Peroxidase-mimicking DNAzyme Research Articles

Homogeneous label-free protein binding assay using small-molecule-labeled DNA nanomachine with DNAzyme-Based chemiluminescence detection

Detecting the interactions between small molecules and proteins was critical for disease theranostics and drug development. Here we propose a novel universal assay strategy for monitoring small molecule-protein interactions in solution using strand displacement amplification (SDA) mediated by protein binding to small molecule with DNAzyme-based chemiluminescence detection. The DNA polymerase and nicking enzyme assisted SDA could yield a great amount of peroxidase-mimicking DNAzyme sequences which cause significantly chemiluminescence signals, while protein binding to the small molecule label would prevent DNA polymerase from extending nick site and DNAzyme sequence, and thus the chemiluminescence signals would obviously decrease. This strategy was demonstrated using folate and its binding protein (folate receptor), and the results revealed that the developed strategy enable offer a label-free, homogeneous, and highly sensitive chemiluminescence detection of folate receptor with a detection limit of 1pM. At the same time, it has been successfully used for folate receptor detection in human serum. The proposed chemiluminescence sensing method might provide a generic, robust, and high-throughput platform for detecting various small molecule-protein interactions for biological applications.

Electrochemical sandwich aptasensor for the carcinoembryonic antigen using graphene quantum dots, gold nanoparticles and nitrogen doped graphene modified electrode and exploiting the peroxidase-mimicking activity of a G-quadruplex DNAzyme.

A sandwich-type electrochemical aptasensor has been constructed and applied for sensitive and selective detection of the carcinoembryonic antigen (CEA). The surface of a glassy carbon electrode (GCE) was first modified with nitrogen-doped graphene and then gold nanoparticles and graphene quantum dots electrodeposited on it to obtain an architecture of type GQD/AuNP/NG/GCE. In the next step, the CEA-binding aptamer was immobilized on the modified GCE. Hemin intercalates in the amino-modified hemin aptamer to form a hemin-G-quadruplex (hemin-G4) DNAzyme. The amino modified CEA aptamer II is connected to hemin-G4 by glutaraldehyde (GA) as a linker to produce CEAaptamerII/GA/hemin-G4 (=ApII/GA/DNAzyme). Through a sandwich mode, the ApII/GA/DNAzyme bioconjugates are captured on the modified GCE. Subsequently, the hemin-G4 acts as peroxidase-mimicking DNAzyme and rapidly catalyzes the electroreduction of hydrogen peroxide. The quantitative determination of CEA was achieved by differential pulse voltammetry, best at a working potential of around -0.27V vs. Ag/AgCl. Under optimized conditions, the assay has a linear response in the 10.0fgmL-1 to 200.0ngmL-1 CEA concentration range and a lower detection limit of 3.2fgmL-1. Graphical abstract Schematic presentation of a sandwich-type electrochemical aptasensor based on nitrogen doped graphene (NG), gold nanoparticles (AuNPs) and graphene quantum dots (GQDs) modified glassy carbon electrode, and thehemin-G4 DNAzyme for femtomolar detection of thecarcinoembryonic antigen.

Electrocatalytic Efficiency Regulation between Target-Induced HRP-Mimicking DNAzyme and GOx with Low Background for Ultrasensitive Detection of Thrombin

Herein, an efficient target-activated enzyme cascade electrocatalysis with low background signal was employed to establish electrochemical biosensor for ultrasensitive detection of thrombin via regulating electrocatalytic efficiency between target-induced hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme (HRP-mimicking DNAzyme) and glucose oxidase (GOx).Impressively, only when the target thrombin was introduced, the HRP-mimicking DNAzyme acting simultaneously as electrochemical signal probe would be formed to activate high-efficiency enzyme cascade electrocatalysis for reducing background signal significantly, which could overcome the defect of inevitable high background signal during the detection of target in the traditional cascade electrocatalysis of two existing bioenzymes. In addition, the detection sensitivity could be further improved by regulating the side length of rigid DNA tetrahedron (TDN) scaffold anchored HRP-mimicking DNAzyme and GOx at adjacent vertices for high enzyme cascade electrocatalytic efficiency. Consequently, the proposed biosensor demonstrated a low detection limit down to 0.3 fM for target thrombin, which provided a promising method for ultrasensitive monitoring of biomolecules in sensing analysis and disease diagnosis.

