DNAzyme Sensor Research Articles

A Syringe‐Based DNAzyme Sensor for Bacterial Detection

AbstractWe present a simple approach for bacterial detection that uses a bead‐bound RNA‐cleaving DNAzyme (RcD) and a syringe. The RcD used in this study, F‐EC1, is fluorescently labelled and can be specifically activated by a protein target released specifically by E. coli. Upon immobilization on agarose beads, the kinetic behaviour of F‐EC1 remains similar to that in solution. The F‐EC1‐agarose system was able to detect as few as 1,000 CFUs of E. coli and retained high recognition specificity. A syringe‐based biosensor was subsequently designed, where F‐EC1‐bearing agarose beads were loaded onto a filter‐containing column. Upon incubation with an E. coli containing reaction mixture, the cleavage fragment released from the beads was filtered onto a nitrocellulose paper to be detected. The sensor was used to achieve the detection of E. coli in water, milk, and apple juice. This method is cost‐effective, simple to use, and can be adapted to other ligand‐responsive RNA‐cleaving DNAzymes.

Cover Feature: A Syringe‐Based DNAzyme Sensor for Bacterial Detection (2/2021)

Cover Feature: A Syringe‐Based DNAzyme Sensor for Bacterial Detection (2/2021)

Detection and Quantification of Tightly Bound Zn2+ in Blood Serum Using a Photocaged Chelator and a DNAzyme Fluorescent Sensor.

DNAzymes have emerged as a powerful class of sensors for metal ions due to their high selectivity over a wide range of metal ions, allowing for on-site and real-time detection. Despite much progress made in this area, detecting and quantifying tightly bound metal ions, such as those in the blood serum, remain a challenge because the DNAzyme sensors reported so far can detect only mobile metal ions that are accessible to bind the DNAzymes. To overcome this major limitation, we report the use of a photocaged chelator, XDPAdeCage to extract the Zn2+ from the blood serum and then release the chelated Zn2+ into a buffer using 365 nm light for quantification by an 8-17 DNAzyme sensor. Protocols to chelate, uncage, extract, and detect metal ions in the serum have been developed and optimized. Because DNAzyme sensors for other metal ions have already been reported and more DNAzyme sensors can be obtained using in vitro selection, the method reported in this work will significantly expand the applications of the DNAzyme sensors from sensing metal ions that are not only free but also bound to other biomolecules in biological and environmental samples.

DNAzyme biosensors for the detection of pathogenic bacteria

• DNAzymes can be used for the detection and diagnosis of pathogenic bacteria. • DNAzymes can be used to detect pathogenic bacteria in food and environments. • DNAzymes have potential applications in molecules detection. • RNA-cleaving DNAzymes as a molecular beacon have great advantages in fabrication of sensors. Pathogenic bacteria contamination can cause many problems, such as infections in humans and animals and environmental pollution. Early prevention and detection of pathogenic bacteria outbreaks is crucial to managing these problems. Some traditional detection methods such as cell culturing and polymerase chain reaction (PCR) are time-consuming, labor-intensive, insensitive, and inconvenient for detection. Therefore, simple and efficient pathogenic bacteria detection methods involving DNAzymes signaling mechanisms have been reported, including fluorescence and color detection. DNAzymes are divided into cases as a molecular recognition element (RNA-cleaving DNAzyme) and as a reporter element (peroxidase mimicking DNAzyme), which have proven compatible with isothermal amplification technology for pathogenic bacteria detection. In this review, we summarize the current methods of detecting pathogenic bacteria based on various DNAzyme sensors. We include the design principles, working mechanisms, and detection applications of these sensors and discuss the experimental and theoretical bases for their application in the diagnosis and prevention of diseases caused by pathogenic bacteria.

An Enzyme-Activatable Engineered DNAzyme Sensor for Cell-Selective Imaging of Metal Ions.

There is intense interest in imaging intracellular metal ions because of their vital roles in many cellular processes. The challenge has been to develop chemical approaches that can detect metal ions in a cell-type-specific manner. Herein we report the design of an enzyme-activatable DNAzyme sensor technology that can distinguish metal-ion signals in tumor cells from those in normal cells both in vitro and in vivo. Specifically, the sensing activity of a traditional DNAzyme sensor was inhibited by engineering with a blocking sequence containing an abasic site that can be cleaved by cancer-specific enzymes and thus enables the selective recovery of metal-ion sensing capability in cancer cells. We demonstrated that the DNAzyme sensor not only enables cancer-cell-selective sensing and imaging of metal ions through an enzymically activated pathway, but also precise control over its metal-ion sensing activity in tumor-bearing mice. We envision the use of this biosensing technology to probe the biological roles of diverse metal ions in specific diseases.

