A Novel DNA Fluorescence Sensing Method And Its Application In Biotin Detection

Fluorescent sensors based on strand displacement not only have the advantages of simple design and high sensitivity,but also have certain advantages in the programmability of fluorescent probes.Biotin can participate in the metabolism of lipids,nucleic acids,and carbohydrates,and act as a coenzyme in the reaction of carboxylase.Lack of biotin in the human body can cause problems such as loss of appetite,seborrheic dermatitis,and hair loss.Therefore,the development of biotin detection sensors is of great significance to pharmaceuticals,clinical diagnosis and treatment.In this study,a fluorescent sensor based on the toehold-mediated strand displacement reaction based on the base stacking effect was designed.By regulating the strand displacement reaction,a streptavidin-biotin reaction system was constructed to detect biotin.This design has a relatively simple method for regulating the reaction balance of strand displacement,and only needs to change one strand to change the fluorescence intensity.The feasibility and practicability of the fluorescence sensor were proved by fluorescence spectrum analysis and polyacrylamide gel electrophoresis experiments.The specific research contents are as follows:(1)Construction of a new DNA fluorescence sensor based on base stacking:We designed a DNA fluorescence sensing device based on the base stacking effect of GC and TC.The principle is that the fluorescent strand F and the quenching strand Q hybridize to form a substrate duplex.Fluorescence response in two cases:(1)adding different concentrations of target strands to replace the Q chain;(2)first adding different G chains to hybridize with the single-stranded part of the substrate duplex,and then adding different concentrations of target strands to replace the Q chain,forming GC and TC base stacking in the toehold region.By comparing the reaction differences with and without base stacking,it is found that when the toehold is 5nt,the GC base stacking has a greater driving force on the strand displacement reaction,and when the toehold is 6nt,the TC base stacking has a smaller driving force on the strand displacement reaction,this is because when the toehold is 6nt,the strand displacement is sufficiently thorough.And changing the G chain structure(single-base overhang,single-strand overhang,double-strand overhang)has different effects on the reaction of strand displacement.In the stacking of GC bases,the stacking position increases the overhang of single-strand and double-strand,and the reaction equilibrium occurs.The effect of double-stranded overhang on the equilibrium constant is greater than that of single-stranded overhang;when the number of bases in G1 chain is reduced,the overhang of T bases at the stacking site increases,the reaction rate decreases,and the reaction tends to proceed in the reverse direction.In the TC base stacking,there is no significant difference in the effect of base stacking on the equilibrium constant.The response of G2 chains with different structures is examined.At the base stacking place,the overhang of a single base has little effect on the reaction,and the maximum fluorescence values were all around 5(n.u.),and there was no difference in the overhang of T bases.Poly-A bases and double-stranded overhangs had a greater impact on the equilibrium constant.In summary,the follow-up work selects the fluorescence sensing based on GC base stacking to verify the practicality.(2)Biotin detection application based on novel fluorescent sensing strand displacement: We designed a biotin-labeled DNA strand for use in a fluorescent sensor.First,the three-chain substrate F·Q·G1-Biotin was detected,and the detection results were basically consistent with the reaction of F·Q·G1;then different concentrations of avidin were added for detection,and the results showed that when the avidin concentration reached 20 nM,it can combine with the labeled biotin on the DNA chain to form a stable steric hindrance,which affects the equilibrium movement;On this basis,different concentrations of biotin are added,when the biotin concentration is 20 nM,biotin can bind to avidin 1:1,a stable avidin-biotin detection system is formed;In the selective detection of biotin,vitamin C tablets,thiamine hydrochloride,estradiol,progesterone,and adenosine triphosphate,the system has better selectivity to biotin.In the test of pure milk with biotin added,the recovery rate of biotin was more than 90 %,which proved that the fluorescence sensor constructed by us can be used for the determination of biotin in actual samples.The RNA fragment is embedded in DNA,so that the enzyme can specifically recognize and cut the RNA fragment.Compared with the sample group with no enzyme added,the reaction rate was faster,and when the enzyme concentration reached 10 μg/mL,the fluorescence intensity reached saturation;Adding 2 μL of goat serum during the digestion process interfered with the digestion,which was different from that without the enzyme.There was almost no difference in the fluorescence detection results of the sample groups added with goat serum,which proved that the strategy has strong anti-interference ability.