Novel Methods For Biomarkers Detection Based On Enzyme Assisted Signal Amplification And DNA Self-assembled Nanostructures

Simple,rapid and sensitive detection of biomolecules?such as nucleic acids,proteins,enzymes,cells and biological molecules?is of great significance in disease diagnosis,food and drug analysis and environmental monitoring.With the need of social development,improving and developing the novel biosensing technology,which is simpler,more sensitive,more specific and high throughput,has become a hot research topic in bioanalysis.In current research,aim to simplify the experimental steps and improve the detection sensitivity and specificity,some novel biosensing systems for the detection of nucleic acids and proteins have been developed by means of isothermal enzyme assisted signal amplification and DNA catalytic self-assembly.The analysis of real samples?serum and cell lysate?confirmed the feasibility,reliability and applicability of these methods.In order to further study the expression and distribution of multiple targets in a single cell,two important RNAs in single cell were,respectively,studied by DNA tetrahedron nanostructures and in-situ rolling circle amplification?RCA?,the corresponding detection mechanism and performance of each research have been carefully studied.These methods have achieved good experimental results in terms of detection sensitivity,selectivity and suitability.The studied contents are mainly as follows:1,Quadratic recycling amplification for label-free and sensitive visual detection of HIV DNAVisual detections have attracted great research attentions recently due to the irconvenient monitoring of the target analytes without using any advanced instruments.However,achieving visual detection of trace amounts of biomolecules with PCR-like sensitivity remains a major challenge.In current work,we describe a new quadratic signal amplification strategy for sensitive visual detection of HIV DNA biomarkers based on exonuclease III?ExoIII?-assisted DNA recycling amplification and DNAzymes.The presence of the target HIV DNA leads to two independent and simultaneous DNA recycling processes to achieve quadratic signal amplification with the assistance of Exo III.This quadratic signal amplification results in catalytic cleavage of the G-quadruplex sequence-locked hairpin probes to release numerous active G-quadruplex sequences,which further associate with hemin to form DNAzymes and cause significantly intensified color change for sensitive and visual detection of HIV DNA down to 2.5 pmol L^-1.The proposed visual detection method employs un-modified hairpin DNA as probes,avoids using any complex and expensive instruments for signal transduction and is essentially simple.This method also shows single-base mismatch discrimination capability as well.All these features make our developed DNA detection methodholds great potential for visual monitoring of various DNA biomarkers at ultralow levels with careful and proper probe designs.2,Target-Triggered Quadratic Amplification for Label-Free and Sensitive Visual Detection of Cytokines Based on Hairpin Aptamer DNAzyme ProbesThe employment of DNAzyme probes for visual biodetections has received increasing interest recently due to the simple nature of this type of assay.However,achieving high sensitivity and detecting targets beyond nucleic acids remain two major challenges in DNAzyme-based visual detections.In this work,based on a new quadratic amplification strategy,we developed a sensitive and visual detection method for cytokines by using hairpin aptamer DNAzyme probes.The target cytokine,interferon??IFN-??,associates with the aptamer sequences and unfolds the hairpin structure of the probes,leading to simultaneous recycling of the target IFN-??assisted by Bst-polymerase?and the DNA sequences?aided by?exonuclease?to achieve quadratic amplification.This quadratic amplification results in the generation of numerous peroxidase-mimicking DNAzymes,which cause significantly intensified color change of the probe solution for highly sensitive detection of IFN-?by the naked eye down to 50 pmol L^-1.The proposed visual sensing method shows also high selectivity toward the target IFN-?and can be performed in homogeneous solutions with using completely unmodified,synthetic aptamer DNAzyme probes.These distinct advantages of our developed assay protocol make it a potential platform for detecting various types of biomolecules with careful probe designs.3,RNA responsive and catalytic self-assembly of DNA nanostructures for highly sensitive fluorescence detection of miRNA from cancer cellsMicroRNA?miRNA?has been used as a novel tumor marker for early detection and related research of tumors in recent years.Due to the short miRNA sequence,high similarity of family homologous sequences,low expression level and intrinsic characteristics of easy degradation,the high specificity and accurate quantitative analysis of miRNAs becomes complicated.Catalytic self-assembly formation of“firework-like”DNA nanostructures triggered by miRNA 21?miRNA-21?is achieved through isothermal toe-hold strand displacement reactions.The miRNA-21is autonomously recycled during the self-assembly process,which makes the generation of the DNA nanostructures proceed in a catalytic fashion.The self-assembly process and the effect of assembly time on the formation of the DNA nanostructures are analyzed by non-denatured polyacrylamide gel electrophoresis,and the morphologies of the self-assembled DNA nanostructures are verified by Atomic Force Microscope imaging as well.The miRNA-21-triggered self-assembly of DNA nanostructures can also serve as a tremendous signal amplification platform to achieve ultrasensitive detection of miRNA-21.By monitoring the miRNA-21 extracted from human breast cancer cells?the MCF-7 line?,as low as 10 cells can be detected by using the developed method.The work demonstrated here thus opens new opportunities for the application of DNA nanostructures in early diagnosis of various cancers.4,Multicolor-Encoded Reconfigurable DNA Nanostructures Enable Multiplexed Sensing of Intracellular MiRNAs in Living CellsDespite the widespread utilization of gold nanoparticles and graphene for in vivo applications,complex steps for the preparation and functionalization of these nanomaterials are commonly required.In addition,the cytotoxicity of such materials is currently still under debate.In this work,by taking the significant advantages of DNA in terms of biocompatibility,nontoxicity,and controllability as building blocks for DNA nanostructures,we describe the construction of a reconfigurable,multicolor-encoded DNA nanostructure for multiplexed monitoring of intracellular miRNAs?miRNAs?in living cells.The DNA nanostructure nanoprobes containing two fluorescently quenched hairpins can be obtained by simple thermal annealing of four ssDNA oligonucleotides.The presence of the target miRNAs can unfold the hairpin structures and recover fluorescent emissions at distinct wavelengths to achieve multiplexed detection of miRNAs.Importantly,the DNA nanostructure nanoprobes exhibit significantly improved stability over conventional DNA molecular beacon probes in cell lysates and can steadily enter cells to realize simultaneous detection of two types of intracellular miRNAs.The demonstration of the self-assembled DNA nanostructures for intracellular sensing thus offers great potential application of these nanoprobes for imaging,drug delivery and cancer therapy in vivo.5,A Programmable DNA Ring/Hairpin Constrained Nanostructure Enables Highly Sensitive and Multiplexed Imaging of mRNAs in Single CellsIn situ mapping mRNAs with high spatial resolution and sequence specificity in single cells would greatly facilitate the research on mRNA regulated biological processes and human diseases,especially malignant tumors.Given that many tumors are associated with multiple mRNAs.Herein,we construct a powerful method for simultaneous in situ detection of TK1 and Survivin mRNA with near-single-molecule resolution in single cells by using target RNA-initiated rolling circle amplification?RCA?.Superior to the traditional RCA methods,this system successfully avoided complicated reverse transcription and padlock DNA ligation process.Moreover,this approach significantly reduced the background noise by using the fluorescently quenched partial dsDNA signal probes and enhanced the detection selectivity with the hairpin recognition probes.With the advantages of in situ signal amplification and multiple detection ability,the current strategy could availably suppress false positive signals and provide more accurate and complete information for early cancer diagnosis.