Ion-dependent DNAzyme Research Articles

A sensitive tobramycin electrochemical aptasensor based on multiple signal amplification cascades

The residue of tobramycin, a broad spectrum antibiotic commonly used in animal husbandry, has evitable impact on human health, which may cause kidney damage, respiratory paralysis, neuromuscular blockade and cross-allergy in humans. Sensitive monitoring of tobramycin in animal-derived food products is therefore of great importance. Herein, a new aptamer electrochemical biosensor for sensing tobramycin with high sensitivity is demonstrated via exonuclease III (Exo III) and metal ion-dependent DNAzyme recycling and hybridization chain reaction (HCR) signal amplification cascades. Tobramycin analyte binds aptamer-containing hairpin probe to switch its conformation to expose the toehold sequence, which triggers Exo III-based catalytic digestion of the secondary hairpin to release many DNAzyme strands. The substrate hairpins immobilized on the Au electrode (AuE) are then cyclically cleaved by the DNAzymes to form ssDNAs, which further initiate HCR formation of lots of long methylene blue (MB)-tagged dsDNA polymers on the AuE. Subsequently electro-oxidation of these MB labels thus exhibit highly enhanced currents for sensing tobramycin within the 5–1000 nM concentration range with an impressive detection limit of 3.51 nM. Furthermore, this strategy has high selectivity for detecting tobramycin in milk and shows promising potential for detect other antibiotics for food safety monitoring.

DNA-modified Prussian blue nanozymes for enhanced electrochemical biosensing.

Prussian blue nanoparticles exhibit the potential to be employed in bioanalytical applications due to their robust stability, peroxidase-like catalytic functionality, straightforward synthesis, and biocompatibility. An efficient approach is presented for the synthesis of nucleic acid-modified Prussian blue nanoparticles (DNA-PBNPs), utilizing nanoparticle porosity to adsorb nucleic acids (polyT). This strategic adsorption leads to the exposure of nucleic acid sequences on the particle surface while retaining catalytic activity. DNA-PBNPs further couple with functional nucleic acid sequences and aptamers through complementary base pairing to act as transducers in biosensors and amplify signal acquisition. Subsequently, we integrated a copper ion-dependent DNAzyme (Cu2+-DNAzyme) and a vascular endothelial growth factor aptamer (VEGF aptamer) onto screen-printed electrodes to serve as recognition elements for analytes. Significantly, our approach leverages DNA-PBNPs as a superior alternative to traditional enzyme-linked antibodies in electrochemical biosensors, thereby enhancing both the efficiency and adaptability of these devices. Our study conclusively demonstrates the application of DNA-PBNPs in two different biosensing paradigms: the sensitive detection of copper ions and vascular endothelial growth factor (VEGF). These results indicate the promising potential of DNA-modified Prussian blue nanoparticles in advancing bioanalytical sensing technologies.

Selective detection of Pb2+ ions based on a graphene field-effect transistor gated by DNAzymes in binding mode

Heavy metal contamination has become a severe threat to dairy products through contaminated feed and the environment water. Among them, Pb(II) is highly toxic to the human body even under minimal exposure. Therefore, establishing a fast and sensitive Pb2+ detection technology is significant for rapid screening of vast number of dairy products. Hererin, we report the development of a sensitive and selective Pb(II) biosensor based on a solution-gated graphene transistor (SGGT) with the gate modified by Pb2+-dependent DNAzyme probes. It has also been explored that the DNAzymes working in simple binding mode integrate better with the SGGT than those working in normal catalytic mode, showing significantly stronger channel current responses and lower detection limit down to 0.39 μg/L (or 1.9 nM). Finally, the biosensor was practicably applied to the detection of lead ions in pure milk samples with a high recovery rate. We believe that this work reveals the best strategy for integrating metal ion dependent DNAzyme probes with SGGT sensing platforms to selectively and sensitively detect many metal ions.

