Insights of Life Molecules' Dynamic Distribution in Live Cells via Sequence-Structure Bispecific Fluorescent RNA.

Life molecules' distributions in live systems construct the complex dynamic reaction networks, whereas it is still challenging to demonstrate the dynamic distributions of biomolecules in live systems. Herein, we proposed a dynamic analysis strategy via sequence-structure bispecific RNA with state-adjustable molecules to monitor the dynamic concentration and spatiotemporal localization of these biomolecules in live cells based on the new insight of fluorescent RNA (FLRNA) interactions and their mechanism of fluorescence enhancement. Typically, computer-based nucleic acid-molecular docking simulation and molecular theoretical calculation have been proposed to provide a simple and straightforward method for guiding the custom-design of FLRNA. Impressively, a novel FLRNA with sequence and structure bispecific RNA named as a structure-switching aptamer (SSA) was introduced to monitor the real-time concentration and spatiotemporal localization of biomolecules, contributing to a deeper insight of the dynamic monitoring and visualization of biomolecules in live systems.

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.

Three-in-One System Based on Multi-Path Nucleic Acid Amplification for Bioanalysis of Pre-miRNA/miRNA and Dicer Activity.

Today, a lot of attention is being paid to the pre-miRNAs/miRNAs or activity of Dicer due to their important functions in various physiological processes. Especially, the intrinsic relationship among these associated targets is of significant importance for more in-depth research on the mechanism of disease formation and early diagnosis. Herein, a strategy for simultaneous bioanalysis of miRNAs/pre-miRNAs and Dicer enzyme based on the self-designed multi-path nucleic acid amplification technology was proposed. Typically, in the presence of pre-miRNA-155, it can hybridize with Helper to generate a structure with two new toeholds, one of which could react with H1, H2, and H3, performing a modified CHA reaction with obvious fluorescence responses of FAM, and another of which could hybridize with H4, H5, and H6 to construct the [H4-H5-H6]n DNA nanosphere with obvious fluorescence responses of Cy5. Similarly, miRNA-155 could just hybridize with H1, H2, and H3 to generate the same modified CHA reaction with obvious fluorescence responses of FAM. Due to the successful multi-path nucleic acid amplification, the proposed bioanalysis strategy could be successfully employed for miRNA-155 and pre-miRNA-155 analysis in the range from 500 pM to 100 nM and 1 to 300 nM, respectively. The proposed strategy could be applied to explore another inter-related nucleic acid relationship also, providing great potential in bioanalysis of various nucleic acids.

Proximity hybridization-induced competitive rolling circle amplification to construct fluorescent dual-sensor for simultaneous evaluation of glycated and total hemoglobin.

The glycated hemoglobin (HbA1c) level, defined as the ratio of HbA1c to total hemoglobin (tHb), has been considered as a standard index for diabetes mellitus diagnosis, avoiding any short-term fluctuation of the blood glucose level. However, it is still a key challenge that some complicated separation procedures are required to detect HbA1c and tHb in a single experiment. Herein, we developed a dual-sensing fluorescent assay for HbA1c and tHb based on proximity hybridization-induced rolling circle amplification (RCA) and T7 exonuclease aided signal cycle to calculate the HbAlc level in one single experiment. Typically, four DNA-tagged probes were used for immunoreactions of anti-HbA1c antibodies (Ab1) and anti-Hb antibodies (Ab2) with HbA1c and Hb, respectively. The RCA1 and RCA2 were induced by the complexes of assistant DNAs and the products of proximity hybridization with anti-HbA1c-DNA1 (2) and anti-Hb-DNA3 (4), respectively. Meanwhile, the tHb in solution could prevent the existing RCA2 based on its competing with Hb-DNA3 for Ab2-DNA4. Thus, RCA1 and RCA2 routes create "signal-on" and "signal-off" readouts, respectively. After the hybridization of signal probes and RCA products, the quenched fluorescence was recovered by the digestion of T7 exonuclease. Under optimized conditions, the method displayed high sensitivity with a limit of detection 2.17 ng/mL and 33.4 ng/mL for HbA1c and tHb, respectively, and the real sample analysis results were found to match well with the theoretical data, holding feasibility for rapid, homogeneous, and easy HbA1c level evaluation and great promise as a potential alternative tool to analyze HbA1c level clinically.

No-nonspecific recognition-based amplification strategy for endonuclease activity screening with dual-color DNA nano-clew.

