Research On Biosensor And Logic Calculation Model Based On G-quadruplex Probe

With the development of biotechnology and nanomaterials science,more and more new nanomaterials have been applied in many fields,such as biocomputing,biomedicine,biosensors and biochemical analysis.In recent years,DNA nanomolecules,as the carriers of life genetic substances,have become a research hotspot in the field of biomolecular logic gates and biosensors,due to the Watson-Crick complementary condition,great parallel processing ability,super storage capacity and better editability.Among them,G-quadruplex probe is a kind of functional DNA molecule.It has not only structural polymorphism and technological diversity,but also important significance in the biological genome.These characteristics stimulate researchers to develop many biochemical analysis models based on G-quadruplex probes,which has realized the detection of metal ions,microRNAs,key proteins and other biological analytes.Meanwhile,graphene oxide(GO)has been found to be a monoatomic layer nanomaterial.It has unparalleled chemical and physical properties,and can maintain biocompatibility and stability.Hence,GO,a darling of new nanomaterials,has also being introduced into the research work of logic computing,intelligent diagnosis,drug loading and other fields.On the basis of fully studying nanomaterials and nanotechnology,we have mainly carried out the following work.(1)In this paper,a non-label and enzyme-free biosensor based on G-quadruplex probe has been constructed to detect thrombin.Taking the advantages of the higher selectivity and affinity of aptamers,the free initiation strand triggers and multi-round participates in catalyzed hairpin assembly reaction,resulting in a large number of G-quadruplex structures.And these special G-quadruplex structures can be detected by various methods.According to the principle of model design,NUPACK simulation combined with manual adjustment is used to complete model updating and sequence design.We also explain it from the point of view of molecular thermodynamics.Then,the simulation experiments are carried out and the preliminary analysis of the results were made.The reaction simulations by Visual DSD prove the theoretical feasibility of the model and optimize the conditions of the toehold length,the DNA concentration and so on.It provides a reference for setting the initial conditions of in vitro biological experiments.Next,we verify the feasibility and effective of the model through polyacrylamide gel electrophoresis,colorimetric detection and fluorescent detection method.Finally,on the basis of optimizing the experimental conditions,we measure the performance of the biosensor,such as sensitivity,serum samples detection and so on,and further improve the sensor model designed in this paper.(2)Taking advantage of the adsorption characteristics with ssDNA and fluorescence quenching properties of GO,we approach a non-label,enzyme-free and universal logic operation platform based on G-quadruplex special structure in our work.The feasibility and universality of the platform has been preliminarily proved by six kinds of simple logic gates and logic devices.Among them,the ssDNA strand(BGG)with G-quadruplex splitting sequence at both ends was anchored on the GO,working as the original platform.Whether different DNA strands exist or not is the corresponding input truth value.The DNA hybridization or DNA strand replacement reaction between different inputs strands and BGG strand on the platform is a signal converting process.Finally,the fluorescence intensity of G-quadruplex/NMM is used as the reporting signal,and its output value is judged according to the set threshold.Based on this design principle,we skillfully utilize G-quadruplex/NMM complex and GO as two natural fluorescent materials and fluorescent quencher to complete the design.A series of basic logic gates(OR,AND,INHIBIT and XOR)have been artfully designed and verified by simulations and biological experiments.Subsequently,two combinatorial logic gates based on the same BGG platform are successfully realized conceptually with the help of AgNCs,including half adder and half subtractor.The researches lay the foundation for the construction of cascaded and feedback complex molecular logic circuits.