DNA Assembly On Electrode Surface And Its Biomedical Application

DNA,a fundamental molecule of life,not only plays a key role in vivo as the carrier of genetic information,but also displays unique predominance in vitro for nanostructure fabrication.With the development of self-assembly technique and electrochemical methods,interfacial behavior of DNA can be studied deeply and assembling DNA on electrode surface could be further widely applied in various biomedical aspects.Herein,to broaden molecular intelligent system and biosensor system,we build a series of biomedical devices based on DNA assembled on electrode surface,which can be used in logic gate fabrication,ribozyme activity display,controllable molecular release as well as the detection of nucleic acid,protein and small molecule.1.A set of logic gates fabricated with G-quadruplex and heminLogic gates are physical devices,which may determine their meaningful output depending on input to implement Boolean function.In this work,six kinds of meaningful logic gates including a set of basic gates capable of generating any Boolean function and two more complicated gates,"INHIBIT" gate and"IMPLICATION" gate,are designed based on G-quadruplex and hemin.The gates are fabricated based on the facts that the conformation of G-quadruplex can be changed by certain ions as well as thermal denaturation,while hemin,an electroactive probe,is able to specifically bind to G-quadruplex with high affinity and the formed G-quadruplex-hemin complex can exihibit electrochemical response.By utilizing these principles,six kinds of electrochemical logic gates are constructed,which can be further and combined into a network depending on their logic relationship.Since the whole network is comparatively integral,uniform and reversible,this work may provide a new perspective for the practical application of molecular-scale logic system for molecular computing.2.Fabrication of three kinds of hammerhead ribozyme on a solid surfaceRibozyme is an RNA molecule that is capable of catalyzing specific biochemical reactions.Of ribozymes reported,hammerhead ribozyme(HHRz)is the smallest but most studied member with nucleolytic activity induced by divalent metal ions.In the present work,we successfully fabricate three kinds of HHRzs,i.e.,an intact HHRz,a split RNA-RNA HHRz and a split DNA-RNA HHRz on a solid surface for the first time and study the nucleolytic behavior of them.Results show that surface-tethered HHRzs retain the Mg ions induced self-cleavage activity,which is well characterized by using electrochemical technologies.The split DNA-RNA HHRz is proven to have some remarkable advantages,which make this DNA-RNA hybridization model favorable for the study of ribozyme on surface.On the one hand,the splict DNA-RNA HHRz can keep over 80%of its self-cleavage activity,even after the DNA capture has been fabricated on the electrode surface for 12 h,while the intact HHRz and the split RNA-RNA HHRz begin to lose its self-cleavage activity in a very short time,and is nearly inactivity after 1.5 h.On the other hand,the splict DNA-RNA HHRz can reveal its self-cleavage activity repeatedly,once DNA capture is immobilized on electrode surface.So,this work deepens the understanding of ribozyme construction and do favor to fabricate ribozymes on solid surface.3.Controllable molecular transport and release by a restorable surface-tethered DNA deviceIn this paper,we report a novel surface-tethered DNA device.Fabricated on an electrode surface by sequential self-assembly of two delicately designed DNA single strands,this DNA device may present three states("duplex state","single state" and"i-motif-triplex state")under the operation of pH.Desirably,with the unidirectional changes of pH,the diverse DNA structural regions integrated into this device undergo sequential and concerted conformational changes through the three states.To make full use of this DNA device and the electrochemical advantages,ferrocene(Fc)has been further employed for the fabrication of the device.On the one hand,the state switches of the DNA device can be characterized conveniently and reliably by the obtained electrochemical signals from Fc.On the other hand,Fc can introduce?-cyclodextrin-ferrocene(?-CD-Fc)host-guest system.Based on different electrochemical behaviors of ?-CD under different states,this DNA device can actualize directional loading,transporting and unloading of ?-CD under controllable conditions.Moreover,convenient regulation and manipulation of chemical environments on the electrode surface may render the construction and operation of this DNA device restorable.Therefore,this DNA device displays uncommon features of reversibility and reparability,and bares promising applications in controllable molecular transport and release,which are of great value to molecular device design.4.Fabrication of an electrochemical biosensor for miRNA detection Detection of miRNA is extremely important in clinical diagnosis.However,current detection method is defective in timeliness,which impedes miRNA as effective clinical diagnostic marker.In this work,based on the selective insertion of redox probe,methylene blue(MB),into dual duplex strand formed by RNA and DNA,we have designed a novel electrochemical biosensor for miRNA detection.The feature of dual signal amplification ensures low detection limit of this sensor.On the one hand,one duplex strand can bind more than one MB.On the other hand,horseradish peroxidase(HRP)in the test system will catalyze these MBs,making a large electrochemical signal.Experimental results show that this biosensor has high selectivity and can distinct not only single strand and duplex strand effectively,but also single nucleotide polymorphisms(SNP)in miRNA.More important,the time required in whole detection process is only about 30 min.Considering that RNA is easy to degradation,this biosensor is suitable to be applied in practical miRNA detection.5.Fabrication of functional grapheme oxide and its application to the detection of biomarker proteinGraphene Oxide(GO)-based materials are very attractive for electrochemical studies and applications,because of the wide range of reactive surface-bound functional groups of GO,high surface-to-volume ratio and high dispersibility in water.In this work,we have fabricated GO-based silver nanoparticle nano-hybrids(GO-AgNPs).GO-AgNPs can interact with nucleobase of the DNA aptamer immobilized on electrode through both ?-? bond and N(or O)-Ag bond.And amplified signals can be obtained through the oxidation of silver nanoparticles by electrochemical methods.When the DNA aptamer bind its target protein firstly,the interaction between GO-AgNPs and nucleobase of the DNA aptamer will be inhibited,resulting in significant signal attenuation.Based on this principle,we can analyse human a-thrombin protein with high sensitivity and selectivity.A linear detection range of 1-10000pM and limit of detection to 0.70pM can be obtained.More importantly,only microliter of sample is needed.This method can be used for the detection of other proteins or small molecules and nucleic acid by changing the aptamer sequence.6.DNA-templated silver nanoclusters formation at electrode surface and its application to hydrogen peroxide detectionIn this work,we have established a biomineralization system to mineralize inorganic nanoclusters at gold electrode surface.This biomineralization is formed by a thiol-modified oligodeoxynucleotide consisting of eighteen cytosine deoxyribonucleotides(polyC18)as biomineralization template and silver ions(Ag+)as biomineralization ions.Experimental results obtained by electrochemical methods show that not only Ag nanoclusters(Ag NCs)can be formed around the template polyC 18,but the formed Ag NCs may also display high catalytic activity for oxygen reduction reaction.So,this biomineralization system can be further used for the detection of hydrogen peroxide(H2O2).Considering that this biomineralization process is quite simple and signals of H2O2 decomposition caused by Ag NCs is very distinct,this work has provided a new method for H2O2 detection.