Fluorescent Biosensor For Biomarkers Detection Based On Enzyme And DNAzyme Assisted Signal Amplification

The cure rate of the disease has gradually increased with the development of medical technology,but the overall death toll is still increasing.Some experts have shown that the incidence of chronic diseases will increase significantly in the next ten years.As the diagnosis of early disease is closely related to the lives of most patients,more and more scientists are paying attention to this.They have proposed a variety of feasible detection methods for this field,which provide powerful help for medical staff to identify or exclude suspicious patients.Among them,nucleic acid detection as an accurate and effective test strategy has also been widely used in food safety,environmental monitoring,disease diagnosis and other fields.Numerous studies have shown that nucleic acid detection has the advantages of simplicity,speed and sensitivity at the molecular level.Besides,DNA biosensor based on fluorescent signal amplification is a very effective detection methods in nucleic acid research.Not only can it be used for the analysis of a variety of disease markers,but also it has many advantages such as time saving,convenience,high sensitivity,good selectivity,and low cost.However,in actual detection,the concentration of the tested sample is usually at a low level,so the help of some signal amplification strategies is usually required to achieve the detection of low-level samples.In this article,adenosine triphosphate(ATP),thrombin,and microRNA-1246 were used as the disease marker models.With the help of biological enzymes and DNAzyme,we skillfully combined three signal amplification strategies,rolling circle amplification(RCA)and toehold strand displacement(TSDR),loop-mediated isothermal amplification(LAMP),to build three fluorescent biosensors for sensitive detection.The specific research work is summarized as follows: Part 1.Polymerization nicking-triggered LAMP cascades enable exceptional signal amplification for aptamer-based label-free detection of trace proteins in human serumDetecting molecular biomarkers in high sensitivity plays an important role in the diagnosis of various diseases at the early stage.Here,by combining the target-induced polymerization nicking reaction(TIPNR)with the LAMP,we describe an ultrasensitive and label-free aptamer-based sensing method for detecting low levels of proteins in human serum by using thrombin as the model target analyte.The target thrombin binds and causes spontaneous assembly of two distinct aptamer probes to form the templates for the polymerization nicking reaction recycling amplification to produce many forward inner primer sequences.Subsequently,downstream LAMP reactions are initiated by these sequences for the generation of tremendous DNA hairpins with various lengths via automated cyclic strand displacement reactions.The SYBR Green I organic dye further binds the many hairpins to show drastically amplified fluorescence for ultrasensitive detection of thrombin down to 3.6 fmol/L in the linear range from 0.01 pmol/L to 10 nmol/L.Such a sensing method based on aptamers has high discrimination capability for the target molecules against other non-specific proteins and is applicable for diluted serum samples.With the successful demonstration of the substantial signal amplification ability and simplicity feature of this assay approach,highly sensitive and convenient detection of other disease biomarkers with this method can be envisioned in the near future.Part 2.An Efficient and Exponential Rolling Circle Amplification Molecular Network Leads to Ultrasensitive and Label-Free Detection of MicroRNAMicroRNAs(miRNAs)are useful biomarkers for the diagnosis of a variety of cancers.However,it is of a major challenge to detect miRNAs,considering their high sequence similarity,low concentration and small size nature.With the establishment of an efficient RCA molecular network by target-driven polymerization/nicking reactions,we present here an exponential signal amplification strategy for label-free monitoring of miRNA with ultrahigh sensitivity.The target miRNA sequences can bind two ssDNA probes to form a junction structure to initiate a dual polymerization/nicking cyclic reaction for the production of many primers,which further trigger multiple RCA reactions in a drastically amplified sequence replication and extension mode for the yield of substantial dsDNAs with various sizes.The SYBR Green I then binds these dsDNAs to induce significantly magnified fluorescence emission for detecting the target miRNA sequences with a detection limit down to 0.86 fM in the linear range between 1 fmol/L and 10 pmol/L.Because of the involvement of the presynthesized circular DNA template,the RCA efficiency is further improved,and such a method can also be used for detecting miRNA in diluted human serum samples,demonstrating its great potential and universality for detecting different nucleic acid sequences for biochemical research and clinical diagnosis applications.Part 3.Target-catalyzed assembly formation of metal-ion dependent DNAzymes for nonenzymatic and label-free amplified ATP detectionIn this work,on the basis of a dual recycling amplification means with the involvement of toeholdmediated strand displacement reaction(TSDR)and metal-ion dependent DNAzymes,we describe the establishment of a non-enzymatic and label-free method for sensitively detecting ATP in human serums.The target ATP binds the corresponding aptamer recognition probes and causes structure switching of the aptamers to expose the toehold regions for TSDR-based recycling of ATP and subsequent catalytic assembly formation of many DNAzymes with the assistance of DNA fuel strands.The corresponding metal ions further cyclically catalyze the cleavage of the G-quadruplex containing hairpin substrate strands of the DNAzymes,releasing lots of free G-quadruplex segments,which associate with the thioflavin T dye to result in drastically enhanced fluorescent signals for detecting ATP with high sensitivity.Such an ATP sensing method shows a dynamic range of 50~600 nmol/L and a detection limit of 20 nmol/L.The developed sensing system also has a high discrimination capability between ATP and other interfering analogue molecules,and can realize the detection of human serum ATP molecules,demonstrating its potential for convenient monitoring of low levels of ATP for the diagnosis of different diseases.