Detection Of MiRNA And PARP-1 Based On Dark Field Scattering Imaging

Label-free localized surface plasmon resonance（LSPR）technique can realize single-particle dark-field scattering imaging.It has many advantages,such as high temporal and spatial resolution,little background interference,and photostability.It has been used as a new means for highly sensitive detection and imaging of cancer biomarkers and in situ monitoring of intracellular active molecules.Noble metal nanoparticles,such as gold and silver,are ideal plasmon nanoprobes due to their significant LSPR optical properties.The plasmon resonance absorption band of nanoparticles can be regulated by changing the size,morphology,composition and localized refractive index.In addition,metal nanoparticles have good biocompatibility,and they can be easily functionalized by oligonucleotides,enzymes,antibodies,streptavidin,and biotin molecules,which promote their wide applications in biosensors and imaging.However,the scattering spectrum shift generated by an individual plasmon nanoprobe is very small.Several nanostructures,such as core-satellites structure and silver coated gold nanorods,have been designed for significantly improving the signal-to-background ratio and detection sensitivity because of the generated strong plasmon coupling effect,exhibiting abundant color changes and large scattering spectrum shifts,which provides the feasibility for constructing biosensors at the cell level.Herein,a kind of label-free plasmon nanoprobe with strong LSPR scattering signals was constructed for extracellular analysis at single-particle level and in situ imaging of poly（ADP-ribose）polymerase-1（PARP-1）in cancer cells.A designed strategy for imaging of intracellular PARP-1 and H₂O₂was instrumental in improving the accuracy of cancer diagnosis.A multifunctional nanoprobe was prepared for micro RNA-21（mi RNA-21）and caspase-3 detection,drug delivery,and apoptotic process monitor.The details are as follows:（1）Based on dark-field microscopy（DFM）,a spectral-resolved single-particle detection（SPD）method was established to not only detect the extracellular PARP-1 activity with high sensitivity,but also image PARP-1 in living cells in situ.First,gold nanoparticles with a diameter of 50 nm were modified with active double stranded DNA(Au₅₀-ds DNA),which was used as the scattering probe.PARP-1 was activated by ds DNA to cleave the substrate nicotinamide adenine dinucleotide（NAD⁺）into nicotinamide and ADP-ribose for forming poly（ADP-ribose）polymer（PAR）by repeated catalytic polymerization.PAR with rich negative charges could absorb the positively charged gold nanoparticles（Au₈）on the surface of Au₅₀.The closer distance between Au₅₀and Au₈affected the localized dielectric environment of Au₅₀,resulting in large LSPR scattering spectrum shift and significant scattering color change,which realized the single-particle detection of PARP-1 from 0.2 to 10m U.Compared with the reported fluorescence methods for imaging PARP-1,this label-free single-particle imaging strategy exhibits high signal-to-background ratio,which opens the way for clinical cancer diagnosis and PARP-1 inhibitor research.（2）Gold nanorods coated with silver（Au@Ag NRs）were synthesized as plasmon probes for imaging of PARP-1 and H₂O₂in living cells by dark-field microscope.Au@Ag NR was used not only to distinguish tumor cells from normal cells but also to induce the apoptosis of cancer cells owing to the etching of Ag shell by H₂O₂,accompanied with the color change from green to orange.On the other hand,Au@Ag NRs modified with active ds DNA could be utilized to image PARP-1 in cancer cells and to quantitatively detect PARP-1 in vitro by colorimetry or dark-field scattering.The reason was that PARP-1 polymerized NAD⁺into large and hyperbranched PAR on the surface of Au@Ag NRs,preventing the Ag shell from being etched by H₂O₂.As the PARP-1 activity increased,a blue shift of the adsorption peak occurred along with a color change from pale pink to green,which could be recognized by naked eye.Under DFM,its scattering light varied obviously from red to green.The single-particle analysis of PARP-1 was based on the large blue shift of LSPR peak.The proposed single-particle imaging strategy could be used for intracellular PARP-1 and H₂O₂imaging respectively,which improves cancer diagnosis accuracy and holds good prospect in biosensing and cancer diagnosis.（3）The anticancer drug doxorubicin（Dox）was loaded with a multifunctional plasmon core-satellites（CS）nanostructure for mi RNA-21 detection,targeting drug release and therapy evaluation.Plasmonic core-satellites nanoprobe was constructed with uniformly distributional50 nm（core）and 13 nm（satellites）Au NPs.Au₅₀and Au₁₃were assembled to form CS nanoprobes based on the formed GC base rich double-stranded DNA by DNA hybridization.Anticancer drug Dox was loaded into the CS nanostructure by intercalating into the GC-rich double strands.Target mi RNA-21 hybridized with L-DNA,and the constructed CS nanostructure was disassembled,producing characteristic LSPR signals and releasing Dox.With the increase of mi RNA-21 concentration ranging from 0.01 to 1000 f M,a more obvious blue shift of scattering spectra peak occurred along with distinct color change from orange to green under a dark-field microscope,which could be used to detect mi RNA-21 at single-particle level.Meanwhile,released Dox induced apoptosis.Caspase-3 involved in apoptosis was then activated to cleave the specific peptide substrate DEVD,releasing fluorophore FAM from Au NPs.As a result,caspase-3 was detected based on restored fluorescence intensity,which was used to evaluate the therapy effectiveness by confocal fluorescence imaging.