Apamer-based sensitive and label-free electrochemical detection of neutrophil gelatinase-associated lipocalin via recycling amplification cascades

Sensitive and selective detection of neutrophil gelatinase-associated lipocalin (NGAL) is critical for the prediction and early diagnosis of acute renal injury. In this work, the establishment of an aptamer-based, highly sensitive and label-free method for detecting NGAL in diluted human serums via metal ion-dependent DNAzyme- and exonuclease III (Exo III)-triggered recycling signal amplification cascades is described. NGAL binds with the aptamer strands in the DNAzyme/aptamer duplexes and results in the liberation of the metal ion-dependent DNAzyme sequences to cleave the hairpin signal probes on the electrode to liberate the G-quadruplex and intermediate strands. The released intermediate strands further complement with the DNAzyme/aptamer duplexes to form favorable substrate for Exo III, which digests the duplexes to release the DNAzyme strands to initiate the cascaded recycling cycles for the yield of plenty of G-quadruplex strands. Hemin can associate with G-quadruplex strands to produce many G-quadruplex/hemin complexes and electrochemical reduction of hemin thus generates highly amplified current for detecting NGAL with the detection limit of 4.45 ng mL−1. Such biosensor also shows high selectivity and can be utilized for monitoring NGAL spiked in diluted serum, indicating its extension potential for detecting various protein biomarkers with different aptamers for disease diagnosis.

Cascaded dissipative DNAzyme-driven layered networks guide transient replication of coded-strands as gene models

Dynamic, transient, out-of-equilibrium networks guide cellular genetic, metabolic or signaling processes. Designing synthetic networks emulating natural processes imposes important challenges including the ordered connectivity of transient reaction modules, engineering of the appropriate balance between production and depletion of reaction constituents, and coupling of the reaction modules with emerging chemical functions dictated by the networks. Here we introduce the assembly of three coupled reaction modules executing a cascaded dynamic process leading to the transient formation and depletion of three different Mg2+-ion-dependent DNAzymes. The transient operation of the DNAzyme in one layer triggers the dynamic activation of the DNAzyme in the subsequent layer, leading to a three-layer transient catalytic cascade. The kinetics of the transient cascade is computationally simulated. The cascaded network is coupled to a polymerization/nicking DNA machinery guiding transient synthesis of three coded strands acting as “gene models”, and to the rolling circle polymerization machinery leading to the transient synthesis of fluorescent Zn(II)-PPIX/G-quadruplex chains or hemin/G-quadruplex catalytic wires.

A Self-Made Optical Tweezers Integrated Upconversion Luminescence Confocal Scanning Instrument Enables Stable and Noninvasive Long-Term In Situ Imaging a Single Suspension Cell Under Exceptionally Efficient Luminescent Resonance Energy Transfer Sensing.

It is necessary to explore labeling probes with worthy optical properties and a noninvasive fluorescence imaging manner for stable long-term in situ measuring a single suspension cell. In response to these goals, we herein make a breakthrough on two fronts. On one hand, a co-sensitizer-induced efficient 808 nm near-infrared light-excited luminescence-confined upconversion nanoparticle with a low thermal effect is fabricated by employing a layer-by-layer seed growing approach to develop a sandwich structure, under which the luminescence domain is vastly restricted into an extremely thin inner shell (∼ 2.77 nm) to finally bring about a high-efficiency luminescent resonance energy transfer (LRET) sensing behavior. On the other hand, a self-made optical tweezers integrated upconversion luminescence confocal scanning instrument is applied to enhance the imaging accuracy, after which the liquid viscous force is sufficiently overcome by the resulting single beam gradient force and the analyzed suspension cell is always immobilized to the focal plane to ensure a constant luminescence excitation condition. By making use of a metal ion-dependent DNAzyme and a hairpin DNA strand to design a corresponding LRET sensing system, our nanoprobe shows satisfactory assay performance for two model biomolecules (Ca2+ and TK1 messenger RNA). Following the optical trapping-assisted imaging, this exceptional measurement method is capable of effectively monitoring the intracellular target changes in different physiological states, endowing a powerful toolbox for single cell analysis.

