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In this work, an ultrasensitive electrochemiluminescence (ECL) biosensor was constructed based on DNA-stabilized Au Ag nanoclusters (DNA-Au Ag NCs) as the efficient luminophore and Au NPs@Ti 3 C 2 as a new coreaction accelerator for determining microRNA-221 (miRNA-221) related to liver cancer. Impressively, DNA-Au Ag NCs were stabilized by the high affinity of the periodic 3C sequence, exhibiting an excellent ECL efficiency of 27% compared with classical BSA-Au Ag NCs (16%). Moreover, the Au NPs@Ti 3 C 2 nanocomposites, as a new coreaction accelerator, were first introduced to accelerate the production of abundant sulfate free radicals (SO 4 ) for promoting the ECL efficiency of DNA-Au Ag NCs in the DNA-Au Ag NCs/Au NPs@Ti •- ) for promoting the ECL efficiency of DNA-Au Ag NCs in the DNA-Au Ag NCs/Au NPs@Ti 3 C 2 /S 2 O 8 2- ternary system due to the energy band of Au NPs@Ti 3 C 2 . Furthermore, the trace target (miRNA-221) could drive the rolling circle amplification to generate an amount of output DNA with periodic 3C and 10A sequences. Through covalent bonds on the surface of poly A and Au NPs, the distance between the luminophor and the coreaction accelerator could be narrowed to further enhance the detection sensitivity. As a result, the constructed sensor has been applied for the ultrasensitive detection of miRNA-221 with a low detection limit of 50 aM and successfully monitored miRNA-221 in MHCC-97L and HeLa cell lysates. This strategy could be utilized for guiding the synthesis of light-emitting DNA-metal NCs, which has great potential in the construction of ultrasensitive biosensors for the early diagnosis of diseases.2 O 8 2- . Furthermore, the trace target (miRNA-221) could drive the rolling circle amplification to generate an amount of output DNA with periodic 3C and 10A sequences. Through covalent bonds on the surface of poly A and Au NPs, the distance between the luminophor and the coreaction accelerator could be narrowed to further enhance the detection sensitivity. As a result, the constructed sensor has been applied for the ultrasensitive detection of miRNA-221 with a low detection limit of 50 aM and successfully monitored miRNA-221 in MHCC-97L and HeLa cell lysates. This strategy could be utilized for guiding the synthesis of light-emitting DNA-metal NCs, which has great potential in the construction of ultrasensitive biosensors for the early diagnosis of diseases.

Herein, a fluorescence light-up 3D DNA walker (FLDW) was powered and accelerated by endogenous adenosine-5'-triphosphate (ATP) molecules to construct a biosensor for sensitive and rapid label-free detection and imaging of microRNA-221 (miRNA-221) in malignant tumor cells. Impressively, ATP as the driving force and accelerator for FLDW could significantly accelerate the operation rate of FLDW, reduce the likelihood of errors in signaling, and improve the sensitivity of detection and imaging. When FLDW was initiated by output DNA H1-op transformed by target miRNA-221, G-rich sequences in the S strand, anchored to AuNP, were exposed to form G-quadruplexes (G4s), and thioflavin T (ThT) embedded in the G4s emitted intense fluorescence to realize sensitive and rapid detection of target miRNA-221. Meanwhile, the specific binding of ThT to G4 with a weak background fluorescence response was utilized to enhance the signal-to-noise ratio of the label-free assay straightforwardly and cost-effectively. The proposed FLDW system could realize sensitive detection of the target miRNA-221 in the range of 1 pM to 10 nM with a detection limit of 0.19 pM by employing catalytic hairpin assembly (CHA) to improve the conversion of the target. Furthermore, by harnessing the abundant ATP present in the tumor microenvironment, FLDW achieved rapid and accurate imaging of miRNA-221 in cancer cells. This strategy provides an innovative and high-speed label-free approach for the detection and imaging of biomarkers in cancer cells and is expected to be a powerful tool for bioanalysis, diagnosis, and prognosis of human diseases.

Herein, a giant-sized DNA nanoarray was subtly assembled by two kinds of independent tetrahedral DNA structures as the DNA track for a multi-armed three-dimensional (3D) DNA nanomachine to perform signal transduction and amplification efficiently, which was developed as an electrochemical biosensor for the rapid and ultrasensitive detection of matrix metalloproteinase 2 (MMP-2). Impressively, in contrast to conventional DNA walkers with inefficiency, which walked on random DNA tracks composed of a two-dimensional (2D) probe or a one-dimensional (1D) single-stranded (ss)DNA probe, the multi-armed 3D DNA nanomachine from exonuclease III (Exo III) enzyme-assisted target recycling amplification would be endowed with faster reaction speed and better walking efficiency because of the excellent rigidity and orderliness of the tetrahedral DNA nanoarray structure. Once the hairpin H 3 -label with the signal substance ferrocene (Fc) was added to the modified electrode surface, the multi-armed 3D DNA nanomachine would be driven to move along the well-designed nanoarray tracks by toehold-mediated DNA strand displacement, resulting in most of the ferrocene (Fc) binding to the electrode surface and a remarkable increase in electrochemical signals within 60 min. As a proof of concept, the prepared biosensor attained a low detection limit of 11.4 fg/mL for the sensitive detection of the target MMP-2 and was applied in Hela and MCF-7 cancer cell lysates. As a result, this strategy provided a high-performance sensing platform for protein detection in tumor diagnosis.

Herein, a novel two-dimensional ladder-like DNA nanostructure (LDN)-mediated cascade catalytic nanomachine (LDN-CCN) with a higher catalytic efficiency beyond those of a conventional one-dimensional hybrid chain reaction (HCR) nanostructure-mediated CCN was constructed and applied to design an electrochemical biosensing platform with first-rank performance for ultrasensitive detection of target miRNA-21. First, output DNA S1' and S2' were produced through the DNAzyme recycle amplification when the target miRNA-21 existed. Then, the controllable LDN-CCN was constructed on S1-S2 modified electrodes by the subsequent reaction triggered by S1' and S2' with H1-AuNPs, H2, H3-AuNPs, and H4 with the assistance of K + and hemin, in which the hemin/G-quadruplexes could produce a prominent electrochemical signal response to the substrate glucose. The best performance of cascade catalysis was acquired when the distance of Au nanoparticles (glucose oxidase-like activity) modified on H1 and H3 and hemin/G-quadruplexes (peroxidase-like activity) formed by the sticky ends of H2 and H4 was roughly 9 nm (27 bp) in LDN-CCN. The constructed electrochemical platform realized the sensitive detection of target miRNA-21 with the linear range from 100 aM to 10 nM and with a detection limit as low as 48.5 aM, which provided novel insights to explore the new functional DNA nanostructure and well-performing mimic enzyme cascade catalytic platforms for applications in biological fields and early diagnosis of diseases.

Herein, with skillfully engaging stable T-Hg 2+ -T bonding, a " Crab Claw "-like DNA nanomachine with concise and highly efficient assembly and enhanced recognition/conversion efficiency was engineered as a super signal amplifier, which was united with Pd@Cu@Pt multimetallic mesoporous nanomaterials (Pd@Cu@Pt MMNs) for ultrasensitive electrochemical assay of mercury ions (Hg 2+ ). Specifically, the formed " Crab Claw "-like DNA nanomachine could simultaneously trigger four same cascade DNAzyme cleavage reactions with the help of Mg 2+ DNAzyme for markedly converting target Hg 2+ to enormous DNA segments labeled with ferrocene (Fc), improving the detection sensitivity. Subsequently, the prepared Pd@Cu@Pt MMNs could not only show commendable electrochemical catalysis to Fc but also act as an excellent immobilization matrix for capturing and accumulating abundant Fc around them to further strengthen the electrochemical signal. As a result, the well-designed electrochemical sensor could achieve a low limit of detection of 3.58 fM in the range from 10 fM to 100 nM for Hg 2+ detection. This strategy offers a simple and rapid avenue to detect heavy metal ions and shows promising application potential for environmental pollutant monitoring.

