Herein, a unique and versatile immobilization-free electrochemical nucleic acid biosensor architecture is proposed for the first time based on the catalyzed release of a methylene blue (MB)-tagged mononucleotide by exonuclease III (Exo III) and the successive enrichment onto a dodecanethiol monolayer, which can be attributed to the hydrophobic force between the alkyl chain of the dodecanethiol monolayer and the hydrophobic part of the MB-tagged mononucleotide. The fabricated biosensor demonstrates considerable advantages including assay simplicity, rapidness, and high sensitivity owing to its immobilization-free and homogenous operation for the biorecognition and amplification process. A low detection limit of approximately 1 pM toward the target DNA could be achieved with an excellent selectivity. The proposed immobilization-free electrochemical biosensing strategy was also extended for the assay of Exo I and III activity. Furthermore, it might be easily extended for the detection of a wide spectrum of targets and thus provide a promising avenue for the development of immobilization-free and sensitive electrochemical biosensors.

The traditional sensitive electrochemical biosensors are commonly confronted with the cumbersome interface operation and washing procedures and the inclusion of extra exogenous reagents, which impose the challenge on the detection simplicity, reliability, and reusability. Herein, we present the proof-of-principle of a unique biosensor architecture based on dynamic DNA assembly programmed surface hybridization, which confers the single-step, reusable, and enzyme-free amplified electrochemical nucleic acid analysis. To demonstrate the fabrication universality three dynamic DNA assembly strategies including DNA-fueled target recycling, catalytic hairpin DNA assembly, and hybridization chain reaction were flexibly harnessed to convey the homogeneous target recognition and amplification events into various DNA scaffolds for the autonomous proximity-based surface hybridization. The current biosensor architecture features generalizability, simplicity, low cost, high sensitivity, and specificity over the traditional nucleic acid-related amplified biosensors. The lowest detection limit of 50 aM toward target DNA could be achieved by hybridization chain reaction-programmed surface hybridization. The reliable working ability for both homogeneous solution and heterogeneous inteface facilitates the target analysis with a robust reliability and reproducibility, also making it to be readily extended for the integration with the kinds of detecting platforms. Thus, it may hold great potential for the biosensor fabrication served for the point-of-care applications in resource constrained regions.

•A highly sensitive and selective electrochemical DNAzyme sensor for Pb2+ was developed.•The 8–17 DNAzyme cleavage-induced template-independent extension was utilized for amplification.•An impressive detection limit of 0.043 nM toward Pb2+ could be achieved.•It opens a promising avenue for the detection of metal ions and other nucleic acid-related analytes.In this article, a simple, highly sensitive and selective electrochemical DNAzyme sensor for Pb2+ was developed on the basis of a 8–17 DNAzyme cleavage-induced template-independent polymerization and alkaline phosphatase amplification strategy. The hairpin-like substrate strand (HP DNA) of 8–17 DNAzyme was firstly immobilized onto the electrode. In the presence of Pb2+ and the catalytic strand of 8–17 DNAzyme, the HP DNA could be cleaved to expose the free 3′-OH terminal, which could be then utilized for the cascade operation by terminal deoxynucleotidyl transferase (TdTase) for the base extension to incorporate biotinylated dUTP (dUTP-biotin). The further conjugated streptavidin-labeled alkaline phosphatase (SA-ALP) then catalyzed conversion of electrochemically inactive 1-naphthyl phosphate (1-NP) for the generation of electrochemical response signal. The currently fabricated Pb2+ sensor effectively combines triply cascade amplification effects including cyclic Pb2+-dependent DNAzyme cleavage, TdTase-mediated base extension and enzymatic catalysis of ALP. An impressive detection limit of 0.043 nM toward Pb2+ with an excellent selectivity could be ultimately obtained, which was superior than most of the electrochemical methods. Thus, the developed amplification strategy opens a promising avenue for the detection of metal ions and may extend for the detection of other nucleic acid-related analytes.

The programmable DNA polymerization across the two branches of the assembled Y-shaped junction was ingeniously manipulated for modular target recycling and cascade lambda exonuclease cleavage, which afforded the one-pot, isothermal and ultrasensitive detection of target DNA. A low detection limit of 28.2 fM of target DNA with an excellent selectivity could be obtained.

