Construction Of Electrochemiluminescence Microrna Biosensor Based On Nucleic Acid Amplification Strategy And DNA Nanomachines

With the growth of population,the aging of population and multiple pathogenic factors,cancer has become one of the most threats toward human health all over the world.It has been reported that microRNAs are widely found in cancer cells,and their abnormal expression can cause abnormal differentiation,proliferation and apoptosis of the cells.Therefore,it is an urgent need to study the detecting method for microRNA with high efficiency and sensitivity,which lays a foundation for the evaluation of anti-cancer drugs and early diagnosis of cancer.Nucleic acid-based electrochemiluminescence?ECL?analytical technology combined the high sensitivity and controllability of ECL to high specific complementary base paring within DNA duplex,which can improve the sensitivity and selectivity of microRNA determination and bring a broad prospect in the development of biological and clinical analysis.However,there still remains challenges in the field of ECL biomarker detection.?1?Meet the requirement of high detecting sensitivity of microRNA due to its inherent virtues of small size and low abundance in cancer cells.?2?Realize multiple ECL detection within one luminophore,which directly break through the influence between two different ECL luminophore.?3?Realize the regeneration of the biosensing platform,which is easy to operate and low cost.?4?Accomplish the quantitative detection of microRNA in rapid single step.Account of that,this thesis has significantly improved the detection sensitivity of microRNA biosensor with the aid of nucleic acid amplification strategies and figured out other issues by using DNA nanomachies.Based on that,a series of ECL biosensor with excellent stability and selectivity have been constructed,realizing the ultrasensitive detection of biomarker.The studies of this thesis are shown as follow:1.An“off-on”Electrochemiluminescent Biosensor Based on DNAzyme-Assisted Target Recycling and Rolling Circle Amplifications for Ultrasensitive Detection of MicroRNAIn this study,an off-on switching of a dual amplified electrochemiluminescence?ECL?biosensor based on Pb²⁺-induced DNAzyme-assisted target recycling and rolling circle amplification?RCA?was constructed for microRNA detection.First,the primer probe with assistant probe and microRNA formed Y junction which was cleaved with the addition of Pb²⁺to release microRNA.Subsequently,the released microRNA could initiate the next recycling process,leading to the generation of numerous intermediate DNA sequences?S2?.Afterward,bare glassy carbon electrode?GCE?was immersed into HAuCl₄ solution to electrodeposit a Au nanoparticle layer?depAu?,followed by the assembly of a hairpin probe?HP?.Then,dopamine?DA?-modified DNA sequence?S1?was employed to hybridize with HP,which switching off the sensing system.This is the first work that employs DA to quench luminol ECL signal,possessing the biosensor ultralow background signal.Afterward,S2 produced by the target recycling process was loaded onto the prepared electrode to displace S1 and served as an initiator for RCA.With rational design,numerous repeated DNA sequences coupling with hemin to form hemin/G-quadruplex were generated,which could exhibit strongly catalytic toward H₂O₂,thus amplified the ECL signal and switched the ON state of the sensing system.The liner range for microRNA detection was from 1.0 fM to 100 pM with a low detection limit down to 0.3 fM.Moreover,with the high sensitivity and specificity induced by the dual signal amplification,the proposed microRNA biosensor holds great potential for analysis of other interesting tumor markers.2.Electrochemiluminescent Graphene Quantum Dots as A Sensing Platform:A Novel Dual Amplification for MicroRNA AssayGraphene quantum dots?GQDs?with an average diameter as small as 2.3 nm were synthesized to fabricate an electrochemiluminescence?ECL?biosensor based on T7 exonuclease-assisted cyclic amplification and three-dimensional?3D?DNA-mediatedsilver enhancement for microRNA analysis.Herein,toovercomethebarrierinimmobilizingGQDs,aminated-3,4,9,10-perylenetetracarboxylic acid?PTCA-NH₂?was introduced to load GQDs through?-?stacking?GQDs/PTCA-NH₂?,realizing the solid-state GQDs application.Furthermore,Fe₃O₄-Au core-shell nanocomposites?Au@Fe₃O₄?was adopted as probe anchor to form a novel electrochemiluminescent?ECL?signal tag of GQDs/PTCA-NH2/Au@Fe₃O₄.The prepared ECL signal tag was decorated on the electrode surface,exhibiting excellent film-forming performance,good electronic conductivity and favorable stability,all of which overcame the obstacle for applying GQDs in ECL biosensing and showed a satisfactory ECL response under the co-reactant of S₂O₈^2-.Afterwards,hairpin probe modified on the electrode was opened by helper DNA,followed by assembling target to hybridize with the exposed stem of helper DNA.Significantly,T7 exonuclease was employed to digest DNA/RNA duplex and trigger the target recycling without asking for a specific recognition site in the target sequence,realizing a series of RNA/DNA detection by changing the sequence of the complementary DNA.At last,ECL signal was further enhanced by AgNPs-based 3D DNA networks.After the two amplifications,the ECL signal of GQDs was extraordinarily increased and the prepared biosensor achieved a high sensitivity with the detection limit of 0.83 fmol L^-1.The biosensor was also explored in real samples and the result was in good accordance with the performance of qRT-PCR.Considering the excellent sensitivity and applicability,we believe that the proposed biosensor is a potential candidate for nucleic acid biosensing.3.Nanomachine Based Regenerated Sensing Platform:A Novel Electrochemiluminescence Resonance Energy Transfer Strategy for Ultra-high Sensitive Detection of MicroRNA from Cancer CellsThe construction of DNA nanomachine holds great meanings