An “on-off” signal-switchable electrochemiluminescence biosensor for ultrasensitive detection of dual microRNAs based on DNAzyme-powered DNA walker

Herein, an “on-off” signal-switchable electrochemiluminescence (ECL) biosensor for dual miRNAs (miRNA-21 and miRNA-155) detection was constructed based on carbon dots (CDs)-labeled DNA walker, which can effectively avoid problems of the signal overlap or limited available labels that general exist in multiplex assays. In the presence of miRNA-21, the CDs-DNA walker were captured on the electrode via nucleic acid hybridization, producing a strong ECL signal corresponding to miRNA-21 (signal on). For miRNA-155, the biosensor was pre-incubated with CDs-DNA walker and assistant DNA (DNA-21). Target miRNA-155 could trigger metal ion-dependent DNAzyme cleavage. With the assistance of Mn2+, the substrate strands were cleaved and CDs detached from the electrode, resulting in a reduced ECL signal (signal off). The interference of miRNA-21 was eliminated because no more CDs-DNA walker was added. As a result, benefiting from the signal amplification offered by the CDs-DNA walker and the inherent properties of ECL technique, this biosensor achieved low detection limits of 33 fM for miRNA-21 and 33 aM for miRNA-155. This paper also demonstrates the biosensor’s bioanalytical application for multiplex detection of miRNA-21/155 in human serum samples and the total RNA extraction solution from the human cancer cell lines.

Light-Activated and Self-Driven Autonomous DNA Nanomachine Enabling Fluorescence Imaging of MicroRNA in Living Cells with Exceptional Precision and Efficiency.

Owing to their favorable design flexibility and eminent signal amplification ability, DNA nanomachine-supported biosensors have provided an attractive avenue for intracellular fluorescence imaging, especially for DNA walkers. However, this promising option not only suffers from poor controllability but also needs to be supplied with additional driving forces on account of the frequent employment of metal ion-dependent DNAzymes. Aiming at overcoming these obstacles, we introduce some fruitful solutions. On one hand, innovative light-activated walking behavior induced by a photocleavage mode is established on the surfaces of gold nanoparticles, and such a photoselective sensing system can be perfectly prevented from pre-activating during the intracellular delivery process and made to achieve target identification only under irradiation using a moderate ultraviolet light source. On the other hand, this light-switchable sensing frame is encapsulated within a dissociable metal-organic framework (ZIF-8) to facilitate endocytosis and ensure sufficient internal cofactors (Zn2+) to realize a self-driven pattern in the acidic environment of the cell lysosome. Based on the abovementioned efforts, the newly constructed autonomous three-dimensional DNA walkers present satisfactory sensitivity (a limit of detection of down to 19.4 pM) and specificity (even distinguishing single-base changes) toward a model biomarker (microRNA-21). More importantly, the sensing method allows determination of the variations in targets in living cancer cells with exceptional precision and efficiency, offering a powerful assay platform for intracellular imaging.

Three kinds of porphyrin dots as near-infrared electrochemiluminescence luminophores: Facile synthesis and biosensing