In this work, an ultrasensitive photoelectrochemical (PEC) assay was established for sensitive DNA detection based on a novel SnS 2 /Co 3 O 4 sensitized structure as a photoactive matrix and benzo-4-chlorohexadienone (4-CD) precipitate as a signal quencher. Noticeably, the photoelectric conversion efficiency of the SnS 2 /Co 3 O 4 sensitized structure was dramatically enhanced due to the effective sensitization of Co 3 O 4 toward SnS 2 , thus attaining an intense photocurrent response, which was sixfold higher than that of pristine SnS 2 . Additionally, with the assistance of Nt.BstNBI enzyme-assisted target cycling process, a limited amount of target DNA (a fragment of p53 gene) could be converted into extensive output DNA, which could hybridize with capture DNA to yield abundant DNA duplex for loading mimetic enzyme manganese porphyrin (MnPP). Subsequently, 4-chloro-1-naphthol (4-CN) could be catalyzed to form 4-CD precipitate by MnPP on the modified electrode surface with the existence of H 2 O 2 . Then, the 4-CD precipitate severely hampered electron transfer, causing a prominently diminished photocurrent response for DNA determination. The elaborated PEC assay not only extended the application of SnS 2 in the PEC biosensing field but also manifested a wide linear range of 100 aM to 1 nM with a low detection limit of 30 aM, exhibiting enormous potential for the detection of various biomarkers or other targets in bioanalysis and disease diagnosis fields.

Herein, a simple and regulable DNA dimer nanodevice was obtained by the assembly of two hairpin DNA monomers of H1 and H2 to control the distance between model enzymes horseradish peroxidase (HRP) and glucose oxidase (GOx) for sensitive electrochemical detection of microRNA. In detail, auto-terminated DNA polymerization reaction was designed on H1 and H2 monomers that decorated with HRP and GOx, respectively, to produce two half-released DNA monomers, which were spontaneously hybridized to each other, thereby obtaining a DNA dimer nanodevice with a rigid dsDNA linker between two DNA monomers. By varying the length of the dsDNA linker on the DNA dimer nanodevice, the distance between GOx and HRP had been regulated to the optimum and the most efficient enzyme cascade reaction was acquired for constructing a sensitive electrochemical microRNA-21 biosensor with a detection limit of 0.03 pM. In summary, the proposed DNA dimer nanodevice avoided the disadvantages of poor biocompatibility and controllability originated from traditional scleroid scaffolds and showed obvious advantages in terms of better assembly yield than previous complicated DNA scaffolds, which provided a novel strategy for developing a high-efficiency enzyme cascade catalytic system and showed great potential in other clinical diagnosis and bioanalysis application.

Herein a photoelectrochemical (PEC) and electrochemical (EC) dual-mode biosensor with cationic N , N -bis(2-(trimethylammonium iodide)propylene)perylene-3,4,9,10-tetracarboxydiimide (PDA + )-decorated multifunctional DNA spheres in situ generated on an electrode was proposed for sensitive and accurate detection of miRNA-141. By employing a target-related ternary "Y" structure cleavage cycling reaction, the target DNA was converted into massive output DNA anchored on a TiO 2 substrate, and hence triggering the rolling circle amplification (RCA) reaction. Upon addition of magnesium ions and PDA + , the long DNA tails of the RCA product were condensed in situ to form multifunctional DNA spheres. Notably, the distance between DNA spheres and TiO 2 substrate was short, thus forming an effective PDA + -TiO 2 sensitization structure with fast electron transfer for acquiring an extremely enhanced PEC signal with assistance of ascorbic acid (AA). Meanwhile, cationic PDA + with a large planar π-π skeleton enabled favorable redox-activity and substantial loading on DNA spheres, directly producing an obviously well-defined cathodic peak for implementing EC biodetection on the same sensing platform. This approach not only avoided difficult assembly of diverse signal indicators but also significantly improved the sensitivity by utilizing cleavage cycling amplification and RCA strategies. Moreover, the distinct dual-response signals from two different transduction mechanisms and independent signal transduction can mutually support accuracy improvement. As a result, detection ranges of 0.1 fM to 1 nM for PEC and 2 fM to 500 pM for EC were obtained for miRNA-141, providing a universal and efficient biosensing method with promising applications in bioanalysis and early disease diagnosis.

In this work, the array arrangement of cascade enzymes was implemented by alternately and equidistantly anchoring two model enzymes glucose oxidase (GOx) and horseradish peroxidase (HRP) to the vertexes of rigid DNA tetrahedron units in lattice-like nucleic acid scaffold, in which the distance between any adjacent cascade enzymes had been regulated to the optimum for obtaining high enzyme cascade catalytic efficiency. Compared to the enzyme cascade system with no-array arrangement of cascade enzymes, the proposed enzyme cascade system allowed the intermediate H 2 O 2 produced by GOx catalyzing substrate glucose to concurrently and equidistantly diffuse toward the four adjacent HRP enzyme surfaces. In this case, the invalid diffusion effect of intermediate H 2 O 2 between cascade enzymes could be effectively avoided, thereby promoting the enzyme cascade reaction with high catalytic efficiency. The specific catalytic efficiency ( k cat / K m ) of the cascade enzyme system with array arrangement had been evaluated, which exhibited catalytic efficiency about 3.6 times higher than that of the randomly arranged cascade enzyme system. As a result, this strategy provided a new avenue for constructing a highly efficient enzyme cascade system with ultimate applications in biosynthesis, bioanalysis, and biodiagnostics.

Here, a target-induced three-dimensional DNA network structure (T-3D Net) produced by catalytic hairpin assembly (CHA) was proposed as a novel signal amplifier to fabricate an ultrasensitive electrochemiluminescence (ECL) biosensor for microRNAs detection. Usually, conventional CHA can produce only one output DNA in each target cycle, while the proposed strategy could produce multiple output DNA by using DNA-functionalized magnetic beads (MBs) and gold nanoparticles (AuNPs) to form T-3D Net. Then, the T-3D Net with high loading capacity could be completely collapsed by dissolving AuNPs to efficiently convert trace microRNA-21 into a large amount of output DNA. Furthermore, the nanocomposite containing Ru(bpy) 3 as luminophore and boron nitride quantum dots (BNQDs) as coreactant provided a strong initial ECL response owing to the short electron transfer distance between luminophore and coreactant (signal-on). Next, the DNA duplex probes labeled with  2+ as luminophore and boron nitride quantum dots (BNQDs) as coreactant provided a strong initial ECL response owing to the short electron transfer distance between luminophore and coreactant (signal-on). Next, the DNA duplex probes labeled with  N -(4-aminobutyl)- N (signal-off). In the presence of the output DNA with enzyme-assisted self-recycling, S2-DA was displaced and detached from the electrode surface to achieve ECL signal recovery. Simultaneously, S1-ABEI restored a stable hairpin structure, making ABEI close to the electrode surface for more effective resonance energy transfer (RET) between ABEI and Ru(bpy) 3 2+ (signal-off). In the presence of the output DNA with enzyme-assisted self-recycling, S2-DA was displaced and detached from the electrode surface to achieve ECL signal recovery. Simultaneously, S1-ABEI restored a stable hairpin structure, making ABEI close to the electrode surface for more effective resonance energy transfer (RET) between ABEI and Ru(bpy) 3 2+ , which greatly improved the final ECL response (signal-super on). Thus, the ECL biosensor demonstrated superior performance for ultrasensitive detection of microRNA-21 with low detection limit (0.33 aM) and was successfully applied to monitor the expression of microRNA-21 in human cancer cell lysates. This strategy provided an ultrasensitive way for the detection of biomolecules and revealed an effective avenue for diseases diagnosis.