The catalytic hairpin DNA assembly-programmed active Mg2+-dependent DNAzyme was proposed for dual-signal amplified detection toward protein and DNA. The protein detection was implemented with the further combination of an additional terminal protection strategy. The detection limit toward avidin and target DNA could be achieved as 2 pM and 0.5 pM, respectively, with a high selectivity.

Label-free and ultrasensitive electrochemical assays of target DNA and T4 polynucleotide kinase phosphatase (PNKP) were developed, which took full advantage of three enzymes to realize the signal readout and amplification. It can ultimately achieve the low detection limits of 10 fM and 1 mU mL−1 for target DNA and PNKP, respectively.

A responsive hairpin DNA aptamer switch was ingeniously designed for enzyme-free, sensitive and selective electrochemical detection of ATP. It takes full advantage of the target-triggered liberation effect of the toehold region and the concomitant proximity effect with the branch-migration region to execute the toehold-mediated strand displacement reaction on the electrode surface.

•A highly sensitive fluorescence DNA biosensing platform was developed.•Exo III-assisted target recycling and DNAzyme amplification strategy was used.•A low detection limit of 20 fM toward target DNA could be achieved.Because of the intrinsic importance of nucleic acid as bio-targets, the simple and sensitive detection of nucleic acid is very essential for biological studies and medical diagnostics. Herein, a simple, isothermal and highly sensitive fluorescence detection of target DNA was developed with the combination of exonuclease III (Exo III)-assisted cascade target recycling and DNAzyme amplification. A hairpin DNA probe was designed, which contained the 3′-protruding DNA fragment as target recognition unit, the caged DNA fragment in the stem region as target analogue, and the caged 8–17 DNAzyme sequence in the loop region as signal response unit. Upon sensing of target DNA, the 3′-strand of hairpin DNA probe could be stepwise removed by Exo III, accompanied by the releasing of target DNA and autonomous generation of new target analogues for the successive hybridization and cleavage process. Simultaneously, the 8–17 DNAzyme unit could be exponentially released from this hairpin DNA probe and activated for the cyclic cleavage toward the ribonucleotide-containing molecular beacon substrate, inducing a remarkable fluorescence signal amplification for target detection. A low detection limit of 20 fM with an excellent selectivity toward target DNA could be achieved. The developed cascade amplification strategy may be further extended for the detection of a wide spectrum of analytes including protein and biological small molecules by combining DNA aptamer technology.

The phosphorylation of nucleic acid catalyzed by polynucleotide kinase is an indispensible procedure involved in many vital cellular activities such as DNA recombination and DNA repair. Herein, a novel strategy for the sensitive determination of T4 polynucleotide kinase (PNK) activity and inhibition was proposed, which combined exonuclease enzyme reaction and bimolecular beacons (bi-MBs)-based signal amplification. A hairpin probe (HP) with 5′-hydroxyl termini and two different types of molecular beacons (MBs), MB1 and MB2, is designed. Taking advantage of the efficient enzyme reactions, namely the phosphorylation of HP by PNK and the λ exonuclease cleavage reaction, the trigger DNA fragment can be released from HP and is used to trigger the catalytic assembly of bimolecular beacons, resulting in a remarkably amplified fluorescence signal toward PNK activity detection. The detection limit of this method toward PNK was obtained as 1 mU/mL, which was superior or comparable with the reported methods. Furthermore, the facile and sensitive method can also be used to screen the inhibition effects toward several common inhibitors. It provides a promising platform for sensitive determination of nucleotide kinase activity and inhibition, and also shows great potential for biological process research, drug discovery, and clinic diagnostics.

A novel electro-active material was successfully prepared with Fe(CN)63− ions loaded by electrostatic interaction onto the layer of poly(allylamine) hydrochloride (PAH), which was first assembled on prepared poly(sodium 4-styrenesulfonate) (PSS)-doped porous calcium carbonate (CaCO3) microspheres. Further, an electrochemical sensor for use in ascorbic acid (AA) detection was constructed with the use of the above electro-active materials embedded into a chitosan (CS) sol–gel matrix as an electron mediator. The electrocatalytic oxidation of AA by ferricyanide was observed at the potential of 0.27 V, which was negative-shifted compared with that by direct electrochemical oxidation of AA on a glassy carbon electrode. The experimental parameters, including the pH value of testing solution and the applied potential for detection of AA, were optimized. The current electrochemical sensor not only exhibited a good reproducibility and storage stability, but also showed a fast amperometric response to AA in a linear range (1.0 × 10−6 to 2.143 × 10−3 M), a low detection limit (7.0 × 10−7 M), a fast response time (<6 s), and a high sensitivity (−4.5127 μA mM−1).

