Fabrication Of Novel Upconversion Nanoprobes For Intracellular Imaging Assay

Ultra-sensitive detection of biomolecules has become more and more important in the fields of food detection,clinical diagnosis,environmental monitoring and anti-bioterrorism.Because of its high sensitivity,good selectivity,high spatial-temporal resolution and easy preparation,fluorescent probe has become one of the powerful tools for molecular sensing and imaging.Compared with traditional fluorescent materials,upconversion materials such as rare earth upconversion nanoparticles?UCNPs?and two-photon fluorescence molecules have the advantages of low light damage,weak background fluorescence and deep penetration.They have received extensive attention and application in biomedical and diagnostic fields.However,in the practical applications,upconversion materials also face some urgent problems,such as poor water dispersion,lack of biocompatibility,hard to synthesize the probe with functional groups and so on.In order to solve the above problems,in this dissertation,UCNPs,mesoporous silica nanoparticles?mSiO₂?,?-cyclodextrin??CD?,two-photon fluorescence materials,spherical nucleic acid?SNA?are combined respectively to construct upconversion nanoprobes for the detection and imaging of biomolecules in vivo.The specific contents are as follows:1 By taking advantage of the optical properties of UCNPs,we have designed a luminescence ratiometric nanosensor for measuring nitric oxide?NO?in biological fluids,live cells,and tissues.The UCNPs coated with mesoporous silica greatly improved the water solubility and biocompatibility of the probe.?CD was used as gatekeeper,which not only improved the biocompatibility of the probe,but also improved the selectivity of the probe based on the size effect.The assay is validated under clean buffer conditions as well as inserum and liver tissue slices obtained from mouse models.2 We have designed a novel UCNPs-based ratiometric luminescence nanosensor to monitor carban monoxide?CO?in real time.This upconversion nanoprobe consists of a UCNP core,CO-responsed palladium ion-bounded rhodamine B-derived molecules?Pd-RdMs?encapsulated within the mesopores of the mSiO₂ shell of the UCNPs,and?CD layer on the exterior of the particle,which can improve the selectivity and biocompatibility of the probe.In addition to working in clean buffer environments with known[CO],the assay has been evaluated using living cells and tissue slices,and also successfully used these probes to study the protective effect of octreotide in the course of hepatic ischemia-reperfusion?HIR?in mice.3 Herein,we describe the first example of a novel two-photon excitation?TPE?-based ratiometric fluorescent pH nanoprobe?UCNP@DMI-mSiO₂@?CD?by conjugating pH-sensitive two-photon dyes?TP dyes?of trans-4-[p-?N,N-diethylamino?styryl]-N-methylpyridinium iodide?DMI?with a pH-insensitive Tm³⁺-doped UCNPs.The probe has good stability and high sensitivity.This TPE nanoprobe has been successfully applied for the fluorescence imaging of the lysosomal pH values,as well as ratiometrically visualizing redox chemicals-stimulated changes of intracellular pH in living cells and tissues.4 Herein,we report here a two-photon nanoprobe for the detection of RNase H activity by combining a two-photon dye with a SNA featuring DNA/RNA duplex corona and gold nanoparticle core.The dye molecule is highly fluorescent when associated with the duplex,but the fluorescence is quenched by the tethered gold nanoparticle.Degradation of the RNA sequence by RNase H releases the dye molecules,thus turning on the fluorescence emission.Taking advantage of EBMVC and SNA,The detection of RNase H is validated in buffer as well as in live cells and liver tissues obtained from rat models.5 Herein,we designed a novel TPE nanosensor for DNase activity assay in vitro and in ex-vivo.Due to the mechanism of hydrolysis of DNA by DNases,Successful DNase assay in cell and liver tissue slices obtained from rat models was demonstrated with this sensing strategy.This nanosensor provides a new opportunity to screen new anti-HIRI drugs and explore the drug protection mechanisms.6 DNAzyme has significant signal amplification ability,and the gold nanoparticle?AuNP?-based SNA has the properties of resistance to enzymatic degradation,enhanced nucleic acid binding and excellent cellular uptake.In this work,we constructed a novel SNA-based TPE fluorescent DNAzyme probe for the selective and sensitive detection of target miRNA in living cells.Taking advantage of DNAzyme and SNA,the probe ultimately achieved the detection of the miRNA through amplification of the fluorescence signals.The assay was successfully validated with clean buffer conditions as well as with cells.