Design Of G-quadruplex-based Nucleic Acid Fluorescent Probes And Their Applications In Small Molecule And Metal Ion Detection

Information about certain biochemical substances in the human body and the environment is of great practical significance for assessing human health.In this process,it is essential to monitor and detect the distribution of certain ions and molecules,such as metabolites,heavy metal ions and other toxins in the human body or the environment.Therefore,it is important to develop fast and sensitive analytical methods for detecting related targets.Functional nucleic acid,a new type of molecular recognition element,has the ability of specific binding to the target,and can be used as detection probe.G-quadruplex,a special nucleic acid with fluorescence quenching ability and fluorescence amplification function,is an important medium for sensing platforms.Based on G-quadruplex and fluorescent dyes,three types of G-quadruplex-based nucleic acid fluorescent probes were designed to detect tetracycline in milk and honey,D-penicillamine in urine and river water,and Ag⁺in tap water and antibacterial agent,respectively.The main contents are as follows:?1?A label-free fluorescence aptasensor was developed for the rapid detection of tetracycline?TET?based on G-quadruplex structure of TET aptamers and Thioflavin T.The fluorescence intensity of free ThT is weak,whereas it selectively identifies the G-quadruplex of aptamers to form the G-quadruplex/ThT conjugates,thus greatly increasing the fluorescence intensity of ThT.However,the fluorescence intensity of G-quadruplex/ThT conjugates was drastically suppressed due to the release of free ThT from G-quadruplex/ThT conjugates after the addition of TET via specific binding with TET aptamers.The key factors affecting sensitivity and selectivity including the reaction medium,intercalation time of ThT to TET aptamers,incubation time between TET aptamers and TET,concentration of ThT and TET aptamers were investigated in detail.The optimal conditions were as follows:ultrapure water as reaction medium,binding time of 5 min,incubation time of 1 min,9.0?mol/L ThT and 0.03?mol/L aptamers.A good linear relationship?correlation coefficient R² of 0.9947?was obtained between the fluorescence quenching efficiency?F₀-F?/F₀ and the logarithm of TET concentration in the range of 0.01-1.0?mol/L.The limit of detection?S/N=3?was 0.001?mol/L.The proposed assay was applied to the detection of TET in the spiked honey and milk samples with recoveries ranging from 93.5%to 106.9%.The developed label-free fluorescence aptasensor showed advantages of high specificity,low cost and short time-consuming,illustrating potential application for on-site detection of TET in foodstuffs.?2?Based on the target-regulated G-quadruplex structural transformation,a label-free“turn on”fluorescence sensing platform using berberine as the fluorescent signal transduction molecule was constructed for the determination of D-penicillamine.The fluorescence intensity of free berberine is weak.However,the fluorescence of berberine increased significantly when combining with the oligonucleotide with G-quadruplex structure.After the addition of Hg²⁺,T-Hg²⁺-T base pair was formed between Hg²⁺and thymine,resulting in the conversion of the G-quadruplex structure to the hairpin structure and fluorescence quenching of berberine.The stable complex formed between Hg²⁺and D-penicillamine destroyed the T-Hg²⁺-T structure.Thus,the G-quadruplex structure reformed by the oligonucleotide probe with hairpin structure,promoting the fluorescence recovery of berberine.Therefore,the conformational change of G-quadruplex would affect the fluorescence intensity of berberine,thus achieving the quantitative and sensitive detection of D-penicillamine.The key factors affecting the performance of the fluorescence sensing method were studied in detail,including dye type,the arm length of oligonucleotide,reaction time,concentration of berberine and Hg²⁺.Under the optimal conditions,a good linear relationship was obtained between the fluorescence intensity??F?of the system and the concentration of D-penicillamine in the range of0.2-4.0?mol/L with correlation coefficient?R²?of 0.9983.The limit of detection?S/N=3?was 0.039?mol/L.The established method was applied to the analysis of D-penicillamine in urine and river water samples with the recoveries ranging from94.0%to 102.0%.The label-free fluorescence method was easy and sensitive,showing potential prospects in environmental and biological process monitoring.?3?A fluorescence sensing method for Ag⁺was established by designing a fluorescent probe with labeled fluorescein?FAM?based on the fluorescence quenching ability of G-quadruplex and specific binding between cytosine and Ag⁺.In the presence of Ag⁺,the polycytosine sequence in the oligonucleotide probe folded into hairpin structure,causing the G-quadruplex and FAM to be in close proximity to each other and thereby significantly quenching FAM fluorescence.Based on the above principles,parameters affecting the sensitivity and selectivity of the method,such as reaction time,p H of the buffer solution,and the cytosines number of 5?-end,were optimized.The optimal conditions were as follows:reaction time of 8 min,buffer pH of 8.0,and 5?-end cytosines of 10 bases.Under the optimal conditions,there was a good linear relationship between the fluorescence quenching efficiency?F₀-F?/F₀ and the concentration of Ag⁺?5.0-80.0 nmol/L?with correlation coefficient?R²?of 0.9851.The limit of detection?S/N=3?was 1.95 nmol/L.The method was applied to the detection of Ag⁺in tap water and antibacterial agent samples with the recoveries ranging from 93.3%to 105.2%with relative standard deviation less than6.1%.The developed fluorescence sensing method has the advantages of simple operation,short analysis time,good sensitivity and higher selectivity.