A DNA nanopillar as a scaffold to regulate the ratio and distance of mimic enzymes for an efficient cascade catalytic platform.

Herein, a rigid 3D DNA nanopillar was used to investigate the influence of spatial organization on the cascade activity in multienzyme systems, realizing controllable regulation of the mimic enzyme ratio and spacing for acquiring a high-efficiency enzyme cascade catalytic platform. Initially, the ratio of mimic enzyme AuNPs (glucose oxidase-like activity) and hemin/G-quadruplex DNAzyme (peroxidase-like activity) fixed at the designed position was adjusted by changing the number of edges in a DNA polyhedron, resulting in an optimal mimic enzyme ratio of 1 : 4 with a quadrangular prism as the scaffold. Notably, the DNA nanopillar formed by quadrangular prism layer-by-layer assembly acted as a track for directional and controllable movement of a bipedal DNA walker based on the toehold mediated strand displacement reaction (TSDR), which endowed the assay system with continuous enzyme spacing regulation compared with previous enzyme cascade systems that induced inflexible operation. Furthermore, enzyme mimetics in this work circumvented the drawbacks of natural enzymes, such as time-consuming purification processes and poor thermal stability. As a proof of concept, the proposed dual regulation strategy of cascade enzymes was applied in the ultrasensitive electrochemical detection of Pb2+, which provided a new route to fabrication of high-performance artificial enzyme cascade platforms for ultimate application in bioanalysis and biodiagnostics.

PtNPs as Scaffolds to Regulate Interenzyme Distance for Construction of Efficient Enzyme Cascade Amplification for Ultrasensitive Electrochemical Detection of MMP-2.

The high catalytic efficiency of enzyme cascade reaction mainly depends on optimal interenzyme distance regulated by the special scaffolds. In this work, the rigid PtNPs with different sizes were employed as scaffolds to regulate interenzyme distance for efficient enzyme cascade amplification to construct electrochemical biosensor for sensitive detection of matrix metalloproteinases-2 (MMP-2), which overcame the drawbacks of instable construction and sophisticated preparation induced by conventional scaffolds such as metal-organic frameworks (MOFs), DNA nanostructures. Here, cucurbit[7]uril functionalized PtNPs (CB[7]@PtNPs) was utilized to load ferrocene (Fc)-labeled horseradish peroxidase (HRP) and glucose oxidase (GOx) via host-guest interaction between Fc and CB[7], respectively, resulting in the formation of a stable three-dimensional netlike structure containing amounts of enzymes. Interestingly, the enzyme cascade reaction regulated by 10 nm PtNPs as scaffold showed highly catalytic efficiency. Meanwhile, the PtNPs could also serve as catalyst to accelerate the enzyme cascade reaction with further enhanced catalytic efficiency. As a result, the proposed biosensor exhibited excellent sensitivity with a wide linear range of 0.1 pg·mL-1 to 20 ng·mL-1 and a detection limit of 0.03 pg·mL-1 for MMP-2. Such a strategy opened a new avenue for adopting metal nanoparticles to regulate interenzyme distance for efficient enzyme cascade amplification, thus providing a universal and easy operating method for sensitively detecting various targets such as DNA, metal ion, and protein.

DNA Enzyme-Decorated DNA Nanoladders as Enhancer for Peptide Cleavage-Based Electrochemical Biosensor.

Herein, we developed a label-free electrochemical biosensor for sensitive detection of matrix metalloproteinase-7 (MMP-7) based on DNA enzyme-decorated DNA nanoladders as enhancer. A peptide and single-stranded DNA S1-modified platinum nanoparticles (P1-PtNPs-S1), which served as recognition nanoprobes, were first immobilized on electrode. When target MMP-7 specifically recognized and cleaved the peptide, the PtNPs-S1 bioconjugates were successfully released from electrode. The remaining S1 on electrode then hybridized with ssDNA1 (I1) and ssDNA2 (I2), which could synchronously trigger two hybridization chain reactions (HCRs), resulting in the in situ formation of DNA nanoladders. The desired DNA nanoladders not only were employed as ideal nanocarriers for enzyme loading, but also maintained its catalytic activity. With the help of hydrogen peroxide (H2O2), manganese porphyrin (MnPP) with peroxidase-like activity accelerated the 4-chloro-1-naphthol (4-CN) oxidation with generation of insoluble precipitation on electrode, causing a very low differential pulse voltammetry (DPV) signal for quantitative determination of MMP-7. Under optimal conditions, the developed biosensor exhibited a wide linear ranging from 0.2 pg/mL to 20 ng/mL, and the detection limit was 0.05 pg/mL. This work successfully realized the combination of DNA signal amplification technique with artificial mimetic enzyme-catalyzed precipitation reaction in peptide cleavage-based protein detection, offering a promising avenue for the detection of other proteases.