GR-5 DNAzyme Research Articles

Extended GR-5 DNAzyme-based Autonomous isothermal Cascade machine: An efficient and sensitive one-tube colorimetric platform for Pb2+ detection

Development of simple, rapid and highly sensitive platforms for heavy metal ions on-site visual monitoring is extremely challenging and appealing for addressing environmental pollution and public health issues. Herein, we present an extended GR-5 DNAzyme-based autonomous isothermal cascade machine (EGD-AICM) for visible detection of trace Pb2+ in a single tube system. A home-designed GR-5 complex is assembled from an extended GR-5 DNAzyme (GR-5E, integrating GR-5 DNAzyme with a DNA track) and its corresponding extended substrate (GR-5S). Once Pb2+ triggers GR-5S cleavage, the machine automatically initiates isothermal strand displacement amplification to synthesize numerous EAD2 DNA copies. The released copies further integrate with hemin to form horseradish peroxidase (HRP)-mimicking DNAzymes, which efficiently promotes H2O2-mediated oxidation of ABTS and generates amplified colorimetric signals. EGD-AICM enables label-free and one-tube determination of Pb2+ with a short assay time (∼50 min), excellent selectivity and high sensitivity (LOD = 35.25 pM). In addition, the proposed EGD-AICM has been employed to measure Pb2+ in real water and shrimp samples with satisfactory accuracy and precision. Such a straightforward and economical EGD-AICM platform demonstrates great potential for routinely screening metal ions in environmental monitoring, food safety assessment, and public safety fields.

Ultrasensitive Detection of Pb2+ Based on a DNAzyme and Digital PCR.

In this study, an ultrasensitive detection method for aqueous Pb2+ based on digital polymerase chain reaction (dPCR) technology and a Pb2+-dependent DNAzyme was developed. In the presence of Pb2+, the Gr-5 DNAzyme was activated and catalyzed the hydrolytic cleavage of the substrate strand, resulting in an increase in the amount of template DNA available for dPCR and a resultant change in the number of droplets showing a positive signal. Moreover, the detection system was found to be sensitive and stable in environmental sample detection. In summary, an ultrasensitive quantitative detection method for Pb2+ within environmental substrates was established.

A new impedimetric sensor based on anionic intercalator for detection of lead ions with low cost and high sensitivity

A novel impedimetric sensor, based on DNA intercalator, GR-5 DNAzyme and the low-cost gold interdigitated electrodes (GID), was studied to detect lead ions in this paper. It takes GR-5 DNAzyme as the recognition element immobilized on GID and anthraquinone-2-sulfonic acid (AQMS) as the DNA intercalator for improving its sensitivity of lead ions detection. When the GID modified with GR-5 DNAzyme were incubated in intercalator solution, AQMS will intercalate into the base-pairing regions of GR-5 DNAzyme. In the presence of Pb2+ ions, the GR-5 DNAzyme was activated and the substrate strand was cleaved into two parts at the RNA site (rA), which results in the releasing of AQMS from the GR-5 DNAzyme. With the decrease of AQMS, the magnitudes of the impedance increased, which relates to the Pb2+ concentration, was obtained. Our results demonstrate that the proposed sensor has a detection limit of 0.33 nM, and the linear range from 1 nM to 100 nM with a high selectivity. Finally, the sensor shows an average recovery of 99.31–107.85% when it was used to detect Pb2+ in water samples. Additionally, the GID were made on printed circuit board (PCB) which was cost-effective and mass production because of the mature produce technology. Furthermore, the application of AQMS leads to significantly decrease of the detection limit of the Pb2+. Therefore, the strategy shown that a promising applications in environmental and food-field monitoring.

