DNAzyme Nanowires Research Articles

Chemiluminescence resonance energy transfer-based multistage nucleic acid amplification circuits for MiRNA detection with low background.

Chemiluminescence resonance energy transfer (CRET)-based assays have shown great potential in biosensing due to their negligible background autofluorescence, yet are still limited by their low sensitivity and short half-life luminescence. Herein, a multistage CRET-based DNA circuit was constructed with amplified luminescence signals for accurate miRNA detection and fixed reactive oxygen species (ROS) signals for cell imaging. The DNA circuit is designed through an ingenious programmable catalytic hairpin assembly (CHA), hybridization chain reaction (HCR), and the use of DNAzyme to realize target-triggered precise regulation of distance between the donor and acceptor for CRET-mediated excitation of photosensitizers. In detail, the analyte catalyzes the hybridization of CHA reactants, which leads to the assembly of multiple HCR-mediated DNAzyme nanowires. Subsequently, DNAzymes catalyze the oxidation of luminol by H2O2, and the adjacent photosensitizer chlorin e6 (Ce6) anchored on the DNA nanostructure is stimulated by the CRET process, resulting in the amplified long-wavelength luminescence and the generation of single oxygen signals through further energy transfer to oxygen. The biomarker miRNA can be detected with great sensitivity by integrating the recognition module into a universal platform. Furthermore, the DNA circuit enables CRET-mediated intracellular miRNA imaging, by detecting singlet oxygen signals through a ROS probe. The significant amplification effect is attributed to the robust multiple recognition of the target and the guaranteed transduction of the CRET signal through programmable engineering of DNA nanostructures. The CRET-based DNA circuit achieves amplified long-wavelength luminescence for accurate miRNA detection with low background and ROS-mediated signal fixation for cell imaging, making it a promising candidate for early diagnosis and theranostics.

Target-triggered assembly of functional G-quadruplex DNAzyme nanowires for sensitive detection of miRNA in lung tissues

MicroRNAs (miRNAs) are important biomarkers for multiple diseases including cancers, neurodegenerative diseases, and cardiovascular diseases. Accurate detection of miRNAs is imperative for early diagnosis and biomedical research. Herein, we demonstrate target-triggered assembly of G-quadruplex DNAzyme nanowires for sensitive detection of miRNAs in lung tissues by integrating primer generation-mediated rolling circle amplification (PG-RCA) with primer exchange reaction (PER). Target miRNA hybridizes with the circular template to initiate PG-RCA, producing numerous primers. The obtained primers then react with the catalytic hairpin DNA template to trigger PER, producing the polymer G-quadruplex DNAzyme nanowires. Eventually, thioflavin T (ThT) as a fluorescence indicator selectively interacts with the G-quadruplex DNAzyme to generate an amplified signal. This assay is capable of detecting miRNA over a large dynamic range (up to 6 orders of magnitude) and it exhibits high sensitivity with a limit of detection (LOD) of 61.34 aM. Moreover, this assay displays excellent specificity and it can distinguish even a single-base mutation in miR-486–5p. Furthermore, this method can quantify the miR-486–5p concentration in different types of cells, and it enables the identification of differentially expressed miR-486–5p in lung tissues of normal persons and nonsmall-cell lung carcinoma (NSCLC) patients, holding promising applications in biomedical analysis and cancer prognosis.

A highly-efficient 3D DNAzyme motor for sensitive biosensing analysis

Herein, driven by the need of highly-efficient DNAzyme-amplified detection strategy, a novel 3D DNAzyme motor was designed as a biosensor platform for realizing sensitive detection of target DNA. The 3D DNAzyme motor was composed of target-activated DNAzyme nanowires and substrates H1-Fc that co-immobilized on Au@Fe3O4 nanoparticles (Au@Fe3O4NPS) surface, possessing high local concentration of DNA reactants and shortened distance between DNAzyme and substrates for enhancing electrochemical signal. Compared with traditional DNAzyme-powered machines, the target-activated DNAzyme nanowires of 3D DNAzyme motor had greater flexibility and more powerful cleavage capability without troublesome sequence optimization, which overcame the space limitation and simultaneously interacted with adjacent and distant substrates H1-Fc to output a large amount of cleavage products with high signal response. Therefore, on account of the above-mentioned merits of nanoparticles localization DNA design and DNAzyme nanowires, the reported 3D DNAzyme motor ingeniously overcame many defects existing in traditional DNAzyme-amplified detection strategies such as low reactants concentration, limited flexibility of DNAzyme and small DNAzyme swing range, realizing the sensitive detection of target DNA with a detection limit of 1.7 fM ranging from 5 fM to 50 nM. Impressively, the 3D DNAzyme motor here presented a new strategy to achieve effective DNAzyme signal amplification and provided a reference for the assembly of various and functional 3D DNA machines in the future.