Three kinds of porphyrin dots, including TCPP dots, ZnTCPP dots and TCPP-based metal organic framework (pMOF) dots, have been subtly synthesized for the first time by a universal straightforward exfoliation of bulk porphyrin aggregates or pMOF. To explore their ECL application, a novel label free “signal-on” ECL biosensor for ultrasensitive detection of Pb 2+ was developed based on TCPP dots as DNA intercalators and DNAzyme-based hybridization chain reaction signal amplification. • Three kinds of porphyrin dots are subtly prepared by a simple and universal exfoliation. • The novel porphyrin dots show excellent annihilation ECL activity. • They all exhibit stable and robust near-infrared co-reactant ECL performance. • A label free ECL biosensor is established for trace Pb 2+ detection based on TCPP dots. • The porphyrin dots as ECL emitters give a new proof of concept. Exploring novel near-infrared (NIR) luminophore with highly efficient and benign is strongly anticipated for electrochemiluminescence (ECL) evolution. Here, three kinds of porphyrin dots including 5,10,15,20-tetrakis (4-carboxyphenyl) porphyrin (TCPP) dots, ZnTCPP dots and TCPP-based metal organic framework (pMOF) dots have been subtly synthesized for the first time by a universal straightforward exfoliation of bulk porphyrin aggregates or pMOF. These as-designed porphyrin dots perform excellent annihilation ECL activity, also exhibit stable and robust cathodic or anodic NIR ECL properties with different coreactants (potassium peroxydisulfate, hydrogen peroxide or tri-n-propylamine) in aqueous phase. Especially, the ECL efficiency of three kinds of porphyrin dots in S 2 O 8 2− cathodic system are 56, 44, 39-fold higher than that of the Ru(bpy) 3 2+ standard, respectively. As a new type of ECL luminophores, TCPP dots coupled with a lead ion-dependent DNAzyme and hybridization chain reaction cycle amplification are used to fabricate a label free “signal-on” ECL biosensor for ultrasensitive detection of Pb 2+ . A dynamic range from 10 pmol·L −1 to 1 μmol·L −1 with a low limit of detection (1.2 pmol·L −1 ) is obtained. These porphyrin dots provide evidence for the use of organics-based dots as new NIR ECL luminophores for fundamental ECL research, and highlight the significance of small-sized organic nanomaterials in ECL sensing applications.

Constitutional Dynamic Networks-Guided Synthesis of Programmed "Genes", Transcription of mRNAs, and Translation of Proteins.

Inspired by nature, where dynamic networks control the levels of gene expression and the activities of transcribed/translated proteins, we introduce nucleic acid-based constitutional dynamic networks (CDNs) as functional modules mimicking native circuits by demonstrating CDNs-guided programmed synthesis of genes, controlled transcription of RNAs, and dictated transcription/translation synthesis of proteins. An auxiliary CDN consisting of four dynamically equilibrated constituents AA', AB', BA', and BB' is orthogonally triggered by two different inputs yielding two different compositionally reconfigured CDNs. Subjecting the parent auxiliary CDN to two hairpins, HA and HB, and two templates TA and TB and a nicking/replication machinery leads to the cleavage of the hairpins and to the activation of the nicking/replication machineries that synthesize two "genes", e.g., the histidine-dependent DNAzyme g1 and the Zn2+-ion-dependent DNAzyme g2. The triggered orthogonal reconfiguration of the parent CDN to the respective CDNs leads to the programmed preferred CDN-guided synthesis of g1 or g2. Similarly, the triggered reconfigured CDNs are subjected to two hairpins HC and HD, the templates I'/I and J'/J, and the RNA polymerase (RNAp)/NTPs machinery. While the cleavage of the hairpins by the constituents associated with the parent CDN leads to the transcription of the broccoli aptamer recognizing the DFHBI ligand and of the aptamer recognizing the malachite green (MG) ligand, the orthogonally triggered CDNs lead to the CDNs-guided enhanced transcription of either the DFHBI aptamer or the MG aptamer. In addition, subjecting the triggered reconfigured CDNs to predesigned hairpins HE and HF, the templates M'/M and N'/N, the RNAp/NTPs machinery, and the cell-free ribosome t-RNA machinery leads to the CDNs-guided transcription/translation of the green fluorescence protein (GFP) or red fluorescence protein (RFP).