Task-specific ionic liquid-enabled mercury sensor for sensitive detection of total mercury in food digestion solution

Heavy metal sensors are limited to the applications for environmental and drinking waters due to matrix interference. We report here a task-specific ionic liquid enabled DNAzyme-based mercury sensor (TSIL-Hg-sensor), allowing the sensitive detection of mercury in solid food samples. In TSIL-Hg-sensor, TSILs sequentially extracted mercury ion from microwave acid digestion solution with high efficiency, and then peroxidase-mimicking DNAzyme (PMD) that was used for the following signal amplification. Hg2+ and PMD shared the extraction capacity of TSILs. The greater amount of Hg2+ extracted from the first extraction would results in the less PMD from the second extraction, enabling the signal-on colorimetric detection of mercury. The detection of limit was 0.5 nM when [OMIM][PF6] was used for the extractions, 100 times more sensitive than the previously reported PMD-based signal-off sensor. The sensor with [Opy][BF4] was less sensitive, but highly specific for Hg2+. The practical applications were demonstrated by challenging TSIL-Hg-sensor with 5 standard solid foods containing mercury from 5.3 to 850 μg/kg with the recover percentage of 102 ˜115%. TSILs are designable for almost any molecules besides metal ions. Nucleic acids are compatible with numerous signal amplification means. TSIL-Hg-sensor therefore represents a universal design for sensors with high matrix interference resistance.

Graphene oxide-based NET strategy for enhanced colorimetric sensing of miRNA

MicroRNAs (miRNAs), short non-coding RNAs, have emerged as promising next-generation biomarkers of diverse diseases. In general, conventional quantitative real-time PCR and miRNA microarray methods have been utilized for the quantitative detection of miRNA. However, owing to their complicated procedures and expensive reagents/instruments, these methods cannot be widely applied by medical experts such as doctors and nurses, and are thus limited for practical clinical application. Here, we established a new graphene oxide (GO)-based NET strategy for facile miRNA detection with enhanced sensitivity, which is applicable for point-of-care testing (POCT). The proposed miRNA sensing strategy is fundamentally based on the peroxidase-mimicking DNAzyme (Dz) mediated colorimetric assay in response to target miRNA using rationally designed duplex molecular beacon (dMB). The application of GO as a net in this basic system (designated GONET) allows for gathering of the target-dependently activated Dz, which enhances the sensitivity and extends the applicability for POCT. The GONET system provides clear visualization of the target at the 10−9 M scale with the naked eye without any complicated amplification steps.

Temporal and Reversible Control of a DNAzyme by Orthogonal Photoswitching.

The reversible switching of catalytic systems capable of performing complex DNA computing operations using the temporal control of two orthogonal photoswitches is described. Two distinct photoresponsive molecules have been separately incorporated into a split horseradish peroxidase-mimicking DNAzyme. We show that its catalytic function can be turned on and off reversibly upon irradiation with specific wavelengths of light. The system responds orthogonally to a selection of irradiation wavelengths and durations of irradiation. Furthermore, the DNAzyme exhibits reversible switching and retains this ability throughout multiple switching cycles. We apply our system as a light-controlled 4:2 multiplexer. Orthogonally photoswitchable DNAzyme-based catalysts as introduced here have potential use for controlling complex logical operations and for future applications in DNA nanodevices.

Controlling the Catalytic and Optical Properties of Aggregated Nanoparticles or Semiconductor Quantum Dots Using DNA-Based Constitutional Dynamic Networks.

