Molecular beacon mediated circular strand displacement strategy for constructing a ratiometric electrochemical deoxyribonucleic acid sensor

A novel ratiometric electrochemical sensor for sensitive and selective determination of deoxyribonucleic acid (DNA) had been developed based on signal-on and signal-off strategy. The target DNA hybridized with the loop portion of ferrocene (Fc) labeled hairpin probe immobilized on the gold electrode (GE), the Fc away from the surface of GE and the methylene blue (MB) was attached to an electrode surface by hybridization between hairpin probe and MB labeled primer. Such conformational changes resulted in the oxidation peak current of Fc decreased and that of MB increased, and the changes of dual signals are linear with the concentration of DNA. Furthermore, with the help of strand-displacement polymerization, polymerase catalyzed the extension of the primer and the sequential displacement of the target DNA, which led to the release of target and another polymerization cycle. Thus the circular strand displacement produced the multiplication of the MB confined near the GE surface and Fc got away from the GE surface. Therefore, the recognition of target DNA resulted in both the “signal-off” of Fc and the “signal-on” of MB for dual-signal electrochemical ratiometric readout. The dual signal strategy offered a dramatic enhancement of the stripping response. The dynamic range of the target DNA detection was from 10⁻¹³ to 10⁻⁸ mol L⁻¹ with a detection limit down to 28 fM level. Compared with the single signaling electrochemical sensor, the dual-signaling electrochemical sensing strategy developed in this paper was more selective. It would have important applications in the sensitive and selective electrochemical determination of other small molecules and proteins.     

A ratiometric electrochemical biosensor for sensitive detection of Hg based on thymine–Hg–thymine structure

In this paper, a simple, selective and reusable electrochemical biosensor for the sensitive detection of mercury ions (Hg²⁺) has been developed based on thymine (T)-rich stem–loop (hairpin) DNA probe and a dual-signaling electrochemical ratiometric strategy. The assay strategy includes both “signal-on” and “signal-off” elements. The thiolated methylene blue (MB)-modified T-rich hairpin DNA capture probe (MB-P) firstly self-assembled on the gold electrode surface via Au–S bond. In the presence of Hg²⁺, the ferrocene (Fc)-labeled T-rich DNA probe (Fc-P) hybridized with MB-P via the Hg²⁺-mediated coordination of T–Hg²⁺–T base pairs. As a result, the hairpin MB-P was opened, the MB tags were away from the gold electrode surface and the Fc tags closed to the gold electrode surface. These conformation changes led to the decrease of the oxidation peak current of MB (I_MB), accompanied with the increase of that of Fc (I_Fc). The logarithmic value of I_Fc/I_MB is linear with the logarithm of Hg²⁺ concentration in the range from 0.5 nM to 5000 nM, and the detection limit of 0.08 nM is much lower than 10 nM (the US Environmental Protection Agency (EPA) limit of Hg²⁺ in drinking water). What is more, the developed DNA-based electrochemical biosensor could be regenerated by adding cysteine and Mg²⁺. This strategy provides a simple and rapid approach for the detection of Hg²⁺, and has promising application in the detection of Hg²⁺ in real environmental samples.     

An Electrochemical Sensor for the Detection of Cu2+ Based on Gold Nanoflowers‐modifed Electrode and DNAzyme Functionalized Au@MIL‐101 (Fe)

In this study, we developed an electrochemical sensor for sensitive detection of Cu²⁺ based on gold nanoflowers (AuNFs)‐modified electrode and DNAzyme functionalized Au@MIL‐101(Fe) (MIL: Materials of Institute Lavoisier). The AuNFs‐modified indium tin oxide modified conductive glass electrode(AuNFs/ITO) prepared via electrodeposition showed improved electronic transport properties and provided more active sites to adsorb large amounts of oligonucleotide substrate (DNA1) via thiol‐gold bonds. The stable Au@MIL‐101(Fe) could guarantee the sensitivity because of its intrinsic peroxidase mimic property, while the Cu²⁺‐dependent DNA‐cleaving DNAzyme linked to Au@MIL‐101(Fe) achieved the selectivity toward Cu²⁺. After the DNAzyme substrate strand (DNA2) was cleaved into two parts due to the presence of Cu²⁺, the oligonucleotide fragment linked to MIL‐101(Fe) was able to hybridize with DNA1 adsorbed onto the surface of AuNFs/ITO. Due to the peroxidase‐like catalytic activity of MIL‐101(Fe) and the affinity recognition property of DNAzyme toward Cu²⁺, the electrochemical biosensor showed a sensitive detection range from 0.001 to 100 μM, a detection limit of 0.457 nM and a high selectivity, demonstrating its potential for Cu²⁺ detection in real environmental samples.     