Fluorescent aptasensors for parallel analysis of biomolecules based on interlocked DNA catenane nanomachines

Stimuli-responsive DNA catenane nanomachines have received considerable interest in the area of DNA nanotechnology. However, the sensing and bio-sensing applications of DNA catenane nanomachines are rarely explored. Herein, the biological small molecule and protein responsive DNA catenane nanomachines were designed by utilizing the specific aptamer/target interaction. In the presence of the target, the blocked catalytically inactive DNA catenane can be activated, while generating the metal ion-dependent DNAzyme as the signal output unit. The method allows the fluorescent detection of ATP and lysozyme with a detection limit of 20 nM and 200 pM, respectively. More importantly, through the use of different fluorophores as labels, the parallel analysis of ATP and lysozyme in the mixed solution was demonstrated based on two interlocked DNA catenanes. Compared with linear scaffold, the interlocked DNA rings showed enhanced stability against enzyme degradation and easily realized intramolecular reconfiguration. Due to its inherent advantages, the interlocked DNA catenane holds great promise in the field of DNA based sensors and nanodevices.

Guanine-wire based walking machine for highly sensitive and selective detection of circulating microRNA

A novel DNA machine-based fluorescent system is designed for highly sensitive and selective detection of circulating microRNA. It is based on topological assembly of Guanine (G)-tetrads and Mg2+ ion-dependent DNAzyme. With the aid of Mg2+ and K+ ions, G-rich sequences first form parallel G-tetrads and further assemble into aggregated G-wires. G-wire is used as not only the walking track but also the quencher of the fluorophore immobilized on its 5′-end. DNAzyme serves as the walker and is embedded in the stem of the tailored hairpin probe in which a recognition element of target microRNA is designed in the loop region. In the absence of target microRNA, the hairpin probe keeps closed to block the DNAzyme walker. In contrast, the presence of target microRNA results in the open of the hairpin probe by hybridizing with the recognition element and the release of the DNAzyme walker. The released walker binds with and cleaves the fluorescence substrate to free the labeled-fluorophore from the track and then steps toward the neighbored substrate. The cycles of this process continuously turn on the fluorophore for the amplified detection. Using this “signal on” strategy, a detection limit of 57.26 fM is realized with five dynamic ranges and high selectivity. Besides, this detection is featured with null protein enzyme and free of labeled-quencher. Thus our system provides a promising candidate for microRNA detection in clinical diagnosis.

Active generation of nanoholes in DNA origami scaffolds for programmed catalysis in nanocavities

DNA origami tiles provide nanostructures for the spatial and temporal control of functional loads on the scaffolds. Here we introduce the active generation of nanoholes in the origami scaffolds using DNAzymes or light as triggers and present the programmed and switchable catalysis in the resulting nanocavities. We engineer “window” domains locked into the origami scaffolds by substrates of the Zn2+-ion- or Pb2+-ion-dependent DNAzymes. Using Zn2+ ions and/or Pb2+ ions, the programmed unlocking of the “window” domains is demonstrated. The tailored functionalization of the origami scaffolds allows the programmed operation of catalytic processes in the confined nanocavities. Also, the “window” domain is integrated into the origami scaffold using photoisomerizable azobenzene-modified locks. The cyclic photoisomerization of the locks between the cis and trans states leads to a reversible opening and closure of the nanoholes and to the cyclic light-induced switching of catalytic processes in the nanocavities.

Electrochemical lead(II) biosensor by using an ion-dependent split DNAzyme and a template-free DNA extension reaction for signal amplification.

A voltammetric biosensor for lead(II) (Pb2+) is described that is based on signal amplification by using an ion-dependent split DNAzyme and template-free DNA extension reaction. The Pb2+-dependent split DNAzyme was assembled on gold nanoparticles (Au@Fe3O4),and this nanoprobe then was exposed to Pb2+ which causes the split-off of DNAzymes to release primers containing 3'-OH groups (S1 and S2). The template-free DNA extension reaction triggers the generation of long ssDNA nanotails, which thencan bind the free redox probe N,N'-bis(2-(trimethylammonium iodide)propylene)perylene-3,4,9,10-tetracarboxyldiimide (PDA+) via electrostatic adsorption. Hence, the concentration of PDA+ in solution is reduced. Therefore, less free PDA+ can be immobilized on a glassy carbon electrodemodified with electrodeposited gold nanoparticles (depAu) to produce an electrochemical signal, typically measured at ∼0.38V (vs. SCE) for quantitation of Pb2+. The use of a Pb2+-dependent split DNAzyme avoids the usage of a proteinic enzyme. It also increases the sensitivity of the sensor which has a lower detection limit of 30 pM of Pb2+. Graphical abstract Novel electrochemical biosensor based on the amplification of ion-dependent split DNAzyme and template-free DNA extension reaction for trace detection of Pb2+.

