One-Step Digital Droplet Auto-Catalytic Nucleic Acid Amplification with High-Throughput Fluorescence Imaging and Droplet Tracking Computation.

Digital droplet technology has emerged as a powerful new tool for biomarker analysis. Temperature cycling, enzymes, and off-chip processes are, nevertheless, always required. Herein, we constructed a digital droplet auto-catalytic hairpin assembly (ddaCHA) microfluidic system to achieve digital quantification of single-molecule microRNA (miRNA). The designed continuous chip integrates droplet generation, incubation, and fluorescence imaging on the chip, avoiding the requirement for extra droplet re-collection and heating operations. Clearly, the digital readout was obtained by partitioning miRNA into many individual pL-sized small droplets in which the target molecule is either present ("positive") or absent ("negative"). Importantly, the suggested enzyme-free auto-catalytic hairpin assembly (aCHA) in droplets successfully mitigated the effects of the external environment and thermal cycling on droplets, and its reaction rate is significantly superior to that of traditional CHA. We got excellent sensitivity with a linear correlation from 1 pM to 10 nM and a detection limit of 0.34 pM in the fluorescence spectrum section, as well as high selectivity to other miRNAs. Furthermore, the minimum target concentration could be reduced to 10 fM based on the high-throughput tracking computation of fluorescent droplets with a self-developed Python script, and the fluorescence intensity distribution agreed well with the theoretical value, demonstrating that it is feasible to detect miRNA efficiently and accurately, which has great potential applications in clinical diagnostics and biochemical research.

Preparation of diameter-controlled free-standing MWCNT membranes and their application for dye adsorption.

The synthesis of multi-walled carbon nanotubes (MWCNTs) was carried out over different Ni-loaded metallic oxide catalyst nanoparticles and under different reduction times to control the outside diameter of the nanotubes. Moreover, high-purity, free-standing membranes were fabricated by a simple filtration of the as-grown MWCNTs. Furthermore, the dye-adsorption properties of the nanotubes depended on the diameter of the carbon nanotubes (CNTs). The adsorption isotherms and kinetics of anionic dyes could be described by Freundlich and pseudo-second-order models, respectively. Thermodynamic studies suggested that the adsorption processes were spontaneous and exothermic. This work provides new insights into the synthesis and application of MWCNTs with the selective adsorption properties of carbon-based materials for the removal of organic dyes.

Rapid self-disassembly of DNA diblock copolymer micelles via target induced hydrophilic-hydrophobic regulation for sensitive MiRNA detection.

In this work, a novel DNA nanostructure with a shorter assembly time and larger loading capacity was constructed using amphiphilic DNA-alkane group (Spacer C12)10 conjugates encapsulating plentiful fat-soluble fluorescent dyes into the hydrophobic core to form the DNA micelles, which could be rapidly self-disassembled via target induced hydrophilic-hydrophobic regulation to release fluorescent dyes from micelles to the organic phase, realizing the fast and sensitive detection of microRNA.

Aggregation-Induced Synergism by Hydrophobic-Driven Self-Assembly of Amphiphilic Oligonucleotides.

The evident contradiction between high local-concentration-based substrate reactivity and free-diffusion-based high reaction efficiency remains one of the important challenges in chemistry. Herein, we propose an efficient aggregation-induced synergism through the hydrophobic-driven self-assembly of amphiphilic oligonucleotides to generate high local concentration whereas retaining high reaction efficiency through hydrophobic-based aggregation, which is important for constructing efficient DNA nanomachines for ultrasensitive applications. MicroRNA-155, used as a model, triggered strand displacement amplification of the DNA monomers on the periphery of the 3D DNA nanomachine and generated an amplified fluorescent response for its sensitive assay. The local concentration of substrates was increased by a factor of at least 9.0×105 through hydrophobic-interaction-based self-assembly in comparison with the traditional homogeneous reaction system, achieving high local-concentration-based reactivity and free-diffusion-based enhanced reaction efficiency. As expected, the aggregation-induced synergism by hydrophobic-driven self-assembly of amphiphilic oligonucleotides created excellent properties to generate a 3D DNA nanomachine with potential as an assay for microRNA-155 in cells. Most importantly, this approach can be easily expanded for the bioassay of various biomarkers, such as nucleotides, proteins, and cells, offering a new avenue for simple and efficient applications in bioanalysis and clinical diagnosis.

A dynamic 3D DNA nanostructure based on silicon-supported lipid bilayers: a highly efficient DNA nanomachine for rapid and sensitive sensing.

Herein, by anchoring cholesterol-labelled DNA probes to silicon-supported lipid bilayers via cholesterol-lipid interaction, a dynamic three-dimensional (3D) DNA nanostructure could be facilely assembled, which is applied as a microRNA (miRNA)-induced self-powered 3D DNA nanomachine with high movement efficiency. Once the self-powered 3D DNA nanomachine is triggered by target miRNA, it achieves autonomous operation without external addition of fuel DNA strands or protein enzymes. Impressively, the biocompatible lipid bilayers not only preserve the biological character of the DNA probes, but also improve the movement efficiency of the DNA nanomachine, which directly solves the key challenge of the steric barrier effect of traditional rigid surfaces (Au or silicon) for DNA probe diffusion. As a proof of concept, our proposed DNA nanomachine is successfully applied in rapid and sensitive detection of miRNAs, which gives a new idea for the construction of highly efficient DNA nanomachines for biosensing and clinic diagnosis.

A novel fluorescent assay for the ultrasensitive detection of miRNA-21 with the use of G-quadruplex structures as an immobilization material for a signal indicator.

In this work, a novel fluorescent assay was proposed for the ultrasensitive detection of microRNA-21 (miRNA-21) based on the efficient immobilization of protoporphyrin IX (PPIX) as signal indicators in massive G-quadruplex structures obtained by target recycling, three-dimensional DNA walker and a rolling circle amplification (RCA) coupled cascade nucleic acid amplification strategy.