Simultaneous electrochemical determination of two analytes based on nuclease-assisted target recycling amplification

In the present study, a method for simultaneous determination of two different DNAs is developed based on nuclease-assisted target recycling and nanoparticle amplification. The target recycling process is accomplished by taking advantage of the cleavage property of nicking endonuclease (NEase) for specific nucleotide sequences in duplex. In the presence of target DNA, the linker DNA in our detection system can hybridize with the target and be cleaved to form short fragments. Thus the target DNA is released and recognized by another linker DNA, activating the next round of cleavage reaction. On the other hand, two bio-barcode probes, a PbS nanoparticles (NPs)-DNA probe and a CdS NPs-DNA probe, are used for tracing two target DNAs to further amplify the detection signals. Based on a sensitive differential pulse anodic stripping voltammetry (DPASV) method for the simultaneous detection of Pb²⁺ and Cd²⁺ obtained by dissolving two probes, two different target DNAs are determined with high sensitivity and single-base mismatch selectivity.     

Signal-on electrochemical Y or junction probe detection of nucleic acid

A methylene-blue (MB)-labeled molecular beacon junction probe allows for a signal-on electrochemical detection of nucleic acids via target recycling using endonucleases. Electron transfer is reduced when the MB is intercalated in the stem of the molecular beacon, but then electron transfer from MB to a gold electrode is enhanced upon cleavage of the junction probe due to increased probability of MB approaching the electrode when attached to the more flexible ssDNA.     

Near-Infrared Photothermally Activated DNAzyme–Gold Nanoshells for Imaging Metal Ions in Living Cells

DNAzymes have enjoyed success as metal ion sensors outside cells. Their susceptibility to metal-dependent cleavage during delivery into cells has limited their intracellular applications. To overcome this limitation, a near-infrared (NIR) photothermal activation method is presented for controlling DNAzyme activity in living cells. The system consists of a three-stranded DNAzyme precursor (TSDP), the hybridization of which prevents the DNAzyme from being active. After conjugating the TSDP onto gold nanoshells and upon NIR illumination, the increased temperature dehybridizes the TSDP to release the active DNAzyme, which then carries out metal-ion-dependent cleavage, resulting in releasing the cleaved product containing a fluorophore. Using this construct, detecting Zn²⁺ in living HeLa cells is demonstrated. This method has expanded the DNAzyme versatility for detecting metal ions in biological systems under NIR light that exhibits lower phototoxicity and higher tissue penetration ability.     

Signal-amplification detection of small molecules by use of Mg2+- dependent DNAzyme

Because small molecules can be beneficial or toxic in biology and the environment, specific and sensitive detection of small molecules is one of the most important objectives of the scientific community. In this study, new signal amplification assays for detection of small molecules based on Mg²⁺-dependent DNAzyme were developed. A cleavable DNA substrate containing a ribonucleotide, the ends of which were labeled with black hole quencher (BHQ) and 6-carboxyfluorescein (FAM), was used for fluorescence detection. When the small molecule of interest is added to the assay solution, the Mg²⁺-dependent DNAzyme is activated, facilitating hybridization between the Mg²⁺-dependent DNAzyme and the DNA substrate. Binding of the substrate to the DNAzyme structure results in hydrolytic cleavage of the substrate in the presence of Mg²⁺ ions. The fluorescence signal was amplified by continuous cleavage of the enzyme substrate. Ochratoxin A (OTA) and adenosine triphosphate (ATP) were used as model analytes in these experiments. This method can detect OTA specifically with a detection limit as low as 140 pmol?L^?1 and detect ATP specifically with a detection limit as low as 13 nmol?L^?1. Moreover, this method is potentially extendable to detection of other small molecules which are able to dissociate the aptamer from the DNAzyme, leading to activation of the DNAzyme.     

G-quadruplex-assisted enzyme strand recycling for amplified label-free fluorescent detection of UO22+

A G-quadruplex-assisted enzyme strand recycling strategy was developed for amplified label-free fluorescent detection of uranyl ion (UO₂²⁺).     