In this work, a bipedal DNA walker was employed to mediate an efficient enzyme-free exponential isothermal DNA signal amplification. On the basis of the bipedal DNA walker mediated enzyme-free exponential isothermal signal amplification, an electrochemiluminescence (ECL) biosensor was constructed for sensitive and rapid detection of microRNA (miRNA) with a limit of detection down to 0.24 fM and requiring less than 40 min.

In this protocol, a photoelectrochemical (PEC) biosensor based on methylene blue (MB) sensitized fullerene (C 60 ) was designed for ultrasensitive DNA detection. First, C 60 nanoparticles (C 60 NPs) as photoactive material was modified on the electrode surface to obtain an initial photocurrent signal. Additionally, the nicking endonuclease (Nb.BvCI enzyme)-assisted signal amplification strategy was implemented to transform limited target to plenty of products, which hybridized with DNA2 to form plentiful DNA duplex for immobilizing numerous MB as the sensitizer, thus accomplishing the effective sensitization toward C 60 NPs and achieving an enhanced photocurrent for quantitatively detecting target DNA. This established PEC strategy realized the target DNA detection with the detection limit down to 3.3 fM, which paved a new avenue for DNA determination and exhibited huge application potential for other biomarkers in clinical analysis and disease diagnosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

DNA amplification strategy has been a valuable tool for improving the sensitivity of biosensors. However, the freely diffusing reactants in most DNA amplification strategies limit the rate of DNA reaction, which further affects the amplification efficiency with unsatisfactory sensitivity. In the present work, a novel localized DNA cascade reaction (LDCR) in a DNA nanomachine was designed for high-efficiency target conversion to construct an electrochemiluminescence (ECL) biosensor for ultrasensitive microRNA-21 detection. The DNA nanomachine was constructed by using three-footholds DNA scaffold to immobilize two metastable hairpins and reporter probe and confine them in a localized space. In the presence of microRNA-21, it initiated the LDCR and produced large amounts of mimic target (ferrocene labeled DNA, Fc-DNA) due to the locality effect. Thus, sensitive detection of microRNA-21 could be realized since Fc could effectively quench the ECL intensity of graphitic carbon nitride nanosheets (CNNS) due to the energy and electron transfer from the excited state of CNNS to oxidized species of Fc. Moreover, compared with the other two developed DNA cascade reactions with freely diffusing reactants, the proposed LDCR benefits by shortening the reaction time and improving the amplification efficiency with enhanced sensitivity of the biosensor. Therefore, the proposed LDCR could be used as a highly efficient amplification strategy for ultrasensitive determination of biomarkers with low abundance, which may promote the diagnostic efficiency of disease.

In this work, a classified cargo-discharge DNA robot with only two DNA strands was designed and driven by an analogous proximity ligation assay (aPLA)-based enzyme cleaving for fast walk to construct a novel electrochemical biosensor for simultaneously ultrasensitive detection of microRNA-155 (miRNA-155) and miRNA-21. Compared with traditional DNA nanomachines, the multifunctional DNA robot possessed simple structure, high self-assembling efficiency and walking efficiency. Once it interacted with target miRNAs, this DNA robot could walk fast on the electrode surface and realize the classified cargoes discharging including beacons methylene blue (MB) and ferrocene (Fc), respectively labeled in the double-stranded DNA (A1-A2) for ultrasensitive detection of multiple miRNAs simultaneously. As a result, the wide linearity ranging from 100 aM to 100 pM and low detection limits of 42.7 and 51.1 aM were obtained for miRNA-155 and miRNA-21 detection, respectively. As a proof of concept, the present strategy initiates a novel and highly efficient walking platform to realize the ultrasensitive detection of biomarkers and possesses potential applications in the clinical diagnosis of disease.

In this work, a novel ratiometric SERS biosensor with only one Raman probe was fabricated in combination with the DNA hydrogel-captured glucose oxidase (GOx) amplification method to realize an accurate and sensitive assay for microRNA (miRNA) 122, which could overcome the complex operational process and chemical waste issues of the traditional ratiometric approach with two different signal probes. Here, 3-mercaptophenylboronic acid (3-MPBA) as a standard reference with a unique Raman peak at 996 cm -1 was first connected to silica@Au nanoflower (Si@AuNF) SERS substrates. When the DNA hydrogel containing GOx was opened by the released DNA (R) from the target miRNA-induced cycle amplification process, GOx could be released from the DNA hydrogel. Therefore, GOx could catalyze the oxidation of glucose to produce H 2 O 2 on Si@AuNFs. Subsequently, the acquired H 2 O 2 could further react with 3-MPBA to produce 3-hydroxythiophenol (3-HTP) with a new Raman peak at 883 cm -1 . During this time, the intensity of the peak at 996 cm -1 almost remained the same, which could act as a reference standard. The intensity ratio of 883 cm -1 to 996 cm -1 increased with the increase in the concentration of target miRNA 122, thus achieving quantitative detection of miRNA 122. As a consequence, our SERS biosensor could sensitively detect miRNA 122 from 10 aM to 100 pM, and the detection limit was 7.75 aM. Our strategy adopts a novel ratiometric method with one Raman probe to detect miRNA, opening a new avenue for the detection of trace amounts of biological samples with high sensitivity and accuracy.

Intelligent DNA walking machines have become a great hot spot in biosensing, but the walking efficiency of DNA walking machines was still limited due to the low local concentration of substance DNA and the derail of leg DNA. Herein, a Zn 2+ -driven DNA rolling machine was proposed to overcome the above shortages and applied as a electrochemiluminescence (ECL) biosensor for speedy ultrasensitive detection of microRNA-21. First, the original DNA rolling machine was synthesized by numbers of leg DNA modified on Au nanoparticle which matched with the high concentration of track DNA on the sensing platform and could roll efficiently through Zn 2+ driving. By this way the DNA rolling machine not only increased the local concentration of leg DNA and track DNA to improve walking efficiency but also changed the motion mode from step-by-step walking to high-speed rolling, weakening the derailment of leg DNA and shortening the moving time. Second, target-induced recycling and acid dissolution could convert a finite amount of target microRNA into a large amount of Zn 2+ , which greatly improved the sensitivity of biosensor and overcame the drawbacks of enzyme cleavage or polymerization in common nucleic acid amplification methods. Lastly, the obtained Zn 2+ was employed to drive the DNA rolling machine through specific sites recognizing and track DNA cutting to remove a quencher of ferrocene, recovering ECL emission of CdS:Mn QDs for microRNA-21 detection with a detection limit of 0.28 fM. Besides, the biosensor was successfully applied in microRNA-21 analysis from human cancer cell lysates, offered a controllable and ultrasensitive strategy for speedy detection of microRNA, and revealed a new avenue for clinical analyses.

In this work, a rigid DNA tetrahedron (TDN) scaffold was synthesized to precisely control the interenzyme distance by randomly anchoring two pairs of horseradish peroxidase (HRP)/glucose oxidase (GOx) at the vertices of TDN, which could not only avoid the drawbacks of poor controllability and biocompatibility from traditional scleroid skeletons, but also overcome the defect of imprecise regulation for interenzyme distance caused by DNA origami. Impressively, by varying the side length of TDN scaffold, the interenzyme distance was precisely regulated, thus, an optimal TDN scaffold with highest catalytic efficiency was acquired and subsequently applied for constructing an ultrasensitive biosensor for DNA detection with a low detection limit of 3 fM. This strategy paved an avenue for developing new reliable scaffold to precisely regulate the catalytic efficiency of enzyme cascade reaction with ultimate applications in bioanalysis and clinical diagnosis.