Cooperative In Situ Assembly of G-Quadruplex DNAzyme Nanowires for One-Step Sensing of CpG Methylation in Human Genomes.

CpG methylation is one the most predominant epigenetic modification that has been recognized as a molecular-level biomarker for various human diseases. Taking advantage of methylation-dependent cleavage and encoding flexibility in nucleic acid functions and structures, we demonstrate the cooperative in situ assembly of G-quadruplex DNAzyme nanowires for one-step sensing of CpG methylation in human genomes. This nanodevice displays good specificity and high sensitivity with a limit of detection (LOD) of 0.565 aM in vitro and 1 cell in vivo. It can distinguish 0.001% CpG methylation level from excess unmethylated DNA, quantify different CpG methylation targets from diverse human cancer cells, and even discriminate CpG methylation expressions between lung tumor and precancerous tissues. Importantly, this nanodevice can be performed isothermally in one step within 2 h in a label-free manner without any bisulfite conversion, fluorescence tagging, and PCR amplification process, providing a new platform for genomic methylation-related clinical diagnosis and biomedical research.

A proteinase-free DNA replication machinery for in vitro and in vivo amplified MicroRNA imaging.

The construction of robust, modular and compact DNA machinery facilitates us to build more intelligent and ingenious sensing strategies in complex biological systems. However, the performance of conventional DNA amplifiers is always impeded by their limited in-depth amplifications and miscellaneously enzymatic requirements. Here, a proteinase-free reciprocal DNA replication machinery is developed by exploiting the synergistic cross-activation between hybridization chain reaction (HCR) and DNAzyme. The DNAzyme provides an efficient way to simplify the sophisticated design of HCR machinery and simultaneously to promote the amplification capacity. And the HCR-assembled tandem DNAzyme nanowires produce numerous new triggers for reversely stimulating HCR amplifier as systematically explored by experiments and computer-aided simulations. The reciprocal amplifier can be executed as a versatile and powerful sensing platform for analyzing miRNA in living cells and even in mice, originating from the inherent reaction accelerations and multiple-guaranteed recognitions. The reciprocal catalytic DNA machine holds great potential in clinical diagnosis and assessment.

Autocatalytic DNAzyme assembly for amplified intracellular imaging.

The autocatalytic HCR-DNAzyme platform was constructed as a versatile amplification platform for intracellular microRNA imaging by integrating hybridization chain reaction (HCR) circuit with DNAzyme biocatalysis. The HCR-assembled multifunctional DNAzyme nanowires produce new HCR triggers and numerous transducer DNAzyme amplifier, and thus shows great promise in earlier cancer diagnosis.

Sensitive electrochemical assay of alkaline phosphatase activity based on TdT-mediated hemin/G-quadruplex DNAzyme nanowires for signal amplification

Taking TdT-mediated hemin/G-quadruplex DNAzyme nanowires as NADH oxidase and HRP-mimicking DNAzyme, a novel DNA-based electrochemical method has been developed for sensitive and selective assay of alkaline phosphatase (AP) activity. The double-stranded DNA (dsDNA) probe consisted of thiol-functionalized DNA1 and 3′-phosphorylated DNA2, was immobilized on a gold nanoparticles (AuNPs) modified glassy carbon (GC) electrode. In the presence of AP, 3′-phosphoryl end of DNA2 was dephosphorylated. Terminal deoxynucletidyl transferase (TdT) catalyzed the sequential addition of deoxynucleotides (dTTPs) at 3′-OH end of DNA2 to extend DNA2 with a poly-T sequence. Then, G-rich DNA3 strand hybridized with the poly-T sequence of DNA2. Upon addition of hemin, the hemin/G-quadruplex DNAzyme was formed. In the presence of NADH, the hemin/G-quadruplex DNAzyme oxidased NADH to NAD+, accompanied by the formation of H2O2 which was further catalyzed by hemin/G-quadruplex DNAzyme (served as a HRP-mimicking DNAzyme) with the thionine (Thi) as electron transfer mediator, leading to the amplified electrochemical signal. Under optimized conditions, the response peak current was linear with the concentration of AP in the range from 0.1UL−1 to 5UL−1 with the detection limit of 0.03UL−1. Also, the developed biosensor possessed good selectivity, reproducibility and stability, and simple sensing structure, showing promising practical applications in AP activity assay.