Nucleic acid-based constitutional dynamic networks (CDNs) attract growing interest as a means to mimic complex biological networks. The triggered stabilization of the CDNs allows the dictated guided reversible reconfiguration and re-equilibration of the CDNs to other CDN configurations, where some of the constituents are up-regulated, while other constituents are down-regulated. Although substantial progress in controlling the adaptive dynamic control of the compositions of networks by means of auxiliary triggers was demonstrated, the use of CDNs as active ensembles for controlling chemical functionalities is still a challenge. We report on the assembly of signal-triggered CDN systems that guide the switchable aggregation of Au nanoparticles (NPs), thereby controlling their plasmonic properties and their catalytic functions (Au NPs-catalyzed oxidation of l-DOPA to dopachrome). In addition, we demonstrate that the triggered and orthogonal up-regulation and down-regulation of the constituents of the CDNs leads to the dictated aggregation of different-sized CdSe/ZnS quantum dots (QDs), cross-linked by K+-ion-stabilized G-quadruplex units. The incorporation of hemin into the G-quadruplex cross-linking units yields horseradish peroxidase-mimicking DNAzyme units that catalyze the generation of chemiluminescence via the oxidation of luminol by H2O2. The resulting chemiluminescence stimulates the chemiluminescence resonance energy transfer (CRET) process to the QDs, resulting in the luminescence of the two-sized QDs. By the application of appropriate triggers, the CDN-dictated up-regulation and down-regulation of the different-sized QDs aggregates are demonstrated, and the control over the photophysical functions of the different-sized QDs, by means of the CDNs, is highlighted.

Chemiluminescent Determination of MicroRNA-141 Using Target-Dependent Activation of the Peroxidase-Mimicking DNAzyme

A chemiluminescent method was developed for microRNA-141 (miRNA-141) detection based on the target-dependent activation of peroxidase-mimicking DNAzyme. The structure of the probes was optimized which allowed the development of a sensitive method for miRNA-141. Under the optimized conditions, the detection limit and the linear range were 100 pM and 0.1–50 nM, respectively. The sensitivity of the assay was 270,000 nM−1. The values of coefficient of variation measured within the working range varied less than 2%, which indicates excellent precision for the proposed method.

DNAzyme-embedded hyperbranched DNA dendrimers as signal amplifiers for colorimetric determination of nucleic acids.

A DNAzyme-embedded hyperbranched DNA dendrimer is used as a colorimetric signal amplifier in an ultrasensitive detection scheme for nucleic acids. The hyperbranched DNA dendrimers were constructed by single-step autonomous self-assembly of three structure-free DNA monomers. A cascade of self-assembly reactions between the first and second strands leads to the formation of linear DNA concatemers containing overhang flank fragments. The third strand, which bears a peroxidase-mimicking DNAzyme domain, serves as a bridge to trigger self-assembly between the first and second strands across the side chain direction. This results in a chain branching growth of the DNAzyme-embedded DNA dendrimer. This signal amplifier was incorporated into the streptavidin-biotin detection system which comprises an adaptor oligonucleotide and a biotinylated capture probe. The resulting platform is capable of detecting a nucleic acid target with an LOD as low as 0.8 fM. Such sensitivity is comparable if not superior to most of the reported enzyme-free (and even enzyme-assisted) signal amplification strategies. The DNA dendrimer based method is expected to provide a universal platform for extraordinary signal enhancement in detecting other nucleic acid biomarkers by altering the respective sequences of adaptor and capture probe. Graphical abstract Schematic of an assembly of a DNAzyme-embedded hyperbranched DNA dendrimer which operates as a signal amplifier for nucleic acids detection. The nanostructure is constructed by autonomous self-assembly of three DNA monomers. Colored letters represent each domain, and complementary domains aremarked by asterisk. Domain d represents the DNAzyme sequence.

A DNA as a Substrate and an Enzyme: Direct Profiling of Methyltransferase Activity by Cytosine Methylation of a DNAzyme.

Inhibiting DNA methyltransferase (MTase) activity is crucial for cancer treatment. Evaluating drug candidates as inhibitors of MTase requires the measurement of its activity. However, direct profiling of MTase activity remains an analytical challenge, since a complicated hydrolysis step using methylation-sensitive restriction enzymes (msRE) was inevitable in almost all previously reported methods. Taking advantage that DNA is the substrate of MTase and certain DNA sequences, known as DNAzymes, can also have enzyme-like activities, we herein developed an enzyme-free and label-free route for direct assaying MTase activity. Specifically, adding a cytosine at the meta-position of a peroxidase-mimicking DNAzyme can improve the catalytic activity of DNAzyme up to 5-fold. After methylation of the cytosine cap, the activity is further doubled. Based on these findings, direct assaying of MTase inhibitors was performed without the extra and complicated hydrolysis step. These new findings provide a helpful tool for screening drugs for cancer therapy. The idea of using the same DNA as a substrate and an enzyme could be a general way for assaying other enzymes.

