| In recent years,the incidence of diseases caused by cancer and viral infections has been increasing,which has posed a huge threat to human health,therefore the early accurate diagnosis and sensitive detection of the disease is of great significance.As an important biomarker for disease detection,nucleic acid has shown great application prospects in disease early warning and post-treatment.Most of the current nucleic acid detection methods suffer the problems such as low sensitivity,cumbersome detection process,and poor versatility.Therefore,we urgently need to design a simple,rapid,and sensitive method to detect nucleic acid disease biomarkers.Based on this,we hope to achieve the purpose of rapid and sensitive detection of nucleic acids from the following two aspects:(1)The introduction of resonance energy transfer(RET)pairs through the fluorescence resonance energy transfer(FRET)between the dye and dye,the energy transfer between dye and nanomaterial surface(nanomental surface energy transger,NSET),or plsmon resonance energy transfer(PRET),etc.,which can reduce the background signal and increase the sensitivity;(2)DNAzyme and exonuclease has the unique characteristic of rapid amplification as the detection target can be circulated in the system by enzyme digestion to achieve signal amplification.Based on this,we have built a series of universal fluorescent probes for nucleic acid detection with high sensitivity.The main contents are as follows:(1)By taking use of the local surface plasmon resonance(LSPR)property of gold nanoparticles(Au NPs),we constructed a random 3D DNA nanomachine by combining NSET and PRET process with the restriction enzyme digestion and amplification effect of exonuclease III(Exo III)to realize highly sensitive detection of HIV DNA.The highlight of this work is that hairpin DNA was used instead of traditional straight-stranded DNA to construct the 3D DNA nanomachines,so it can be "up right" on the surface of the nanoparticle which has a stronger affinity with Exo III and is beneficial for enzyme digestion.Both ends of the DNA were modified with sulfhydryl(-SH)and TAMRA dyes.At this time,the distance between TAMRA and Au NPs was close,so NSET and PRET can occur between them,causing the quenching of fluorescence.When the target HIV DNA was present,it can act as a walking chain under the cutting action of Exo III.The hairpin DNA on the surface of Au NPs was gradually cleaved and the fluorescence was released.The degree of fluorescence recovery has a linear relationship with HIV DNA in the concentration range of 0.05-1.2n M,and the detection limit is 12.7 p M.Experiments such as enzymatic reaction kinetics and dark-field light scattering imaging have proved that the DNA walker gradually walks on the surface of the 3D nanomachine.This method has the advantages of simplicity,rapidity,and good selectivity for the detection of HIV DNA.The addition and recovery experiments in human serum samples showed that this method was expected to be used for accurate detection of HIV DNA in complex samples.The programmable design of DNA walkers has broad prospects in the development of highly scalable nano-and micro-scale nanomachines.(2)In order to improve the reaction speed and shorten the reaction time,this chapter combined the cyclic cutting of DNAzyme and the strand displacement reaction of the catalytic chain to achieve double amplification of the signal,which was used for the rapid and sensitive detection of mi RNAlet7 a.TAMRA dye and black hole quencher2(BHQ2)were modified in both ends of the hairpin DNA,so FRET occurred,which reduced the background signal.Mi RNAlet7 a can trigger the cyclic cleavage of DNAzyme and the strand displacement reaction of the catalytic chain,then release the TAMRA dye which caused the recovery of fluorescence signal.Therefore,dual signal amplification strategies were used to improve the sensitivity of detection.More importantly,the local target concentration was increased owing to the occurrence of circulation,which greatly shorten the detection time to only 30 minutes to complete the reaction.The experimental results showed that the recovery of fluorescence signal has a good linear relationship with the concentration of mi RNAlet7 a in the linear range of2-50 n M,with the detection limit of 64 p M.Control experiments showed that when there was no catalytic chain,the linear range was 10-90 n M,with the detection limit of670 p M,indicating that the presence of the catalytic chain can significantly reduce the detection limit.Therefore,the combining of the cyclic cutting of DNAzyme and the strand displacement reaction of the catalytic chain can greatly reduce the detection time while improving the sensitivity,which provides a new idea for the development of portable,simple and rapid nucleic acid detection sensors in the future.(3)In order to further improve the accuracy of disease diagnosis,this chapter developed a simple fluorescent probe based on DNAzyme cycle amplification strategy for simultaneous detection of two biomarkers of pancreatic ductal adenocarcinoma,mi RNA21 and mi RNA10 b.By constructing two fluorescent probes,FRET occured between the fluorescent dyes FAM,Cy5 and BHQ,causing the fluorescence quenching.In the absence of target,the DNAzyme cannot be released,so the background was low.When the target was present,the DNAzyme was activated to cut the substrate strand which realized the recovery of fluorescence.By this way,we can achieve accurate detection of the two mi RNAs.The feasibility of the detection method was proved by fluorescence signal recovery and gel electrophoresis.The experiment found that the recovery of fluorescence signal showed a good linear relationship with the concentration of mi RNA21 and mi RNA10 b in the range of 10-60 n M,and the detection limit was 468 p M and 893 p M,respectively.Therefore,the simultaneous detection of two mi RNAs was realized through the simple design,which greatly improved the accuracy of disease diagnosis and avoided the false positive problem of single target detection in disease diagnosis.In summary,in this thesis,three probes for the detection of nucleic acid disease biomarkers were designed based on energy transfer and enzymatic signal amplification,which achieved the purpose of high-sensitivity and rapid detection of nucleic acid disease biomarkers.It has the advantages of simple design,rapid detection,low background,good selectivity,etc.,which has great potential for the detection of disease biomarkers in complex samples.At the same time,the design of this probe has good versatility and it can be applied to different kinds of nucleic acid detection,which provides a good idea for the development of detection sensors for nucleic acid disease biomarkers. |
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