Solid-State Nanopore Single-Molecule Sensing of DNAzyme Cleavage Reaction Assisted with Nucleic Acid Nanostructure

The detection and investigation of biomolecules at a single-molecule level is important for improving diagnosis in biomedicine. Solid-state nanopores are a unique tool that have the potential to accomplish this task because they are label-free and require only low sample consumption. However, the event-readouts of current small polymer molecules are still limited because of its relatively large size and low signal-to-noise ratios. Here, we present a rapid sensing approach for the detection of GR-5 DNAzyme cleaving specific substrate reactions using relatively larger size silicon nitride nanopores by introducing a type of nucleic acid nanostructure (DNA tetrahedron) as a carrier. The proposed method is convenient and sensitive enough to detect the cleavage reactions by identifying translocation events before and after reactions with nanomolar concentrations of the target sample. Furthermore, this assay was also carried out by using larger size nanopores (60 nm diameter) to achieve the DNAzyme cleavage sensing with the same sample concentration. This approach can improve event detectability of other smaller molecules' translocation, which opens up a wide range of applications for analytes detection by incorporating solid-state nanopores. Nucleic acid nanostructure-assisted nanopore sensing can promote the development of single-molecule studies.

Graphene oxide-assisted Au nanoparticle strip biosensor based on GR-5 DNAzyme for rapid lead ion detection.

This study has reported that a GR-5 DNAzyme based lead ion strip biosensor could exhibit an enhanced specificity with the assistance of graphene oxide (GO). This enhancement results from the specific π-stacking interaction between the ribose rings of the nucleobases and the carbon hexagons in GO which can reduce the false positive interference by removing unhybridized ssDNA during the annealing of GR-5 DNAzyme. Meanwhile, conjugate pad was sprayed with two kinds of AuNP-DNA probes, and nitrocellulose membrane test zone and control zone were immobilized with two kinds of biotin-DNA probes, respectively. The limit of detection of this strip biosensor was estimated to be about 0.05 nM (S/N = 3) and 1 nM (with naked eyes) with a linear range from 0.01 to 100 μM. Furthermore, the strip biosensor exhibited excellent selectivity toward Pb2+ in the presence of other divalent metal ions. For real soil samples, the obtained recoveries were in the range from 91.5% to 113.1%.

A protease-free and signal-on electrochemical biosensor for ultrasensitive detection of lead ion based on GR-5 DNAzyme and catalytic hairpin assembly

A simple protease-free and signal-on electrochemical biosensor based on GR-5 DNAzyme and catalytic hairpin assembly (CHA) for sensitive lead ion (Pb2+) assay was constructed in this work. In the presence of Pb2+, GR-5 DNAzyme specifically reacted with Pb2+ and cleaved the substrate strand into two free DNA fragments, and one of the two fragments then opened the hairpin capture DNA (Hc) that was assembled on the Au electrode surface and triggered the CHA reaction. Exploiting the CHA strategy, a large number of the hairpin signal DNA (Hs) labeled with two methylene blues (Mbs) were captured on the Au electrode surface and generated an efficient electron transfer, resulting in the dramatic increase of methylene blue current. In this method, Pb2+ could be detected quantitatively in the range of 4 × 10−11–3 × 10−6 M, and the detection limit was as low as 2.7 × 10−11 M (S/N = 3). Experimental results demonstrated that the biosensor was highly specific for Pb2+ and exhibited remarkable improvements of electrochemical analytical performance. This biosensor was also used for the analysis of Pb2+ in the serum sample spiked with Pb2+, and the obtained result in good agreement with the correct values. The excellent performance of the biosensor shows its promising potential for the clinical diagnosis of lead poisoning.

A label–free GR–5DNAzyme sensor for lead ions detection based on nanoporous gold and anionic intercalator

A label–free electrochemical sensor, based on a classic lead ions (Pb2+)–dependent GR–5DNAzyme as the catalytic unit, disodium–anthraquinone–2,6–disulfonate (AQDS) as DNA intercalator, and nanoporous gold (NPG) for signal amplification, was designed for sensitive and selective detection of Pb2+. Firstly, NPG modified electrode surface were employed as a platform for the immobilization of thiolated probe DNA, and then, hybridized with DNAzyme catalytic beacons. The Pb2+–induced catalytic reaction makes the substrate strand break at the cleavage sitGe irreversibly, which disturbs the formation of DNA strands. AQDS served as an indicator that intercalated into the base–pairing regions of DNAzyme, resulting in a strong electrochemical signal. In the presence of Pb2+, the complementary regions were reduced, due to the fracture of the DNA strand, resulting in the release of AQDS. And a decreased current was obtained, corresponding to Pb2+ concentration. Taking advantage of the amplification effect of NPG electrode for increasing the reaction sites of thiol modified capture probe, the proposed electrochemical biosensor could detect Pb2+ quantitatively, in the range of 1–120nM, with a limit of detection as low as 0.02nM, which is much lower than the maximum contamination level for Pb2+ in drinking water defined by the U.S. Environmental Protection Agency. The electrochemical sensor was also used to detect Pb2+ from real water samples, and the results showed excellent agreement with the values determined by inductively coupled plasma mass spectroscopy. This biosensor showed a promising potential for on–site detecting Pb2+ in aqueous environment.

