Highly Sensitive Detection Of Mercury Ions Using Graphene Oxide-Phosphorothioated DNA Based Sensors

Mercury ion(Hg²⁺)is a kind of common heavy metal ions with high toxicity and can cause serious pollution to the environment.Due to the poor degradation of Hg²⁺,it will be accumulated in the biological chain,and eventually will reach a very dangerous concentration in the human body,threatening the life and health.When the concentration of Hg²⁺exceeds the standard,it will cause irreversible damage to the human organs such as causing functional disorders to the kidneys and muscles,and seriously damaging the nerves.Therefore,it is important to develop the methods for detecting mercury ions.Graphene oxide（GO）plays an important role in the field of biosensors and collaborates with DNA to form an effective sensing system for fast and efficient detection of analytes.In this study,a few graphene oxide（GO）-DNA biosensors were designed based on the principle of fluorescence resonance energy transfer（FRET）and the mechanism based on Hg²⁺-induced cleavage of phosphorothioate modified RNA（Phosphorothioated cleavage site or PS site）.Highly sensitive detection of Hg²⁺was achieved based on the change of fluorescence intensity.1.Detection of mercury ions based on graphene oxide physically adsorbing DNA modified with multiple PS cleavage sites.One,two,three PS cleavage sites were designed in the DNA sequence to improve the sensitivity of detecting Hg²⁺by increasing the number of PS cleavage sites.The physical adsorption of GO combined with FRET between GO and DNA achieved highly sensitive detection of Hg^2+.The limits of detection（LOD）were respectively 13.43 nM,8.14 nM and 6.83 nM.2.Detection of mercury ions based on GO-binding polycytosine DNA-assisted silica coated silver（Ag@SiO₂）.The 15 mer of polycytosine designed at the end of DNA has a strong affinity to GO.Therefore,DNA can be firmly adsorbed on the surface of GO.Then GO quenched the fluorescence of FAM from DNA.The other end of DNA was designed with one and two PS cleavage sites.Hg²⁺can cleave the PS sites to restore the fluorescence of FAM.The LOD of the Poly-C PS 1 site sensor was 1.11 nM,and the LOD of the Poly-C PS 2 sites sensor was 0.84 nM.Ag@SiO₂ can amplify the signals of fluorescence to these two sensors.The LOD of the Poly-C PS 1site sensor was reduced to 0.36 nM.And the LOD of the Poly-C PS 2 sites sensor was reduced to 0.15nM.3.Detection of mercury ions based on graphene oxide and DNA modified with carbon quantum dots（CDs）and PS cleavage sites.One end of the DNA-modified PS cleavage site was covalently bound to CDs using NHS/EDC.Therefore,the whole DNA sequence was labeled with CDs.The CDs of DNA were quenched by GO based on FRET.After the addition of Hg²⁺,the PS site in the DNA was cleaved.Then the CDs were detached from the surface of GO and the fluorescence was restored.The LOD of the sensors with one cleavage site and two cleavage sites were respectively 10.84 nM and 9.05 nM.To reduce the fluorescent background caused by CDs,gold nanoparticles（AuNPs）were introduced.DNA modified with CDs and PS sites was fixed on the surface of AuNPs and formed the spherical ssDNA-AuNPs by Au-S bond.The fluorescent background was reduced with centrifugation.The LOD of the sensors with one cleavage site sensor and two cleavage sites were 6.35 nM and 4.94 nM.4.Detection of mercury ions based on DNA modified with multiple PS cleavage sites on the microarray of graphene oxide（GO）.The GO was immobilized on the surface of glass modified with amino group.Then DNA modified with amino group was immobilized on the surface of GO on the glass.Hg²⁺can cleave the PS sites in the DNA and result in releasing of the Cy5.Then this was removed and resulted in a lower fluorescence intensity in the remaining DNA on surface of microarray.The LOD of the DNA chip with one cleavage site and two cleavage sites hybridized by the short complementary sequence was 1.75 nM and 1.49 nM,and the LOD of the DNA chip of the two cleavage sites hybridized by the long complementary sequence was 0.38 nM.This method has the advantages of high-throughput,highly sensitive detection and so on.This can provide the relevant experimental and theoretical basis for the detection of Hg²⁺on the microarray in the future.Although the four methods for detecting Hg²⁺have different advantages and disadvantages through the above researches,they all achieve high-sensitivity detection of Hg²⁺,which provides some solutions for the problems and drawbacks in the current method for detecting Hg²⁺,and also provides support and help for human health.