Construction Of Functional Nanomaterials-based Fluorescent Biosensors And Their Application In Determination Of MicroRNA-167 In Plants

Micro RNA-167 is one of the few important micro RNAs that regulate signaling pathway of auxin indole-3-acetic acid（IAA）through auxin transcription factor（ARF）.The misuse of growth hormones can lead to excessive levels in crops and affect crop growth and development,which will further affect food and environmental safety and threaten human health.The effect of auxin on plant growth can be reflected by monitoring the change of the expression level of micro RNA-167 in plants.Therefore,it is very important to develope methods for detecting micro RNA-167 in plant.Here,we prepared fluorescent nanomaterials and fluorescent quenching nanomaterials,and applied them to fabricated fluorescent biosensors.Using fluorescence resonance energy transfer（FRET）method and ratio fluorescence method,the biosensors realized quatitive dermination of micro RNA-167.The research consists of the following two main parts:1. Calcium ion assisted fluorescence determination of micro RNA-167 using carbon dots–labeled probe DNA and polydopamine-coated Fe₃O₄ nanoparticlesIn this part,a fluorescence biosensor was described for determination of micro RNA-167 using fluorescent resonant energy transfer（FRET）strategy.The FRET system comprised carbon dots（CDs,donor）labeled with probe DNA（p DNA）and polydopamine（PDA）-coated Fe₃O₄ nanoparticles（Fe₃O₄@PDA NPs,acceptor）.With the enhanced sensitivity by Ca²⁺assisted interaction and application of twice magnetic separation,the biosensor showed a wide linear range of 0.5–100 n M to micro RNA-167 with a 76 p M detection limit.With good selectivity,the method was applied to the determination of micro RNA-167 in samples of total micro RNA extractions from A.thaliana seedlings.2. Preparation of DNA templated gold nanoclusters（DNA-AuNC）and its application in construction of ratio fluorescence biosensor for determination of micro RNA-167In this experiment,AuNC was prepared using the DNA template method.By optimizing the DNA template sequence and the preparation conditions,we successefully prepared the DNA-templated AuNC（DNA-AuNC）with good fluorescence performance,high fluorescence intensity and good stability.It was then designed to applied in fabrication of ratio fluorescence biosensor for micro RNA-167.In the presence of the target micro RNA-167,the DNA sequence on the probe was hybridized with micro RNA-167 to form a double-stranded structure.SYBR Green（SG）was embedded into the double-chain,and a ratiometric fluorescence sensor was constructed to enable the determination of the target micro NRA-167.The sensor showed a linear range of 5–500 n M in determination of micro RNA-167 with a detection limit of 1.26 n M.