Sensitive and rapid aptasensing of chloramphenicol by colorimetric signal transduction with a DNAzyme-functionalized gold nanoprobe

By combination of the aptamer biorecognition with the colorimetric signal transduction of a DNAzyme-functionalized nanoprobe, a new biosensing method was developed for the rapid and sensitive detection of chloramphenicol (CAP). The nanoprobe was prepared through the functionalization of gold nanoparticles with the complementary oligonucleotide against aptamer and high-content hemin/G-quadruplex DNAzyme. When one-step incubating the nanoprobe and CAP at a constructed aptamer-magnetic bead (MB) biosensing platform, due to the competitive biorecognition reaction, the nanoprobes related with CAP amounts were quantitative captured onto the MB surface. Based on the catalytic reaction of the peroxidase-mimicking DNAzyme, a colored substance was produced for the colorimetric signal transduction of the method. Due to the great signal amplification of the nanoprobe, a very low detection limit down to 0.13 pg/mL was obtained. Considering the excellent performance of the aptasensing method and satisfactory results for milk sample experiments, it indicates good reliability for practical applications.

Target-triggered three-way junction in conjugation with catalytic concatemers-functionalized nanocomposites provides a highly sensitive colorimetric method for miR-21 detection

With the great advances in DNA nanotechnology, scientists have shown interest in developing dynamic nanostructures for theranostic applications, analyte sensing and cargo delivery. Here, we present a specific enzyme-free ultrasensitive platform based on a multilayer coupled signal amplification strategy to quantify miR-21 molecule. The biosensor was integrated based on three signal amplification gadgets, namely a translator-mediated catalytic hairpin assembly (CHA), a multilayer DNA concatemer on the surface of gold decorated magnetic nanoparticle (GMNP), and a DNAzyme-mediated catalytic signal amplification. MiR-21 mediates the release of a DNA translator from an immobilized duplex to engage in a CHA reaction using three hairpins, including a GMNP-conjugated hairpin 1 (H1), biotin-labeled hairpin 2 (H2) and a GMNP-conjugated hairpin 3 (H3) to form a three-way junction (3WJ). Meanwhile, a plenty of initiator strand 0 (S0) on GMNPs – each of which has been bifunctionalized with S0/H1 or S0/H3 – drive several multilayer peroxidase-mimicking DNAzyme concatemers in the presence of two accessory oligonucleotides; strand 1 (S1) and strand 2 (S2). Since a G-rich sequence was attached at the 5′-end of S1 strand, in the presence of hemin cofactor, an active G-quadruplex DNAzyme with peroxidase activity was formed. The concatemers on the surface of GMNPs can convert a colorless substrate to a green product. The biosensor can detect as low as 1 aM of miR-21 and provide an excellent capability to discriminate single-base mismatches. The required time for the formulation of the assay reagents is about three days and the reaction time for the detection of miR-21 takes place in less than four hours.

Label-free ochratoxin A electrochemical aptasensor based on target-induced noncovalent assembly of peroxidase-like graphitic carbon nitride nanosheet

The development of simple, fast and sensitive aptasensor for OTA plays an important role in food safety due to its severe toxicity. In this research, taking advantage of the intrinsic peroxidase-like activity of g-C3N4 nanosheet (g-CNNS) and its high affinity toward signal-strand DNA, a label-free electrochemical OTA aptasensor was fabricated. There is an outstanding property that the aptasensor did not require labeled aptamer and immobilization of g-CNNS compared with previous g-CNNS-based aptasensors. The presence of OTA induced the conformational change of the aptamer, and then g-CNNS noncovalently assembled with the free complementary strand on the electrode via π-π interaction. The captured g-CNNS catalyzed the oxidation of H2O2 to amplify current signal. As a result, this aptasensor realized the detection limit of 0.073 nM, and was also employed to assay OTA in the real samples, including red wines, juices and corns. More importantly, the development of this label-free electrochemical aptasensor opens up the application of g-CNNS as the peroxidase-mimicking DNAzyme.

