Nanomaterials Combined With Cyclic Amplification Reactions For The Analysis Of Metal Ions And Nucleic Acid Molecules

Metal ions such as copper,zinc ions and biomolecules such as RNA and DNA play an important role in various biological processes.In recent years,highly sensitive detection of metal ions and biomolecular microRNAs（miRNAs）which are indicative of disease have attracted considerable interest in research.However,it has been proven that high concentrations of these metal ions are toxic to human health.Therefore,there is an urgent need to develop highly sensitive and selective methods for detecting metal ions in the human body and in the daily diet.And disease-associated biomarkers in tissues or body fluids are usually expressed at lower levels in the early stages of the disease.It is difficult to detect them by using conventional detection methods.Therefore,there is an urgent requirement for the development of new ultra-sensitive detection strategies and bioanalytical technologies.This paper mainly introduces several methods for efficient detection of metal ions and miRNAs.The main contents are as follows:1.A fluorometric assay for zinc ion is described that relies（a）on the use of an isothermal cycle to amplify the fluorescence signal,and（b）of magnetic beads（MBs）to completely remove unreacted DNA detection probes.Biotin and fluorophore-labeled substrate（Zn-Sub）strands acting as detection probes were first assembled on MBs.Next,Zn（Ⅱ）-specific DNAzyme（Zn-Enz）strands were hybridized with the Zn-Sub strands.In the presence of Zn（Ⅱ）,the Zn-Sub strands are cleaved.This results in the release of the shorter DNA fragments（containing fluorescent label）and the dissociation of Zn-Enz strands.The dissociated Zn-Enz strands then hybridize with the residual Zn-Sub strands and cleave them in a similar fashion.This leads to a target recycling amplification mechanism and in a cumulative signal amplification process.A strongly amplified signal is thus obtained in the presence of Zn（Ⅱ）.The use of MBs warrants that unreacted Zn-Sub strands can be magnetically separated from the solution.The method has a detection limit as low as33 fM at a signal-to-noise ratio of 3 and a linear response in the 100 fM to 11 nM Zn（Ⅱ）concentration range.It was applied to the determination of Zn（Ⅱ）in spiked tap water and seawater samples,and the results compared well with data obtained by ICP-MS analysis.The method was also applied to the determination of Zn（Ⅱ）in infant milk powder and breast milk.2.A sensitive copper（Cu（Ⅱ））sensor was developed on the basis of Cu（Ⅱ）-specific DNAzyme and Ni/Fe-layered double hydroxide（LDH）/G-quadruplex peroxidase mimic.Briefly,in the presence of Cu（Ⅱ）,short guanine（G）-rich fragment-labeled substrate（Cu-Sub）strands hybridized with and were cleaved by the Cu（Ⅱ）-specific DNAzyme（Cu-Enz）strands,resulting in the release of the G-rich DNA fragments.Meanwhile,the Cu-Enz strands were disassociated and participated in the next hybridization-cleavage cycle.G-rich fragments were then accumulated and further folded into the quadruplex structure.Afterwards,the streptavidin-coated magnetic beads were used to conveniently separate out all the unreacted Cu-Sub strands,the cleaved Cu-Sub strands and the Cu-Enz strands.Finally,Ni/Fe-LDH nanosheets combinded with G-quadruplex（without hemin）,for the first time,presenting an enhanced peroxidase-like activity,were applied to catalyze a colorimetric reaction.The results of this new detection method for Cu（Ⅱ）provided the detection limit to 0.29 pM with a linear range from 1 pM to 10μM（r²>0.997）.The established dual DNAzymes-based sensor was validated by applying to determine Cu（Ⅱ）in serum samples and obtained comparable results of using ICP-MS.Ni/Fe-LDH/G-quadruplex was demonstrated to be a new and efficient DNAzyme for analytical application.3.A pH-dependent selective ion exchange coupled with catalytic polymerization of aniline has been developed for sensitive detection of copper(Cu²⁺)and ferric ions(Fe³⁺).Ethylenediamintetraacetic acid（EDTA）chelated with nickel ion(Ni²⁺)were intercalated in a layered double hydroxide via a co-precipitation reaction.The product was subsequently applied as sorbent for the enrichment of Cu²⁺at pH 6.5 and Fe³⁺at pH 4.5.Since both Cu²⁺and Fe³⁺have stronger complex formation constants with EDTA,Ni²⁺exchanges with Cu²⁺/Fe³⁺selectively.The resulting sorbent containing Cu²⁺/Fe³⁺was transferred to catalyze the aniline polymerization reaction,since Cu²⁺/Fe³⁺could be released by the sorbent effectively at different pH values and have high catalytic abilities for the polymerization reaction.The resulting polyaniline with different colors were produced at different pH values,an observation that was utilized to distinguish between the colorimetric signals of Cu²⁺and Fe³⁺.The extraction temperature,extraction time,catalysis time and pH were optimized.The results showed that this method provided low limits of detection of 0.1 nM（6.4 ng/L）for Cu²⁺,1 nM(56 ng/L for Fe³⁺),wide linear ranges（0.0005-2.5μM,and 0.005-5μM,respectively）,and good linearities（r² values of 0.9904,and 0.9965,respectively）.The optimized method was applied to river water samples.Using Cu²⁺/Fe³⁺as examples,this work provided a new and interesting approach for the convenient and efficient detection of metal ions in aqueous samples.4.A highly sensitive triple-amplification assay for the detection of microRNA（miRNA）let-7a is reported in this work.The assay relies on the formation of magnetic DNA/Fe₃O₄ nanosheet networks initiated by the target miRNA-associated hybridization chain reaction（HCR）.The Fe₃O₄ nanosheets in the DNA/Fe₃O₄networks display peroxidase-like catalytic activity towards a colorimetric reaction,thereby producing a highly sensitive signal for the quantification of let-7a.Under optimal conditions,the assay achieved a detection limit of 13 aM at a signal-to-noise ratio of 3 and a linear calibration plot between 0.05 fM and 12 nM.Successful attempts were made in the quantification of let-7a in serum samples.This method demonstrated to be a new,fast and efficient miRNA analysis approach through the triple-amplification strategy based on HCR,networking and catalytic reaction.