Study On Highly Sensitive Electrochemical Biosensors Based On The Combination Of Functional DNA Nanostructures With Multiple Signal Amplification

In recent years,the combination of target analyte recognition elements and electrochemical signal conversion elements has been widely concerned to construct electrochemical biosensors.Because of its advantages such as simple operation,economy,sensitivity,portability,and simple structure,it has shown great potential application value in the fields of biochemical analysis,clinical medical diagnosis,environmental monitoring,and food quality safety.In addition,DNA nanomaterials with different functionalities and structures are widely used in the construction of electrochemical biosensor detection platforms due to their good programmability,biocompatibility,and high specificity.Our work focuses on functionalized DNA nanostructures?such as i-motif and "Y-shaped" structures?and multiple signal amplification methods?such as loop-mediated isothermal amplification,enzyme-assisted recycle amplification and enzyme cascade catalysis?.We want to develope electrochemical biosensors with high selectivity,high specificity,low background signal and high sensitivity,and to be used for the rapid detection of target DNA of different disease markers.The main research work of this thesis includes three parts:1.LAMP-generated H⁺ions-induced dimer i-motif as signal transducer for ultrasensitive electrochemical detection of DNABased on i-motif structure conversion,DNA amplification,and enzyme-assisted recycle amplification,we constructed an ultra-sensitive electrochemical biosensor for the detection of the Flu A virus biomarker DNA?fDNA?.Hydrogen ions?LAMP-H⁺?generated during the loop-mediated isothermal amplification?LAMP?using fDNA as a template induced the formation of a dimer i-motif structure?DiMS?.DiMS as a signal transducer,combined with exonuclease III?ExoIII?-assisted DNA walking,to achieve dual-amplification for electrochemical signals.Firstly,when LAMP-H⁺was introduced,two cytosine?C?-rich affinity strands?AS1 and AS2?were bound together through semi-protonated C·CH⁺pairs,allowing the stable construction of DiMS as signal transducer at slightly acidic condition.The unpaired overhangs in DiMS hybridized with a protection strand?PS?confined by a flexible sequence-specific moving arm that was tethered in a walking leg?W?.In turn,the liberated W unfolded ferrocene-tagged hairpin signal probe?Fc-SP?.At exonuclease III?ExoIII?-recognizable sites,the opened Fc-SP was cleaved,releasing the walking leg for continuous unfolding and cleavage of other Fc-SP hairpins.Resultantly,all the cleaved segments with Fc were taken from the modified electrode surface,leading to significantly decreased electrochemical response.With ExoIII-assisted signal amplification,the electrochemical current of Fc was linearly varied in a wide concentration range of fDNA,endowing this method with simplification,convenience,high specificity and sensitivity down to 0.018 fg·?L^-1,and great potential in wide-spread biosensing and bioanalysis.2.Proximity binding-induced DNA assembly as signal translator and enzyme-catalyzed cleavage recycle as signal amplifier for highly sensitive electrochemical assay of target DNABased on the target DNA?tDNA?-dependent proximity ligation to develop unique DNA assembly as output translator,an electrochemical impedimetric biosensor was developed for the amplified detection of tDNA related to Alzheimer's disease?AD?.Herein,this binding-induced DNA assembly is constructed by the simultaneous ligation of tDNA with two affinity probes?A1 and A2?on Au-Fe₃O₄ nanocomposites,which are previously functionalized with A1,a linker DNA?L1?,and DNA transduction probes?TP?.In this context,A2 contains a polythymine spacer,and two specific domains?C1*and C2*?complementary to tDNA and L1,respectively.The proximal binding of tDNA with A1 and A2 brings C2*close to TP:L1 and displace TP.The hybridization of C2*:L1 forms a complete recognition site of T7 exonuclease?T7?.Under the T7-powered catalysis,C2*liberated from C2*:L1 is available for the next displacement of TP from adjacent TP:L1 hybrids,again releasing TP.In this case,T7-catalyzed iterative cleavage drives C2*in A2 to stepwise move along the assembled DNA structure surface until all cleavage events are complete,resulting in the dissociation of DNA assembly.In response to a single-target binding event,the releasing of hundreds of TP realizes the amplified translation of tDNA input.The liberated TP is recognizable for hairpin DNA?NH₂-HP?modified in the electrode surface.NH₂-HP containing C-rich sequence repeats is unfolded through the hybridization of TP and NH₂-HP,which provides a nicking site of exonuclease III?ExoIII?.ExoIII-catalyzed cleavage recycle is activated,allowing for the retaining of only C-rich repeats in random coil configuration at pH 7.0 in the electrode surface.At decreased pH,these C-rich sequences with negative charges are switched into more stable i-motif conformation with positive charges.So,redox pairs[Fe?CN?₆]^3-/4-were electrostatically attracted close to the electrode sensing interface,consequently decreasing the electron transfer impedance.Thus,based on binding-triggered DNA assembly as signal translator and integrating enzymes-catalyzed recycle amplification,the developed impedimetric biosensor for tDNA is highly specific and sensitive with a limit of detection as low as 31 fmol·L^-1,which would be interesting and promising to open up a new analytical route for sensitive monitoring diverse targeted DNAs in disease diagnosis.3.Bienzymatic cascade catalysis confined in an oriented "Y-shaped" DNA nanostructure for amplified and renewable electrochemical biosensorIn addition to the traditional enzyme-catalyzed cleavage recycle and DNA amplification techniques,enzymatic cascade catalysis can also amplify the signal.An amplified and renewable electrochemical biosensor was developed via bienzymatic cascade catalysis of glucose oxidase?GOx?and horseradish peroxidase?HRP?,which were confined in a functional "Y-shaped" DNA nanostructure oriented by a dual-thiol-ended hairpin probe?dSH-HP?with paired stem as rigid scaffold and unpaired loop as enclosed binding platform.For proof-of-concept assay of sequence-specific biomarker DNA related to Alzheimer's disease?aDNA?,GOx and redox ferrocene-modified HRP were chemically conjugated in two enzyme strands?GOx-ES1 and Fc@HRP-ES2?,respectively.The repeated recycling of aDNA was powered by the displacement of GOx-ES1 by aDNA and exonuclease III?ExoIII?-assisted cleavage reaction for amplified output of numerous GOx-ES1 as dependent transducers,together with Fc@HRP-ES2 which was simultaneously hybridized with dSH-HP to assemble this "Y-shaped" nanostructure.Rationally,the bienzymatic cascade catalysis was motivated through GOx-catalyzed glucose oxidization to in situ generate H₂O₂ and overlapped HRP-catalyzed H₂O₂decomposition to greatly promote the electron transfer,achieving significantly enhanced electrochemical signal of Fc with an ultrahigh sensitivity down to 0.22fmol·L^-1 of aDNA.Benefited from the unique design of dSH-HP-oriented bienzymatic cascades,this one-step strategy without non-specific blockers passivation was simple and renewable,and would pave a promising avenue for sensitive electrochemical assay of biomolecules.