Quantum Dot Nanobeads-Labelled Lateral Flow Immunoassay Strip for Rapid and Sensitive Detection of Salmonella Typhimurium Based on Strand Displacement Loop-Mediated Isothermal Amplification

Rapid,sensitive,point-of-care detection of pathogenic bacteria is important for food safety.In this study,we developed a novel quantum dot nanobeads-labelled lateral flow immunoassay strip(QBs-labelled LFIAS)combined with strand displacement loop-mediated isothermal amplification(SD-LAMP)for quantitative Salmonella Typhimurium(ST)detection.Quantum dot nanobeads(QBs)served as fluorescence reporters,providing good detection efficiency.The customizable strand displacement(SD)probe was used in LAMP to improve the specificity of the method and prevent by-product capture.Detection was based on a sandwich immunoassay.A fluorescence strip reader measured the fluorescence intensity(FI)of the test(T)line and control(C)line.The linear detection range of the strip was 10^(2)–10^(8) colony forming units(CFU)·mL^(-1).The visual limit of detection was 10^(3) CFU·mL^(-1),indicating that the system was ten-fold more sensitive than AuNPs-labelled test strips.ST specificity was analyzed in accordance with agarose gel outputs of polymerase chain reaction(PCR)and SD-LAMP.We detected ST in foods with an acceptable recovery of 85%–110%.The method is rapid,simple,almost equipment-free,and suitable for bacterial detection in foods and for clinical diagnosis.

Toeless and reversible DNA strand displacement based on Hoogsteen-bond triplex

Strand displacement reaction is a crucial component in the assembly of diverse DNA-based nanodevices,with the toehold-mediated strand displacement reaction representing the prevailing strategy.However,the single-stranded Watson-Crick sticky region that serves as the trigger for strand displacement can also cause leakage reactions by introducing crosstalk in complex DNA circuits.Here,we proposed the toeless and reversible DNA strand displacement reaction based on the Hoogsteen-bond triplex,which is compatible with most of the existing DNA circuits.We demonstrated that our proposed reaction can occur at pH 5 and can be reversed at pH 9.We also observed an approximately linear relationship between the degree of reaction and pH within the range of pH 5-6,providing the potential for precise regulation of the reaction.Meanwhile,by altering the sequence orientation,we have demonstrated that our proposed reaction can be initiated or regulated through the same toeless mechanism without the requirement for protonation in low pH conditions.Based on the proposed reaction principle,we further constructed a variety of DNA nanodevices,including two types of DNA logic gates that rely on pH 5/pH 9 changes for initiating and reversing:the AND gate and the OR gate.We also successfully constructed a DNA Walker based on our proposed reaction modes,which can move along a given track after the introduction of a programmable DNA sequence and complete a cycle after 4 steps.Our findings suggest that this innovative approach will have broad utility in the development of DNA circuits,molecular sensors,and other complex biological systems.

Toehold-mediated strand displacement reaction-propelled cascade DNAzyme amplifier for microRNA let-7a detection

DNAzyme amplifiers have been extensively explored as a useful sensing platform,but single DNAzyme amplifier is limited in biosensing applications by its low sensitivity.Herein,a cascade DNAzyme amplifier was designed by exploiting concurrent amplification cycle principles of toehold-mediated strand displacement reaction(TSDR)and Zn^(2+)-assisted DNAzyme cycle with lower cost and simpler procedures.Compared with single DNAzyme amplifier,the proposed TSDR-propelled cascade DNAzyme amplifier exhibited higher sensitivity by releasing more DNAzyme through TSDR to cleave substrate strand during the DNAzyme cycle.Base on this,let-7a could be sensitively detected in the range of 5-50 nmol/L with a detection limit of 64 pmol/L.Furthermore,the dual signal amplification strategy of the cascade DNAzyme amplifier exhibited excellent selectivity to distinguish single-base mismatched DNA strands,which has been successfully applied to the determination of let-7a in blood serum,showing high promise in early cancer diagnosis.