Allosteric Control of Peroxidase-Mimicking DNAzyme Activity with Cationic Copolymers

Control of protein conformation and function, induced by the binding of an effector, plays significant roles in modulating biochemical reaction. Although the DNAzymes catalytic activity is similar to protein-based enzymes, reports of allosterically controlled DNAzymes are still limited except for aptamer-DNAzymes hybrrids. Here, we report allosteric control of peroxidase-mimicking DNAzyme activity using cationic copolymers. The DNAzyme requires a structured G-quadruplex core and hemin for activity, and the DNAzyme with a parallel G-quadruplex core has higher DNAzyme activity than DNAzymes based on other types of structure. We previously reported that a cationic copolymer composed of a cationic backbone and hydrophilic dextran side chains selectively stabilizes parallel G-quadruplex structures. In this study, we investigated effects of the cationic copolymer on peroxidase-mimicking DNAzyme activity. The cationic copolymer enhanced the DNAzyme activity by more than 30-fold by stabilizing the parallel G-quadruplex structure. Furthermore, reversible allosteric control of DNAzyme activity was achieved by adding cationic and anionic polymers.

A Paper Sensor Printed with Multifunctional Bio/Nano Materials.

We report a paper-based aptasensor platform that uses two reaction zones and a connecting bridge along with printed multifunctional bio/nano materials to achieve molecular recognition and signal amplification. Upon addition of analyte to the first zone, a fluorescently labelled DNA or RNA aptamer is desorbed from printed graphene oxide, rapidly producing an initial fluorescence signal. The released aptamer then flows to the second zone where it reacts with printed reagents to initiate rolling circle amplification, generating DNA amplicons containing a peroxidase-mimicking DNAzyme, which produces a colorimetric readout that can be read in an equipment-free manner or with a smartphone. The sensor was demonstrated using an RNA aptamer for adenosine triphosphate (a bacterial marker) and a DNA aptamer for glutamate dehydrogenase (Clostridium difficile marker) with excellent sensitivity and specificity. These targets could be detected in spiked serum or feacal samples, demonstrating the potential for testing clinical samples.

Homogeneous Chemiluminescent Determination of Mercury(II) Using a Peroxidase-Mimicking DNAzyme Assay

ABSTRACTEnvironmental pollution in manufacturing sectors is often accompanied by the release of diverse forms of pollutants including heavy metals. Mercury is one of the most toxic heavy metals. Here, we describe a homogeneous chemiluminescent method for Hg2+ detection based on allosteric activation of peroxidase-mimicking DNAzyme and formation of Hg2+-thymine bonds in DNA duplex with T–T mismatches in the presence of mercury. The formation of such duplex increased the activity of peroxidase-mimicking DNAzyme. The analysis conditions and structures of probes were optimized. Under the favorable conditions, the limit of detection and a linear range of the assay were 12 and 12–600 nM, respectively. The values of coefficient of variation measured within the working range varied from 0.7 to 3.0%. The study of cross-reactivity of Hg2+, Ag+, Pb2+, Ca2+, Zn2+, Bi3+, Ni2+, Co2+, Ba2+, Mn2+, Cd2+, Mg2+, and Cr3+ showed that only mercury in concentration nanoscale activates peroxidase-mimicking DNAzyme that indicates high specificity of the developed Hg2+ assay. Thus, an easy-to-use, specific, rapid, and sensitive method for Hg2+ detection was developed.

Aptamer–integrated DNA nanoassembly: A simple and sensitive DNA framework to detect cancer cells

The development of powerful techniques to detect cancer cells at early stages plays a notable role in diagnosing and prognosing cancer patients and reducing mortality. This paper reports on a novel functional DNA nanoassembly capable of detecting cancer cells based on structural DNA nanotechnology. DNA nanoassemblies were constructed by the self-assembly of a DNA concatemer to a plenty of sticky-ended three-way junctions. While an aptamer moiety guided the nanoassembly to the target cancer cell, the peroxidase-mimicking DNAzymes embedded in the nanoassemblies were used as the sensing element to produce colorimetric signals. As proof-of-concept, as low as 175 cancer cells were detected by the assay, and color change was clearly distinguished by the naked eyes. The proposed system enjoys potential applications for point-of-care cancer diagnosis, with its excellent sensitivity and selectivity.