In this article, a simple strategy of electroless deposition for gold nanoparticle (Au NP) modification on the conductive substrate is developed. The morphology of Au NP modified electrode could be controlled to some extent by choosing different solution concentrations, deposition times, etc. The Au NP modification increased the electrode surface area largely, and the surface area after Au NP modification on the polyelectrolyte multilayer (PEM) assembled electrode was about 3.3 times that of the planar gold electrode. The enhancement of DNA immobilization and hybridization on the Au NP modified electrode were characterized by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) with the use of Ru(NH3)63+ as an electrochemical redox indicator. With this approach, the sensitivity of Au NP modified PEM electrode for target DNA could reach 1 × 10− 11 M. Compared with that of planar gold electrode, the detection limit was increased to be about 3 orders of magnitude.

The complex of rutin–Cu (C81H86Cu2O48, abbreviated by Cu2R3, R = rutin) was synthesized and characterized by elemental analysis and IR spectra. Cyclic voltammetry (CV) and fluorescence spectroscopy were used to investigate the interaction of Cu2R3 with salmon sperm DNA. It was revealed that Cu2R3 could interact with double-stranded DNA (dsDNA) by a major intercalation role. Using Cu2R3 as a novel electroactive indicator, an electrochemical DNA biosensor for the detection of specific DNA fragment was developed and its selectivity for the recognition with different target DNA was assessed by differential pulse voltammetry (DPV). The target DNA related to coliform virus gene could be quantified ranged from 1.62 × 10−8 mol L−1 to 8.10 × 10−7 mol L−1 with a good linearity (r = 0.9989) and a detection limit of 2.3 × 10−9 mol L−1 (3σ, n = 7) was achieved by the constructed electrochemical DNA biosensor.

In this article, colloidal gold nanoparticles (Au NPs) and carboxyl group-functionalized CdS Nanoparticles (CdS NPs) were immobilized on the Au electrode surface to fabricate a novel electrochemical DNA biosensor. Both Au NPs and CdS NPs, well known to be good biocompatible and conductive materials, could provide larger surface area and sufficient amount of binding points for DNA immobilization. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) experiments were performed to follow the whole electrode fabrication process. DNA immobilization and hybridization were characterized with differential pulse voltammetry (DPV) by using [Co(phen)2(Cl)(H2O)]Cl·2H2O as an electrochemical hybridization indicator. With this approach, the target DNA could be quantified at a linear range from 2.0 × 10− 10 to 1.0 × 10− 8 M, with a detection limit of 2.0 × 10− 11 M by 3σ. In addition, the biosensor exhibited a good repeatability and stability for the determination of DNA sequences.

The programmable DNA polymerization across the two branches of the assembled Y-shaped junction was ingeniously manipulated for modular target recycling and cascade lambda exonuclease cleavage, which afforded the one-pot, isothermal and ultrasensitive detection of target DNA. A low detection limit of 28.2 fM of target DNA with an excellent selectivity could be obtained.

The catalytic hairpin DNA assembly-programmed active Mg2+-dependent DNAzyme was proposed for dual-signal amplified detection toward protein and DNA. The protein detection was implemented with the further combination of an additional terminal protection strategy. The detection limit toward avidin and target DNA could be achieved as 2 pM and 0.5 pM, respectively, with a high selectivity.

Label-free and ultrasensitive electrochemical assays of target DNA and T4 polynucleotide kinase phosphatase (PNKP) were developed, which took full advantage of three enzymes to realize the signal readout and amplification. It can ultimately achieve the low detection limits of 10 fM and 1 mU mL−1 for target DNA and PNKP, respectively.

An autonomous target recycling and cascade circular exponential amplification strategy was proposed to construct an autocatalytic DNA machine, which afforded one-pot, isothermal and ultrasensitive detection of nucleic acids. A low detection limit of 0.61 fM toward the target DNA with an excellent selectivity could be obtained.