Visually Intracellular Detection of Telomerase Activity Based on DNA Strand Displacement Reaction and Gold Nanoparticle Labeling

Telomerase,which is regarded as a common biomarker for early cancer diagnostics and a potential target for clinical therapies,has attracted considerable interests concerning its detection and monitoring.Herein,we propose a sensitive method by designing a gold nanoparticle(AuNP)probe for visually intracellular detection of telomerase activity.The AuNPs were functionalized with a telomer-ase substrate primer(SH-prime).A 6-carboxy-fluorescein(FAM)modified strand(FAM-probe)was attached to the surface of AuNP through its complementary stand(SH-attach).In the absence of telomerase,the fluorescence resonance energy transfer(FRET)from FAM to AuNPs results in efficient fluorescence quenching.In the presence of telomerase,SH-primers on AuNPs were extended with the repeat units(TTAGGGG)n.The extension sequence triggered the strand displacement of FAM-probe to restore the fluorescence signals.It is worth mentioning that the proposed strategy does not need to design complex hairpin structure and allows the meas-urement of telomerase in crude cell extracts down to 0.5 HeLa cells/μL in 2 h.In addition,the present sensing platform can be ap-plied to the visually intracellular detection of telomerase activity in living cells.

A primer-initiated strand displacement amplification strategy for sensitive detection of 5-Hydroxymethylcytosine in genomic DNA

5-Hydroxymethylcytosine(5 hmC),an intermediate product of DNA demethylation,is important for the regulation of gene expression during development and even tumorigenesis.The challenges associated with determination of 5 hm C level include its extremely low abundance and high structural similarity with other cytosine derivatives,which resulted in sophisticated treatment with large amount of sample input.Herein,we developed a primer-initiated strand displacement amplification(PISDA)strategy to quantify the global 5 hm C in genomic DNA from mammalian tissues with high sensitivity/selectivity,low input and simple operation.This sensitive fluorescence method is based on 5 hmC-specific glucosylation,primer ligation and DNA amplification.After the primer was labeled on 5 hm C site,DNA polymerase and nicking enzyme will repeatedly act on each primer,causing a significant increase of fluorescence signal to magnify the minor difference of 5 hm C content from other cytosine derivatives.This method enables highly sensitive analysis of 5 hm C with a detection limit of 0.003%in DNA(13.6 fmol,S/N=3)from sample input of only 150 ng,which takes less than 15 min for determination.Further determination of 5 hmC in different tissues not only confirms the widespread presence of 5 hmC but also indicates its significant variation in different tissues and ages.Importantly,this PISDA strategy exhibits distinct advantages of bisulfite-free treatment,mild conditions and simple operation without the involvement of either expensive equipment or large amount of DNA sample.This method can be easily performed in almost all research and medical laboratories,and would provide a promising prospect to detect global 5 hmC in mammalian tissues.

Recent advances in molecular machines based on toehold-mediated strand displacement reaction

Background： The DNA strand displacement reaction, which uses flexible and programmable DNA molecules as reaction components, is the basis of dynamic DNA nanotechnology, and has been widely used in the design of complex autonomous behaviors. Results： In this review, we first briefly introduce the concept of toehold-mediated strand displacement reaction and its kinetics regulation in pure solution. Thereafter, we review the recent progresses in DNA complex circuit, the assembly of AuNPs driven by DNA molecular machines, and the detection of single nucleotide polymorphism （SNP） using DNA toehold exchange probes in pure solution and in interface state. Lastly, the applications of toehold-mediated strand displacement in the genetic regulation and silencing through combining gene circuit with RNA interference systems are reviewed. Conclusions： The toehold-mediated strand displacement reaction makes DNA an excellent material for the fabrication of molecular machines and complex circuit, and may potentially be used in the disease diagnosis and the regulation of gene silencing in the near future.

Cascade Toehold-Mediated Strand Displacement Reaction for Ultrasensitive Detection of Exosomal MicroRNA

MicroRNA(miRNA)in exosomes is a powerful molecular signature for early diagnosis of cancers with the merits of high specificity and high stability.Herein,we report an ultrasensitive electrochemical assay to measure miRNA using a cascade toeholdmediated strand displacement reaction(SDR).In SDR,the trapped exosomal miRNA releases a large amount of single-stranded DNA in the solution.The product then triggers the downstream SDR at the electrode surface.

