Development Of Label-free Assays For Nucleic Acid Detection

Viruses exist widely in nature and threatens human life and health all the time.Rapid and accurate detection of viruses is of great significance to prevent disease transmission and pandemics.Nucleic acid detection is the gold standard for judging the presence of viruses.Many fluorescence-based detection platforms(such as real-time PCR)have been applied to the detection of viruses with high sensitivity and high specificity.However,most of the existing fluorescence-based methods require additional fluorophore labeling of the reporters,which limits their wide applications.The label-free fluorescence detection method can effectively reduce the background signal,simplify the reagent preparation process and reduce the cost,and can even be used for in-situ detection in special scenarios,which is very attractive.Based on this,this thesis developed two new methods for label-free detection of nucleic acids based on G-triple(G3)and G-quadruplex(G4),and further combined with microfluidics technology to establish a platform for fast and label-free detection of nucleic acids.The contents are as follows:1.In this paper,a system based on G3 and Thioflavin T(ThT)was developed for label-free detection of RNA targets.Although human telomere G4(ht-G4)has been widely studied and used in biosensing,there are few reports investigating the properties of ht-G3.First,it was found that ht-G3 could significantly enhance the fluorescence of ThT,Then,a DNA molecular beacon(ht-G3MB)that can form a hairpin structure is designed based on the ht-G3 sequence.If there is no target,ht-G3MB will not form the G3 structure;when the RNA target exists,it will bind with ht-G3MB and promote the formation of the G3 structure,and further enhance ThT fluorescence.It is found that the sensitivity can reach 1 nM based on this method,and the specificity of this method was also proved.Finally,in order to further improve the detection sensitivity,a dualspecificity nuclease(DSN)was introduced into the system to cut the DNA that is complementary to the RNA target,so that the target RNA can be recycled to amplify the signal The detection sensitivity was improved 10-fold to 0.01 nM.This ht-G3/ThTbased label-free detection strategy is expected to be widely used in RNA detection.2.A new method based on G4/ThT and CRISPR/Cas 12a was developed for labelfree detection of DNA target.Our group recently reported that CRISPR/Cas12a can trans-cleave G4 structures.Based on this,this work first found out the optimal G4 sequence(ht-G4)and the ionic condition for the trans-cleavage of Cas12a.When Cas12a is activated by the DNA target,G4 can be efficiently cleaved,and thus weaken the fluorescence of ThT.Using the Cas12a-G4/ThT-based method,the HPV18 plasmid was successfully detected and the sensitivity could reach 0.1 nM,similar to that of a fluorescent reporter-based detection system.3.This thesis further developed a method for rapid fabrication of microfluidic chip without photolithography,and combined the microchip prepared by this method with the above Cas12a-G4/ThT detection strategy to realize the label-free detection of HPV18.Microfluidic chips are generally fabricated based on complex processes(such as lithography,development,etc.)in an ultra-clean room.The fabrication of a photoresist mold always takes a long time and the cost is high,which limits the wide use of microchips in common biochemical laboratories.In this paper,a laser cutting machine is used to ablate micro-patterns on the transparent tape,and the tape is then transferred to a silicon wafer.Next,a drop of positive photoresist is added to the micropattern and scratched with a cover glass to make it flat.After 5-minute heating,the mold will be obtained.Finally,a PDMS device can be obtained quickly.This method can achieve a resolution of 100 μm for the microchannel.In addition,the height of the mold can be easily controlled by changing the type and the tape layers.After testing the device performance in common biochemical reactions,it was further applied for labelfree detection of HPV18 with one step.The method developed in this chapter for making microchips shows advantages of low cost,simple operation and short time,which is expected to be widely used in general biochemical laboratories.In this thesis,new label-free detection methods were developed based on htG3/ThT and Cas12a-ht-G4/ThT,respectively.A novel strategy for preparing microfluidic chips without photolithography was also developed and used for the rapid detection of HPV18.The above label-free detection methods are expected to be extended to rapid and low-cost detection of viruses.