Studies And Applications Of Novel Imaging Approaches For MRNA Analysis In Tumor

Cancer is still the main disease that threats human life seriously.Recent studies have shown that the survival rate and cure rate were attached to the early diagnosis and treatment of malignant tumor.However,the conventional detections with low selectivity and sensitivity are difficult for early diagnosis.Besides,most of the commonly used antineoplastic drugs cannot discern between diseased and healthy cells and have serious side-effects on healthy cells.Hence,developing analytical technologies with significant performance for early tumor marker analysis and design site-specific stimuli-responsive controlled drug delivery systems are two of the most important factors in the fields of cancer diagnosis and treatment.With the fast development of imaging techniques and the widely application of nanomaterials,the new thoughts and opportunities have appeared for resolution of the key problem mentioned above.Nanomaterials with large surface areas,good biocompatibility,large loading capacity and high stability have been widely used in the fields of biosensing,disease diagnosis,cell imaging,drug delivery,etc.Based on the combination of unique nanomaterials with high-sensitive imaging techniques,the main work of this current study focuses on the construction of sensitive detection methods and design of multifunctional drug delivery system.The details are summarized as follows:1.On-chip Selective Capture of Cancer Cells and Ultrasensitive Fluorescence Detection of Survivin mRNA in Single Living CellRapid recognition of cancer cells and detection of tumor biomarker survivin mRNA play a critical role in the early diagnosis of many cancers.Based on the integration of specific cancer cell capture and intracellular survivin mRNA detection,this work presents a novel and sensitive on-chip approach for the bioanalysis of survivin mRNA in single living cell.The microchannel surface was firstly modified with prostate stem cell antigen(PSCA)monoclonal antibody as the recognition element for prostate cancer cell(PC-3).As a result of the antigen-antibody specific affinity interactions,PC-3 cells could be selectively captured on the microchannel surface.After cell capture,nano-sized graphene oxide-poly(ethylene glycol)bis(amine)(NGO-PEG)is employed as a quencher and carrier of signal tag,fluorescein isothiocyanate(FITC)-labeled antisense oligonucleotide(F-S1)complementary to part of survivin mRNA(target survivin mRNA),to transfect into the captured PC-3 cells.Upon the selectively binding of S1 to intracellular survivin mRNA,F-S1 will be released from NGO-PEG,inducing the fluorescence recovery of FITC.This antibody-based microfluidic device enables simple and inexpensive monitoring of survivin mRNA amount in single captured cell without the need for sample pretreatment.The survivin mRNA content in each PC-3 cell was estimated to be(4.8±1.8)×106 copies.This strategy opens a different perspective for ultrasensitive survivin mRNA detection,which may facilitate the early screening of malignancy.2.Functional Oriented Probes with Invisibility:Towards Highly Sensitive Mapping of mRNA in Living Single CellHere,we demonstrate a novel strategy for monitoring single mRNA in living cells.We use single plasmonic nanoparticle as an independent invisible probe for quantitative genetic profiling of survivin mRNA in single cell.Each asymmetrically PEGylated gold nanoparticle(AuNPs)serves as invisible monomer probes.Hybridization of these monomer probes to a single target mRNA resulted in the formation of a geometrical Y-shaped DNA and DNA origami-assembled AuNPs dimers.The dimer formation induced a significant plasmon resonance coupling effect,which in turn leads to a noticeable dimer scattering resonance and a turn-on colorimetric signal from zero background.The zero-background and strong plasmon coupling enabled this method for monitoring and mapping distribution of survivin mRNA in living single cell with single-molecule precision.3.Tumor Marker Mediated "on-demand" Drug Release and Real-Time Monitoring System Based on Multifunctional Mesoporous Silica Nanoparticles"On-demand" drug release can maximize therapeutic efficacy on specific malignancies state and minimize drug toxicity to healthy cells.Meanwhile there is lack of a real-time monitoring platform to accurately investigate the release amount of anticancer drug,especially nonfluorescent drugs.So it is significant to integrate both of them in one ideal drug delivery system.To achieve this,here we present a novel stimuli-responsive controlled drug delivery system towards the tumor marker survivin mRNA,using real-time monitoring approach based on fluorescence resonance energy transfer(FRET)strategy to quantify the process of drug release.First,7-Amino-4-methlcoumarin(AMCA)dye-terminated short oligonucleotide(FlareA)will hybridize with fluorescein isothiocyanate(FITC)labelled long oligonucleotide(S1f)which contains a recognition element to a specific RNA transcript,to form a FRET pair capped on the pores of mesoporous silica nanoparticles(MSNs).Followed by target recognition reaction,the target with longer strand displaces the FlareA strand to form a longer and more stable duplex with S1F,which leads to the removal of the capped oligonucleotide from the MSNs and triggers the entrapped cargo release while FRET between AMCA and FITC is broken.The relevant change in donor and acceptor fluorescence signal can be used to monitor the unlocking and release event in real-time.Further investigations have also demonstrated that,this release system possesses the capacity of modulating the extent of drug release according to the cell states,making the platform with an equally broad spectrum of applications in anti-cancer therapy.