Cooperative Amplification of Au@FeCo as Mimetic Catalytic Nanozymes and Bicycled Hairpin Assembly for Ultrasensitive Electrochemical Biosensing.

Exploring the cooperative amplification of peroxidase-like metal nanocomposites and cycled hairpin assembly is intriguing for sensitive bioanalysis. Herein, we report the first design of a unique electrochemical biosensor based on mimicking Au@FeCo nanozymes and bicycled hairpin assembly (BHA) for synergistic signal amplification. By loading the enzyme-like FeCo alloy in Au nanoparticles (AuNPs), the as-synthesized Au@FeCo hybrids display great improvement of electronic conductivity and active surface area and excellent mimic catalase activity to H2O2 decomposition into •OH radicals. The immobilization of Au@FeCo in an electrode sensing interface is stabilized via the resulting electrodeposition in HAuCl4 while efficiently accelerating the electron transfer of electroactive ferrocene (Fc). Upon the immobilization of a helping hairpin (HH) via Au-S bonds, a specific DNA trigger (T*) is introduced to activate BHA operation through competitive strand displacement reactions among recognizing hairpin (RH), signaling hairpin (SH), and HH. T* and RH are rationally released to catalyze two cycles, in which the transient depletion of dsDNA intermediates rapidly drives the progressive hairpin assemblies to output more products SH·HH. Thus, the efficient amplification of Au@FeCo mimic catalase activity combined with BHA leads to a significantly increased current signal of Fc dependent on miRNA-21 analogous to T*, thereby directing the creation of a highly sensitive electrochemical biosensor having applicable potential in actual samples.

Target DNA-Activating Proximity-Localized Catalytic Hairpin Assembly Enables Forming Split-DNA Ag Nanoclusters for Robust and Sensitive Fluorescence Biosensing.

Proximity-localized catalytic hairpin assembly (plCHA) is intriguing for rapid and sensitive assay of an HIV-specific DNA segment (T*). Using template-integrated green Ag nanoclusters (igAgNCs) as emitters, herein, we report the first design of a T*-activated plCHA circuit that is confined in a three-way-junction architecture (3WJA) for the fluorescence sensing of T*. To this end, the T*-recognizable complement is programmed in a stem-loop hairpin (H1), and two split template sequences of igAgNCs are separately overhung contiguous to the paired stems of H1 and another hairpin (H2). The hybridization among H1, H2, and two single-stranded linkers (L1 and L2) allows the stable construction of 3WJA. Upon presenting the input T*, the 3WJA-localized plCHA is operated through toehold-mediated strand displacements of H1 and H2 reactants, and T* is rationally displaced and repeatably recycled, analogous to a specific catalyst, inducing more hairpin assembly events. Resultantly, the hybridized products enable the collective combination of two splits in the parent scaffold for hosting igAgNCs, outputting T*-dependent fluorescence response. Because of 3WJA structural confinement, the spatial proximity of two reactive hairpins yielded high local concentrations to manipulate the plCHA operation, achieving rapider reaction kinetics via T*-catalyzed recycling than typical catalytic hairpin assembly (CHA). This simple assay strategy would open the arena to develop various plCHA-based circuits capable of modulating the fluorescence emission of igAgNCs for applicable biosensing and bioanalysis.

Amplifiable ratiometric fluorescence biosensing of nanosilver multiclusters populated in three-way-junction DNA branches.

To explore the fluorescence bio-responsiveness of emissive silver nanoclusters (AgNCs) populated in DNA-branched scaffolds is intriguing yet challenging. In response to a desired targeting model (T*) as a vehicle, herein a customized three-way-junction DNA construct (TWJDC) is assembled via competitive hybridizing cascade among three stem-loop hairpins with specific base sequences, where the repeated recycling of T* enables the exponentially amplifiable output of rigid TWJDC. As designed, these stable hybridization products are highly T*-stimulated responsive and constructing-directional. In the three branched-arms, the unpaired sticky ends provide isotropic binding sites for a signaling hairpin encoded with two C-rich templates of green- and red-AgNCs clustering. The identical ligation of signal probe with three arms of TWJDC liberates its locked stem, enabling the separate growth of red-clusters in three branches. As demonstrated, three clusters of red-AgNCs possess advantageous self-enhancing fluorescent performance relative to single or two cluster(s), good biocompatibility and low cytotoxicity. Utilizing the bicolor AgNCs as dual-emitters with reversely changed emission intensity, we developed an innovative ratiometric strategy displaying sensitively linear dose-dependence on variable T* down to 1.9 pM, which can afford a promising platform for biosensing, bioanalysis, cell imaging, or even clinical theranostics.

LAMP-H+-responsive electrochemical ratiometric biosensor with minimized background signal for highly sensitive assay of specific short-stranded DNA.

Herein, the sequence-specific short-stranded biomarker DNA (hDNA, 21-nt) is acted as targeting out-primer to implement the loop-mediated isothermal amplification for releasing hydrogen ions (LAMP-H+). Using LAMP-H+ as signaling transducer, we report a highly sensitive electrochemical ratiometric biosensor for hDNA with minimized background signal, which is achieved via magnetic separation using AuNPs-modified Fe3O4 (Au@Fe3O4) as micro-reactor. In Au@Fe3O4, a double-stranded complex of a pH-responsible strand (I*) and a substrate strand (S*) is bound via Au-N bonds, where the treatment with LAMP-H+ leads to I* folding into i-motif conformation and S* dehybridization. The S* further hybridizes a catalytic strand (C*) to assemble Mg2+-DNAzymes that are cleaved by Mg2+, releasing C* for repeated formation and robust nicking of Mg2+-DNAzymes. The resultant output fuel strands (F*) are introduced in a modified electrode to drive the strand displacement of two hairpins individually labeled with two electron mediators. Through F*-mediated recycled amplification, the ratio of their electrochemical currents changed in opposite is highly sensitive to the varied hDNA down to 2.1 fM. By integrating LAMP-H+-stimulated i-motif switching with Mg2+-DNAzyme cleavage, this logic transduction of LAMP-H+(i-motif/Mg2+-DNAzyme)F* efficiently minimizes the inherent background of traditional LAMP-based assays. Resultantly, our electrochemical ratiometric strategy would be applicable to diverse short-stranded DNAs or even RNAs as targeting primers of LAMP.