Though preferable progresses have been achieved to improve the photoelectric performance of fullerene (C 60 NPs) by sensitized structure in photoelectrochemical (PEC) field, further application inevitably suffers from the inherent scarcities of heavy metal-involved quantum dots as sensitizers containing restricted sensitization effect, complex preparation and biological toxicity. In this work, a PEC biosensor based on [Ru(dcbpy) 2 dppz] 2+ /Rose Bengal dyes co-sensitized C 60 NPs was constructed for ultrasensitive DNA (a fragment sequence of p53 gene) detection. With the merits of low toxicity and accessible operation, [Ru(dcbpy) 2 dppz] 2+ /Rose Bengal dyes exhibited a further sensitization efficiency towards C 60 NPs. Through modifying wide band gap C 60 NPs with two narrower band gap dyes ([Ru(dcbpy) 2 dppz] 2+ and Rose Bengal) to form a cascade-type energy band structure, the photoelectric conversion of C 60 NPs was significantly improved and the visible light absorption was markedly promoted, leading to an exceptional photocurrent signal. Additionally, Nt.BstNB I enzyme-assisted target recycling amplification was employed to convert a limited quantity of target to numerous SiO 2 NPs-labeled DNA sequences (the signal quencher), resulting in a sharp decrement of photocurrent since a dramatic increment of steric hindrance on the modified electrode surface, which was performed to quantitatively estimate target. The proposed PEC biosensor for DNA detection possessed a wide linear range from 0.1 fM to 1 nM with a calculated detection limit of 37 aM. This work opened up an intriguing avenue for determination of various targets such as DNAs, microRNAs and proteins, and exhibited desirable application potential in the clinic researches, cancer therapies and other related subjects., (Copyright © 2018. Published by Elsevier B.V.)

In this work, a new label-free biosensor based on highly effective decomposition of a branched DNA polymer and target-transition recycling amplification was designed for ultrasensitive electrochemical determination of lead ion (Pb 2+ ). The branched DNA polymer formed by hybridization chain reaction (HCR) was first immobilized on electrode and served as the carrier for loading abundant silver nanoparticle (AgNPs) to generate an extremely high initial current signal output. Then, the molecular triggers T 1 and T 2 , which were produced through a target-transition recycling amplification, could respectively disassemble the backbone and side chain of the branched DNA polymer with remarkably decreased current for sensitive detection of Pb 2+ . Compared with traditional one-trigger induced disassembly strategy, the proposed approach exhibited higher decomposition efficiency. The experimental data showed that this developed biosensor had a superior performance for Pb 2+ detection with a low detection limit down to 0.24 pM. Furthermore, the established strategy set up a new way to achieve the ultrasensitive detection of other metal ions., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Traditional scaffolds such as metal nanoparticles and DNA origami remain a considerable challenge to regulate the enzyme cascade catalytic efficiency dynamically and reversibly on account of their irreversible conformation. To address these issues, a regenerated DNA tweezer was designed to dynamically regulate the interenzyme spacing for high-efficiency enzyme cascade amplification for homogeneous determination of target DNA related to cancer diseases. Initially, the enzyme-functionalized DNA tweezer was maintained at the opened state with a relatively distant interenzyme distance (19-24 nm), leading to a low catalytic efficiency. Benefiting from target induced Mg 2+ -dependent DNAzyme cleavage recycling, the one input target could be transduced to multiple corresponding methylene blue (MB) labeled DNA (S5), which served not only as the signal probe to provide a detectable electrochemical signal but also fuel to switch the DNA tweezer from the opened to closed state, leading to cascaded enzymes close enough (5-10 nm) for enhancing the catalytic efficiency for sensitive target DNA analysis with a low detection limit down to 30 fM. In the presence of antifuels, the closed DNA tweezer easily switched back to the opened state via a one-step strand displacement, and the obtained DNA tweezer achieved regeneration for subsequently recycling target detection. With the dynamical regulation of interenzyme distance in an "open-close-open" way, the enzyme cascade catalytic efficiency became dynamically controllable, and the DNA tweezer realized simple reutilization over five times, overcoming the drawbacks of inflexible, time-consuming operation and false positive signal induced by traditional scaffolds. More importantly, this method opened a new avenue for employing the arbitrary change of enzyme cascade catalytic efficiency for sensitive detection various biomolecules.

In this work, the highly efficient target recycling-based netlike Y-shaped DNA (Y-DNA), which regulated the electrocatalysis of Fe 3 O 4 @CeO 2 -Pt nanoparticles (Fe 3 O 4 @CeO 2 -PtNPs) toward methylene blue (MB) for signal amplification, was developed to prepare a sensitive DNA biosensor for detecting the DNA associated with oral cancer. Specifically, with the help of highly efficient enzyme-assisted target recycling (EATR) amplification strategy, one target DNA input was converted to corresponding plenty of DNA strands S1-Fe 3 O 4 @CeO 2 -Pt and S2-MB output, which could be employed to interact with HP2 immobilized on the electrode surface to form stable netlike Y-DNA without any waste of recycling products. Meanwhile, the formation of netlike Y-DNA could regulate electrocatalytic efficiency of Fe 3 O 4 @CeO 2 -PtNPs, inducing the proximity of Fe 3 O 4 @CeO 2 -PtNPs to MB and significantly enhancing electrochemical signal. Further, the signal could also be amplified by Fe 3 O 4 @CeO 2 -PtNPs modified on the electrode surface. By virtue of this ingenious design, a novel netlike Y-DNA structure based on highly efficient EATR was simply constructed and successfully applied to an electrochemical DNA biosensor along with electrocatalysis of Fe 3 O 4 @CeO 2 -PtNPs, achieving the sensitive detection of target DNA ranging from 10 fM to 50 nM with a detection limit of 3.5 fM. Impressively, the biosensor here demonstrates an admirable method for regulating the electrocatalysis of NPs toward substrates to enhance signal, and we believe that this biosensor is a potential candidate for the sensitive detection of target DNA or other disease-related nucleic acids.

Usually, photoelectrochemical (PEC) assays were devoted to the direct modification of photoactive materials on sensing interface, thereby producing high initial signal and unneglected background noise, which could further result in low sensitivity and restricted detection limit during the detection of targets. In this work, a PEC biosensor with near-zero background noise was established for ultrasensitive microRNA-141 (miRNA-141) detection based on DNA tetrahedron (TET) as nanocarrier for efficient immobilization of CdTe quantum dots (QDs)-Methylene Blue (MB) (TET-QDs-MB complex) as signal probe. First, CdTe QDs as PEC signal indicator was bound to the TET through DNA hybridizations. Then, massive MB as PEC signal enhancer was attached to DNA duplex of the TET immobilized with CdTe QDs via intercalation. Thereafter, the as-prepared TET-QDs-MB complex was considered as an efficient PEC signal probe owing to its excellent photovoltaic properties, thereby avoiding direct modification of photoactive materials on sensing interface and producing a near-zero background noise to improve the sensitivity of this PEC biosensor. Besides, the detection sensitivity could be further improved with the help of the duplex specific nuclease (DSN) enzyme-assisted target cycling amplification strategy. The proposed PEC biosensor performs a wide linear range from 50 aM to 50 pM with a low detection limit of 17 aM for miRNA-141, paving a new and promising horizon for highly accurate and ultrasensitive monitoring of multifarious analytes such as proteins, DNAs, and miRNAs in bioanalysis and disease diagnosis.

In this work, an elegantly designed electrochemical biosensor was constructed for platelet-derived growth factor (PDGF) detection based on homogeneous entropy catalytic-induced DNA hydrogel as a strong signal blocker to significantly inhibit the electrochemical signal of g-C 3 N 4 @Au@Fc-NH 2 nanomaterials as signal tag. First, the good film-forming nanomaterials of g-C 3 N 4 @Au@Fc-NH 2 , containing large numbers of Fc-NH 2 with low resistance and high electric conductivity, were directly immobilized on an electrode surface to provide a strong original electrochemical signal, then the DNA hydrogel blocker formed by target-induced homogeneous entropy catalytic amplification was captured onto the modified electrode surface for significantly reducing the electrochemical signal, in which both the efficient conversion of the single protein to large numbers of DNA strands and the amplification of cycling products could doubly improve the detection sensitivity. As a result, the detection limit could reach 3.5 fM at the range of 0.01 pM to 10 nM. The present strategy by integration of a strong signal blocker to sharply reduce the electrochemical signal of signal tag initiates a new thought to realize the highly sensitive detection of biomarkers and possesses potential applications in clinical diagnosis, sensing, and other related subjects.

