with high spatial and temporal resolution.Nonetheless,the minor scattering spectral shift occurring in an individual nanoparticles limits their applications in biosensing.Several strategies toward the development of plasmonic nanoparticles with different shapes and architectures such as nanoflowers,nanorods,nanocubes,and core satellites nanostructures have been developed to enhance the scattering spectral shift.The prominent and amplified LSPR performances of these plasmonic nanoparticles with excellent structure greatly improve the detection sensitivity and signal-to-noise ratio,and pushed the feasibility for sensing in long-duration and real-time single cell studies.The LSPR technology was used for in-situ detection and intracellular imaging of tumor biomarker at single particle level.The research included in situ detection and imaging of telomerase activity in cancer cell lines,detection and imaging of cellular alkaline phosphatase activity,the design of diagnostic and therapeutic probe for multimodal assay and imaging of ROS and caspase-3 activity.The details are given below:?1?In the present work,a core-satellites assembled nanostructure of Au₅₀@Au₁₃was designed for in-situ detection and intracellular imaging of telomerase activity by combining plasmonic resonance Rayleigh scattering spectroscopy with dark-field microscope?DFM?.The Au₅₀@Au₁₃ was fabricated by using 50 nm gold nanoparticles(Au₅₀)as core and 13 nm gold nanoparticles(Au₁₃)as satellites,both of them were functionalized with single chain DNA and gathered proximity through the highly specific DNA hybridization with a nicked hairpin DNA?O1?containing a telomerase substrate?TS?primer as linker.In the presence of telomerase,the telomeric repeated sequence of?TTAGGG?_n extended at the 3'-end of O1 would hybridized with its complementary sequences at 5'-ends.This led the telomerase extension product of O1be folded to form a rigid hairpin structure.As a result,the Au₅₀@Au₁₃ was disassembled with the releasing of O1 and Au₁₃-S from Au₅₀-L,which dramatically decreased the plasmon coupling effect.The remarkable LSPR spectral shift was observed accompanied with a detectable color change from orange to green with the increase of telomerase activity at single particle level with a detection limit of 1.3×10^-13 IU.The ability of Au₅₀@Au₁₃3 for in-situ imaging intracellular telomerase activity,distinguishing cancer cells from normal cells,in-situ monitoring the variation of cellular telomerase activity after treated with drugs were also demonstrated.?2?In this work,a simple and ultrasensitive localized surface plasmon resonance?LSPR?method that use Au nanoflowers?AuNFs?as probe was designed for in-situ monitoring of alkaline phosphatase?ALP?activity.The AuNFs was fabricated by hydrogen tetrechloroaurate-induced oxidative disruption of polydopamine-coated Au nanoparticles?AuNPs?and subsequently growth of Au nanopetals on AuNPs.The as-prepared AuNFs showed a much higher LSPR capability and stronger scattering color change than AuNPs.The strategy for in-situ cellular ALP activity detection relied on the deposition of Ag on AuNFs surface,which changed the morphology of AuNFs and led to a tremendous LSPR response and scattering color change.The deposition of Ag shell on AuNFs was related to ALP activity,where ALP catalyzed the hydrolysis of L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate to form L-ascorbic acid?AA?,then AA reduced Ag⁺to Ag and deposited onto AuNFs.With this concept,the ALP activity could be monitored with a detection limit of 0.03?U L^-1.Meanwhile,the ALP activity of single HepG2 cells and HEK 293 cells was tracked with proposed approach,which indicated the trace expression level of ALP in HEK 293T cell and overexpressed level of ALP in HepG2 cells.After treated with drugs,the cellular ALP activity of HepG2 cells was decreased with the treating time and dose increasing.Therefore,the proposed strategy could be used for tracking the cellular ALP activity,which paved a new avenue for cell studies,and hold great potential for discovering novel ALP-based drugs applications.?3?Herein,we fabricated a multifunctional nanocomposite for cancer cell treatment,as well as intracellular imaging.The composite consists of AgNPs and hematoporphyrin monomethyl ether?HMME?as apoptosis inducing agents and Au nanoflowers?AuNFs?as both a nanocarrier and a LSPR monitoring marker.HMME and AgNPs were bonded at the surface of the AuNFs to form Au-Ag probe.Under irradiation at 638 nm laser,ROS generated from the photosensitizers induced cancer cells undergoing apoptosis while normal cells survive.The release of silver ions on the probe led to a tremendous LSPR response and scattering color change,which was related to ROS activity.In addition,caspase-3 produced in the apoptosis of tumor cells can break the DEVD peptide and separate HMME with AuNFs,resulting in the significant fluorescence recovery,which was related to caspase-3 activity.On the basis of the above results,the probe could be applied as a new strategy to distinguishing cancer cells from normal cells,photodynamic therapy and chemotherapy for cancer cells,and monitoring and imaging the variation of cellular ROS and caspase-3 after treated with drugs.