Construction And Application Of Quantum Dot@nanogel Fluorescent Probe

Quantum dots(QDs),also called nanocrystals,have unique physical and chemical properties,such as high brightness,good photobleaching resistance,broad-band absorption and narrow-band emission,prone to energy/charge transfer and facile surface functional modification.The QDs based fluorescent probes have so far been widely used in labeling,sensing and bioimaging.Nanogel(NG)is a biocompatible nanoscale three-dimensional network structure formed by physical or chemical cross-linking of polymers,showing the advantages such as high-water content,anti-fouling ability,and target-enrichment capability.By encapsulating probes into biologically localized embedding,the so-called PEBBLE probes,are thus advantageous in terms of combinatorial excellent biocompatibility,high stability and high detection sensitivity,desirable for various biosensing and bioimaging applications.In this thesis,we report the preparation of QD-micelle spherical nucleic acid(QM-SNA)probes that feature three-dimensional DNAzyme walker mediated signal amplification,the construction of PEBBLE probes by embedding QM-SNA probes into a core-shell-structured NG,and their application for sensitive detection of circulating miRNAs in crude serum samples as well as in situ,high-resolution imaging of miRNAs in living cells.The main contents are listed as follows:1.Preparation and characterization of QM-SNA probes.Based on green-,yellow-,red-emissive QDs,respectively,triple-colored micelles were obtained by thin-film hydration method.QM-SNA was then prepared by coupling the DNAzyme and quencher-modified substrate onto the micelles via typical EDC/NHS condensation reaction.The infrared spectra,fluorescence spectra and TEM measurements showed that the QM-SNA was indeed formed,spherical in shape and quenched in fluorescence.2.QM-SNA-based “AND” gate-controlled DNAzyme walker-mediated signal amplification strategy.The signal amplification strategy includes: a AND logic gate with target miRNA and zinc ion as two inputs,an amplifier operated with a DNAzyme walker,and the QM fluorescence as the output.That is,the binding of target miRNA makes DNAzyme recognize the nearby substrate and the binding of zinc ion can activate enzymatic activity of the DNAzyme;by enzymatic hydrolysis of the specific sites on substrate,the DNAzyme drives itself autonomously walking along DNA track(substrate)on the QM surface,resulting in the release of quenchers;the “one target-to-many quenchers”-type operation leads to fluorescence recovery of QM.Gel electrophoresis results indicated that the DNAzyme walking is indeed co-initiated by the presence of target miRNA and zinc ion.The real-time fluorescence measurements defined that the DNAzyme walking time was about 40 min.3.Construction of QM-SNA@NG PEBBLE probe and its use for circulating miRNA detection.The PEBBLE probes were constructed by 4 steps: preparation of ~70 nm in diameter silica core by St(?)ber method and subsequent amination,coupling of P(MVE-β-CD)on the surface of silica via EDC/NHS reaction,assembly of Ad-anchored QM-SNA onto P(MVE-β-CD)-modified silica core via the host-guest reaction,and cross-linking of P(MVE-β-CD)with NH2-PEG-NH2 to form the hydrogel shell.TEM measurements clearly showed the core-shell structure of the QM-SNA@NG PEBBLE probe.The DLS,Zeta potential,stability and cytoxicity measurements showed that the probe was ~100 nm in size,neutral in charge,stable in complex biological environment,and non-toxic toward living cells.With target enrichment by NG shell and QM-SNA-based signal amplification,the PEBBLE probe features miRNA detection in a sensitive and specific manner.The obtained limit of detection limit was as low as 1 f M,and the linear dynamic range was 3.5 orders of magnitude.With an array of triple-colored PEBBLE probes,multiplexing of miRNAs was readily achieved.The probe array was demonstrated to be capable of simultaneously quantifying target miRNAs(mi R-196 a,mi R-25 and mi R-21)in crude serum samples.4.The use of PEBBLE probes for fluorescent imaging of multiplex miRNAs in living cells.Fluorescence colocalization experiments indicated that the PEBBLE probe enters the cytoplasm through cell enytosis and is unable to enter the nucleus due to the ~100 nm size.Based on endogenous miRNA and exogenous zinc ion-co-initiated,DNAzyme walker-mediated signal amplification,the QM-SNA@NG PEBBLE probes feature multi-channel fluorescence imaging of intracellular miRNAs with remarkable-high resolution.Via the multiplexed fluorescence imaging,non-small cell lung cancer(NSCLC)cells can be distinct from normal lung cells due to different expression levels of target miRNAs(mi R-196 a,mi R-25 and mi R-21).Moreover,the cells for the subtypes of NSCLC can be precisely differentiated,ascribing to the high resolution of fluorescence imaging.