Gold Nanoparticle Based Fluorescent Nucleic Acid Probes For Intracellular Sensing

Variety kinds of biological molecules in the cells have special physiological functions and play vital roles in the life process.The essential information on intracellular biological molecules obtained accurately and sensitively has great significance for understanding the regularity and mechanism of interactions between the biological molecules,researching the development mechanism of major diseases and their treatment.So far,variety new types of fluorescent probe based on nanomaterials have been estabilished and applied for living cell studies.However,research works in living cells are still in its infancy,there are challenges to overcome interferences from intracellular environment,get higher sensitivity.This dissertation mainly focuses on two important scientific problems,which are false positive and false negative signals.In this dissertation,a series of flourecent probes based on DNA functionalized AuNP-fluorescent probes are estabilised to study biological molecules in living cells.The main researches of this dissertation are summarized as follows:1.FRET Nanoflares for intracellular mRNA detection:avoiding false positive signals and minimizing effects of system fluctuationsA new type of intracellular nanoprobe,termed fluorescence resonance energy transfer(FRET)nano flares,has been developed to sense mRNA in living cells.First,the AuNP functionalized with recognition sequences hybridized to flares are designed as hairpin structures and fluorescently labeled at their 5' and 3' termini with donors and acceptors,respectively.In the absence of targets,the flares are captured by binding with the recognition sequences,separating of the donor and acceptor,and inducing low FRET efficiency.However,in the presence of targets,the flares are gradually displaced from the recognition sequences by the targets,subsequently forming hairpin structures that bring the donor and acceptor into close proximity and result in high FRET efficiency.In this closed state,the fluorescence of the acceptor can be detected.Thus,the fluorescence emission ratio of acceptor to donor(A/D)can be used as a signal for quantitation of target sequences.Compared to the conventional single-dye nanoflares,the upgraded FRET nanoflares can avoid false positive signals by chemical interferences(such as nuclease and GSH)and thermodynamic fluctuations.Moreover,the signal generation in FRET nanoflares can be easily made with ratiometric measurement,minimizing the effect of system fluctuations.2.Aptamer-based FRET nanoflares for imaging potassium ions in living cellsBased on the aforementioned work,combining a aptamer and AuNP,we design an aptamer-based FRET nanoflare,for sensing K+ values in living cells.The aptamer-based FRET nanoflares consist of an AuNP,short single-stranded oligonucleotides,and dual-fluorophore-labeled aptamer sequences,in which FAM acts as donor(D)and TAMRA(A)as acceptor.The complementary oligonucleotides are designed to bind with the aptamer sequences(flares)and immobilized on the AuNP surface via an Au-S bond.In the absence of target K+,the aptamer sequences are captured by binding with the complementary strands,separating of the donor(FAM)and acceptor(TAMRA),and inducing a low FRET efficiency.In this open state,only the fluorescence of donors can be detected.However,in the presence of target K+,the flares are gradually displaced from the complementary strands,subsequently forming G-quadruplex structures that bring the donor and acceptor into close proximity and result in a high FRET efficiency.In this closed state,the fluorescence of the acceptor can be detected.Thus,the fluorescence emission ratio of acceptor to donor(A/D)can be used as a signal for detection of target K+.It is demonstrated that this probe could be applied for K+ detection and imaging in living cells.3.Aptazyme-gold nanoparticle sensor for amplified molecular probing in living cellsWe construct an aptazyme-AuNP fluorescent probe for sensitive molecular detection in living cells.In this design,a gold nanoparticle(AuNP)is modified with substrate strands hybridized to aptazyme strands.Only the target molecule can activate the aptazyme and then cleave and release the fluorophore-labeled substrate strands from the AuNP,resulting in fluorescence enhancement.The process is repeated so that each copy of target can cleave multiplex fluorophore-labeled substrate strands,amplifying the fluorescence signal.Results show that the detection limit is about 200 nM,which is 2 or 3 orders of magnitude lower than that of the reported aptamerbased adenosine triphosphate(ATP)sensors used in living cells.Furthermore,it is demonstrated that the aptazyme sensor can readily enter living cells and realize intracellular target detection.4.Gold nanoparticle based hairpin-locked-DNAzyme probe for amplified miRNA imaging in living cellsA new class of intracellular nanoprobe,termed AuNP-based hairpin-locked-DNAzyme probe,has been developed to sense miRNA in living cells.It consists of an AuNP and hairpin-locked-DNAzyme strands.In the absence of target miRNA,the hairpin-locked-DNAzyme strand formed a hairpin structure by intramolecular hybridization,which could inhibit the catalytic activity of DNAzyme strand and the fluorescence is quenched by the AuNP.However,in the presence of target,the target-probe hybridization could open the hairpin and form the active secondary structure in the catalytic cores to yield an 'active' DNAzyme,which then cleaves the self-strand with the assist of Mg2+.The cleaved two shorter DNA fragments were separated with the target.As a result,the fluorophores were released from the AuNP and the fluorescence was enhanced.Meanwhile,the target was also released and binds to another hairpin-locked-DNAzyme strand to drive another cycle of activation.In such a way,each target can be recycled multiple times,which leads to the cleavage of multiple DNAzyme strands and thus offers high detection sensitivity.In addition,results show that the probe can readily enter living cells and realize intracellular miRNA imaging.