Lineal DNA logic gate for microRNA diagnostics with strand displacement and fluorescence resonance energy transfer

Designing molecular logic gates to operate programmably for molecular diagnostics in molecular computing still remains challenging.Here,we designed a novel linear DNA logic gates for microRNA analysis based on strand displacement and fluorescence resonance energy transfer（FRET）.Two labeled strands closed each other produce to FRET through hybridization with a complementary strand to form a basic work unit of logic gate.Two indicators of heart failure（microRNA-195 and microRNA-21） were selected as the logic inputs and the fluorescence mode was used as the logic output.We have demonstrated that the molecular logic gate mechanism worked well with the construction of YES and AND gates.

Research progress and prospects of nucleic acid isothermal amplification technology

Nucleic acid(DNA and RNA)detection and quantification methods play vital roles in molecular biology.With the development of molecular biology,isothermal amplification of DNA/RNA,as a new molecular biology technology,can be amplified under isothermal condition,it has the advantages of high sensitivity,high specificity,and high efficiency,and has been applied in various fields of biotechnology,including disease diagnosis,pathogen detection,food hygiene and safety detection and so on.This paper introduces the progress of isothermal amplification technology,including rolling circle amplification(RCA),nucleic acid sequence-dependent amplification(NASBA),strand displacement amplification(SDA),loop-mediated isothermal amplification(LAMP),helicase-dependent amplification(HDA),recombinase polymerase amplification(RPA),cross-priming amplification(CPA),and its principle,advantages and disadvantages,and application development are briefly summarized.

New insight into the mechanism of DNA polymerase I revealed by single-molecule FRET studies of Klenow fragment

We use single-molecule FRET and newly-developed D-loop techniques to investigate strand displacement activity of Klenow fragment(exo-)of DNA polymerase I in DNA sequences rich in guanine and cytosine(GC)bases.We find that there exist in the FRET traces numerous ascending jumps,which are induced by the backsliding of Klenow fragment on DNA chains.Our measurements show that the probability of backsliding is closely related to the GC-richness and d NTP concentration:increasing the GC-richness leads to an increase in the backsliding probability,and increasing the d NTP concentration however leads to a decrease in the backsliding probability.These results provide a new insight into the mechanism of DNA polymerase I.

Triplex-structure based DNA circuits with ultra-low leakage and high signal-to-noise ratio

DNA circuits are powerful tools in various applications such as logical computation,molecular diagnosis and synthetic biology.Leakage is a major problem in constructing complex DNA circuits.It directly affects the output signal and harms the circuit’s performance significantly.In the traditional DNA circuits,the gate complex is a duplex structure.There are insufficient energy barriers to prevent spontaneous detachment of strands,resulting in a leak prone.Herein,we have developed triplex-structure based DNA circuit with ultra-low leakage and high signal-to-noise ratio(SNR).The triplex structure improves the stability in the absence of input.At the same time,the driving force of the strand displacement cascades reduces the influence of the triplex structure on the desired reaction.The SNR of the DNA circuit was increased to 695,while the desired reaction rate remained 90%of the conventional translator circuit.The triplex-structure mediated leakage prevention strategy was further tested at different temperatures and in DNA translator and seesaw circuits.We also constructed modular basic logic gates with a high efficiency and low leakage.On this basis,we further constructed triplex-structure based tertiary DNA logic circuits,and the SNR reached 295,which,to the best of our knowledge,was among the highest of the field.We believe that our scheme provides a novel,valid,and general tool for reducing leakages,and we anticipate that it will be widely adopted in DNA nanotechnology.