A label-free DNAzyme fluorescence biosensor for amplified detection of Pb2+-based on cleavage-induced G-quadruplex formation

DNAzyme-based catalytic beacons have been widely studied for both in vivo and in vitro molecular detection. However, only a few label-free catalytic beacons with excellent analytical performance have been reported so far. In this work, by combining a catalytic DNAzyme for amplified sensing through enzymatic turnover with cleavage-induced G-quadruplex formation, a label-free DNAzyme biosensor was developed for amplified “turn-on” fluorescence detection of Pb2+ with a detection limit of 3nM. The method is very competitive compared to many other labeled or label-free methods with or without signal amplification. Due to the inherent specificity of the GR-5 DNAzyme, the method also exhibits excellent selectivity. This biosensor successfully detected Pb2+ in river water samples with high sensitivity and selectivity. Such a method might provide a universal DNAzyme-based sensing platform for sensitive detection of various targets both in environmental and biomedical fields.

Nanoparticles-free fluorescence anisotropy amplification assay for detection of RNA nucleotide-cleaving DNAzyme activity.

Fluorescence anisotropy is a homogeneous, sensitive, ratiometric, and real-time analytical technology. However, it is a great challenge to produce a large fluorescence anisotropy change upon the presence of target small molecules without nanoparticles-dependent amplification. This work reports a nanoparticle-free and multiple G-enhanced fluorescence anisotropy assay for detection of DNAzyme activity. A Pb(2+)-dependent GR-5 DNAzyme was used as a model. We hybridized the rA-cleavable substrate strand containing a TMR label at the 5'-end with the DNAzyme strand containing an extended three G bases at the 3'-end. By this design, we demonstrate that both fluorescence quenching and the enhanced DNAzyme activity contribute to a Pb(2+)-induced large fluorescence anisotropy change (|Δr| = 0.168). The limit of detection for Pb(2+) is estimated to be about 100 pM with a dynamic range from 200 pM to 100 nM. The interference from the other nine divalent metal ions of 1000-times excess amount is negligible. Moreover, we show an extended assay for evaluation of the interactions of Pb(2+) with cysteine and glutathione by the detection of GR5 DNAzyme activity. Collectively, we developed a novel fluorescence anisotropy amplification assay, enabling us to detect DNAzyme activity and associated cofactors and inhibitors and to characterize the Pb(2+)-chelation capability of free thiols.

Photocaged DNAzymes as a general method for sensing metal ions in living cells.

DNAzymes, which are sequences of DNA with catalytic activity, have been demonstrated as a potential platform for sensing a wide range of metal ions. Despite their significant promise, cellular sensing using DNAzymes has however been difficult, mainly because of the "always-on" mode of first-generation DNAzyme sensors. To overcome this limitation, a photoactivatable (or photocaged) DNAzyme was designed and synthesized, and its application in sensing Zn(II) in living cells was demonstrated. In this design, the adenosine ribonucleotide at the scissile position of the 8-17 DNAzyme was replaced by 2'-O-nitrobenzyl adenosine, rendering the DNAzyme inactive and thus allowing its delivery into cells intact, protected from nonspecific degradation within cells. Irradiation at 365 nm restored DNAzyme activity, thus allowing the temporal control over the sensing activity of the DNAzyme for metal ions. The same strategy was also applied to the GR-5 DNAzyme for the detection of Pb(II), thus demonstrating the possible scope of the method.