The portable and rapid detection of biomolecules via pH meters to monitor the concentration of hydrogen ions (H + ) from biological reactions (e.g. loop-mediated isothermal amplification, LAMP) has attracted research interest. However, this assay strategy suffered from inherent drawback of low sensitivity, resulting in great limitations in practical applications. Herein, a novel electrochemical biosensor was constructed for highly sensitive detection of Nosema bombycis gene DNA (PTP1) through transducing chemical stimuli H + from PTP1-based LAMP into electrochemical output signal of electroactive ferrocene (Fc). With use of target PTP1 as the template, the H + from LAMP induced the conformational switch of pH-responsive DNA nanostructures (DNA NSs, Fc-Sp@Ts) that was assembled by the hybridization of Fc-labeled signal probe (Fc-Sp) with DNA-based receptor (Ts). Due to the folding of Ts into stable triplex structure at decreased pH, the configuration change of Fc-Sp@Ts led to the releasing of Fc-Sp, which was subsequently immobilized in the electrode interface through the hybridization with the capture probe modified with -SH (SH-Cp), generating amplified electrochemical signal from Fc. The developed biosensor for PTP1 exhibited a reliable linear range of 1 fg µL -1 to 50 ng µL -1 with the limit of detection of 0.31 fg µL -1 . Thus, by the regulation of H + from LAMP reaction on DNA NSs allostery, this novel and simple transduction scheme would be interesting and promising to open up a novel analytical route for sensitive monitoring of different target DNAs in related disease diagnosis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In this work, one stimuli-responsive DNA microcapsule was designed to combine duplex-specific nuclease (DSN) amplification strategy and surface-enhanced Raman spectroscopy (SERS) technology for sensitive detection of microRNA 155 (miRNA 155). First, toluidine blue (TB) as Raman dye and CaCO 3 as core templates co-precipitated to form TB@CaCO 3 composite. Then, DNA networks were layer by layer constructed with oligonucleotide layers cross-linked by linker ssDNA L to lock TB@CaCO 3 inside. In the presence of ethylenediaminetetraacetic acid, the core CaCO 3 would be dissolved to form TB-loading DNA microcapsule. With target miRNA 155-induced DSN signal amplification, a large amount of simulative target ssDNA D was obtained, which can completely complement with the linker L on the DNA networks, destroying the microcapsule to release TB and obtain a strong Raman signal. So by this smart design, a SERS platform was fabricated on the basis of the stimuli-responsive DNA microcapsule to detect miRNA 155 from 1 fM to 10 nM with a detection limit of 0.67 fM. In the present study, the programmable property and rapid response speed of DNA microcapsule, which helped in fabrication of a new potential biosensing technology for miRNA detection that is anticipated to be applied for clinical diagnosis.

The DNA nanocrane with functionalized manipulator and fixed-size base offered a programmable approach to modulate the luminous efficiency of copper nanoclusters (Cu NCs) for achieving remarkable electrochemiluminescence (ECL) enhancement, further the Cu NCs as signal label was constructed in biosensor for ultrasensitive detection of microRNA-155. Herein, the DNA nanocrane was first constructed by combining binding-induced DNA assembly as manipulator and tetrahedral DNA nanostructure (TDN) as base, which harnessed a small quantity of specific target (microRNA (miRNA)-155) binding to trigger assembly of separate DNA components for producing numerous AT-rich double-stranded DNA (dsDNA) on the vertex of TDN. Upon the incubation of Cu 2+ on the AT-rich dsDNA, each DNA-stabilized Cu NCs probe could be in situ electrochemically generated on an individual TDN owing to the A-Cu 2+ -T bond. Thus, the generation of Cu NCs was highly regulated with AT-rich dsDNA as the template, and its lateral distance was tuned by the TDN size, which were two key factors to influence the luminous efficiency of Cu NCs. By coordinate modulation, the detection limit of the ultrasensitive biosensor for miRNA-155 down to 36 aM and the programmable modulation strategy paved the way for comprehensive applications of DNA nanomachines and metal nanoclusters in biosensing and clinical diagnosis.

In this work, an elegantly designed host-guest recognition-assisted electrochemical release was established and applied in a reusable electrochemical biosensor for the detection of microRNA-182-5p (miRNA-182-5p), a prostate cancer biomarker in prostate cancer, based on the DNA cross configuration-fueled target cycling and strand displacement reaction (SDR) amplification. With such a design, the single target miRNA input could be converted to large numbers of single-stranded DNA (S1-Trp and S2-Trp) output, which could be trapped by cucurbit[8]uril methyl viologen (CB-8-MV 2+ ) based on the host-guest recognition, significantly enhancing the sensitivity for miRNA detection. Moreover, the nucleic acids products obtained from the process of cycling amplification could be utilized sufficiently, avoiding the waste and saving the experiment cost. Impressively, by resetting a settled voltage, the proposed biosensor could release S1-Trp and S2-Trp from the electrode surface, attributing that the guest ion methyl viologen (MV 2+ ) was reduced to MV +· under this settled voltage and formed a more-stable CB-8-MV +· -MV +· complex. Once O 2 was introduced in this system, MV +· could be oxidized to MV 2+ , generating the complex of CB-8-MV 2+ for capturing S1-Trp and S2-Trp again in only 5 min. As a result, the simple and fast regeneration of biosensor for target detection was realized on the base of electrochemical redox-driven assembly and release, overcoming the challenges of time-consuming, burdensome operations and expensive experimental cost in traditional reusable biosensors and updating the construction method for a reusable bisensor. Furthermore, the biosensor could be reused for more than 10 times with a regeneration rate of 93.20%-102.24%. After all, the conception of this work provides a novel thought for the construction of effective reusable biosensor to detect miRNA and other biomarkers and has great potential application in the area requiring the release of nucleic acids or proteins.

In this study, an electrochemiluminescence (ECL) regenerated biosensor was reported to sensitively detect microRNA through 3D DNA walking machine and "on-off-super on" strategy. First, 3D DNA walking machine with higher efficiency of payload releasing and superior signal amplification than those of the traditional DNA walking machine was initially introduced in the ECL system for converting target microRNA to intermediate DNA and achieving significant signal amplification. Second, the distance between CdS:Mn quantum dots and Au nanoparticles was increased with the hybridization of intermediate DNA and Au nanoparticles modified S2, which weakened the energy transfer for ECL signal recovering and excited the surface plasma resonance for further enhancing the signal to construct the on-off-super on biosensor. Such an on-off-super on strategy not only reduced the ECL background signal but also increased the detection sensitivity. Impressively, the elaborately designed biosensor could be regenerated by Lambda exonuclease hydrolyzing the intermediate DNA to make Au nanoparticles modified S2 recover to their original hairpin structure. With the amazing signal amplification of 3D DNA walking machine and sensitive distance control of the on-off-super on strategy, the biosensor showed excellent performance for microRNA-141 detection with a low detection limit of 3.3 fM and could be applied to human prostate cancer cells analysis. Furthermore, this work established a foundation to apply 3D walker in ECL methodology and provided an effective way for analysis of other microRNA or cancer cells.

Usually, SERS technology requires labeling of the Raman reporter to obtain characteristic spectra for detection of biological samples. However, the number of labeled Raman reporters is often limited, resulting in the restricted improvement for sensitivity of SERS biosensor. In this work, switchable target-responsive 3D DNA hydrogels were introduced to precisely control trapping and release of Raman reporter toluidine blue (TB), which not only avoid labeling signal molecule but also improve the sensitivity of miRNA detection due to immobilization of abundant TB. In the absence of target miRNA, the DNA hydrogel presented a weak Raman signal because TB was far away from SERS substrates with an "OFF" status. However, the DNA hydrogel can be opened by the target miRNA to release TB producing strong Raman signal with an "ON" status. On the basis of this sensitive strategy, this switchable DNA hydrogel-based SERS platform can achieve quantitative detection of miRNA 155. Simultaneously, flexible SERS substrate (leaf@nafion@Ag) and target miRNA-induced duplex-specific nuclease signal amplification strategy were employed to significantly improve the sensitivity of the SERS platform. As a result, the as-proposed SERS platform can sensitively and selectively detect miRNA 155 with a wide linear range of 0.1 fM to 100 pM and low detection limit of 0.083 fM, which indicated that the platform has great potential to be applied in miRNA-related clinical diagnostics and biochemical researches.