A Universal Electrochemical Biosensor Using Nick-HCR Nanostructure as Molecular Gate of Nanochannel for Detecting Chromium(III) Ions and MicroRNA.

Solid-state nanochannels demonstrating excellent mechanical properties and chemical stability combined with programmable DNA provide an opportunity to control on-demand ion transport. However, poor functionalization of the nanochannels limits the types of detected targets, as well as its universality in the sensing field. To solve these issues, a universal nanochannel sensing platform was developed by employing a nick hybridization chain reaction (nHCR) nanostructure as a molecular gate, which could generally respond to the universal sequence Y. Metal ion-dependent DNAzyme cleavage was used to transfer the chromium(III) (Cr3+) ions into nucleic acid X, which was further amplified and converted into universal sequence Y. Upon adding sequence Y into the nHCR nanostructure-functionalized nanochannel, the disassembly of the nHCR molecular gate turned on the ionic current signal inside the nanochannel. The ON-OFF ratio displayed a linear relationship with the Cr3+ concentration in the range from 200 fM to 20 nM. In less than 66 min, the nanochannel-based biosensing platform successfully detected Cr3+ ions as low as 200 fM. In addition, the detection of microRNA with a concentration as low as 1 pM was achieved by only regulating the sequence of template X'-Y'.

Triggered Interconversion of Dynamic Networks Composed of DNA-Tetrahedra Nanostructures.

Constitutional dynamic networks (CDNs) consisting of DNA tetrahedra allow the dynamically triggered adaptive control over the compositions and structures of the constituents. In one system, a CDN consisting of four tetrahedra constituents is orthogonally triggered by two alternative triggers, T1 or T2, to reconfigure into two different CDNs, revealing adaptive control-over the tetrahedra compositions in the two CDNs. In the presence of the counter triggers T1' or T2', the parent CDN is regenerated. In the second system, the assembly of a CDN consisting of four dimeric tetrahedra exhibiting variable sizes and shapes is described. The orthogonal triggering of the CDN by two different triggers T3 or T4, leads to the adaptive reconfiguration of the CDN into new equilibrated CDNs exhibiting control-over the compositions and shapes of the dimeric tetrahedra comprising the CDNs. Mg2+-ion-dependent DNAzyme units conjugated to the tetrahedra nanostructures and complementary electrophoretic experiments provide means to quantitatively evaluate the compositions of the different CDN systems. By the functionalization of the four-tetrahedra-based CDN system with two fluorophor donor-acceptor pairs and the orthogonal reconfiguration of the CDN in the presence of two alternative triggers, the control-over the FRET functions of the CDN systems is demonstrated.

Three-Dimensional Nucleic-Acid-Based Constitutional Dynamic Networks: Enhancing Diversity through Complexity of the Systems.