Coupling hybridization chain reaction with DNAzyme recycling for enzyme-free and dual amplified sensitive fluorescent detection of methyltransferase activity

Aberrant DNA methylation originated from changes in DNA methyltransferase activity can lead to many genetic diseases and tumor types, and the monitoring of methyltransferase activity is thus of great importance in disease diagnosis and drug screening. In this work, by combing hybridization chain reaction (HCR) and metal ion-dependent DNAzyme recycling, we have developed a convenient enzyme-free signal amplification strategy for highly sensitive detection of DNA adenine methyltransferase (Dam MTase) activity and its inhibitors. The Dam MTase-induced methylation and subsequent cleavage of the methylated hairpin DNA probes by DpnI endonuclease lead to the release of ssDNA triggers for HCR formation of many Mg²⁺-dependent DNAzymes, in which the fluorescently quenched substrate sequences are catalytically and cyclically cleaved by Mg²⁺ to generate remarkably amplified fluorescent signals for highly sensitive detection of Dam MTase at 7.23 × 10⁻⁴ U/mL. In addition, the inhibition of different drugs to Dam MTase activity can also be evaluated with the developed method. With the advantages of simplicity and significant signal amplification over other common methods, the demonstrated biosensing approach thus offers great potential for highly sensitive detection of various methyltransferases and provides a convenient platform for drug screening for therapeutic applications.     

A regenerative ratiometric electrochemical biosensor for selective detecting Hg based on Y-shaped/hairpin DNA transformation

Inspired by dual-signaling ratiometric mechanism which could reduce the influence of the environmental change, a novel, convenient, and reliable method for the detection of mercury ions (Hg²⁺) based on Y-shaped DNA (Y-DNA) was developed. Firstly, the Y-DNA was formed via the simple annealing way of using two different redox probes simultaneously, omitting the multiple operation steps on the electrode. The Y-DNA was immobilized on the gold electrode surface and then an obvious ferrocene (Fc) signal and a weak methylene blue (MB) signal were observed. Upon addition of Hg²⁺, the Y-DNA structure was transformed to hairpin structure based on the formation of T-Hg²⁺-T complex. During the transformation, the redox MB gets close to and the redox Fc gets far away from the electrode surface, respectively. This special design allows a reliable Hg²⁺ detection with a detection range from 1 nM to 5 μM and a low detection limit down to 0.094 nM. Furthermore, this biosensor exhibits good selectivity and repeatability, and can be easily regenerated by using l-cysteine. This study offers a simple and effective method for designing ratiometric biosensors for detecting other ions and biomolecules.     

Near‐Infrared Photothermally Activated DNAzyme–Gold Nanoshells for Imaging Metal Ions in Living Cells

DNAzymes have enjoyed success as metal ion sensors outside cells. Their susceptibility to metal‐dependent cleavage during delivery into cells has limited their intracellular applications. To overcome this limitation, a near‐infrared (NIR) photothermal activation method is presented for controlling DNAzyme activity in living cells. The system consists of a three‐stranded DNAzyme precursor (TSDP), the hybridization of which prevents the DNAzyme from being active. After conjugating the TSDP onto gold nanoshells and upon NIR illumination, the increased temperature dehybridizes the TSDP to release the active DNAzyme, which then carries out metal‐ion‐dependent cleavage, resulting in releasing the cleaved product containing a fluorophore. Using this construct, detecting Zn²⁺ in living HeLa cells is demonstrated. This method has expanded the DNAzyme versatility for detecting metal ions in biological systems under NIR light that exhibits lower phototoxicity and higher tissue penetration ability.     

Enzyme spheres as novel tracing tags coupled with target-induced DNAzyme assembly for ultrasensitive electrochemical microRNA assay

