Resonance Energy Transfer System for Detection and Imaging of miRNA

Author:Chai Shui Qin

Supervisor:huang cheng zhi

Database:Doctor

Degree Year:2019

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Pages:113

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In the past few decades,Resonance Energy Transfer(RET)has been widely used as a spectroscopy technique for structural identification and interaction of biomolecules,in vitro analysis,in vivo molecular monitoring,nucleic acid detection,signal transduction,light harvesting and other aspects of research.RET is a non-radiative energy transfer process through the dipole-dipole interaction when the energy donor and the acceptor are in close proximity.The appropriate donor-acceptor pairs in this process is critical to improving energy transfer efficiency.Graphene quantum dots(GQDs)are great energy donors due to their simple preparation,good water solubility,adjustable photoluminescence,strong photostability and good biocompatibility.In recent years,GQDs have been gradually used as energy donors in many fields such as biochemical analysis,bioimaging,and disease diagnosis.GQDs modification is the key to construct energy transfer systems.At present,the highly specific covalent modification method requires a large number of carboxyl groups or amino groups on the surface of GQDs.Therefore,the synthesis of functional groups-riched GQDs is very important for the construction of energy transfer systems.At present,energy transfer-based fluorescent nanoprobes have been widely used for detection and imaging analysis of biomarker miRNAs.Conventional energy transfer nanoprobes contain one donor and one acceptor.As a result of the limitation of energy transfer efficiency,for cell imaging,there are many problems such as high background signal and false positive signal,which affects the sensitivity and accuracy of imaging.In addition,intracellular low-abundant miRNAs have higher requirements for detection sensitivity.Some conventional amplification strategies,including RT-PCR,rolling circle amplification,and isothermal exponential amplification,have satisfied the need of low-abundance miRNAs detection.However,these methods require the participation of DNA polymerase or endonuclease,thereby reducing the repeatability of miRNA detection.In order to solve these problems,we synthesized a single-layer graphene quantum dot with good light stability,high biocompatibility and abundant carboxylic acid functional groups,which was applied for the detection of phosphate by fluorescence off-on type.The covalent modification method was successfully applied for the energy transfer system construction and applied to detect miRNA.In addition,a dual energy transfer probe was constructed for low background imaging analysis of miRNAs.Finally,highly-sensitively detection of miRNA was achieved through double signal amplification with catalyzed hairpin assembly(CHA)and X-shaped DNA.The main works are as follows:1.The synthesis of single-layer graphene quantum dots and their detection for phosphate and miR-21.Surface functional groups play a crucial role in the functionalization of GQDs,so it is very meaningful to synthesize functional groups-riched GQDs.Single-layer graphene quantum dots(s-GQDs)have a simple structure and a large surface area which is more helpful to the functionalization of the both sides of functional group.We used existing carbon quantum dots(CQDs)as precursor to synthesize s-GQDs with the 86.9%of absolute QY by methanol exfoliating.The morphology and particle size were characterized by transmission electron microscopy and high-resolution transmission electron microscopy.The thickness was characterized by atomic force microscopy.Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy were used to characterize the surface functional groups.The oxygen content of the as-prepared s-GQDs was 47.5%,which was obviously higher than that of the precursor CQDs(24.8%).And the phenomenon of rare earth ion Dy3+induced s-GQDs aggregation emission quenching proved that the surface of s-GQDs was abundant in carboxylic acid functional groups,and phosphate could destroy the Dy3+-induced aggregates of s-GQDs,inducing the emission recovery of the s-GQDs,which can be applied to detect phosphate.In addition,carboxylic acid groups provide favorable conditions for the functionalization of s-GQDs.The s-GQDs were functionalized by DNA using covalent modification methods.s-GQDs were used as donors and BHQ1 was used as acceptors to construct energy transfer system through DNA ligation,which was applied to detect miR-21 by means of CHA cycle.2.A dual energy transfers-based fluorescent nanoprobe for imaging miR-21 in non-alcoholic fatty liver cells with low background.Traditional energy transfer system mostly