Study On A Novel Fluorescence Biosensor Based On Toehold Strand Displacement Reactions Aided Target Recycling Amplification

Fluorescent biosensor is a equipment based on a specific biological recognition event in combination with a transducer for fluorescence signal processing.Since its inception,fluorescent biosensors have been expected to play a significant analytical role in diseases diagnostics,environmental monitoring and food safety because of their unique advantages in terms of simple equipment,good selectivity and fast response.However,it has been generally recognized that the concentrations of the analytes are usually on a relatively low level in actual testing,which requires researchers to design signal amplification strategies to improve the sensitivity of the sensor.Considering that the enzyme-assisted recycling amplification is susceptible to environmental factors such as temperature and pH,this research mainly focus on the development of simple,convenient and non-enzymatic signal amplification approaches to detect different targets,and has obtained a series of effective results.The specific content of our research work are summarized as follows: Part 1.MicroRNA-activated molecular machine for non-enzymatic target recycling amplification detection of microRNA from cancer cells.The dysregulated levels of microRNAs?miRs?can be useful biomarkers for early diagnosis of different cancers.Based on a new microRNA-activated DNA machine for non-enzymatic target recycling amplification,the development of a simple and highly sensitive method for fluorescent detection of miR-141 from human prostate cancer cells is described.The hybridization of the target miR-141 with the terminal toehold region of the fluorescently quenched DNA complex probe through toehold strand displacement reaction?TSDR?leads to the release of the FAM-labeled signal probes from the complex probes,and produces another toehold on the DNA complex probe for subsequent hybridization with the fuel strands.Upon binding to the subgenerated-toehold of the DNA complex probe,the DNA fuel strand displaces the hybridized miR-141 through the second TSDR to activate the DNA machine,which enables the target recycling of miR-141 and results in the release of plenty of the signal probes for amplified detection of miR-141 down to 620 cancer cells.This method also shows high selectivity and can be employed to monitor miR-141 from cancer cells.The developed method avoids the use of enzymes for target recycling amplification and therefore provides significant future applications in bioanalysis.Part 2.Target responsive aptamer machine for label-free and sensitive non-enzymatic recycling amplification detection of ATP.The abnormal concentration of ATP is associated with many diseases,and the development of simple and sensitive methods for the identification and detection of ATP is important in clinical diagnosis.In this work,based on a new ATP-responsive molecular aptamer machine,we developed a label-free and non-enzymatic signal amplification approach for sensitive detection of ATP in human serum.The binding of the target ATP with the corresponding aptamer of a three-strand DNA duplex probe changes its conformation and leads to the exposure of a toehold region.The fuel DNA strand binds to the toehold region and liberates a G-quadruplex active sequence and the ATP molecule through the toehold-mediated strand displacement reaction.Therefore,the target ATP molecules can be cyclically reused to release many G-quadruplex active sequences,which associate with N-methylmesoporphyrin IX to generate significantly amplified fluorescent signals for sensitive detection of ATP down to 25 nM.Besides,this developed method shows high selectivity toward ATP against other control molecules,and can be employed for detecting ATP in diluted serum samples.Moreover,our approach avoids the use of enzymes and nanomaterial labels for signal amplification and the modification of the probes for signal readout.Thus,the sensor proposed here holds great potential for the construction of simple and sensitive platform for the detection of other small molecules with careful probe designs in a homogeneous fashion.Part 3.Metallo-Toehold-Activated Catalytic Hairpin Assembly Formation of Three-Way DNAzyme Junctions for Amplified Fluorescent Detection of Hg²⁺.Because of their irreversible toxicological impacts on the environment and human body,the development of reliable and sensitive Hg²⁺ detection methods with high selectivity is of great significance.On the basis of the substantial signal amplification by metallo-toehold-triggered,catalytic hairpin assembly?CHA?formation of three-way DNAzyme junctions,we have constructed a highly selective and sensitive fluorescent sensing system for the determination of Hg²⁺ in different environmental water samples.The presence of the target Hg²⁺ ions can lead to the generation of T-Hg²⁺-T base mismatched metallo-toeholds,which trigger the catalytic assembly of three split-DNAzyme containing hairpins to form many Mg²⁺-dependent DNAzyme junction structures upon binding to the fluorescently quenched substrate sequences.The Mg²⁺ ions then cyclically cleave the fluorescently quenched substrate sequences of the Mg²⁺-dependent DNAzymes to generate drastically enhanced fluorescent signals for sensitively detecting Hg²⁺ at the low 4.5 pM level.The developed sensing method offers high selectivity toward the target Hg²⁺ over other possible competing metal ions due to the specific T-Hg²⁺-T bridge structure chemistry in the metallo-toehold domain,and reliable detection of spiked Hg²⁺ in environmentally relevant water samples with this method is also verified.Considering the nucleic acid nature of the trigger and assembly sequences,the developed approach thus holds great potentials for designing new enzyme-free signal amplification strategies to achieve highly sensitive determination of different DNA and RNA targets.