Manganese Dioxide And CRISPR/Cas12a-based Ratiometric Fluorescent Sensor For Disease Marker Detection

Manganese dioxide nanosheets（MnO₂ NS）are widely used in biosensing field due to its simple and fast synthesis method,large specific surface area,non-toxic and environmental friendly,and low cost.In addition,the excellent catalytic oxidation activity of MnO₂ NS can oxidize nonfluorescent o-phenylenediamine（OPD）to2,3-diaminophenothiazine（DAP）with yellow fluorescence.Therefore,in this paper,we use MnO₂ NS/OPD materials,combined with various signal amplification techniques such as three-dimensional chain-cycling amplification,CRISPR/Cas technology and rolling loop amplification,for the quantitative detection of glutathione（GSH）,ascorbic acid（AA）,alkaline phosphatase（ALP）,p53 gene,HPV-16 and miRNA-33.The specific work is as follows:（1）To achieve highly sensitive detection of GSH,AA and ALP activities,a MnO₂ NS/OPD-based ratiometric fluorescence sensing platform was designed,which achieves highly sensitive detection of GSH,AA and ALP activities by modulating the internal filtration effect（IFE）between persistent luminescent particles（PLPs）and DAP.As a nanomaterial that mimics oxidase,MnO₂ NS can oxidize OPD to DAP and emit a yellow fluorescence emission signal at 558 nm,while the fluorescence of PLPs at 475 nm is bursted by DAP through IFE.The presence of GSH and AA reduced MnO₂ NS to Mn²⁺,which inhibited the oxidation of OPD and effectively preserved the blue fluorescence of PLPs at 475 nm.In addition,ALP catalyzes the generation of AA from L-ascorbic acid-2-phosphate trisodium salt（AAP）,and this method can be used for ALP activity assay as a recent step.The linear ranges of GSH,AA and ALP were 1-100μmol/L,1～100μmol/L and 1～5 m U/m L,respectively,and the lower limits of detection were 0.2μmol/L,0.33μmol/L and 0.3 m U/m L,respectively.Finally,the sensor was used for the detection of human serum and urine samples,and satisfactory recoveries were obtained.In addition,with the help of a portable UV lamp and a color scanning application on a smartphone,the red,green,and blue（RGB）intensity of color images was obtained by scanning measurement analysis,thus enabling real-time in situ detection of ALP activity,GSH,and AA.（2）A highly sensitive,reproducible and accurate three-dimensional DNA bionic scaffold network（S-3D）ratiometric fluorescence platform was proposed for the detection of cancer-related genes（p53 gene）.The sensing platform was constructed as follows:the aminated magnetic beads（MBs）were modified on the capture probe hairpin H1.When the target p53 was present,it specifically binds and opens the hairpin H1,and the remaining single-stranded portion of the opened H1 hybridizes with the 3’end biotin-modified（biotin）H2.H2 had the same single-stranded sequence portion as H1,specifically opens the 5’end biotin-modified H3 neck-loop structure and triggers the hybridization chain reaction（HCR）.Unlike conventional HCR,the 5’end of H2 has 10 palindromic bases,and after annealing,the two H2chains can form a hairpin dimer by hybridizing their palindromic fragments.Streptavidin-labeled alkaline phosphatase（SA-ALP）was attached to MBs by interacting with biotin,thereby preparing an S-3D scaffold network composed of multiple signals.Alkaline phosphatase attached to the stent hydrolyzes AAP to produce AA,which was oxidized by MnO₂ NS to produce dehydroascorbic acid（DHAA）.DHAA reacts with OPD to form 3-（dihydroxyethyl）furano[3,4-b]oxyquinoline-1（DFQ）,which fluoresces intensely blue at 430 nm.In the absence of the p53 gene,OPD was oxidized by MnO₂ NS to generate 558 nm yellow fluorescent DAP.Therefore,in this work,a dual-emission peak ratiometric fluorescence platform was successfully established to detect the p53 gene.Compared with conventional ratiometric fluorescence platforms,this ratiometric fluorescence sensor has the advantages of high stability,low toxicity,and does not require specific DNA template sequences,which can avoid interference from factors such as instrumentation and environment.To provide a cost-effective platform,a smartphone with a built-in camera was also used to record changes in the fluorescence color of the solution.The proposed assay platform accurately identifies the p53 gene by ratiometric output,demonstrating high sensitivity（lower limit of detection of 2 fmol/L）for in situ quantification.（3）A CRISPR/Cas12a-based ratiometric fluorescent biosensing platform for human papillomavirus 16（HPV-16）detection was proposed.This method does not require DNA amplification and directly uses the target HPV-16 to specifically bind to the Cas12a/cr RNA complex,initiating the trans cleavage ability of Cas12a.The sensor was constructed as follows:the reporter short chain S DNA of biotin was modified on the surface of MBs,combined with magnetic separation technology to control the ALP content on the surface of MBs.With the addition of HPV-16,the cleavage ability of Cas12a was activated,S DNA was cleaved,ALP on the surface of MBs was reduced,and AAP could not be hydrolyzed to generate AA.Therefore,the fluorescence emission of DFQ could not be detected at 430 nm,on the contrary,MnO₂ NS would oxidize the OPD and detect the strong fluorescence emission of DAP at 558 nm,and the quantitative detection of HPV-16 was achieved by recording the fluorescence intensity ratio(F₅₅₈/F₄₃₀).In addition,the visualization of HPV-16detection can also be achieved by recording the RGB values of the solution colo r under UV light.The dual-signal ratio output method can further reduce the background interference of the system and reduce the testing error,and the method was used for testing in human serum,which showed satisfactory results.（4）RCA and CRISPR/Cas12a-based ratiometric fluorescence sensing platform was constructed for miRNA-33 detection.When miRNA-33 was present,it binded to the circular template and initiates the RCA reaction in the presence of T4 DNA ligase,phi29 polymerase and d NTPs to replicate a single strand complementary to the circular DNA.The RCA product can bind specifically to the Cas12a/cr RNA complex,activating the trans-cleavage ability of Cas12a and triggering its reverse-strand cleavage activity on S DNA.The S DNA labeled with biotin was sheared off from the surface of MBs,which in turn controled the amount of ALP on the surface of MBs,causing a change in the AA content of the system.The quantitative detection of miRNA-33 was achieved by detecting the change in the fluorescence ratio of DFQ with blue fluorescence emission and DAP with yellow fluorescence emission.The lower limit of detection was 4.6 fmol/L.The ratiometric sensing platform proposed in this work offered significant improvements over the traditional RCA method,while eliminating environmental variations and instrument efficiency interference,ha d better stability,and can be used for the detection of miRNA-33 in human serum,showed good promise in disease diagnosis.