Simultaneous photoelectrochemical detection of dual microRNAs by capturing CdS quantum dots and methylene blue based on target-initiated strand displaced amplification

Herein,we propose a novel photoelectrochemical(PEC) biosensor for dual microRNAs(miRNAs) highly sensitive and simultaneous biosensing based on strand displaced amplification(SDA) reaction.The recognition of HmiR-21 and Hlet-7 a by microRNA-21 and let-7 a leads to their change in hairpin structures,subsequently initiating the immobilization of abundant CdS quantum dots(CdS QD s) and methylene blue(MB) based on SDA reaction.The immobilized CdS QDs and MB produce both high PEC currents under430 nm light and 627 nm light illumination,respectively,and the generated PEC currents are closely relied on target miRNAs amounts.Thus,highly sensitive and simultaneous detection of microRNA-21 and let-7 a was readily achieved with detection limit at 6.6 fmol/L and 15.4 fmol/L based on 3σ,respectively.Further,this PEC biosensor was applied in simultaneous analysis of miRNA-21 and let-7 a in breast cancer patient’s serum with acceptable results.We expect this biosensor will find more useful application in diagnosis of miRNA-related diseases.

Electrochemical analysis of microRNAs with hybridization chain reaction-based triple signal amplification

Selective and sensitive detection of trace microRNA is important for early diagnosis of diseases due to its expression level related to diseases.Herein,a triple signal amplification strategy is developed for trace microRNA-21 (miRNA-21) detection by combining with target-triggered cyclic strand displacement reaction (TCSDR),hybridization chain reaction (HCR) and enzyme catalytic amplification.Four DNA hairpins(H1,H2,H3,H4) are employed to form an ultralong double-strand DNA (dsDNA) structure,which is initiated by target miRNA-21.As H3 and H4 are labeled with horseradish peroxidase (HRP),numerous HRPs are loaded on the long dsDNA,producing significantly enhanced electrocatalytic signals in the hydrogen peroxide (H_(2)O_(2)) and 3,3,5,5-tetramethylbenzidine (TMB) reaction strategy.Compared with single signal amplification,the triple signal amplification strategy shows higher electrochemical response,wider dynamic range and lower detection limit for miRNA-21 detection with excellent selectivity,reproducibility and stability.Taking advantage of the triple signal amplification strategy,the proposed electrochemical biosensor can detect miRNA-21 in 10 He La cell lysates,suggesting that it is a promising method for fruitful assay in clinical diagnosis.

Addressable DNA Information Processing System with a Fluorescent Readout for Rewritable Memory

DNA-based nanostructure allows the construction of molecular devices useful in biological computing and information processing.Herein,an addressable and editable DNA information processing system established on a fluorescence intensity signal detection platform to save and encrypt information is proposed.The system operates by encoding information into distinct and changeable units of the trigger strands decoding by fluorescence intensity signal detection.Through toehold-mediated strand displacement reactions,the trigger strand can be precisely added to and removed from the memorizer and reporter to implement the function of editing,encrypting,and decrypting.Our strategy is simple to implement,requiring only two mixing steps at room temperature for each operation and fluorescence intensity signal detection to read the data.And the system can realize accurate retrieval of specific individual information,eliminating all unnecessary redundant readouts.Because of its point-to-point accurate readout and programmability,the system is expected to become a powerful tool for the future development of information storage and sensing of biological molecules.

Locked nucleic acids based DNA circuits with ultra-low leakage

DNA circuits based on toehold-mediated DNA strand displacement reaction are powerful tools owing to their programmability and predictability.However,performance and practical application of the circuits are greatly restricted by leakage,which refers to the fact that there is no input(invading strand)in the circuit,and the output signal is still generated.Herein,we constructed locked nucleic acids-based DNA circuits with ultra-low leakage.High binding affinity of LNA(locked nucleic acid)-DNA/LNA suppressed the leakage by inhibiting the breathing effect.Based on the strategy,we have built various low-leakage DNA circuits,including translator circuit,catalytic hairpin assembly(CHA)circuit,entropy-driven circuit(EDC),and seesaw circuit.More importantly,our strategy would not affect the desired main reactions:The output signal remained above 85%for all tested circuits,and the signalto-noise ratios were elevated to 148.8-fold at the most.We believe our strategy will greatly promote the development and application of DNA circuits-based DNA nanotechnology.

