Construction Of Trace Copper(â…¡) Ion Detection Strategies And Lateral Flow Biosensor For Histone Methylation Detection

Copper ion (Cu2+) is one of the essential micronutrient elements for human life, and the concentration level of Cu2+directly affects human’s health. As an essential micronutrient element, Cu2+is a necessary cofactor or structural component of numerous enzymes and proteins (such as superoxide dismutase, cytochrome oxidase, dopamine beta hydroxylase, tyrosinase, and ceruloplasmin) needed in metabolic processes, and only trace amount of Cu2+in the human body is able to maintain normal life activities. However, a lower or higher concentration of Cu2+can cause adverse health effects. For instance, in copper deficiency state, accompanied by the lack of other nutrients and excessive intake its biological antagonists, the activity of the enzymes and cell metabolism can be influenced. And a higher concentration of Cu2+retained in the body was usually caused by genetic diseases or the environment pollution, intaking too many foods containing copper or inhaled the gas containing high concentration of copper ion. Both of atmospheric and water pollution by Cu2+can eventually cause soil pollution, which further affects the yields and quality of crops. Copper ion accumulated in the crops and animals was taken into the body through the food chain expansion process step by step, resulting severely harm to human health. The liver is an important place to store copper ions and help Cu2+releasing into the bile, as a consequence, excessive Cu2+will affect the normal metabolism of liver and kidney, causing gastrointestinal disturbance and hemolytic anemia as well as loss of cognition in the elderly and individuals with Wilson’s disease. Hence, considerable efforts have been devoted to the detection of copper ion due to the high concentration of copper ion has high toxicity on human health, ecosystem and food safe. The maximum level of Cu2+in drinking water permitted by the U.S. Environmental Protection Agency (EPA) is20μM.Classical approaches for the detection of Cu2+, such as graphite furnace atomic absorption spectrometry and inductive coupled plasma atomic emission spectroscopy, have been used as standard procedures. However, the requirement of costly instruments and highly trained personnel prevent their use in many laboratories. Some new methods and technologies, including fluorescence methods, colorimetric method based gold nanoparticles, and dynamic light scattering technique, et al., have been developed in recent years. Although these methods are effective, the use of DNA ligase and fluorophore labeled oligonucleotides is not only expensive but also increases the complexity of the assay. Hence, the development of an inexpensive and sensitive method for Cu2+detection is urgently needed.Eukaryotic genome carrys two kinds of genetic information, one is the nucleic acid sequence carrying genetic information in the conventional sense, the other is epigenetic information, which mainly refers to the reversible and heritable variation of gene function without DNA sequence changes. Histone methylation is an important epigenetic modification, and the commonly modified residues are lysine (K) and arginine (R) at the N-terminal tails of core histones. Previous studies have shown that histone methylation plays important roles in the formation of heterochromation, X-chromosome inactivation, transcriptional regulation, maintenance and differentiation of stem cells, and tumorigenesis, all of which depend on the degree and position of histone methylation. Some new techniques for histone methylation detection emerged in recent years with the development of epigenetics, such as western blot, ChIp (chromatin immunoprecipitation), or ChIp combines with PCR and microarray. These strategies are accurate and reliable except for the tedious operation and heavy workload. Hence, it is necessary to develop a much more simple method for the rapid detection of histione methylation.This paper describes two biosensors for Cu2+detection making full use of the catalytic activity of DNAzyme, including a fluorescence biosensor based on self-assembled DNA concatamers for signal amplification, and a colorimetric biosensor using Cu+-catalyzed click chemistry and hemin/G-quadruplex DNAzyme. An enhanced strip biosensor using DNA functionalized gold nanoparticles as an enhancer probe for rapid and sensitive detection of histone methylation was also constructed. Three research themes are included in this paper and listed as follows:(1) An enzyme-free and label-free fluorescence turn on biosensor for amplified Cu2+detection has been constructed based on self-assembled DNA concatamers and SYBR Green Ⅰ. The reaction mechanism is based on the cleavage of Cu2+-specific DNAzyme, the released target DNA triggering the formation of dsDNA concatamers and using SYBR Green I as a signal reporter. The detection limit for Cu2+(12.8pM) is six orders of magnitude lower than the United States EPA limit of Cu2+in drinkable water (20μM). The whole reaction process does not need any protein enzyme and fluorescent-labeled DNA, making the system more simple and cost-effective.(2) The accurate and rapid detection of Cu2+using Cu+-catalyzed click chemistry and hemin/G-quadruplex horseradish peroxidise (HRP)-mimicking DNAzyme by the naked eye without using typically bulky instruments was developed. The reaction mechanism is based on the formation of a G-quadruplex-forming sequence using Cu+-promoted click chemistry between azide-and alkyne-modified short G-rich sequences, followed by the self-assembly of hemin/G-quadruplex DNAzyme in the presence of hemin and K+in aqueous solution. This G-quadruplex DNAzyme can catalyze its colorless substrate TMB into a colored product. Hence, the whole process of color change of the solution can be monitored by the naked eye. For quantitative analysis, the resulting yellow solution was transferred into a96-well microtiter plate. The optical density (OD) value of the yellow solution at450nm in each well was recorded using a microplate reader. The LOD of our strategy (5.9nM) is much lower than the EPA limit of Cu2+in drinkable water (20μM). In comparison with previously reported Cu2+-specific DNAzyme/substrate based methods for Cu2+detection, the presence of Cu2+can be evaluated by the naked eye, and the whole reaction process does not need long manipulation time and complicated procedure, making the system more simple and convenient.(3) An enhanced strip biosensor using DNA functionalized gold nanoparticles (AuNP-DNA) as an enhancer probe for rapid and sensitive detection of histone methylation has been successfully constructed. Another dual labeled AuNPs used in this assay was functional ized with an antibody and another oligonucleotide (c-DNA) simultaneously. The sequence of the c-DNA is complementary to the DNA on the enhancer probe. The mechanism of the enhanced biosensor is based on the formation of an antibody/target/dual labelled AuNP sandwich structure on the test zone, and a red band can be observed firstly, then AuNPs-DNA solution was added onto the strip to hybridize with the c-DNA on the surface of dual labelled AuNPs. In this case, the red band on the test zone and control zone deepened significantly due to accumulation of more AuNPs. Tri-methylated lysine9of histone H3(H3K9me3) in HeLa cells was chosen as our target. With this biosensor, we can visually detect H3K9me3in20ng of histone extract from HeLa cells in15min without instrumentation, which is10-fold and15-fold lower detection limit than the conventional strip biosensor and western blot, respectively. In addition, the principle of the enhanced strip biosensor can be applied to detect other types of histone methylation, such as mono-, di-or tri-methylated H3K4and H3K9, as well as other analytes, such as nucleic acids, proteins, virus, microorganisms, and small molecules.