Amplified electrochemical biosensing based on bienzymatic cascade catalysis confined in a functional DNA structure.

Herein, an amplified and renewable electrochemical biosensor was developed via bienzymatic cascade catalysis of glucose oxidase (GOx) and horseradish peroxidase (HRP), which were confined in a functional Y-shaped DNA nanostructure oriented by a dual-thiol-ended hairpin probe (dSH-HP) with a paired stem as a rigid scaffold and unpaired loop as enclosed binding platform. For proof-of-concept assay of sequence-specific biomarker DNA related to Alzheimer's disease (aDNA), GOx and redox ferrocene-modified HRP (Fc@HRP) were chemically conjugated in two enzyme strands (GOx-ES1 and Fc@HRP-ES2), respectively. The repeated recycling of aDNA was powered by the displacement of GOx-ES1 by aDNA and exonuclease III (ExoIII)-assisted cleavage reaction for amplified output of numerous GOx-ES1 as dependent transducers, together with Fc@HRP-ES2 which was simultaneously hybridized with dSH-HP to assemble this DNA structure. Rationally, the bienzymatic cascade catalysis was motivated through GOx-catalyzed glucose oxidization to in situ generate hydrogen peroxide (H2O2) and overlapped HRP-catalyzed H2O2 decomposition to promote the electron transfer, producing significantly enhanced electrochemical signal of Fc with an ultrahigh sensitivity down to 0.22 fM of aDNA. Benefited from the unique design of dSH-HP-oriented bienzymatic cascades, this one-step strategy without non-specific blockers passivation was simple and renewable, and would pave a promising avenue for sensitive electrochemical assay of biomolecules.

Highly sensitive electrochemical assay for Nosema bombycis gene DNA PTP1 via conformational switch of DNA nanostructures regulated by H+ from LAMP.

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.

Glucose oxidase-initiated cascade catalysis for sensitive impedimetric aptasensor based on metal-organic frameworks functionalized with Pt nanoparticles and hemin/G-quadruplex as mimicking peroxidases.

Based on cascade catalysis amplification driven by glucose oxidase (GOx), a sensitive electrochemical impedimetric aptasensor for protein (carcinoembryonic antigen, CEA as tested model) was proposed by using Cu-based metal-organic frameworks functionalized with Pt nanoparticles, aptamer, hemin and GOx (Pt@CuMOFs-hGq-GOx). CEA aptamer loaded onto Pt@CuMOFs was bound with hemin to form hemin@G-quadruplex (hGq) with mimicking peroxidase activity. Through sandwich-type reaction of target CEA and CEA aptamers (Apt1 and Apt2), the obtained Pt@CuMOFs-hGq-GOx as signal transduction probes (STPs) was captured to the modified electrode interface. When 3,3-diaminobenzidine (DAB) and glucose were introduced, the cascade reaction was initiated by GOx to catalyze the oxidation of glucose, in situ generating H2O2. Simultaneously, the decomposition of the generated H2O2 was greatly promoted by Pt@CuMOFs and hGq as synergistic peroxide catalysts, accompanying with the significant oxidation process of DAB and the formation of nonconductive insoluble precipitates (IPs). As a result, the electron transfer in the resultant sensing interface was effectively hindered and the electrochemical impedimetric signal (EIS) was efficiently amplified. Thus, the high sensitivity of the proposed CEA aptasensor was successfully improved with 0.023pgmL-1, which may be promising and potential in assaying certain clinical disease related to CEA.

A sub-picomolar assay for protein by using cubic Cu2O nanocages loaded with Au nanoparticles as robust redox probes and efficient non-enzymatic electrocatalysts.

In this work, a simple and sensitive electrochemical aptasensor for protein (thrombin - TB used as the model) was developed by using cubic Cu2O nanocages (Cu2O-NCs) loaded with Au nanoparticles (AuNPs@Cu2O-NCs) as non-enzymatic electrocatalysts and robust redox probes. Through the specific sandwich-type reaction between TB and TB aptamers (TBA), the formed AuNPs@Cu2O-NCs bound with NH2-TBA were captured onto the electrode surface modified with SH-TBA. Based on the inherent redox activity of AuNPs@Cu2O-NCs with cubic nanostructures, a detectable electrochemical signal was generated which was dependent on the analyte concentration. Meanwhile, AuNPs@Cu2O-NCs showed an efficient electrocatalytic capability in the reduction of H2O2, resulting in a significant enhancement of the response signal. Thus, the simplification of the proposed strategy and the improvement of analytical performances were easily achieved with a sub-picomolar sensitivity (the limit of detection was 0.066 pmol L-1). The applicability of the simple and sensitive aptasensor was successfully demonstrated by assaying TB in human serum samples. This non-enzymatic detection platform would be potential and promising in clinical diagnostics and protein analysis techniques.