In this work, an ultrasensitive electrochemical microRNA detection strategy was developed based on porous palladium-modified horseradish peroxidase sphere (Pd@HRP) and target-induced assembly of DNAzyme. A highly loaded HRP sphere was prepared by covalent layer-by-layer assembly with CaCO₃ as sacrificial template for the first time, and was further modified with porous Pd. Notably, Pd@HRP composite showed a good redox activity of HRP and electrocatalytic activity toward H₂O₂. The utilization of Pd@HRP as electrochemical signal indicator and enhancer to fabricate biosensor could avoid the need for additional redox mediator and amplify the detection sensitivity. Moreover, target recycling amplification was achieved by Pb²⁺-induced cleavage of ternary “Y” structure, circumventing the use of labile nuclease. Subsequent DNA concatamer synthesized through rolling circle amplification (RCA) reaction with cleaved hairpin probe as primer, hybridized with plentiful Pd@HRP-DNA probes, which led to the increased loading of redox-active and electrocatalytic Pd@HRP for sensitivity improvement. So the proposed electrochemical biosensor detected miRNA-24 down to 0.2 fM (S/N = 3) with a wide linear range from 3 fM to 1 nM. With bifunctional Pd@HRP tag, DNAzyme-aided target recycle and programmable junction probe, this strategy possessed the advantages of high efficiency, high sensitivity, low cost and versatility, and thus held great promise for other low-abundance nucleic acids determination.     

Adenosine Triphosphate Sensing by Electrocatalysis with DNAzyme

Electrocatalysis of redox enzymes shows wide application for biosensing. DNAzymes exhibiting specific catalytic activities have aroused great interest recently. However, there are few studies on the electrocatalysis between DNAzyme and electron mediator. In this paper, based on the electrocatalysis of methylene blue (MB) and horseradish peroxidase mimicking DNAzyme (HRP‐DNAzyme), an amplified electrochemical biosensor for the detection of adenosine triphosphate (ATP) was designed. In the present system, by means of the ATP‐aptamer interaction, two guanine‐rich DNA sequences, one of which was labeled with MB at the 5′ end, were assembled on the gold electrode. In the presence of K⁺ and hemin, the guanine‐rich DNA sequences transferred to HRP‐DNAzyme. The conformational change of the structure resulted in the approaching of MB and HRP‐DNAzyme which made the electrocatalytic process between MB and HRP‐DNAzyme possible. We used cyclic voltammetry and electrochemical impedance spectroscopy to study the electrocatalytic process. The system was therefore utilized for amplified detection of ATP without imposing any new constraints to the platform which showed satisfactory result.     

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 1.0 ng L⁻¹ to 10 mg L⁻¹ Hg²⁺ and a detection limit of 0.5 ng L⁻¹ (2.5 pM) at the 3S_blank level, and it also demonstrated excellent selectivity against other interferential metal ions.     

Ultrasensitive amperometric aptasensor for the epithelial cell adhesion molecule by using target-driven toehold-mediated DNA recycling amplification

An amperometric aptasensor is reported for the electrochemical determination of the epithelial cell adhesion molecule (EpCAM). It is based on a combination of EpCAM-driven toehold-mediated DNA recycling amplification, the specific recognition of EpCAM aptamer, and its binding to EpCAM. Hairpin probe 1 (Hp1) with a toehold region was modified with a 5′-thiol group (5’-SH) and self-assembled onto the surface of a gold electrode. Upon addition of EpCAM, the probe A (a 15-mer) is liberated from the aptamer/probe A complex and then hybridizes with the toehold domain of Hp1. This results in the exposure of another toehold for further hybridizing with hairpin probe 2 (Hp2) to displace probe A in the presence of Hp2 that was labeled with the electrochemical probe Methylene Blue (MB). Subsequently, liberated probe A is hybridized again with another Hp1 to start the next round of DNA recycling amplification by reusing probe A. This leads to the formation of plenty of MB-labeled DNA strands on the electrode surface and generates an amplified current. This 1:N probe-response amplification results in ultrasensitive and specific detection of EpCAM, with a 20 pg·mL^?1 detection limit. The electrode is highly stable and regenerable. It was successfully applied to the determination of EpCAM in spiked human serum, urine and saliva, and thus provides a promising tool for early clinical diagnosis.

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Graphical abstract Schematic illustration of the electrochemical detection for EpCAM. The method is based on aptamer-based recognition and EpCAM-driven toehold-mediated DNA recycling amplification. Hp1: Hairpin probe 1; Hp2: Hairpin probe 2; MB: Methylene blue; MCH: 6-Mercapto-1-hexanol; EpCAM: Epithelial cell adhesion molecule.