An Enzyme‐Activatable Engineered DNAzyme Sensor for Cell‐Selective Imaging of Metal Ions

AbstractThere is intense interest in imaging intracellular metal ions because of their vital roles in many cellular processes. The challenge has been to develop chemical approaches that can detect metal ions in a cell‐type‐specific manner. Herein we report the design of an enzyme‐activatable DNAzyme sensor technology that can distinguish metal‐ion signals in tumor cells from those in normal cells both in vitro and in vivo. Specifically, the sensing activity of a traditional DNAzyme sensor was inhibited by engineering with a blocking sequence containing an abasic site that can be cleaved by cancer‐specific enzymes and thus enables the selective recovery of metal‐ion sensing capability in cancer cells. We demonstrated that the DNAzyme sensor not only enables cancer‐cell‐selective sensing and imaging of metal ions through an enzymically activated pathway, but also precise control over its metal‐ion sensing activity in tumor‐bearing mice. We envision the use of this biosensing technology to probe the biological roles of diverse metal ions in specific diseases.

Inside Back Cover: An Enzyme‐Activatable Engineered DNAzyme Sensor for Cell‐Selective Imaging of Metal Ions (Angew. Chem. Int. Ed. 12/2021)

Cell-selective imaging of metal ions was achieved by Lele Li and co-workers in their Communication on page 6300 by using an enzyme-activatable DNAzyme sensor technology. In the picture, the sensing activity is silent in normal cells and can be restored by an overexpressed enzyme in cancer cells through site-specific cleavage. The DNAzyme sensor allowed distinguishing metal-ion signals in tumor cells from healthy cells both in vitro and in vivo.

Innenrücktitelbild: An Enzyme‐Activatable Engineered DNAzyme Sensor for Cell‐Selective Imaging of Metal Ions (Angew. Chem. 12/2021)

Ein zellselektives Imaging von Metallionen wird von Lele Li et al. in ihrer Zuschrift auf S. 6370 durch den Einsatz einer enzymaktivierbaren DNAzym-Sensortechnologie erreicht. Im Bild ist die Sensoraktivität in normalen Zellen stumm und kann in Krebszellen durch ein überexprimiertes Enzym durch ortsspezifische Spaltung wiederhergestellt werden. Der DNAzym-Sensor ermöglichte die Unterscheidung von Metallionensignalen in Tumorzellen und gesunden Zellen sowohl in vitro als auch in vivo.

Intelligent demethylase-driven DNAzyme sensor for highly reliable metal-ion imaging in living cells.

The accurate intracellular imaging of metal ions requires an exquisite site-specific activation of metal-ion sensors, for which the pervasive epigenetic regulation strategy can serve as an ideal alternative thanks to its orthogonal control feature and endogenous cell/tissue-specific expression pattern. Herein, a simple yet versatile demethylation strategy was proposed for on-site repairing-to-activating the metal-ion-targeting DNAzyme and for achieving the accurate site-specific imaging of metal ions in live cells. This endogenous epigenetic demethylation-regulating DNAzyme system was prepared by modifying the DNAzyme with an m6A methylation group that incapacitates the DNAzyme probe, thus eliminating possible off-site signal leakage, while the cell-specific demethylase-mediated removal of methylation modification could efficiently restore the initial catalytic DNAzyme for sensing metal ions, thus allowing a high-contrast bioimaging in live cells. This epigenetic repair-to-activate DNAzyme strategy may facilitate the robust visualization of disease-specific biomarkers for in-depth exploration of their biological functions.

Visual Detection of SARS-CoV-2 RNA by Conventional PCR-Induced Generation of DNAzyme Sensor.

The gold standard for the diagnosis of SARS-CoV-2, the causative agent of COVID-19, is real-time polymerase chain reaction (PCR), which is labor-intensive, expensive, and not widely available in resource-poor settings. Therefore, it is imperative to develop novel, accurate, affordable, and easily accessible assays/sensors to diagnose and isolate COVID-19 cases. To address this unmet need, we utilized the catalytic potential of peroxidase-like DNAzyme and developed a simple visual detection assay for SARS-CoV-2 RNA using a conventional thermal cycler by the PCR-induced generation of DNAzyme sensor. The performance of RT-PCR DNAzyme-based sensor was comparable to that of real-time PCR. The pilot scale validation of RT-PCR DNAzyme-based sensor has shown ~100% sensitivity and specificity in clinical specimens (nasopharyngeal swab, n = 34), with a good correlation (Spearman r = 0.799) with the Ct-value of fluorescence probe-based real-time PCR. These findings clearly indicate the potential of this inexpensive, sensitive, and specific molecular diagnostic test to extend our testing capabilities for the detection of SARS-CoV-2 to curtail COVID-19 transmission.

