Detection Of Mercury Ion And Circulating Tumor DNA Based On Magnetic Relaxation Switching

Heavy metal pollutants and cancer pose great threats to human health.Heavy metal pollutants,such as Hg²⁺is one of the most dangerous heavy metal ions.Hg²⁺in the natural environment can directly cause diseases after being absorbed by the human body,such as brain injury,kidney failure and dyskinesia;Cancer has become one of the main causes of death worldwide,the incidence and mortality of cancer continue to increase every year.It shows that circulating tumor DNA（ctDNA）carries sequence information of tumor mutations,which can realize early screening,disease detection and prognosis evaluation of cancer.Therefore,the detection of heavy metal Hg²⁺and ctDNA is vital to human health.Due to the sensitivity of optical and electrochemical sensors to impurity interferences,based on optical and electrochemical detection often require complex sample preparation.However,magnetic relaxation switching has deep penetration capability due to the applied radiofrequency radiation,so it can be analyzed without the purification and separation of samples.The developed magnetic relaxation switching based on the aggregation/disaggregation of iron oxide nanoparticles has some limitations.Frist,there are two analysis modes of magnetic relaxation switching:nanoparticles from dispersion to aggregation and aggregation to dispersion,and the relaxation signal trend of the two analysis modes are different.The choice of the two analysis modes is related to many factors,such as the size of the nanoparticles and the the type of target.It is difficult to accurately determine which analysis mode the reaction belongs to,or whether the two analysis modes exist at the same time,thereby affecting the accuracy of target detection.Then,improper ratio of magnetic relaxation probe constructed by iron oxide nanoparticles and target causes the change of relaxation signal change trend also affect the accuracy of target detection.Therefore,we constructed a concentration-dependent magnetic relaxation switching based on iron oxide nanoparticles for the detection of Hg²⁺in complex samples.In addition,current ctDNA detection methods cannot achieve direct detection of ctDNA in some complex samples,a new type of magnetic relaxation switching was constructed based on concentration changes in Mn²⁺and exonuclease III（Exo-Ⅲ）,ctDNA in complex samples was directly detected.The main contents are as follows:Part I:Magnetic relaxation switching assay for mercury ion based on oligonucleotide functionalized iron oxide magnetic nanoparticle.DNA₁ and DNA₂ were coupled with two kinds of iron oxide nanoparticles MB₃₀ and MB₂₀₀,to construct signal probe MB₃₀-DNA₁ and magnetic separation probe MB₂₀₀-DNA₂.Hg²⁺activates the hybridization between MB₃₀-DNA₁ and MB₂₀₀-DNA₂,causing MB₃₀-DNA₁ to aggregate.The T₂ value of MB₃₀-DNA₁ remaining after magnetic separation was measured,and the quantitative analysis of Hg²⁺in the sample was carried out according to the relationship between the change of T₂ value and the concentration of Hg²⁺.The research results show that the detection range of this method is 0.8μM to 50μM,the linear equation isΔT₂=164.06 x+35.5,R²=0.98,and the limit of detection（LOD）is0.23μM.Further research results show that the recovery rate of this method in tap water,lake water and serum of complex samples is 98.6%～101.7%,which has high recovery rate and accurate.Because relaxation signal can ignore the background interference of scattering,absorption,or autofluorescence of biological samples,the magnetic relaxation switching has potential application value for Hg²⁺determination in clinical and environmental monitoring.Part Ⅱ:Direct detection of circulating tumor DNA in whole blood based on Magnetic relaxation switching with Mn²⁺concentration changing.In this work,MB₁₀₀₀is used as a carrier,and the surface of MB₁₀₀₀ is coupled with a single-stranded nucleic acid（DNA₁）that are complementary to the target DNA and alkaline phosphatase（ALP）to obtain the MB₁₀₀₀-DNA₁-ALP.In the presence of target DNA,MB₁₀₀₀-DNA₁-ALP hybridizes with target DNA to form double-stranded DNA,which is specifically recognized by Exo-Ⅲand cyclically cleaved to release ALP.ALP enriched by magnetic separation and reduces ascorbic acid phosphate to produce ascorbic acid,and then ascorbic acid reduces MnO₂ to Mn²⁺,resulting in T₂ signal change.Quantify the amount of target DNA in the sample based on the change in T₂ signal.The research results show that the detection range of this method is 0.5 nM to 1000 nM,the linear equation is y=298.38 x+121.3,R²=0.997,and the limit of detection（LOD）is 0.34 nM.The recovery rate of this method in whole blood is 99.2%～101.3%.This work combined with magnetic separation and Exo-III assisted circulation cleaving make the magnetic relaxation switching based on the change of Mn²⁺concentration become a more sensitive detection method,which has the potential value of clinical application.