in the development of DNA nanostructure,yet the real application of the nanomachine is still in its early stage.Furthermore,a one-step regenerated sensing platform for biomarkers detection in current researches remains a realistic challenge.Herein,a novel electrochemiluminescence resonance energy transfer?ERET?strategy between Alexa Flour 488?AF 488?which is a kind of small molecule dye as the donor and CdSe@ZnS quantum dots?QDs?as the acceptor was reported,which was much easier to enter a cell and was applied for the construction of a DNA nanomachine-based regenerated biosensor for the ultra-high sensitive determination of cancer cells without any enzymes.First,a dual amplification strategy including target recycling and signal transformation was employed to achieve the conversion of a small number of microRNAs to a large amount of universal DNA reporters.Primordially,the DNA tweezer was kept in the“off”state with two arms labeling with QDs and AF488 respectively.Second,in the presence of the DNA reporters,the tweezer transformed to“on”state through the hybridization of reporter DNA and the exposed arms of the tweezer.Simultaneously,QDs and AF488 on the two arms were closed enough to generate ERET,increasing the ECL intensity of QDs remarkably.Impressively,the sensor could be regenerated by a one-step strand displacement and could be cycled for more than seven times.Owing to the dual amplification strategy and the high efficiency of the ERET between QDs and AF488,the proposed biosensor performed the linear range from 10 pmol L^-1 to 0.1 fmol L^-1 with a detection limit of 0.03 fmol L^-1for microRNA determination,and the monitoring of different cancer cells was also achieved.Amazingly,the elaborated biosensor can also realize the sensitive detection of Pb²⁺,which entailed the sensor be potentially used for field environmental analysis and monitoring,offering a new modular platform for the construction of functional DNA nanomachines in the ultra-high sensitive analysis of promising biomarkers and toxic metals.4.Dual MicroRNAs-fueled DNA Nanogears:A Case of Regenerated Strategy for Multiple Electrochemiluminescence Detection of MicroRNAs with Single LuminophoreThe determination of multiple microRNA biomarkers from cancer cells features a considerable step toward early diagnosis of cancers.However,realizing different microRNAs detection with single electrochemiluminescence?ECL?luminophore and regenerating the sensing platform remain a compelling goal in current researches.Herein,with the ingenious integration of DNA nanomachines and distance-based ECL response,dual microRNAs-fueled DNA nanogears were introduced in an enzyme-free ECL biosensor to perform the multiple sensitive detection of the microRNA biomarkers with single luminophore.As shown in the scheme,the nanogears system was assembled on the CdS quantum dots?QDs?modified sensing surface.Using microRNA-21 as the motive power,one of the nanogears?B?which was labeled with Au nanoparticles?AuNPs?could be activated to roll against another nanogear?A?,increasing the distance between AuNPs and CdS QDs.Thus the significant ECL enhancement of CdS QDs was obtained owing to the ECL energy transfer between AuNPs and CdS QDs,simultaneously realizing the detection of microRNA-21.After the incubation of microRNA-155,nanogear B revolved against nanogear A continuously and realized the close-range of AuNPs and CdS QDs,resulting in the quenching of the ECL intensity due to the F?rster energy transfer and realizing the analysis of microRNA-155.The successive locomotion of the nanogears on the sensing surface generated the corresponding response of ECL emission,which led to a significant increasing of ECL intensity for analysis of microRNA-21 down to 0.16 fmol L^-1 and a remarkable suppression of the ECL intensity for determination of microRNA-155 down to 0.33 fM.Impressively,the proposed biosensor performed multiple detection efficiently and exhibited excellent usability owing to the fact that the sensor was convenient and stable enough to be regenerated along with the gears roll against each other.In general,with the integration of the DNA nanotechnology,this enzyme-free strategy initiates a new thought to realize the multiple ECL detection with single luminophore,paving the way for applications of nanomachines in clinical diagnosis,sensing and other related subjects.5.A Smart Self-assembled 3D DNA Nanomachine with Reversible Photoswitching and Its Application for Rapid Single-Step Sensing of MicroRNAsOne-step,ultrasensitive and rapid determination of microRNA from cancer cells is the key factor for cancer diagnosis and treatment.Recently,development of 3D DNA nanomachines has made a progress.However,the overall immobilization of probes on Au surface is disordered and negatively influenced by steric effect and nonspecific interactions between probes/probes-Au,which may limit the movement efficiency of the nanomachine and destroy the biological activities of DNA.Herein,with a simple performance of solution annealing,a smart 3D DNA nanomachine is self-assembled by azobenzene?azo?-incorporated DNA nippers in rapid single-way,which have an improved moving efficiency due to organized and high local concentrated nippers comparing to the traditional Au-based 3D nanomachine.Once microRNA is modified on the 3D nanomachine,the nippers“open”for hybridization of nippers and microRNA.Impressively,the photoisomerization of azo induces dehybridization/hybridization of nippers and microRNA under different wavelength irradiation,which easily solve one main technical challenge for DNA nanotechnology and biosensing:reverse and regeneration in one step within 10 min.As a proof of concept,the elaborated3D machine is successfully applied in rapid one-step detection of biomarker,which gives impetus to design new generation of mechanical devices beyond the traditional ones,with the ultimate purpose of sensing analysis and diagnostic technology.