A single-step DNAzyme sensor for ultra-sensitive and rapid detection of Pb2+ ions

Effective protection from Pb2+ contamination calls for its rapid detection in environmental and biological samples, including water sources. This work demonstrates a rapid, highly sensitive and specific DNAzyme-based Pb2+ biosensor, which is also the first reported label free bioelectronic sensor for catalytic hydrolysis. The sensors are prepared from gold-plated interdigitated electrodes (IDEs), functionalized with oligonucleotide substrate strands and subsequent hybridization with DNAzyme strands. After sample fluid is applied onto a sensor for detection, the sensor's serial capacitance is continuously interrogated by an optimized alternating current (AC) signal for 15 s. If Pb2+ is present, the DNAzyme catalyzes a cleavage reaction of the substrate, releasing the DNAzyme and substrate fragments into the solution. The reaction leaves partial substrate strand at the sensor surface, causing a change in the IDEs’ interfacial capacitance. Another novelty here is that capacitance measurement simultaneously induce a fluidic enrichment effect, AC electrothermal effect, thus concentrating and measuring Pb2+ in a single step. The AC signal is carefully optimized to minimize non-specific collection of macromolecules, including released DNAzyme and substrate fragments. In this work, Pb2+ level can be quantified in 15 s with a detection limit of 1.26 fM and a linear dynamic range from 1 fM to 1 pM in analytical buffer. To demonstrate the sensor's specificity, non-target metal ions are tested, all giving negligible responses. Testing of tap water samples collected under different conditions also yields reasonable results, validating the robustness of this sensor. This rapid and sensitive sensor holds high promise for on-site detection of Pb2+ in practical samples.

DNAzyme Sensor for the Detection of Ca2+ Using Resistive Pulse Sensing.

DNAzymes are DNA oligonucleotides that can undergo a specific chemical reaction in the presence of a cofactor. Ribonucleases are a specific form of DNAzymes where a tertiary structure undergoes cleavage at a single ribonuclease site. The cleavage is highly specificity to co-factors, which makes them excellent sensor recognition elements. Monitoring the change in structure upon cleavage has given rise to many sensing strategies; here we present a simple and rapid method of following the reaction using resistive pulse sensors, RPS. To demonstrate this methodology, we present a sensor for Ca2+ ions in solution. A nanoparticle was functionalised with a Ca2+ DNAzyme, and it was possible to follow the cleavage and rearrangement of the DNA as the particles translocate the RPS. The binding of Ca2+ caused a conformation change in the DNAzyme, which was monitored as a change in translocation speed. A 30 min assay produced a linear response for Ca2+ between 1–9 μm, and extending the incubation time to 60 min allowed for a concentration as low as 0.3 μm. We demonstrate that the signal is specific to Ca2+ in the presence of other metal ions, and we can quantify Ca2+ in tap and pond water samples.

Insight into an Oxidative DNA-Cleaving DNAzyme: Multiple Cofactors, the Catalytic Core Map and a Highly Efficient Variant.

SummaryAn oxidative DNA-cleaving DNAzyme (PL) employs a double-cofactor model “X/Cu2+” for catalysis. Herein, we verified that reduced nicotinamide adenine dinucleotide (NADH), flavin mononucleotide, cysteine, dithiothreitol, catechol, resorcinol, hydroquinone, phloroglucinol, o-phenylenediamine, 3,3′,5,5'-tetramethylbenzidine, and hydroxylamine acted as cofactor X. According to their structural similarities or fluorescence property, we further confirmed that reduced nicotinamide adenine dinucleotide phosphate (NADPH), 2-mercaptoethanol, dopamine, chlorogenic acid, resveratrol, and 5-carboxyfluorescein also functioned as cofactor X. Superoxide anions might be the commonality behind these cofactors. We subsequently determined the conservative change of individual nucleotides in the catalytic core under four different cofactor X. The nucleotides A4 and C5 are highly conserved, whereas the conservative levels of other nucleotides are dependent on the types of cofactor X. Moreover, we observed that the minor change in the PL's secondary structure affects electrophoretic mobility. Finally, we characterized a highly efficient variant T3G and converted its double-cofactor NADH/Cu2+ to sole-cofactor NADH.

