Nuclease-assisted Signal Amplification Method For Highly Sensitive Detection Of Biomolecules

Biological molecules and drug molecules have a huge impact on maintaining the health of life.Therefore,it is of great application value to develop biosensors that can accurately and sensitively detect trace amounts of important molecules.As an analytical tool,nucleic acid molecular probes have provided a new development direction for biosensors.They have been widely used in the fields of analytical chemistry and clinical diagnostics,and have shown an increasingly broad application prospect.However,many biological molecules in the human body are rare,and the sensitivity and precision of traditional nucleic acid biosensors often fail to match the requirements.Therefore,scientists have been working to develop a nucleic acid biosensor that can detect targets more rapidly,accurately,and ultrasensitively.In recent years,scientists have developed a series of signal cyclic amplification strategies based on exonuclease and nicking enzyme degradation.Although these detection strategies have improved sensitivity,their shortages have gradually emerged.For example,exonuclease is less specific and nicking enzymes are less versatile.Traditional nucleic acid biosensors tend to have high background signal and low accuracy,and are difficult to be applied to the direct detection of targets in complex biological systems.As a new attempt,this thesis develops two new schemes,which not only achieve highly sensitive detection of the target,but also avoid the defects of previous work.The main works are summarized as follows:1.Graphene oxide protection/Cryonase-assisted signal amplification for sensitive detection of biological small molecules.We use nucleic acid aptamers as analytical tools,which greatly improves the selectivity of analytical methods.We use graphene oxide to protect nucleic acid aptamers and quench fluorescence,greatly reducing the background fluorescence signal of the system.And the rapid degration of the nucleic acid with Cryonase helps to achieve a cyclic amplification of the signal,and improve detection Sensitivity.Using this method,we successfully achieved high sensitivity detection of theophylline,ATP and kanamycin with detection limits of 47 nM,23 nM and 5 nM,respectively.The method was also applied to catechin in 5%fetal bovine serum,and the detection limit is 52 nM,which is almost consistent with the buffer solution,indicating that the method is still feasible in the complex sample test.2.A T4 DNA ligase-based binary probe for the highly sensitive detection of the cancer-associated gene K-ras.By skillfully using the proximity of T4 DNA ligase,the specificity of the sensor is improved.Fluorescence resonance energy transfer is used as a signal output method to reduce the background fluorescence signal of the system,and the cyclical amplification of the signal is achieved by combining the steady-state conformational changes of nucleic acid molecules at different temperatures.The method was used to detect the pM concentration of K-ras.