Hemin/G-quadruplex DNAzyme nanowires amplified luminol electrochemiluminescence system and its application in sensing silver ions

Herein, a DNA biosensor with a hemin/G-quadruplex DNAzyme amplified luminol electrochemiluminescence (ECL) system was constructed for sensing silver ions (Ag+).

Ultrasensitive electrochemical detection of avian influenza A (H7N9) virus DNA based on isothermal exponential amplification coupled with hybridization chain reaction of DNAzyme nanowires

In this work, a simple and label-free electrochemical biosensor with duel amplification strategy was developed for DNA detection based on isothermal exponential amplification (EXPAR) coupled with hybridization chain reaction (HCR) of DNAzymes nanowires. Through rational design, neither the primer nor the DNAzymes containing molecular beacons (MBs) could react with the duplex probe which were fixed on the electrode surface. Once challenged with target, the duplex probe cleaved and triggered the EXPAR mediated target recycle and regeneration circles as well as the HCR process. As a result, a greater amount of targets were generated to cleave the duplex probes. Subsequently, the nanowires consisting of the G-quadruplex units were self-assembled through hybridization with the strand fixed on the electrode surface. In the presence of hemin, the resulting catalytic G-quadruplex–hemin HRP-mimicking DNAzymes were formed. Electrochemical signals can be obtained by measuring the increase in reduction current of oxidized 3.3′,5.5′-tetramethylbenzidine sulfate (TMB), which was generated by DNAzyme in the presence of H2O2. This method exhibited ultrahigh sensitivity towards avian influenza A (H7N9) virus DNA sequence with detection limits of 9.4fM and a detection range of 4 orders of magnitude. The biosensor was also capable of discriminating single-nucleotide difference among concomitant DNA sequences and performed well in spiked cell lysates.

A novel electrochemical aptasensor for highly sensitive detection of thrombin based on the autonomous assembly of hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme nanowires

In this work, a new signal amplified strategy was constructed based on isothermal exponential amplification reaction (EXPAR) and hybridization chain reaction (HCR) generating the hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme (HRP-mimicking DNAzyme) nanowires as signal output component for the sensitive detection of thrombin (TB). We employed EXPAR’s ultra-high amplification efficiency to produce a large amount of two hairpin helper DNAs within a minutes. And then the resultant two hairpin helper DNAs could autonomously assemble the hemin/G-quadruplex HRP-mimicking DNAzymes nanowires as the redox-active reporter units on the electrode surface via hybridization chain reaction (HCR). The hemin/G-quadruplex structures simultaneously served as electron transfer medium and electrocatalyst to amplify the signal in the presence of H2O2. Specifically, only when the EXPAR reaction process has occurred, the HCR could be achieved and the hemin/G-quadruplex complexes could be formed on the surface of an electrode to give a detectable signal. The proposed strategy combines the amplification power of the EXPAR, HCR, and the inherent high sensitivity of the electrochemical detection. With such design, the proposed assay showed a good linear relationship within the range of 0.1pM–50nM with a detection limit of 33 fM (defined as S/N=3) for TB.

Mediator-free triple-enzyme cascade electrocatalytic aptasensor with exonuclease-assisted target recycling and hybridization chain reaction amplification

The amplified sensitive detection of protein is essential to biomedical research as well as clinical diagnosis. Here, we developed an ultrasensitive mediator-free triple-enzyme cascade electrocatalytic aptasensor for thrombin detection on the basis of exonuclease-assisted target recycling and hybridization chain reaction (HCR) amplification strategy. The double strands constructed by the hybridization of thrombin binding aptamer (S1) with its complementary strand (S2) were firstly assembled on the electrode. Upon addition of target to the system, the S1 recognized thrombin and left off electrode to make space for assembly of hybrid-primer probe (H0). Then, the H0 triggered the HCR to form the multi-functional hemin/G-quadruplex DNAzyme nanowires. In the mediator-free triple-enzyme cascade electrocatalytic amplification system, the hemin/G-quadruplex DNAzyme nanowires here simultaneously played three roles: the redox probe, NADH oxidase and HRP-mimicking DNAzyme, respectively, which effectively avoided the fussy redox probe and enzyme labeling process, serving a useful alternative or supplement to conventional assays that typically suffer from complexity and poor sensitivity. Additionally, in order to improve the assembly amount of hemin/G-quadruplex DNAzyme nanowire, the exonuclease-assisted target recycling amplification was used for the continuous removal of S1. As a result, the proposed method can detect thrombin specifically with a detection limit as low as 20fM.