Herein, a dual microRNA (miRNA) powered bi-directional DNA walking machine with precise control was developed to fabricate an enzyme-free biosensor on the basis of distance-based electrochemiluminescence (ECL) energy transfer for multiple detection of miRNAs. By using miRNA-21 as the driving force, the DNA walker could move forth along the track and generated quenching of ECL response due to the proximity between Au nanoparticles (AuNPs) and Mn 2+ doped CdS nanocrystals (CdS:Mn NCs) film as the ECL emitters, realizing ultrasensitive determination of miRNA-21. Impressively, once miRNA-155 was introduced as the driving force, the walker could move back along the track automatically, and surface plasmon resonance (SPR) occurred owing to the appropriate large separation between AuNPs and CdS:Mn NCs, achieving an ECL enhancement and realizing ultrasensitive detection of miRNA-155. The bi-directional movement of the DNA walker on the track led to continuous distance-based energy transfer from CdS:Mn NCs film by AuNPs, which resulted in significant ECL signal variation of CdS:Mn NCs for multiple detection of miRNA-21 and miRNA-155 down to 1.51 fM and 1.67 fM, respectively. Amazingly, the elaborated biosensor provided a new chance for constructing controllable molecular nanomachines in biosensing, disease diagnosis, and clinical analysis.

The construction of DNA nanomachines holds great significance in the development of DNA nanostructures; however, the real application of nanomachines is still in its early stage. Moreover, one-step regenerated sensing platforms for the detection of biomarkers in the current research remain a practical challenge. Herein, a novel electrochemiluminescence resonance energy transfer (ERET) strategy between Alexa Flour 488 (AF 488), which is a type of small molecule dye, as the donor and CdSe@ZnS quantum dots (QDs) as the acceptor, which easily enter the cells, has been reported and was applied for the construction of a DNA nanomachine-based regenerated biosensor for the ultra-high sensitive determination of cancer cells without any enzyme. First, a dual amplification strategy, including target recycling and signal transformation, was employed to achieve the conversion of a small number of miRNAs into a large amount of universal DNA reporters. Initially, the DNA tweezer was kept in the "off" state with two arms labeled with QDs and AF488, respectively. Second, in the presence of DNA reporters, the tweezer transformed to the "on" state through the hybridization of the reporter DNA and exposed the arms of the tweezer. Simultaneously, QDs and AF488 on the two arms were close enough to generate ERET, which remarkably increased the ECL intensity of the QDs. Impressively, the sensor could be regenerated by a one-step strand displacement and could be cycled for more than seven times. Owing to the dual amplification strategy and the high efficiency of the ERET between the QDs and AF488, the proposed biosensor performs in the linear range from 10 pM to 0.1 fM with a detection limit of 0.03 fM for miRNA determination, and the monitoring of different cancer cells was also achieved. Moreover, the elaborated biosensor can also realize the sensitive detection of Pb 2+ , which indicates that it can be potentially used for field environmental analysis and monitoring, thus offering a new modular platform for the construction of functional DNA nanomachines in the ultra-high sensitive analysis of promising biomarkers and toxic metals.

In this work, a label-free electrochemical biosensor was developed for sensitive and selective detection of mercury (II) ions (Hg(2+)) based on in-situ deposition of silver nanoparticles (AgNPs) on terminal deoxynucleotidyl transferase (TdT) extended ssDNA for signal output and nicking endonuclease for cycling amplification. In the presence of target Hg(2+), the T-rich DNA (HP1) could partly fold into duplex-like structure (termed as output DNA) via T-Hg(2+)-T base pairs and thus exposed its sticky end. The sticky end of output DNA could then hybridize with 3'-PO4 terminated capture DNA (HP2) on electrode surface to form output DNA-HP2 hybridization complex with the sequence 5'-CCTCAGC-3'/3'-GGAGTCG-5' (the sequence could be recognized by nicking endonuclease Nt. BbvCI). With the introduction of Nt. BbvCI, output DNA existed in hybridization complex was released from electrode and participated in the next hybridization process, accompanying with the cleave of HP2 to expose substantial 3'-OH group, which could be extended into a long ssDNA nanotail with the aid of TdT and deoxyadenosine triphosphate (dATP). Since the long negatively charged ssDNA nanotail absorbed the positively charged silver ions on the DNA skeleton, the metallic silver could be in-situ deposited on electrode surface for electrochemical signal output upon addition of reduction regent sodium borohydride. Under optimal conditions, the developed electrochemical biosensor presented a good response to Hg(2+) with a detection limit of 3 pM (S/N=3). Furthermore, the biosensor exhibited good reproducibility and high selectivity towards other interfering ions. The proposed sensing system also showed a promising potential application in real sample analysis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Currently, the photoelectrochemical (PEC) strategies can just achieve single analyte detection on a single interface with limited detection efficiency. It is highly valuable but full of challenge to develop a PEC biosensor for multiple analytes evaluation on a single interface. For this point, the wavelength-selective photoactive materials, which could generate separated photocurrents under excitation lights with certain wavelengths, were mainly important to overcome this challenge. Herein, these wavelength-selective photoactive materials were successfully synthesized and served as signal indicators to construct a novel PEC biosensor for multiple analytes evaluation on a single interface for the first time. Moreover, an enzyme-assisted target recycling amplification strategy was introduced for ultrasensitive monitoring. As a result, the proposed PEC biosensor showed excellent analytical performance for both oral cancer (ORVOA 1) gene and p53 gene down to attomolar level. In addition, the fabricated PEC biosensor was employed to evaluate ORVOA 1 gene and p53 gene in Hela cells. This assay has laid the foundation for fabrication of simple, ultrasensitive and economical PEC diagnostic devices to detect multiple analytes in cells, which paved a new avenue for early diagnosis of cancer with higher efficiency and accuracy., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Here, a novel "light-switch" molecule of Ru (II) complex ([Ru(dcbpy)2dppz](2+)-DPEA) with self-enhanced electrochemiluminescence (ECL) property is proposed, which is almost nonemissive in aqueous solution but is brightly luminescent when it intercalates into DNA duplex. Owing to less energy loss and shorter electron-transfer distance, the intramolecular ECL reaction between the luminescent [Ru(dcbpy)2dppz](2+) and coreactive tertiary amine group in N,N-diisopropylethylenediamine (DPEA) makes the obtained "light-switch" molecule possess much higher light-switch efficiency compared with the traditional "light-switch" molecule. For increasing the loading amount and further enhancing the luminous efficiency of the "light-switch" molecule, biotin labeled DNA dendrimer (the fourth generation, G4) is prepared from Y-shape DNA by a step-by-step assembly strategy, which provides abundant intercalated sites for [Ru(dcbpy)2dppz](2+)-DPEA. Meanwhile, the obtained nanocomposite (G4-[Ru(dcbpy)2dppz](2+)-DPEA) could well bind with streptavidin labeled detection antibody (SA-Ab2) due to the existence of abundant biotin. Through sandwiched immunoreaction, an ECL immunosensor was fabricated for sensitive determination of N-acetyl-β-d-glucosaminidase (NAG), a typical biomarker for diabetic nephropathy (DN). The detemination linear range was 0.1 pg mL(-1) to 1 ng mL(-1), and the detection limit was 0.028 pg mL(-1). The developed strategy combining the ECL self-enhanced "light-switch" molecular and DNA nanotechnology offers an effective signal amplification mean and provides ample potential for further bioanalysis and clinical study.

