Optimization Of Guanine Chemiluminescence System And Its Analytical Application

Chemiluminescence(CL)analysis has attracted much attention in analysis and detection due to its advantages of high sensitivity,wide linear range,and simple instrumentation.The guanine CL system has a broad application prospect in nucleic acid-based biological analysis due to easy to combine with the molecular recognition of nucleic acids.However,compared with other classical CL systems,the guanine CL system has not been studied in depth and is not widely used.In this thesis,we focus on improving the luminescence intensity of guanine CL system.The luminescence intensity of guanine CL system is improved by CL energy transfer,and then exonuclease activity is measured.The specific research content is as follows:1.Construction of a highly efficient guanine CL systemRelative to the classical CL system such as luminol,the luminescence efficiency of guanine CL system is relatively low,which limits the analytical application of guanine CL system.In this study,fluorescein-labeled guanine-rich DNA strands were used as CL energy transfer receptors,and CL energy transfer was used to increase the luminescence intensity of the guanine CL system.A series of G-rich DNA strands labeled with fluorescein at various positions were designed and synthesized to study the effect of the relative positions of the donor and the acceptor on the CL energy transfer efficiency of the guanine CL resonance energy transfer system.The experimental results show that the closer the labeling position of fluorescein is to the G-rich sequence,the higher the CL resonance energy transfer efficiency.Relative to the marker at the end of the DNA,the fluorescein donor is tagged in the middle of the G-rich DNA strand,which increases the efficiency of the system by a factor of five.This study proposes a new method to improve the luminescence efficiency of guanine chemical system,which will be helpful for the analysis and application of guanine chemiluminescence system.2.Detection of exonuclease ? activity by guanine CL systemExonuclease ? plays an integral role in ensuring the integrity and correct expression of genes as DNA modification enzyme,it can hydrolyze double-stranded into single nucleotides along the 3' blunt end or concave end of double-stranded DNA to the 5' end.Intact is hydrolyzed.In this work,we designed and synthesized G-dsDNA labeled with fluorescein on the intermediate adenine sequence,in which a single-stranded sequence is AGAGAGAGAGAGA(FAM)GAGAGAGAGAGA,using this DNA as CL probe,exonuclease ? activity was detected.In this fluorescein-labeled G-dsDNA,the receptor fluorescein can react CL resonance energy transfer with guanine to generate a strong luminescent signal.When exonuclease ? is added to the system,exonuclease ? hydrolyzes the designed G-dsDNA,leaving the acceptor fluorescein away from guanine,making it impossible to achieve CL resonance energy transfer and reducing the CL intensity of the system.In this experiment,exonuclease ? activity was detected using the the intensity difference with or without exonuclease ?.The detection limit of exonuclease ? was 0.03 U/mL.The method was simple and rapid,which laid the foundation for the development of highly efficient guanine CL system.3.Detection of exonuclease ? activity by guanine CL resonance energy transfer based on conformational transformationThe CL intensity of G-rich DNA molecule is closely related to its conformation.The formation of a G-quadruplex conformation can significantly reduce the intensity of CL of guanine relative to single-or double-stranded G-rich DNA molecules.In G-rich DNA duplexes,one is designed to form a G-quadruplex single chain,and the other is its non-complementary strand.The 3' end is fully complementary to the G-rich and the other end is concave.The 3' end of the G-rich chain is prominent to prevent exonuclease ? cleavage.When exonuclease ? is present,the semi-complementary strands are hydrolyzed,leaving the G-rich single strands released,and upon the induction of potassium ions,the G-quadruplex is formed,resulting in a weak guanine CL signal;in the absence of exonuclease ?,the presence of a G-rich double strand stably produces a strong CL signal,and exonuclease ? is detected based on the difference in the signals of the two.This method,without magnetic separation,is convenient,simple and rapid,and it contributes to the further application of the guanine CL system.