Sensitive pseudobienzyme electrocatalytic DNA biosensor for mercury(II) ion by using the autonomously assembled hemin/G-quadruplex DNAzyme nanowires for signal amplification

Herein, a novel sensitive pseudobienzyme electrocatalytic DNA biosensor was proposed for mercury ion (Hg2+) detection by using autonomously assembled hemin/G-quadruplex DNAzyme nanowires for signal amplification. Thiol functionalized capture DNA was firstly immobilized on a nano-Au modified glass carbon electrode (GCE). In presence of Hg2+, the specific coordination between Hg2+ and T could result in the assembly of primer DNA on the electrode, which successfully triggered the HCR to form the hemin/G-quadruplex DNAzyme nanowires with substantial redox probe thionine (Thi). In the electrolyte of PBS containing NADH, the hemin/G-quadruplex nanowires firstly acted as an NADH oxidase to assist the concomitant formation of H2O2 in the presence of dissolved O2. Then, with the redox probe Thi as electron mediator, the hemin/G-quadruplex nanowires acted as an HRP-mimicking DNAzyme that quickly bioelectrocatalyzed the reduction of produced H2O2, which finally led to a dramatically amplified electrochemical signal. This method has demonstrated a high sensitivity of Hg2+ detection with the dynamic concentration range spanning from 1.0ngL−1 to 10mgL−1 Hg2+ and a detection limit of 0.5ngL−1 (2.5pM) at the 3Sblank level, and it also demonstrated excellent selectivity against other interferential metal ions.

Amplified colorimetric detection of mercuric ions through autonomous assembly of G-quadruplex DNAzyme nanowires

An amplified colorimetric detection of Hg2+ is proposed by combining T–Hg2+–T base pairs and hybridization chain reaction (HCR). Two hairpins consisting of three-fourths and one-fourth of the horseradish peroxidase (HRP)-mimicking DNAzyme in inactive configuration are used as functional elements. In the presence of Hg2+, one of the hairpins is opened by an assistant probe with the help of T–Hg2+–T base pairs and this triggers an autonomous cross-opening of the two hairpins using the strand displacement principle, resulting in the formation of DNA nanowires consisting of numerous reunited Q-quadruplex DNAzyme units. The resulting catalytically active hemin/G-quadruplex HRP-mimicking DNAzymes catalyze the oxidation of ABTS2− by H2O2 into a green-colored cationic radical ABTS•+ for the colorimetric readout. This “turn-on” sensing system enables the high sensitive and selective detection of aqueous Hg2+ with a detection limit of 9.7pM.

An ultrasensitive electrochemical aptasensor with autonomous assembly of hemin–G-quadruplex DNAzyme nanowires for pseudo triple-enzyme cascade electrocatalytic amplification

We for the first time fabricate a pseudo triple-enzyme cascade electrocatalytic electrochemical aptasensor by using the alcohol dehydrogenase (ADH) as well as the autonomously assembled hemin-G-quadruplex that simultaneously acted as an NADH oxidase and HRP-mimicking DNAzyme.

Amplified Detection of DNA through the Enzyme-Free Autonomous Assembly of Hemin/G-Quadruplex DNAzyme Nanowires

An enzyme-free amplified detection platform is described using the horseradish peroxidase (HRP)-mimicking DNAzyme as an amplifying label. Two hairpin structures that include three-fourths and one-fourth of the HRP-mimicking DNAzyme in caged, inactive configurations are used as functional elements for the amplified detection of the target DNA. In the presence of the analyte DNA, one of the hairpins is opened, and this triggers the autonomous cross-opening of the two hairpins using the strand displacement principle. This leads to the formation of nanowires consisting of the HRP-mimicking DNAzyme. The resulting DNA nanowires act as catalytic labels for the colorimetric or chemiluminescent readout of the sensing processes (the term "enzyme-free" refers to a protein-free catalyst). The analytical platform allows the sensing of the analyte DNA with a detection limit corresponding to 1 × 10(-13) M. The optimized system acts as a versatile sensing platform, and by coaddition of a "helper" hairpin structure any DNA sequence may be analyzed by the system. This is exemplified with the detection of the BRCA1 oncogene with a detection limit of 1 × 10(-13) M.