TNF-αresponsive DNA star trigon formation from four hairpin probes and the analytical application

Tumor necrosis factor-alpha(TNF-α) is a type of critical pro-inflammatory cytokines,which participates in numerous cellular signal pathways and is regarded as a critical protein biomarker for inflammatory based diseases.In this contribution,we have developed a strategy to fabricate multiple DNA star trigon structures with fluorescence signals from four hairpin probes which are detonated by a single molecule of TNF-α.This process causes significant enhancement of fluorescence and a sensitive and selective biosensor for TNF-α assay is constructed.This method is able to achieve the limit of detection(LOD) at 5 pg/mL(0.285 pM).Moreover,some other advantages such as fast response,high selectivity and convenient operation promise the potential use of this method for TNF-α measurement in point of care testing application.Upon further development,this strategy can also be converted to detect other analytes such as small molecules,nucleic acids and other proteins.

一种以上转换和Fe_(3)O_(4)纳米粒子为基础的超灵敏ctDNA检测系统

ctDNA是重要的肿瘤标志物之一,在肿瘤疾病的早期诊断和临床监控方面发挥着重要作用.目前,常用的ct DNA检测方法通常都缺乏足够的灵敏性和特异性,不能满足实际诊疗的需要.因此,我们急需一种新型的、超灵敏的ct DNA检测方法.上转换纳米粒子是一种优异的发光材料,因其独特的发光特性在生物医学方面具有重要应用.因此,我们设计了一种以上转换和Fe_(3)O_(4)纳米粒子为基础的ct DNA检测方法.在该检测系统中,上转换和Fe_(3)O_(4)纳米粒子通过DNA碱基互补配对原则自组装在一起.目标序列可以通过熵驱动链置换(ESDR)反应将连着上转换纳米粒子的核酸链释放出来.经磁性分离后,上清液中上转换纳米粒子在980 nm激光器激发下的荧光强度与目标序列的浓度在1 nmol L^(-1)–100 amol L^(-1)的范围内具有非常好的线性关系,经计算,最低检出浓度为1.6 amol L^(-1).此外,该检测系统在识别单碱基突变、实际细胞和血清样本的检测中均有较好的应用.因此,该检测系统因为其灵敏性和特异性在肿瘤早期诊断、疾病进行过程监控、治疗方法评价和愈后风险评估等方面具有重大应用潜力.

Target Recycling Transcription of Lighting-Up RNA Aptamers for Highly Sensitive and Label-Free Detection of ATP

We describe here a target recycling transcription of lighting-up aptamer strategy for detecting ATP in human serums in a label-free means with high sensitivity.ATP molecules specifically recognize the binding aptamer and result in the structure switching of the DNA assembly probes to imitate the target ATP molecule recycling cycles through the toehold-mediated strand displacement reaction,which causes the formation of many dsDNAs containing the RNA promoter sequences for subsequent transcription generation of large amounts of lighting-up aptamers.The organic dye,malachite green,then associates with these lighting-up aptamers to produce significantly enhanced fluorescence signals,which can sensitively detect ATP within a dynamic range from 10 to 500 nM in a label-free way.The sensing approach shows a detection limit of 7.3 nM and also has an excellent selectivity for ATP analogue molecules.In addition,this method can detect ATP molecules in diluted human serum samples sensitively,which proves the promising potential to diagnose ATP-related diseases.

Ultrasensitive assay of ctDNA based on DNA triangular prism and three-way junction nanostructures

Circulating tumor DNA(ctDNA) refers to a class of acellular nucleic acids carrying genetic features of primary tumor,which can be regarded as a promising noninvasive biomarker for cancer diagnosis.The development of ctDNA assay is an important component of liquid biopsy.In this study,we have fabricated a novel electrochemical strategy for ultrasensitive detection of ctDNA combining the merits of strand displacement amplification and DNA nanostructures.Stable DNA triangular prism is firstly selfassembled and modified on the electrode surface.After target initiated strand displacement polymerization reaction,the generated DNA product helps the formation of three-way junction nanostructure on triangular prism,which localizes electrochemical species.By carefully investigating the electrochemical responses,the limit of detection(LOD) for ctDNA assay as low as 48 amol/L is achieved.This proposed electrochemical biosensor shows great potential for clinical applications.