consists of a donor and a receptor.Due to the limitation of energy transfer efficiency,the energy transfer nanoprobe has a high background signal in biological tissue or cell imaging,reducing the sensitivity of detection.To solve this problem,we developed a dual energy transfer fluorescent nanoprobe containing a donor and two receptors for lowing background and high-sensitive imaging of miR-21 in non-alcoholic fatty liver cells.The energy complex acceptor was constructed by coupling black hole quencher 2(BHQ2)to the surface of gold nanoparticle(AuNPs)and quantum dots(QDs)were choosen as donor of energy transfer.The composite receptor was modified with a single-stranded SH-DNA1through Au-S bonds and the donor was modified with a single-stranded Biotin-DNA2through biotin-streptavidin interactions,respectively.The distance between the receptors and the receptor was narrowed by DNA hybridization.The fluorescence of QDs was quenched from 56.5%to 82.8%simultaneously by the AuNPs and the BHQ2 via nanometal surface energy transfer(NSET)and fluorescence resonance energy transfer(FRET),reducing the background signals for target imaging.After the nanoprobe entered non-alcoholic fatty liver cells,miR-21 target could hybrid with the DNA2,triggering the disassembly of QDs with the composite receptor,ultimately yielding significant fluorescence recovery signals.Moreover,the fluorescence intensity of QDs increased with the increase of target miR-21 concentration.The sensitivity of this nanoprobe has also been enhanced toward detecting miR-21 in the range of 2.0-15.0 nM with the detection limit(3σ)of 0.22 nM,which was 13.5 times lower than that without BHQ2.In addition,the method has better selectivity and is capable of distinguishing miRNAs with only one base mismatch.Therefore,the probe can be applied to low background imaging analysis of miR-21 in nonalcoholic fatty liver cells.3.Target-activated catalytic self-assembly of X-shaped DNA for highly sensitive detection of miR-21.miRNAs are extremely low abundant in cells,thus it is a great challenge for highly sensitive quantitative analysis of miRNAs.We performed signal amplification strategy by target-activated catalytic hairpin self-assembly(CHA)of X-shaped DNA for highly sensitive detection of target miR-21.We used the four hairpin DNA modified with energy donor FAM and receptor BHQ1 at the ends of DNA as the component molecules.When the target miR-21 was absent,the distance between FAM and BHQ1 was narrowed,resulting in the fluorescence of FAM was quenched by BHQ1.When miR-21 was added,the hairpin 0(H0)was first opened under the activation of the target,followed by H1,H2,H3,and H4 sequentially opened through the base complementary pairing to form the X-shaped DNA structure and the miR-21 loaded on H0 entered the next reaction.The fluorescence of FAM was recovered due to the wide distance bewteen donor FAM and acceptor BHQ1.Dual signal amplification strategy was acquired by both aggregating four fluorescent dyes on one X-shaped DNA and the target cycle reaction.With the increase of target miR-21 concentration,the fluorescence intensity of FAM gradually increased,which showed a good linear relationship in the range of 0.1 nM-3.2nM.The detection limit was 0.020 nM(3σ).Compared to only CHA,the detection sensitivity of dual signal amplification was improved by 5.7 times.Compared to a single fluorescent dye on an X-DNA,the detection sensitivity of dual signal amplification was improved by 11.3 times.In addition,the reaction could be completed in 1 hour,which was short to avoid miRNA degradation.Therefore,the method achieves the purpose of highly sensitive detection of miR-21.In summary,this study mainly focused on the energy transfer processes.First,we successfully prepared a s-GQDs energy donor with strong photostability,good biocompatibility and abundant carboxylic acid functional groups by using simple and rapid methanol exfoliating method.The s-GQDs was modified and used in the construction of energy transfer system to detect miR-21.The s-GQDs also could be induced by Dy3+to aggregation fluorescence quenching and phosphate competed with s-GQDs to coordinate Dy3+,making s-GQDs fluorescence recovery,which was used to detect phosphate.Next,we developed a dual energy transfer fluorescent nanoprobe for lowing background and high-sensitive imaging of miR-21 in non-alcoholic fatty liver cells and performed dual signal amplification strategy by target-activated CHA of X-shaped DNA for highly sensitive detection of low abundant miR-21.This study provides a new method for the synthesis of functional groups-rich donors in energy transfer systems,and also provides a new idea for the construction of low background energy transfer systems and the highly sensitive detection of disease marker miR-21.