Fabrication Of Novel DNA Hydrogel And Its Application In Biochemical Analysis

DNA hydrogels are three-dimensional crosslinked networks that are composed of hydrophilic polymers and DNA.DNA hydrogels present such remarkable features as great biocompatibility,precise programmability,degradability,facile synthesis and modification.In addition,the DNA hydrogels not only maintain the original biological characteristics of DNA,but also process the features of gel.Therefore,DNA hydrogel,a representative of three-dimensional macroscopic DNA materials,exhibits a variety of applications in biosensing,drug delivery and release,cell culture,and 3D printing.However,the synthesis methods of DNA hydrogels reported to date depend on high-concentration of DNA as raw material,greatly limiting the fabrication and application of DNA hydrogels.Therefore,our research focuses on this issue and uses rolling circle amplification technology to construct a series of functionalized DNA hydrogels,which have been applied in the field of nanozyme catalysis,interfacial sensing systems,and portable detection.The detailed content was described as follows:(1)Fabrication of nanozyme@DNA hydrogel and its application in biochemicalanalysis:Nanozymes have received intensive attention for the advantages of easy preparation and low cost.Unlike natural enzymes that adapt to physiological environments exactly,the artificial nanozymes are apt to passivate in complex clinical samples(e.g.serum),which may damage the catalytic capability and consequently limit the application in biomedical analysis.To conquer this problem,in this work,we have fabricated novel nanozyme@DNA hydrogel architecture by enwrapping nanozyme into pure DNA hydrogel.Gold nanoparticles(AuNPs)are adopted as a model nanozyme.Results show that AuNPs can retain their catalytic capability in serum,benefited from the protection of DNA hydrogel,whereas in comparison,AuNPs alone totally lose the catalytic capability in the same case.Meanwhile,detection of hydrogen peroxide and glucose in serum is achieved on the basis of the catalysis of the AuNPs@DNA hydrogel.Detection limit of each reaches 1.7μM and 38 μM respectively,which can rival the value by using nature enzymes.Besides,the mechanisms as well as some other advantages such as recyclable availability have also been explored.This nanozyme@DNA hydrogel architecture may have great potential for the utilization of nanozymes as well as the application of nanozymes for biomedical analysis in complex physiological samples.(2)Surface-immobilized and self-shaped DNA hydrogel and its application for biosensing:hydrogel of good biocompatibility,plasticity,and capability working as 3D scaffold has been joined up with surface biosensing systems for broaden the application of hydrogel in biosensing.Here for the first time,surface-immobilized pure DNA hydrogel is synthesized using a surficial primer-induced strategy and successfully constructed on a transparent ITO electrode,which facilitates both colourimetric and electrochemical measurements.Results show that the hydrogel is able to wrap enzymes solidly and form enzyme@DNA hydrogel,which exhibits favourable stability under different conditions.Owing to the free difftusion of micromolecular targets throughout the hydrogel and isolat:ing of the enzymes from those macromolecular interferences outside the hydrogel in the meanwhile,colourimetric and electrochemical detection of hydrogen peroxide and bilirubin directly in serum is achieved.The detection limit of hydrogen peroxide in serum is 22 nM by colourimetric analysis and 13 nM by electrochemical measurement,respectively.The detection limit of bilirubin is 32 nM,a favourable limit that could be used in jaundice diagnosis.Moreover,the enzyme@hydrogel can be easily regenerated with the catalytic activity retained for a few cycles,thus allowing the recycles of the hydrogel-based biosensing system.The successful integration of DNA hydrogel with surface biosensing system greatly expanded the application of hydrogel for diagnostic and environment monitoring purposes.(3)Fabrication of DNAzyme-comprized hydrogel and its application for visible detection of circulating tumor DNA:A macroscopically visual method based on DNAzyme-functionalized DNA hydrogel has been developed for qualitative and quantitative analysis of circulating tumor DNA(ctDNA).In this work,target ctDNA could trigger the rolling circle amplification and finally synthesize a macroscopic DNA hydrogel with G-quadruplex structures,which could be observed by the naked eye.Furthermore,by utilizing the DNAzyme activity of the G-quadruplex/heme complex,the DNA hydrogel could catalysis the colorless ABTS to green oxidative product ABTS*.Consequently,quantitaing ctDNA could be achieved by detecting the color change.The results indicate that the detection of limit of ctDNA is 0.32 pM with a linear range from 1 pM to 10 nM.Moreover,our visual strategy is able to identify single-base mutant of ctDNA and has been successfully applied to the simulate analysis of clinical blood samples.In addition,our method does not require expensive equipments.Therefore,this macroscopically visual method not only exhibits great potential application in poor regions with limited conditions,but also opens new insight into the establishment of new visuable analysis method of DNA.(4)Fabrication of enzyme and electronics transport functionalized DNA hydrogel for electrochemical analysis:A dual functional DNA hydrogel(DRhydrogel)with enzyme activity and three-dimensional electronic transferred capability has been constructed on the electrode surface for electrochemical analysis.Here,the DRhydrogel was fabricated based on surfacial primer induced strategy and nucleic acid isothermal amplification technology.On the one hand,the DRhydrogel possess DNAzyme activity by forming the G-quadruplex structures and binding with heme.On the other hand,the DRhydrogel could absorb electroactive probes to form a three-dimensional electron transport vector that mediates the electron transfer between the DNAzyme and the electrode.The experimental results show that the DRhydrogel has been successfully applied to the electrochemical analysis of hydrogen peroxide.The detection limits of hydrogen peroxide in phosphate buffer and serum are 0.33 μM and 0.41μM,respectively.Notably,the DRhydrogel achieves the conversion of electrochemical signals from conventional one-dimension to three-dimension.Furthermore,the detection could be carried out immediately after the addition of target,shortening the time of measurement.In addition,the DRhydrogel displays great anti-interference,stability and reusability.Most importantly,the dual-functionalizaition of enzyme and electronic transfer realizes the integration of biological and scaffold functions of DNA hydrogels,thus not only has potential application in electrochemical analysis,but also inspires the designation of other multifunctional DNA hydrogels.(5)DNA hydrogel based on DNA conformational change for portable and visual potassium detection:A DNA hydrogel microplate has been fabricated based on the metal ion-induced DNA conformational changes for portable and visual detection of potassium ion(K+).Firstly,the DNA hydrogel was functionalized by embedding G-rich DNA probes.When target K+ is added,G-rich DNA probes converted to G-quadruplex structure,which could bind specific fluorescent dye and produce a fluorescence enhancement effect in situ.Our strategy integrates the whole process of sample injection,signal response and result reading,which can be automatically realized by pointing sample into the micropores of the hydrogel.Furthermore,the quantitating K+ could be realized by calculating the optical density of fluorescence aperture.The detection limit for K+ is 0.34 mM.Moreover,our method exhibts great specificity and expected to be applied to high-throughput screening of K+.Moreover,our proposed method has been expanded to detect Hg2+by replacing DNA probes with other sequence.