Based on a new target-triggered aptamer molecular machine, a label-free and non-enzymatic target recycling amplification strategy for sensitive fluorescence detection of ATP in human serums is described. The presence of the target ATP together with the DNA fuel strand initiates the operation of the aptamer machine and leads to cyclic reuse of ATP and the release of many G-quadruplex sequences, which associate with a fluorescent dye to generate significantly amplified fluorescence signals to achieve sensitive detection of ATP.

The presence of target microRNA triggers cascaded and catalytic self-assembly of two DNA motifs into DNA nanostructures, which serves as a remarkable signal amplification means for the highly sensitive monitoring of the target microRNA and the detection of low numbers of tumor cells.

Catalytic self-assembly of DNA nanostructures triggered by microRNA 21 (miR-21) is achieved through isothermal toe-hold strand displacement reactions. The miR-21 is autonomously recycled during the self-assembly process, which makes the generation of the DNA nanostructures proceed in a catalytic fashion. The miR-21-triggered self-assembly of DNA nanostructures can also serve as a remarkable signal amplification platform to achieve ultrasensitive detection of miR-21 from as low as 10 MCF-7 human breast cancer cells.

We describe here the construction of the DNA self-assembled molecular tweezers and the application of the tweezers for the monitoring of microRNA (miR-141) from human prostate cancer cells. The self-assembly formation of the DNA tweezers and the regulation of the tweezers upon alternative addition of the fuel miR-141 and the anti-fuel strands are characterized by native polyacrylamide gel electrophoresis. The addition of miR-141 to the DNA tweezers turns "off" the tweezers, while subsequent introduction of the anti-fuel strands switches the tweezers back to the "on" state, which verifies the regulatory ability of the tweezers. The miR-141-regulated DNA tweezers are concentration dependent and can be employed for sensitive detection of miR-141 down to 0.6 pM. The DNA tweezers also show high selectivity toward the fuel strand and can be used to monitor miR-141 expression in cancer cells, which provides new opportunities for the application of the dynamic DNA devices in clinical diagnostics., (Copyright © 2015 Elsevier B.V. All rights reserved.)

By coupling target DNA-induced reconfiguration of the dsDNA probes with enzyme-assisted target recycling amplification, we describe the development of a signal-on electrochemical sensing approach for sensitive detection of hereditary tyrosinemia type I gene. The dsDNA probes are self-assembled on the sensing electrode, and the addition of the target DNA reconfigures and switches the dsDNA probes into active substrates for exonuclease III, which catalytically digests the probes and leads to cyclic reuse of the target DNA. The target DNA recycling and the removal of one of the ssDNA from the dsDNA probes by exonuclease III result in the formation of many hairpin structures on the sensor surface, which brings the electroactive methylene blue labels into proximity with the electrode and produces a significantly amplified current response for sensitive detection of the target gene down to 0.24 pM. This method is also selective to discriminate single-base mismatch and can be employed to detect the target gene in human serum samples. With the demonstration for the detection of the target gene, we expect the developed method to be a universal sensitive sensing platform for the detection of different nucleic acid sequences.

Herein, we report on the development of a simple and sensitive biosensor for electrochemiluminescent (ECL) detection of microRNAs (miRNA) based on the intercalation of doxorubicin-conjugated quantum dot nanoparticles (Dox-QDs) into the DNA/RNA hybrids as the new signal acquisition and amplification platform. The thiolated DNA capture probes are self-assembled onto gold electrodes via the formation of Au-S bonds. The sensing surface is then incubated in a target miRNA-containing buffer solution to form the double-stranded duplexes. In this case, massive Dox-QDs can intercalate into the base pairs of the hybrid duplexes, resulting in amplified ECL emissions due to their reactions with the coreactant [Formula: see text] and the dissolved oxygen in the detection buffer. The increase in ECL intensity proportional to the amount of target miRNA in the testing samples serves as the quantitative basis. Different from traditional QDs-based methods such as labeling and embedding, our sensor involves the employment of the intercalation of the Dox-QDs as the signal acquisition and amplification platform. The combination of the QDs intercalation amplification with the high sensitivity of the ECL technique enables us to detect miRNA down to the low femtomolar level. Moreover, our method is also coupled with acceptable selectivity in discriminating the target miRNA and against its family members as well as other interference sequence, and can monitor miRNAs from human prostate carcinoma (22Rv1) cell lysates., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The construction of a restriction enzyme (Nt.AlwI)-powered DNA walking machine and its application for highly sensitive detection of DNA are described. DNA nanostructure tracks containing four overhang sequences with electrochemiluminescence (ECL) labels and complementary to the walker (target DNA) are self-assembled on the sensing electrode. The walker hybridizes with the complementary sequences on the tracks and forms specific recognition sites for Nt.AlwI, which cleaves the overhang sequences, releases the ECL labels and enables directional movement of the walker along the tracks. The formation of the nanostructure tracks and the Nt.AlwI-assisted cleavage of the overhang sequences in the presence of the walker are verified by using polyacrylamide gel electrophoresis analysis and cyclic voltammetry. The successive movement of the walker on the nanostructure tracks leads to continuous removal of massive ECL labels from the sensing electrode, which results in a significantly amplified suppression of the ECL emission for highly sensitive detection of sequence-specific DNA down to 0.19 pM. Results show that this DNA walking machine can also offer single-base mismatch discrimination capability. The successful application of the DNA walking machine for sequence-specific DNA detection can thus offer new opportunities for molecular machines in biosensing applications.

In this work, we proposed a novel electrochemical strategy for sensitive detection of thrombin (TB) based on in situ generation of polyaniline (PANI) as a redox mediator by using a manganese porphyrin-double stranded DNA (MnTMPyP-dsDNA) peroxidase-like artificial enzyme mimic as a powerful catalyst and template.

Based on nicking endonuclease (NEase)-assisted target recycling and rolling circle amplification (RCA) for in situ generation of numerous G-quadruplex/hemin complexes, we developed a new, dual amplified and ultrasensitive electrochemical biosensor for mutant human p53 gene. The target mutant DNA hybridizes with the loop portion of a dithiol-modified hairpin probe (HP) self-assembled on a gold sensing electrode and forms nicking site for the NEase, which cleaves the HP and releases the target DNA. The released target DNA again hybridizes with the intact HP and initiates the DNA recycling process with the assistance of the NEase, leading to the cleavage of a large number of the HPs and the generation of numerous primers for RCA. With rationally designed, G-quadruplex complementary sequence-encoded RCA circular template, subsequent RCA results in the formation of long DNA sequences with massive tandem-repeat G-quadruplex sequences, which further associate with hemin and generate significantly amplified current response for highly sensitive DNA detection down to 0.25 fM. The developed method also exhibits high specificity for the target DNA against single-base mismatched sequence. With the ultrahigh sensitivity feature induced by the dual signal amplification, the proposed method can thus offer new opportunities for the detection of trace amounts of DNA., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Endotoxin, also known as lipopolysaccharide (LPS), is able to induce a strong immune response on its internalization into mammalian cells. To date, aptamer-based biosensors for LPS detection have been rarely reported. This work describes a new signal-on electrochemical aptasensor for the ultrasensitive detection of LPS by combining the three-way DNA hybridization process and nanotechnology-based amplification. With the help of DNA1 (associated with the concentration of target LPS), the capture probe hybridizes with DNA1 and the assistant probe to open its hairpin structure and form a ternary "Y" junction structure. The DNA1 can be released from the structure in the presence of nicking endonuclease to initiate the next hybridization process. Then a great deal of cleaved capture probe produced in the cyclic process can bind with DNA2-nanocomposite, which contains the electroactive toluidine blue (Tb) with the amplification materials graphene (Gra) and gold nanoparticles (AuNPs). Thus, an enhanced electrochemical signal can be easily read out. With the cascade signal amplification, this newly designed protocol provides an ultrasensitive electrochemical detection of LPS down to the femtogram level (8.7 fg mL(-1)) with a linear range of 6 orders of magnitude (from 10 fg mL(-1) to 50 ng mL(-1)). Moreover, the high sensitivity and specificity make this method versatile for the detection of other biomolecules by changing the corresponding sequences of the capture probe and the assistant probe.