Inspired by nature, where gene regulatory networks consisting of intercommunicating constituents, each composed of three or more components, play a central role in the development of living systems, we use the information encoded in the base sequences of nucleic acids to construct three-dimensional constitutional dynamic networks (3D CDNs) consisting of eight three-component constituents (AiBjCk). Upon subjecting the parent 3D CDN I to four auxiliary nucleic acid triggers (T1, T2, T3, or T4), the adaptive reconfiguration of CDN I into four different CDNs (II, III, IV, or V) is demonstrated, and by applying two consecutive triggers or counter triggers, the adaptive reversible hierarchical control over the compositions of new CDN systems (VI, VII, VIII, or IX) is demonstrated. The labeling of the constituents with nine different Mg2+-ion-dependent DNAzyme reporter units and the incorporation of a fluorescent dye/anticocaine aptamer complex into the structure of one of the constituents enable the quantitative evaluation of the contents of the constituents in the different CDNs. The quantification of the compositions of the CDNs is based on the activities of the DNAzymes conjugated to the constituents, the fluorescence signals upon the cocaine-induced separation of the dye/aptamer complex, appropriate calibration curves, and the set of equations. These assessments are further supported by quantitative electrophoretic experiments of the respective CDNs.

Evolution of Nucleic-Acid-Based Constitutional Dynamic Networks Revealing Adaptive and Emergent Functions.

The evolution of networks is a fundamental unresolved issue in developing the area of systems chemistry. We introduce a versatile rewiring mechanism that leads to the emergence of nucleic-acid-based constitutional dynamic networks (CDNs). A two-component constituent AA' functionalized with a Mg2+ -ion-dependent DNAzyme activator unit forms a complex with an intact hairpin HBB' composed of B and B' sequences. Cleavage of HBB' leads to the two-component constituent BB', and its rewiring with AA' yields CDN X composed of the equilibrated constituents AA', AB', BA', and BB'. In analogy, subjecting AA' to an intact hairpin HCC' leads to the formation of CDN Y consisting of AA', AC', CA', and CC'. Subjecting AA' to the mixture of HBB' and HCC' evolves the [3×3] CDN Z, composed of nine constituents, thus demonstrating hierarchical adaptive properties. Furthermore, the DNAzyme units associated with the constituents are applied to tailor emerging catalytic functions from the different CDNs.

Hairpin/DNA ring ternary probes for highly sensitive detection and selective discrimination of microRNA among family members

The detection and quantification of microRNA (miRNA) plays essential roles in clinical and biomedical research. Yet, it is of major challenge to sense miRNA with high degree of selectivity and sensitivity due to its unique characteristics of short length, similarity of sequence among family members and low abundance. Here, with the design of a new hairpin/DNA ring ternary probe, we describe the development of a rolling circle amplification (RCA) method for sensitively and selectively sensing miRNA from cancer cells. The target miRNA binds the hairpin/DNA ring probes through toehold-mediated strand displacement (TSD) to form the ternary structures, in which the bound miRNA and DNA ring are respectively used as the primer and template to realize RCA, leading to the generation of many repeated metal ion-dependent DNAzyme sequences. The fluorescently quenched hairpin signal probes can be cyclically cleaved by these DNAzyme sequences with co-existence of the corresponding metal ions in buffer to show drastically enhanced fluorescence recovery for highly sensitive sensing of miRNA in the range between 10 fM and 10 nM with a detection limit of 1.51 fM. Besides, owing to the high base variation discrimination ability of TSD, selective detection of the target miRNA among the corresponding family members can be achieved by this method. Moreover, such a method can also be employed to differentiate miRNA expression variations in cancer cells for screening potential therapeutic drugs.

Electrochemical selective detection of carnitine enantiomers coupling copper ion dependent DNAzyme with DNA assistant hybridization chain reaction

An electrochemical method for carnitine enantiomers recognition is proposed employing Cu (II)-l-cysteine complexes and signal amplification strategies based on DNAzyme and DNA assistant hybridization chain reaction (HCR). The reported method combined Cu (II)-amino acids complexes with DNAzyme and proposed a smart and efficient electrochemical method to recognize chiral molecules through the difference between homochiral interaction and heterochiral interaction. Moreover, further employment of hairpin DNA assistant HCR greatly enhanced the detecting signal, realizing the selective recognition of carnitine enantiomers in low concentrations. This study creatively provides a general sensing method for sensitive chiral molecules recognition without large-scale precision instruments.