Study On The Functional Nucleic Acids-based Novel Methods For The Detection Of Heavy Metal Ions In Aqueous Solution

With the fast development of industrial and agricultural production,heavy metal pollution in the environment became more and more serious nowadays,of which heavy metal pollution in water is attracting universal attention due to its characteristics such as high toxicity,cannot be decomposed but easy to be bio-concentrated and bio-magnified by microorganism.Traditional methods for heavy metals detection depend on large equipment,tedious sample pretreatment,long detection cycle and high cost,failing to meet the requirements of on-site or on-line monitoring.Therefore,developing simple,reliable,rapid and efficient methods for heavy metal detection in aqueous solution is of great research significance and practical value.Currently,methods based on functional nucleic acids?FNAs?have been widely applied in rapid detection of heavy metals because of their prominent advantages such as wide target range,easy modification and selection process,high stability,specificity and affinity.However,most of the sensing systems of these methods rely on complex design and rare component,and are not easy to be applied in practical use.Thus it is essential to design some novel methods which are simple to operate and compose of common reagents for heavy metal ions detection.In this thesis,several FNAs-based new methods such as gold nanoparticles?AuNPs?-based colorimetry,fluorometry and AuNPs-based lateral flow dipstick for As?III?,Ag⁺,Cu²⁺,Pb²⁺and Hg²⁺detection in aqueous solution were established by choosing/designing arsenic aptamer,silver/copper-specific DNA,G-rich oligonucleotide,integrated functional nucleic acid and proper fluorescent dye,and the conclusions were summarized as follows.?1?A new method for colorimetric detection of As?III?in aqueous solution using arsenic aptamer and AuNPs was developed.The sensing system was composed of AuNPs,arsenic aptamer and NaCl.The optimal detection condition was determined as 200?L AuNPs,0.12 M NaCl and 20 nM arsenic aptamer in the sensing system of500?L,both the incubation time and reaction time were 10 min.This method showed good selectivity and could detect the As?III?in the linear range of 1.26-80 ppb,with recovery of 93.6-105.6%and a detection limit of 1.26 ppb that lower than the maximum contamination level?MCL?for As?III?in drinking water?10 ppb?defined by the U.S.Environmental Protection Agency?EPA?and the World Health Organization?WHO?.?2?By introducing rhodamine B?RhoB?into the classic colorimetric method that based on the AuNPs aggregation,a new method for fluorescent determination of Ag⁺in aqueous solution based on the aggregation of AuNPs mediated by silver-specific DNA?Ag-CC?and its effect on the fluorescence of RhoB was established.The sensing system was composed of AuNPs,Ag-CC,NaNO₃ and RhoB.The optimal detection condition was determined as 250?L AuNPs,0.12 M NaNO₃,15 nM Ag-CC and 8?M RhoB in the sensing system of 500?L.This method showed good selectivity and could detect Ag⁺in the linear range of 2.73-200 ppb,with recovery of99.9-102.7%and a detection limit of 2.73 ppb that lower than the MCL for Ag⁺in drinking water?50 ppb?defined by EPA and WHO.?3?A ssDNA named Cu100 that show unexpected specially recognizing ability to Cu²⁺was discovered and based on which and SYBR Green I?SG?,a new fluorescent assay for Cu²⁺determination in aqueous solution was exploited.The sensing system was composed of Cu100 and SG,and the optimal detection condition was determined as 0.3×SG,20 nM Cu100 and 150?M sodium ascorbate in the sensing system of 500?L.This method showed good selectivity and could detect Cu²⁺in the linear range of5.57-250 ppb,with recovery of 98.5-104.8%and a detection limit of 5.57 ppb that lower than the MCL for Cu²⁺in drinking water?1.3 ppm?defined by EPA.?4?By applying fluorescently labeled or label-free G-rich oligonucleotide,two new fluorescent assays for the direct detection of Pb²⁺in aqueous solution were constructed.The sensing system of the fluorescently labeled method was composed of6-carboxyfluorescein?FAM?labeled T30695 and the optimal detection condition was determined as 25 nM FAM-T30695 in the sensing system of 500?L,with a reaction time of 5 min.While the sensing system of the label-free one was composed of T30695 and SG,with its optimal detection condition determined as 4×SG and 50 nM T30695 contained in the sensing system of 500?L,and an optimal reagent adding order T30695+Pb²⁺+SG.Both of these two methods showed good selectivity,and could detect Pb²⁺in the linear ranges of 0.77-20 ppb and 3.79-100 ppb,with recoveries of 95.3-110.8%and 97.5-110%,and detection limits of 0.77 ppb and 3.79ppb that lower than the MCL for Pb²⁺in drinking water?15 ppb?defined by EPA and WHO,respectively.?5?An integrated FNA named modified T30695 that owns the ability of recognizing Hg²⁺and Pb²⁺was obtained by adding six thymines?Ts?to both terminals of T30695.Based on this modified T30695 and GeneFinder??GF?,a new fluorescent method for the detection of Hg²⁺and Pb²⁺in aqueous solution was designed.The sensing system was composed of modified T30695 and GF,and the optimal detection condition for Hg²⁺detection was determined as 50 nM modified T30695 and 0.3×GF in the sensing system of 500?L and that for Pb²⁺detection was50 nM modified T30695 and 2.4×GF.As a result,Hg²⁺and Pb²⁺could be selectively detected in the linear ranges of 3.23-40 ppb and 2.62-80 ppb,with the recoveries of98.5-106%and 98.6-103.6%,detection limits of 3.23 ppb and 2.62 ppb,respectively.?6?Development of arsenic aptamer and AuNPs based lateral flow dipstick test was carried out.Several design schemes were tried and the scheme in which the arsenic aptamer-AuNPs conjugate was coated on the conjugate pad,the arsenic aptamer was fixed on the test line and the complementary DNA of the arsenic aptamer was immobilized on the control line was chose.As a result,As?III?could be qualitatively detected or semi-quantitatively detected as low as 10 ppb,accompanied with which the intramolecular and intermolecular interaction between As?III?and arsenic aptamer was revealed.As for this thesis,novelty shows in testing Cu²⁺with copper-specific DNA instead of Cu²⁺-dependent DNAzyme,detecting Pb²⁺directly based on G-quadruplex formation instead of on substitution of K⁺by Pb²⁺,designing integrated FNA for double targets detection,and transferring the homogeneous phase sensing system onto solid phase carrier.And limitations remain in that much effort should be carried out before those methods could be applied and popularized in practical application.