Colorimetric detection of microRNA based on DNAzyme and nuclease-assisted catalytic hairpin assembly signal amplification

Accurate and quantitative analysis of microRNA (miRNA) expression is critical for the diagnostics and theranostics of a disease. Herein, a proof-of-concept of a colorimetric horseradish peroxidase-mimicking DNAzyme (HRP-DNAzyme) biosensor for miRNA assay based on nuclease-assisted catalytic hairpin assembly (CHA) signal amplification was demonstrated. Duplex-specific nuclease (DSN) was employed to cleave the single-stranded DNA (ssDNA) chimeric probe (CP) on the magnetic bead (MB) surface via hybridization of the CP and target miRNA. The regenerated miRNA can cleave a large number of ssDNA CP to produce CHA initiator sequence fragments. The CP consists of two main regions: a target miRNA recognition DNA sequence at the 5′ end and a CHA initiator (CI) sequence at the 3′ end. The catalyzed assembly process of CHA produces a large amount of G-rich DNA. In the presence of hemin, the G-rich DNA forms G-quadruplex/hemin complex and mimics the horseradish peroxidase activity, which catalyzes a colorimetric reaction. For the proof-of-concept, microRNA-21 (miR-21) was selected as the model target to authenticate this strategy as a versatile assay platform. The proposed strategy allowed quantitation of the sequence specificity of miRNA-21 with a detection limit of 9.2 fM in a dynamic range from 10 fM–1 nM, with an excellent ability to discriminate the differences in miRNAs. Additionally, the miRNA assay in real samples was satisfactory, thereby confirming its applicability. Therefore, this method exhibited a great potential as a miRNA quantification method in biomedical research and clinical diagnosis.

Chemiluminescent Detection of HIV DNA Based on Allosteric Activation of Peroxidase-Mimicking DNAzyme

Homogeneous chemiluminescent method for HIV DNA detection based on allosteric activation of peroxidase-mimicking DNAzyme (PMDNAzyme) was developed. The probes used in the assay contain PMDNAzyme fragment and the additional oligonucleotide sequence complementary to HIV DNA. The interaction of PMDNAzyme fragment and the additional oligonucleotide sequence results in changes in G-quadruplex structure of the PMDNAzyme and decreases peroxidase-like activity of the probe. In the presence of HIV DNA such interaction was destroyed due to the formation of stable duplex between the additional fragment of the probe and DNA-analyte. Consequently, some reorganizations in G-quadruplex structure of the probe are observed, which are accompanied by enhancement of catalytic activity of the PMDNAzyme. The mechanism of the DNA-dependent activation of PMDNAzyme containing probes was confirmed by CD spectroscopy as well as modeling of the probes and their complexes with DNA target. The calibration curves for HIV DNA determination allowed estimating the analytical parameters of the assay. The detection limit value and the linear range were shown to be 0.3 nM and 0.3–15 nM, respectively. The assay sensitivity was high (190000 nM–1). The values of coefficient of variation (CV) measured within the working range varied less than 4%, which indicates the high accuracy of the proposed assay.

Optical and electrochemical detection of a verotoxigenic E. coli gene using DNAzyme-labeled stem-loops

The activity of a peroxidase-mimicking DNAzyme was optimized to be used as a catalytic label in a stem-loop genosensor construction for quantifying the gene sequence Shiga-like toxin I of verotoxigenic E. coli. Experimental conditions such as pH, buffer composition, potassium ion concentration, and hemin-to-oligonucleotides ratio, were analyzed to maximize optical and electrochemical responses using microvolumes. Different stem-loop constructions were evaluated to obtain the optimum response against the target concentration. Linear ranges of 0.05-0.5 µM and limits of detection of 174 nM and 144 nM were estimated for the optical and electrochemical measurements, respectively. Selectivity was proved by assaying other verotoxigenic, enterotoxigenic and enteroinvasive sequences. The results show that, if a combination of small-volume electrochemical cells and low-cost untreated screen-printed electrodes with a relatively high geometric area is used, electrochemical measurements present similar sensitivity and limits of detection to the more usual optical ones, allowing the development of low-cost electrochemical biosensors based on the use of soluble DNAzymes as labels.