In this work, by integrating multiple signal enhancement approaches, a new cascade signal amplification strategy is described to achieve highly sensitive electrochemical DNA detection. The presence of the target DNA leads to the unfolding of the biotin-modified hairpin probes on the sensor surface. With the addition of the primer sequences and polymerase, the target DNA is recycled and reused through isothermal strand-displacement polymerase reactions (ISDPR) to unfold a large number of the probes, which offer numerous binding sites to capture alkaline phosphatase (ALP)-loaded nanoparticle labels. These surface captured ALP enzymes catalyze the conversion of p-aminophenylphosphate to p-aminophenol, which generates amplified catalytic current responses due to the redox-recycling process during the potential sweep in the presence of the co-reactant NADH. With the synergistic signal amplifications by ISDPR-assisted target recycling, multi-ALP enzyme labels and redox-recycling, the proposed method offers highly sensitive detection of DNA down to 0.1 fM with single-base discrimination capability. Due to the significantly high sensitivity, the developed cascade signal amplification strategy can be potentially extended to detect various DNA targets at ultralow levels for early diagnoses of different diseases.

Here we report a novel electrochemiluminescence (ECL) immunosensor for ultrasensitive detection of carcinoembryonic antigen (CEA) via histidine labeled supersandwich DNA structure to amplify the ECL signal. Histidine is an α-amino acid with an imidazole group, which can act as a co-reactant of tris(2,2'-bipyridyl) ruthenium(II) (Ru(bpy)3(2+)) to amplify ECL signal. To the best of our knowledge, this is the first time to use histidine as co-reactant of Ru(bpy)3(2+) for signal amplification. The ECL substrate is fabricated by locating the complex of Pt nanoparticles (PtNPs) and Ru(bpy)3(2+) (Ru-PtNPs) on Nafion modified electrode surface. In the presence of antigens, the sandwiched immunocomplex can be formed between the primary antibodies on the ECL substrate and the secondary antibodies on the gold nanoparticles. The carried auxiliary probe I trigger a cascade of hybridization events between alternating auxiliary probe I and histidine-modified auxiliary probe II to form the long DNA concatamers. Thus the long DNA concatamers contain multiple histidine-modified auxiliary probe II, which accordingly achieve significant signal amplification. As a result, the sensitivity of proposed immunosensor for CEA detection is improved, the linear range is from 0.1 pg mL(-1) to 100 ng mL(-1) with a low detection limit of 33.3 fg mL(-1). Moreover, with the satisfying stability, selectivity and reproducibility, the proposed supersandwich immunoassay performs good promise in clinical detection., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In this work, by coupling background current reduction with rolling circle amplification (RCA), we describe the development of an ultrasensitive electrochemical sensing method for protein detection based on a small molecule-linked DNA terminal protection strategy. Our detection platform employs a typical streptavidin (STV)-biotin interaction system. Biotin-linked single-stranded DNA (SH-ssDNA-biotin) is self-assembled on a gold electrode to capture the target protein, STV. The binding of STV with the biotin small molecule recognition element protects the SH-ssDNA-biotin against hydrolysis by exonuclease I (Exo I), while the unbound SH-ssDNA-biotin is effectively hydrolyzed and removed from the electrode surface. The bound STV further interacts with long, RCA-amplified biotin DNAs to facilitate the adsorption of numerous electroactive reporters, hexaammineruthenium(III) chloride (RuHex) via electrostatic interactions, which results in significantly amplified signals for the quantitative determination of STV. Moreover, the removal of the unbound SH-ssDNA-biotin probes from the sensing electrode obviates the accumulation of RuHex and leads to a highly minimized background current. The simultaneous RCA signal amplification and background current reduction is expected to significantly enhance the signal-to-noise ratio and to achieve ultrahigh sensitivity. The results reveal that the developed strategy provides a low detection limit of 0.4 pM with high selectivity.

In the present study, an ultrasensitive electrochemical aptasensor for the simultaneous detection of thrombin (TB) and ochratoxin A (OTA) was fabricated by using exonuclease-catalyzed target recycling and DNA concatemers for signal amplification. The previously hybridized double-stranded DNAs (SH-cTBA/TBA and SH-cOBA/OBA) were self-assembled on a gold electrode. In the presence of targets, the formation of aptamer-target complexes would lead to not only the dissociation of aptamers (TBA and OBA) from the double-stranded DNAs but also the transformation of the complementary DNAs (SH-cTBA and SH-cOBA) into hairpin structures. Subsequently, owing to employment of RecJf exonuclease, which is a single-stranded DNA-specific exonuclease to selectively digest the appointed DNAs (TBA and OBA), the targets could be liberated from the aptamer-target complexes for recycling of the analytes. Thereafter, probe DNAs (T1 and T2) were employed to hybridize with SH-cTBA and SH-cOBA respectively to provide primers for the concatemer reaction. After that, when four auxiliary DNA strands S1, anthraquinone-2-carboxylic acid (AQ)-labeled S2, S3, S4, as well as hemin were introduced, extended dsDNA polymers with lots of AQ moieties and hemin-G-quadruplex complexes could form on the electrode surface. Then, based on the signal of the AQ and hemin-G-quadruplex complex, an electrochemical aptasensor for the simultaneous detection of TB and OTA was successfully fabricated.

The commercially available glucometer has been the most successful point-of-care (POC) sensor up to date. However, the glucometer only responds to glucose rather than other species. Extending the use of the glucometer for monitoring different types of targets would potentially revolutionize the applicability of the glucometer. Here we report a new sensing strategy for sensitive and selective detection of Cu(2+) based on multi-invertase conjugated magnetic bead signal amplification labels and a glucometer transducer. The Cu(2+) is in situ reduced to Cu(+) by sodium ascorbate, which catalyzes the click linking between the alkynyl-DNA immobilized on a disposable screen printed carbon electrode and the azido-DNA attached to the invertase/magnetic bead conjugates. The numerous invertase on the magnetic bead labels through Cu(+)-catalyzed click chemistry reaction convert sucrose to glucose, which is monitored by the glucometer and offers amplified digital readings for Cu(2+) detection. By employing the multi-invertase signal amplification, as low as 10nM Cu(2+) can be detected. Our method also shows high selectivity for Cu(2+) against other metal ions owing to the highly specific Cu(+)-catalyzed click chemistry reaction, and is applicable for monitoring Cu(2+) in real river samples. Our strategy can be easily expanded for the monitoring of a wide range of targets when coupled with various recognition events., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In this work, we describe a new sensitive strategy for electrochemical detection of protein via small molecule/protein interactions. This assay is based on a terminal protection mechanism that small molecule-linked single-stranded DNA (ssDNA) is protected against hydrolysis by exonuclease I when the target protein is captured by the corresponding small molecule recognition element. Positively charged gold nanoparticles (AuNPs) are attached to the termini-protected and negatively charged ssDNA through electrostatic interactions. Subsequent AuNP-catalyzed silver enhancement followed by a highly characteristic and sensitive solid-state Ag/AgCl process is introduced to the sensing platform to amplify the signal output. By combining the amplification ability resulting from the silver deposition on the surface-captured AuNPs with the inherent high sensitivity of the electrochemical solid-state Ag/AgCl process, our method expands its range to the detection of macromolecules that bind to specific small molecules and enables low picomolar detection of protein. As a model of biotin/streptavidin interaction, a detection limit of 10 pM for streptavidin is readily achieved with desirable sensitivity and specificity, which indicates that the terminal protection assay coupled with the electrochemical solid-state Ag/AgCl process can offer a promising platform for the determination of various of types of proteins or small molecule-protein interactions., (Copyright © 2012 Elsevier B.V. All rights reserved.)