Construction Of Ratiometric Sensing System Based On DNA Nanomachines And Its Application In Disease Biomarkers Detection

In recent years,nanomaterials have shown great potential in the field of analytical testing due to their unique physicochemical properties.In this work,nanomaterials with different structures,including cobalt oxide nanosheets（Co OOH NS）,manganese dioxide nanosheets（MnO₂ NS）and zirconium-based metal skeletons（UiO-66-NH₂）,were synthesized.Ratiometric fluorescent and electrochemical biosensors were constructed for the quantitative detection of ascorbic acid（AA）,ascorbate oxidase（AAO）and miRNA-21 using Co OOH NS and MnO₂ NS materials with high specific surface area and excellent oxidative activity,and UiO-66-NH₂ material with electrochemical signal response,combined with the signal amplification capability of entropy-driven DNA nanomachines.Simultaneously through a rolling loop amplification reaction and DNA strand substitution strategy,photocontrolled modulation of the self-assembly process of G4 nanoflowers（DNF）.The specific works are as follows:（1）AAO and AA play an important role in delaying the aging and metabolism of life.In this work,a ratiometric fluorescence sensing system based on the internal filtration effect（IFE）between persistent luminescent particles（PLPs）and 2,3-diaminophenoxazine（DAP）was designed for the sensitive detection of AA and AAO activities.Among them,PLPs emit blue fluorescence at 475 nm.The Co OOH NS with oxidase-like activity can oxidize o-phenylenediamine（OPD）to generate DAP with fluorescence emission at 558 nm,while the generated DAP can burst the fluorescence of PLPs through the IFE.When AA was introduced into the system,the Co OOH NS were disrupted and reduced to Co²⁺,which inhibited the oxidation of OPD and effectively maintained the blue fluorescence of PLPs at 475 nm.Therefore,a fluorescence sensing platform with PLPs as the reference signal and DAP as the reporter signal was established for the quantitative detection of AA.Also based on the principle that AAO can specifically hydrolyze AA,this method can be further used for the determination of AAO activity.Under the optimal conditions,the linear ranges for the detection of AA and AAO were 1-45μmol/L and 1-20 m U/m L,respectively,with the detection limits of 0.2μmol/L and 0.25 m U/m L,respectively.Finally,the proposed ratiometric fluorescence analysis method was used for the detection of AA in real samples and the results were satisfactory when compared with commercial kits.（2）A ratiometric electrochemical sensor of entropy-driven DNA nanomachines in homogeneous solution was constructed for the determination of miRNA-21.The constructed entropy-driven DNA nanomachine avoids the problem of waste accumulation during the conventional entropy-driven signal amplification,all the DNA involved in the reaction was effectively utilized,and the whole process was carried out in homogeneous solution,avoiding the conventional electrochemical biosensor layer-by-layer self-assembly process and saving detection time.At the same time,UiO-66-NH₂ with electrochemical response was used as the signal reporting group,and ferrocene（Fc）in the electrolyte was used as the reference signal group,and the current ratio of I_UiO-66-NH2/I_Fc-IR was used as the output.This form of dual signal ratio output can correct the interference of background signal and reduce the detection error.In homogeneous solution,the entropy-driven DNA nanomachines were carried on the surface of magnetic beads（MBs）.In the absence of target miRNA-21,the entropy-driven DNA nanomachines were not activated and biotin-modified F1 strands were retained on the surface of MBs.After magnetic separation,a strong electrochemistry signal of UiO-66-NH₂ at+0.83 V was detected.When miRNA-21 was present,it effectively triggered the signal amplification process of the entropy-driven DNA nanomachine,and the biotin-modified F1 strand was released from the surface of MBs.After magnetic separation,SA-UiO-66-NH₂ could not bind effectively to the surface of MBs,and the IUiO-66-NH₂/IFc-IR ratio was reduced.Under the optimal conditions,the linear range for the detection of miRNA-21 was 50 fmol/L-10 nmol/L and the detection limit was 9.8 fmol/L.（3）In this work,a ratiometric fluorescence sensing system based on MnO₂NS and entropy-driven DNA nanomachines was constructed for the quantitative detection and visual analysis of miRNA-21.Entropy-driven DNA nanomachines were constructed on the surface of MBs,and the entropy-driven signal amplification process could not be triggered when miRNA-21 was not present in the system.The streptavidin-modified alkaline phosphatase（ALP-SA）specifically bound to biotin on top of F1,and ALP was effectively retained on MBs after magnetic separation.When L-ascorbic acid-2-phosphate trisodium salt（AAP）was added,ALP could hydrolyze AAP specifically to AA,which was oxidized by MnO₂ NS to form dehydroascorbic acid（DHAA）,and DHAA reacted with OPD to form 3-（dihydroxyethyl）furano[3,4-b]oxyquinoline-1（DFQ）,which had an intense blue fluorescence at 430 nm,the F₄₃₀/F₅₅₈value was larger.When miRNA-21 was present,an entropy-driven DNA nanomachine was initiated and biotin-modified F1 was shed from the surface of MBs.After magnetic separation,ALP could not be attached to the surface of MBs,and since no AA was produced in the system,all of MnO₂ NS was used to oxidize OPD to DAP,so the fluorescence intensity detected at 558 nm was larger,while the fluorescence intensity at 430 nm was weaker,and the ratio of F₄₃₀/F₅₅₈ decreased.quantification of miRNA-21 by recording the change in the ratio of F₄₃₀/F₅₅₈.The constructed method was sensitive,selective and stable,with a linear range of 10 fmol/L-20 nmol/L and a detection limit of 3.3 fmol/L for the detection of miRNA-21.In addition,visual analysis of miRNA-21 can be achieved by extracting its red,green,and blue（RGB）intensity through smartphones.（4）A light-controlled DNA strand replacement strategy was designed based on the rolling loop amplification（RCA）reaction to regulate the self-assembly process of functionalized G4 helical DNA nanoflowers（G4 DNF）.Upstream part used photodegradable nitrobenzyl structure for"dynamic change"of hairpin structure,The structure can be photolyzed under UV light,and the DNA hairpin was then opened to form an incompletely paired double-stranded structure,exposing a hidden sticky end as a way to achieve light control.The initiator chain A was then released from the double-stranded complex substrate to participate in the downstream reaction by successive chain substitution reactions assisted by the initiator chain I.Downstream the self-assembly of G4 DNFs was achieved by initiating the RCA process by using the initiating chain A as a target.The G4 loop template was prepared with the help of T4ligase,and the double-stranded structure and the remaining padlock probes in the reaction system were removed by nucleic acid exonucleases I and III to ensure that only the initiating strand A generated by the upstream process could initiate the RCA amplification process.Flower-like structures with a diameter of about 1.5μm were observed by scanning electron microscopy（SEM）and transmission electron microscopy（TEM）under the optimum conditions.It was demonstrated that the light-controlled DNA strand replacement strategy can successfully regulate G4 DNF self-assembly,and the strategy was further exported as a logical operation.