Application Of Graphene-based Resonance Energy Transfer In Apoptosis Imaging Analysis

Apoptosis is a highly regulated and complex process of programmed cell death.It is of utmost significance to cell growth and proliferation.And the deregulation of cell apoptosis would result in many diseases,such as neurodegenerative diseases,autoimmune disorders,and cancers.Therefore,the establishment of a series of simple,rapid and sensitive methods for the analysis and detection of apoptosis-related substances is of great significance to reveal the process of apoptosis and the diagnosis and treatment of diseases.Fluorescence imaging analysis based on Resonance Energy Transfer(RET)is simple,intuitive,sensitive,and has high temporal and spatial resolution.It has been widely used in biochemical analysis and clinical diagnosis.However,in the traditional RET system,one donor usually corresponds to one receptor,resulting in low efficiency of RET.As a new two-dimensional carbon nanomaterial,graphene oxide(GO)not only has the advantages of low cytotoxicity,high specific surface area and easy for modification,but also contains a large number of conjugated ? electrons,allowing it to adsorb single-stranded DNA by hydrogen bonding and ?-? stacking,resulting to effective quenching ofthe fluorophores on its surface,so it is of great significance to establish a series of simple,sensitive and accurate RET analysis methods using GO as energy receptor for real-time monitoring of cell apoptosis.Ideally,the single-stranded DNA can be stably absorbed by GO and can only be desorbed from the GO surface by identifying the target.However,the interaction between ssDNA and GO of the physisorption system are usually susceptible to competition from biomolecules,causing a false positive signal.Based on this,this thesis carries out the following three aspects of research:1.Energy transfer-based DNA/graphene oxide nanocomplex probe for accurate detection of miR-21.Single-stranded DNA(ssDNA)bound to GO by physical adsorption is easily displaced by nontarget molecules,causing false positive signals.Based on the fact that purines are adsorbed by GO more tightly than pyrimidines at the individual nucleobase level,FAM–labelled probe DNA(the complementary DNA sequence of mi R-21)was hybridized with bridge dye Cy3-labelled capture DNA(cDNA)to form double-stranded DNA,which was anchored on GO stably through poly-adenine(poly-A)to improve the anti-interference ability.Under the excitation of FAM,FRET occurs between FAM and Cy3,while LrRET occurs between Cy3 and GO,resulting in the quenching of FAM.With the increase of miR-21 concentration,the fluorescence of FAM gradually recovered and showed a good linear relationship in the concentration range of 1-400 nM,with the detection limit of 0.668 nM(3?/k).When detected in complex biological samples,FRET between FAM and Cy3 can be used to judge whether the nanoprobe was non-specifically detached from GO by observing whether the Cy3 signal is recovered,so as to achieve a high accuracy detection of apoptosis-related mi R-21 and avoid false positive signals caused by non-specific adsorption.2.Dual energy transfer-based DNA/graphene oxide nanocomplexprobe for low backgroundand accurate monitoringof apoptosis-related microRNAs.In the traditional RET system,one donor usually corresponds to one receptor,resulting in low energy transfer efficiency between donor and receptor.Therefore,these RET nanoprobes often have problems of high background signal and low sensitivity in cell imaging analysis.Based on the fact that GO and Black hole quencher(BHQ)are both effective quenching agents,we constructed a one donor-two-acceptor RET nanoprobe based on GO and BHQ,which improved the energy transfer efficiency and reduced the background of the probe.On the other hand,the probe was combined with the poly-A anti-interference strategy,and the strong binding ability of poly-A and GO was utilized to adsorb the double-stranded DNA on the GO surface stably,which served as the first insurance for anti-interference of the probe.FRET between BHQ and the donor minimizes false positive signals due to possible nonspecific poly-A DNA desorption,which served as the second insurance for anti-interference of the probe and effectively improves accuracy of the in complex biological samples.Based on the dual receptor design of GO and BHQ and the dual insurance design of GO and poly-A,the probe was used for simultaneous monitoring of apoptosis-related mi R-630 and miR-21 with low background,high accuracy and high selectivity,in which the linear range of miR-630 and miR-21 was 0.5-150 nM and 0.5-100 nM,respectively,with the detection limits of 0.25 nM(3?/k)and 0.12 nM(3?/k),respectively.Notably,the nanoprobe was used to dynamically monitor the changes of mi R-630 during A549 cell apoptosis in situforthe first time.It was found that in the first 120 min of apoptosis,the expression of miR-630 was up-regulated.This method is simple and fast,and has great potential for accurate detection and imaging of various markers in complex biological samples.3.Dual energy transfer-based DNA/graphene oxide nanocomplex probe fortargeted imaging of cancer cells and accurate monitoringofthe apoptosis process.The aboved dual energy transfer-based DNA/graphene oxide nanocomplex probe was demonstrated to have the advantages of low background,high accuracy and high selectivity.In order to achieve targeted imaging of cancer cells,Atto425-labelled nucleolin aptamer(AS1411)was hybridized with BHQ1-DNA to form double-stranded DNA,which was absorbed on GO through poly-A.This nanocomplex probe can be used for accurate diagnosis of cancer cells based on the fluorescence recovery of Atto425.Meanwhile,the Cy5-modified cytochrome c(Cyt c)aptamer was hybridized with BHQ2-DNA to form double-stranded DNA,which was also loaded on GO through poly-A.After internalization into cancer cells,the fluorescence of Cy5 gradually recovered with the increasing of Cyt c concentration in the linear range of 12.5 nM-1 ?M,with the detection limit of 38 nM(3?/k).In this case,monitoring of the apoptosis process can be realized by real-time monitoring the release of Cyt c in the process of cancer cell apoptosis.The design has great potential for accurate diagnosis of cancer cells,apoptotic imaging analysis,evaluation and screening of apoptosis-related anticancer drugs.In summary,in this study,with apoptosis as the research object and RET as the technical means,a nanoprobe with GO as the energy receptor was constructed.By introducing poly-A and the bridge dye Cy3 into the GO-adsorbed DNA energy transfer nanoprobes,the anti-interference ability of such physisorption probes was improved,and the high accuracy detection of apoptosis-related miR-21 was achieved.By constructing a GO-BHQ-based one-donor-two-acceptor RET nanoprobe,the energy transfer efficiency was improved and the background of the probe was reduced.At the same time,on the basis of the introduction of poly-A,the effective quenching of fluorescent dyes by BHQ avoids the false positive signal caused by the possible non-specific desorption of poly-A DNA,and further improves the accuracy of such physisorption probes in the analysis and detection.This method was successfully used to simultaneously monitor and image apoptosis-related mi R-630 and mi R-21 with low background,high accuracy and high selectivity.Based on the design of GO-BHQ-based one-donor-two-acceptor RET nanoprobe,we introduced the nucleolin aptamer and Cyt c aptamer into the probe,which was used for accurate diagnosis of cancer cells and accurate monitoring of cell apoptosis process.This study is universal and can be applied for the analysis of other targets through appropriate replacement of the probe.It also provides a new idea for the construction of low-background,high-accuracy energy transfer system and the analysis and detection of apoptosis related markers.