     

基于核酸切割酶与脱氧核酶的荧光循环放大系统检测铅(Ⅱ)

利用核酸切割酶(Nicking endonuclease)识别特定DNA双链并切割其中某条单链的性质,构建了基于8-17E脱氧核酶(8-17E DNAzyme)的pb2+荧光循环放大检测方法.pb2+可激活8-17E脱氧核酶水解RNA底物,产生并释放出的单链与分子信标探针( Molecular beacon,MB)杂交,导致其茎环结构被破坏,荧光信号恢复;同时形成含有核酸切割酶Nt.BbvCI识别位点的双链区域.在核酸切割酶Nt.BbvCI的作用下,分子信标探针被切割释放,游离出来的单链可与其它分子信标重新杂交,从而触发下一轮酶切,引起荧光检测信号的循环放大.本方法避免了8-17E脱氧核酶与底物链的修饰,最低可以检测出水溶液中1.0×10-10 mol/L Pb2+,并在2倍浓度的Zn2+,以及5倍浓度的其它干扰金属离子存在的情况下对pb2+显示出良好的选择性.本方法对环境水样中pb2+的标准加样回收率为96.1％～108.0％.     

Highly sensitive electrochemical detection of mercury (II) via single ion-induced three-way junction of DNA

A “signal-on” electrochemical sensing strategy was designed for highly sensitive and selective detection of mercury (II) via its induction to three-way junction of DNA (DNA-TWJ). The TWJ consisted of the capture probe that was self-assembled on a gold electrode surface through SAu bond, the signal probe that was labeled with ferrocene (Fc) and contained single T–T mismatch to capture probe, and an assistant probe for the formation of DNA-TWJ upon the presence of mercury (II). This process caused the Fc tag approaching the electrode for fast electron transfer and thus increased the oxidation current. The “signal-on” sensing method could detect Hg^2 + ranging from 0.005 to 100 nM. The assay was simple and fast. It showed potential application in on-site and real-time Hg^2 + detection.     

Ultrasensitive detection of lead ion based on target induced assembly of DNAzyme modified gold nanoparticle and graphene oxide

In this paper, we report a novel colorimetric strategy for the detection of small molecules by using Pb²⁺ ion as an example. In this strategy, DNAzyme duplex modified gold nanoparticles (GNPs) are designed to be unable to interact with graphene oxide (GO). However, in the presence of Pb²⁺, the substrate strand of the DNAzyme is cleaved at its cleavage site, resulting in the disassembly of the DNAzyme duplex modified GNPs into three parts, i.e., the 3′- and 5′-fragments of substrate strand and the DNAzyme strand modified GNPs. By taking advantage of the efficient cross-linking effect of ssDNA-GNPs to GO, colorimetric sensor for the detection of the metal ion can be fabricated with a detection limit of 100 pM, which is much lower than the previous reports. This colorimetric method has also been used for the determination of Pb²⁺ in the tap water of the local city and the water from a reservoir with satisfactory results, so it may have potential applications in the future.     

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 Zn²⁺-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.     

Electrochemiluminescence of Supramolecular Nanorods and Their Application in the “On–Off–On” Detection of Copper Ions

In this work, an “on–off–on” switch system has been successfully applied through the construction of an electrochemiluminscent biosensor for copper ion (Cu²⁺) detection based on a new electrochemiluminescence (ECL) emitter of supramolecular nanorods, which was achieved through supramolecular interactions between 3,4,9,10‐perylenetetracarboxylic acid (PTCA) and aniline. The initial “signal‐on” state with strong and stable ECL emission was obtained by use of the supramolecular nanorods with a new signal amplification strategy involving a co‐reaction accelerator. In addition, ECL quencher probes (Fc‐NH₂/Cu‐Sub/nano‐Au) were fabricated by immobilizing aminoferrocene (Fc‐NH₂) on Cu‐substrate strand modified Au nanoparticles. The quencher probes were hybridized with the immobilized Cu‐enzyme strand to form Cu²⁺‐specific DNAzyme. Similarly, the “signal‐off” state was obtained by the high quenching effect of Fc‐NH₂ on the ECL of the excited‐state PTCA (¹PTCA*). As expected, the second “switch‐on” state could achieved by incubating with the target Cu²⁺, owing to the Cu²⁺‐specific DNAzyme, which was irreversibly cleaved, resulting in the release of the quencher probes from the sensor interface. Herein, on the basis of the ECL intensity changes (ΔI_ECL) before and after incubating with the target Cu²⁺, the prepared Cu²⁺‐specific DNAzyme‐based biosensor was used for the determination of Cu²⁺ concentrations with high sensitivity, excellent selectivity, and good regeneration.