A highly sensitive and selective biosensing strategy for the detection of Pb2+ ions based on GR-5 DNAzyme functionalized AuNPs

In this paper, a novel fluorescence biosensing strategy based on GR-5 DNAzyme has been developed for the simple, rapid, sensitive and selective detection of lead ions (Pb2+) by using gold nanoparticles (AuNPs) as the fluorescence quencher. Data from dynamic light scattering (DLS) and zeta potential measurements demonstrated that GR-5 DNAzyme functionalized AuNPs were successfully prepared with good dispersion in aqueous solution. The carboxyfluorescein (FAM) labeled GR-5 DNAzyme was in close proximity to the AuNPs and the fluorescence signal was quenched, when the DNAzyme was modified on the surface of AuNPs. In the presence of Pb2+ ions, the GR-5 DNAzyme was activated and cleaved the substrate strand at the RNA site (rA) into two parts, which led to a short FAM-linked oligonucleotide fragment being released from the surface of the AuNPs, and the fluorescence intensity was gradually recovered. By taking advantage of the high fluorescence quenching efficiency of AuNPs, our proposed method exhibited a high sensitive detection of Pb2+ ions with a detection limit of 250 pM and a linear range from 1 nM to 100 nM. With the choice of a classic GR-5 DNAzyme instead of 8-17 DNAzyme as the catalytic unit, the selectivity of this strategy was obviously improved compared with previously reported methods. Moreover, this biosensing strategy was also used for the determination of Pb2+ in lake water samples with satisfying results. The strategy would find potential applications in environmental and biomedical fields.

Input-dependent induction of G-quadruplex formation for detection of lead (II) by fluorescent ion logic gate

A label-free fluorescent AND logic gate has been developed utilizing ion-tuned configuration conversion of DNA probe with K+ and Pb2+ as two inputs. A well-designed hairpin DNA which is composed of a poly-G loop and a GR-5 DNAzyme stem serves as a recognition probe, and an derivative of aloe-emodin (AED) was designed and synthesized as signal probe. In the presence of Pb2+, the substrate strand of DNAzyme is irreversibly and specifically cleaved at the cleavage site, which made the poly-G loop form G-quadruplex in the presence of a constant concentration of K+. Such a structural change significantly affects the spectral behaviors of AED, which can be explored to ultra-sensitively detect Pb2+ with a limit of detection of 22.8pM. By combing the high specificity of hairpin DNA and GR-5 DNAzyme, Pb2+ can be highly selectively detected even when coexisted with other metal ions. Circular dichroism (CD), UV–vis absorption spectrometry and fluorescence polarization (FP) measurements further verified the reliability and reasonability of the sensing mechanism. Therefore, it provides a simple and label-free approach to detect ions with high sensitivity and specificity, and promises to provide a solid sensing platform for the detection of targets by altering the specific sequence of nucleic acid probe.

A sensitive and selective DNAzyme-based flow cytometric method for detecting Pb2+ions

A sensitive and selective Pb(2+) sensor based on GR-5 DNAzyme has been developed by a flow cytometric method.

Graphene–DNAzyme Based Biosensor for Amplified Fluorescence “Turn-On” Detection of Pb2+ with a High Selectivity

On the basis of the remarkable difference in affinity of graphene (GO) with ssDNA containing a different number of bases in length, we for the first time report a GO-DNAzyme based biosensor for amplified fluorescence "turn-on" detection of Pb(2+). A FAM-labeled DNAzyme-substrate hybrid acted as both a molecular recognition module and signal reporter and GO as a superquencher. By taking advantage of the super fluorescence quenching efficiency of GO, our proposed biosensor exhibits a high sensitivity toward the target with a detection limit of 300 pM for Pb(2+), which is lower than previously reported for catalytic beacons. Moreover, with the choice of a classic Pb(2+)-dependent GR-5 DNAzyme instead of 8-17 DNAzyme as the catalytic unit, the newly designed sensing system also shows an obviously improved selectivity than previously reported methods. Moreover, the sensing system was used for the determination of Pb(2+) in river water samples with satisfying results.