Construction Of The Fluorescence Biosensor Based On Entropy Driven Circuit And Its Application In The Determination Of Disease Markers

Disease markers are biochemical indicators that mark the changes or possible changes in the structure or function of the system,organs,tissues,cells and subcells.The detection of disease markers,which include proteins,nucleic acids and enzymes,can prevent disease early or prevent disease progression in time.There are many methods used for the detection of disease markers.However,the content of disease markers is generally low,which requires the detection method to have enough sensitivity.Enzyme-free nucleic acid signal amplification technology takes into account both sensitivity and specificity,and is simple in operation and experimental conditions.Therefore,it is a good method to construct the platform for disease marker detection.Varieties of enzyme-free signal amplification techniques based on nucleic acid probes have been developed,including catalyzed hairpin assembly(CHA),hybridization chain reaction(HCR),entropy-driven circuit(EDC),etc.The driving force of EDC reaction comes from the increase of structure entropy.After the reaction,the degree of freedom of the system increases,resulting in the entropy increase.Therefore,this signal amplification technique is insensitive to the changes of factors affecting the intensity of DNA hybridization in the environment and more stable.Fluorescence method has its own unique advantages,such as simple operation,stable signal,low instrument requirements,and it is widely used in practical applications.At present,fluorescent biosensors based on EDC are developing better and better.There are two main signal output modes: one is that functional materials quench the fluorescence of fluorescent dyes;the other is that the fluorescence of fluorescent dyes is quenched by small molecule of quenching agent via fluorescence resonance energy transfer(FRET).In the research of functional materials,the objects of exploration are mostly gold nanoparticles.There are also many functional materials with high fluorescence quenching efficiency that have not been studied,which limits the development of EDC to some extent.The studies that have been done to quench fluorescence by FRET have focused primarily on the use of FRET over short distances,limiting its use over long distances,such as in sensing large structures.In this study,we constructed novel fluorescent biosensors.The difference of fluorescence quenching effect of FAM on single and double stranded DNA by metalorganic gel(MOG)and the change of long range energy transfer efficiency in multi-color FRET cascade were used to detect mi RNA-155(mi R-155)and thrombin,respectively.The specific work was as follows:(1)A novel fluorescence biosensor was constructed by coupling EDC and MOG to measure mi R-155.The nucleic acid probe was modified with the FAM dye.In the prescence of the mi R-155,EDC was triggered,which changed the DNA strand modified with FAM from the hybridized state to the free state.Then the synthetic MOG that can effectively quench the fluorescence of FAM was added.Since the adsorption capacity of MOG on single-stranded DNA was stronger than that on double-stranded DNA,the fluorescence quenching capacity of MOG on single-stranded DNA was much stronger than that on double-stranded DNA.The fluorescence intensity of solution decreased,relazing the detection of mi R-155.This strategy used cheap and easily available MOG as fluorescence quenching agent,expanding the direction for the diversified development of EDC.(2)DNA photonic nanowires with long range energy transfer were constructed using cascade FRET to determine thrombin.As a quenching fluorescence material,MOG also has some problems.For example,the response signal is single signal fluorescence quenching and the detection accuracy needs to be improved.The ratio method of FRET can effectively address this problem.Nevertheless,the current signal output of EDC is mostly based on one-step FRET,which greatly limits the development of sensing over long distances.Cascaded FRET involves multiple steps,more complex energy transfer pathways and theoretical studies,and allows the transfer of energy over long distances.First,we constructed DNA photonic nanowires(DNA PNs)capable of long distance energy transfer using FRET cascades.The substrate in the EDC cycle was designed as DNA nanophotonic wire skeleton,and the organic dyes such as FAM,Cy3 and Cy5 were modified on the DNA.Cy3 acted as a bridge and transferred the energy of FAM to Cy5,realizing the long distance energy transfer between FAM and Cy5.Secondly,in order to explore the application effect of DNA PNs in biosensing,we chosed the thrombin as the target to be tested.We designed two segments of DNA that interacted with thrombin,S1 and S2,which were mainly composed of thrombin aptamer and partial catalytic sequences that triggered the EDC cycle.In the presence of thrombin,S1 and S2 combined with thrombin which close the distance between S1 and S2,forming a complete catalytic sequence which triggered the EDC circulation.Therefore,the FRET between FAM and Cy5 was destroyed,resulting in the increase of FAM fluorescence intensity and the decrease of Cy5 fluorescence intensity,realizing the detection of thrombin.In conclusion,this study used EDC as signal amplification technology and constructed nucleic acid probe based on it.A simple and fast method was used to prepare MOG with significant difference in fluorescence quenching efficiency between single stranded DNA and double stranded DNA.The MOG were applied to the EDC cycle triggered by mi R-155,which realized the detection of mi R-155 and expanded the application of MOG in fluorescence analysis system and the diversified development direction of EDC.Further,we intended to overcome the inaccuracy of the single response signal and the distance limitation of short range FRET.Nevertheless,DNA PNs could achieve long range FRET,and the substrate in the EDC cycle is a good DNA PNs skeleton.Therefore,we constructed a fluorescent biosensor capable of long range energy transfer and realized the detection of thrombin by ratio method.It provided a new idea for the construction of long range energy transfer nucleic acid probe and its application in the detection of disease markers.