Highly sensitive remote biosensor for the determination of lead (II) ions by using nanoporous anodic alumina modified with DNAzyme

Herein, we have designed a biosensor based on interferometric reflectance spectroscopy (IRS) for the lead (II) ion (Pb2+) ions sensing. For this purpose, the electrochemically fabricated nanoporous anodic alumina (NAA) was fabricated and then functionalized with amino-containing silane (NAA-NH2). After that, the amino-terminal G-rich DNA single strands were immobilized into the pores of NAA-NH2 by using glutaraldehyde (Glu). Finally, the NAA modified with G-rich DNA single strands was immersed in a solution containing hemin and potassium ions, fabricating a DNAzyme sensor (NAA-NH-Glu-NH-DNAzyme). The proposed biosensor oxidized 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) solution to green color ABTS˙− anion radical solution in the presence of hydrogen peroxide. The generated green color ABTS˙− anion radical solution absorbed the white light and therefore the intensity of the reflected light from NAA to the CCD decreased. However, in the presence of Pb2+ ions, the catalytic property of the proposed biosensor decreased. In the presence of Pb2+ ions, the concentration of the generated green color ABTS˙− anion radical solution decreased because of releasing the hemin cofactor during the transformation of the K+-stabilized hemin- G-quadruplex DNAzyme into the Pb2+-stabilized G-quadruplex. Therefore, the intensity of the reflected light to the detector increased. This increase in the intensity of the reflected light to the detector consequent on the increase in the peak area of the IRS. The biosensor exhibited a good response to Pb2+ ions with a linear range from 50 to 3200 nM with a low detection limit of 12 nM (2.6 ppb).

Acid-Switchable DNAzyme Nanodevice for Imaging Multiple Metal Ions in Living Cells.

Metal-assisted deoxyribozyme catalysis (DNAzyme) has been a general platform for constructing highly sensitive and selective detection sensors of metal ions. However, the "always on" mode of the traditional DNAzyme sensors greatly limits their application in the visual analysis of endogenous metal ions in a complex physiological microenvironment. To overcome this obstacle, a smart acid-switchable DNAzyme nanodevice is designed to control the DNAzyme activity in living cells and achieve simultaneous visualization of metal ions (Zn2+ and Pb2+) in situ. This nanodevice is built on DNAzyme precursors (DPs) and acid-switchable DNA (SW-DNA), precisely responding to pH variations in the range of 4.5-7.0, and the state of the three-strand hybridization of DPs successfully renders the DNAzymes inactive before being transported into cells. Once the nanodevice is taken up into living cells, the SW-DNA will change the configuration from linear to triplex in the acidic intracellular compartments (lysosomes, pH ∼4.5 to 5.0) and then the strands hybridized with the SW-DNA are liberated and subsequently react with DPs to form the active DNAzyme, which can further realize multi-imaging of intracellular metal ions. Moreover, this strategy has broad prospects as a powerful platform for constructing various acid-switchable nanodevices for visual analysis of multiple biomolecules in living cells.

Highly Sensitive RNA-Cleaving DNAzyme Sensors from Surface-to-Surface Product Enrichment.

The engineering of easy-to-use biosensors with ultra-low detection sensitivity remains a major challenge. Herein, we report a simple approach for creating such sensors through the use of an RNA-cleaving DNAzyme (RcD) and a strategy designed to concentrate its cleavage product significantly. The assay uses micron-sized beads loaded with a target-responsive RcD and a paper strip containing a microzone covered with a DNA oligonucleotide capable of capturing the cleavage product of the RcD through Watson-Crick hybridization. Placing the beads and the paper strip in a target-containing test sample allows the bead-bound RcD molecules to undergo target-induced RNA cleavage, releasing a DNA fragment that is captured by the paper strip. This strategy, though simple, is very effective in achieving high levels of detection sensitivity, being able to enrich the concentration of the cleavage product by three orders of magnitude. It is also compatible with both fluorescence-based and colorimetric reporting mechanisms. This work provides a simple platform for developing ultrasensitive biosensors that take advantage of the widely available RcDs as molecular recognition elements.

Enzyme-Mediated Endogenous and Bioorthogonal Control of a DNAzyme Fluorescent Sensor for Imaging Metal Ions in Living Cells.

Bioorthogonal control of metal-ion sensors for imaging metal ions in living cells is important for understanding the distribution and fluctuation of metal ions. Reported here is the endogenous and bioorthogonal activation of a DNAzyme fluorescent sensor containing an 18-base pair recognition site of a homing endonuclease (I-SceI), which is found by chance only once in 7×1010 bp of genomic sequences, and can thus form a near bioorthogonal pair with I-SceI for DNAzyme activation with minimal effect on living cells. Once I-SceI is expressed inside cells, it cleaves at the recognition site, allowing the DNAzyme to adopt its active conformation. The activated DNAzyme sensor is then able to specifically catalyze cleavage of a substrate strand in the presence of Mg2+ to release the fluorophore-labeled DNA fragment and produce a fluorescent turn-on signal for Mg2+ . Thus I-SceI bioorthogonally activates the 10-23 DNAzyme for imaging of Mg2+ in HeLa cells.

Enzyme‐Mediated Endogenous and Bioorthogonal Control of a DNAzyme Fluorescent Sensor for Imaging Metal Ions in Living Cells

AbstractBioorthogonal control of metal‐ion sensors for imaging metal ions in living cells is important for understanding the distribution and fluctuation of metal ions. Reported here is the endogenous and bioorthogonal activation of a DNAzyme fluorescent sensor containing an 18‐base pair recognition site of a homing endonuclease (I‐SceI), which is found by chance only once in 7×1010 bp of genomic sequences, and can thus form a near bioorthogonal pair with I‐SceI for DNAzyme activation with minimal effect on living cells. Once I‐SceI is expressed inside cells, it cleaves at the recognition site, allowing the DNAzyme to adopt its active conformation. The activated DNAzyme sensor is then able to specifically catalyze cleavage of a substrate strand in the presence of Mg2+ to release the fluorophore‐labeled DNA fragment and produce a fluorescent turn‐on signal for Mg2+. Thus I‐SceI bioorthogonally activates the 10–23 DNAzyme for imaging of Mg2+ in HeLa cells.

Cationic copolymer-chaperoned DNAzyme sensor for microRNA detection

Multi-component nucleic acid enzymes (MNAzymes) are allosteric deoxyribozymes that are activated upon binding of a specific nucleic acid effector. MNAzyme activity is limited due to an insufficient assembly of the MNAzyme and its turnover. In this work, we describe the successful improvement of MNAzyme reactivity and selectivity by addition of cationic copolymers, which exhibit nucleic acid chaperone-like activity. The copolymer allowed a 210-fold increase in signal activity and a 95-fold increase in the signal-to-background selectivity of MNAzymes constructed for microRNA (miRNA) detection. The selectivity of the MNAzyme for homologous miRNAs was demonstrated in a multiplex format in which isothermal reactions of two different MNAzymes were performed. In addition, the copolymer permitted miRNA detections even in the presence of a ribonuclease which is ubiquitous in environments, indicating the protective effect of the copolymer against ribonucleases.

Target-inspired Pb2+-dependent DNAzyme for ultrasensitive electrochemical sensor based on MoS2-AuPt nanocomposites and hemin/G-quadruplex DNAzyme as signal amplifier

In this work, a novel Pb2+ electrochemical DNAzyme sensor was developed for ultrasensitive detection of lead ions (Pb2+) in water environment by coupling with the MoS2–AuPt nanomaterials and hemin/G-quadruplex DNAzyme, which acting as the electrocatalytic signal tag. Streptavidin (SA) modified tin dioxide-functionalized reduced graphene oxide (rGO-SnO2) /gold nanoparticles (AuNPs) served as a sensor platform for enhancing conductivity and immobilizing more Pb2+-specific DNAzyme. In the presence of Pb2+, the Pb2+-dependent DNAzyme specifically reacted with Pb2+, cleaving the substrate strand (SS) into two free fragment and releasing the biotin-modified enzyme strand (Bio-ES) on the electrode. Connecting MoS2–AuPt nanocomposites labeled with G-rich DNA (G-DNA) strand and exposure of Bio-ES through the Helper DNA, as well as adding hemin to form a hemin/G-quadruplex, the biosensor achieved signal amplification. Chronoamperometry was used to record the current signal, which was primarily derived from the cocatalysis reduction of H2O2 by MoS2–AuPt nanocomposites and the hemin/G-quadruplex. Under optimal conditions, the designed biosensor exhibited sensitive detection of Pb2+ from 0.1 pg mL−1 to 1000 ng mL−1, with a lower detection limit of 38 fg mL−1 (based on 3σ). This proposed biosensor is ultrasensitive and specific, representing a potential application for the detection of Pb2+ in a water environment.