The inevitable nonspecific recognition severely restricted widely used nucleic acid amplification strategies, which has become an urgent problem in current scientific research. Herein, we developed a novel no-nonspecific recognition-based amplification strategy to construct dual-color dye loaded nano-clew as ultrabright illuminant for screening endonuclease activity with Escherichia coliRY13 I (EcoR I) as a model, which overcame some major drawbacks such as nonspecific recognition and photobleaching. Typically, the target endonuclease induces cleavage of the customized dumbbell-shape substrate (DSS) to generate two same triggers that can initiate the rolling circle amplification (RCA) to prepare long single-strand DNA (lssDNA), which could self-assemble into irregular DNA nano-clew based on the electrostatic interactions with Mg2+ to furtherly capture the donor and accepter fluorophore proximately, constructing the dye loaded nano-clew with dual-color fluorescence (FL) emission to resist photobleaching. Importantly, in absence of EcoR I, even if the DSS could combine with circular template a little, the reaction system performed hardly RCA reaction due to no cohesive terminus, resulting an extremely low background fluorescence signal because of the prevention of nonspecific RCA reaction. As expected, the proposed sensing platform with a low limit of detection (LOD) of 3.4 × 10-7 U/µL was demonstrated to work well for endonuclease inhibitors screening also. Furthermore, the proposed no-nonspecific recognition strategy could be readily extended to various DNA or RNA enzymes such as DNA methyltransferase, DNA repair-related enzymes and polynucleotide kinase just by simply changing the recognition sequence in the DNA substrate, performing great potential of endonucleases-related clinical diagnosis and drugs discovery.

Successively amplified electrochemical immunoassay based on biocatalytic deposition of silver nanoparticles and silver enhancement.

A successively signal-amplified electrochemical immunoassay has been reported on the basis of the biocatalytic deposition of silver nanoparticles with their subsequent enlargement by nanoparticle-promoted catalytic precipitation of silver from the silver-enhancer solution. The immunoassay was carried out based on a heterogeneous sandwich procedure using polystyrene microwells to immobilize antibody. After all the processes comprising the formation of immunocomplex, biocatalytic deposition of silver nanoparticles and following silver enhancement were completed, the silver on polystyrene microwells was dissolved and quantified by anodic stripping voltammetry (ASV). The effect of relevant experimental conditions, including the concentration of ascorbic acid 2-phosphate (AA-p) substrate and Ag(I) ions, the biocatalytic deposition time, and of crucial importance, the silver enhancement time, were investigated and optimized. The anodic stripping peak current was proportional to the concentration of human IgG in a dynamic range of 0.1-10 ng ml(-1) with a detection limit of 0.03 ng ml(-1). Scanning electron microscope (SEM) was applied to characterize the silver nanoparticles before and after silver enhancement on the surface of polystyrene microplates. By coupling the highly catalytic effect of enzyme and nanoparticles to successively amplify the analytical signal, the sensitivity of immunoassay was enhanced so dramatically that this approach would be a promising strategy to achieve a lower detection limit for bioassays.

An electrochemical amplification immunoassay using bi-electrode signal transduction system.

An electrochemical immunoassay technique has been developed based on the sensitive detection of the enzyme-generated product with a bi-electrode signal transduction system. The system uses two separate electrodes, an immunoelectrode and a detection electrode to form a galvanic cell to implement the redox reactions on two different electrodes, that is the enzyme-generated reductant in the anode region is electrochemically oxidized by an oxidant (silver ions) in the cathode apartment. Based on a sandwich procedure, after immunoelectrode with antibody immobilized on its surface bound with the corresponding antigen and alkaline phosphatase conjugated antibody successively, the immunoelectrode was placed in enzyme reaction solution and wired to the detection electrode which was immerged into a silver deposition solution. These two solutions are connected with a salt bridge. Thus a bi-electrode signal transduction system device is constructed in which the immunoelectrode acts as anode and the detection electrode serves as cathode. The enzyme bound on the anode surface initiates the hydrolysis of ascorbic acid 2-phosphate to produce ascorbic acid in the anode region. The ascorbic acid produced in the anodic apartment is electrochemically oxidized by silver ions coupled with the deposition of silver metal on the cathode. Via a period of 30min deposition, silver will deposited on the detection electrode in an amount corresponding to the quantity of ascorbic acid produced, leading to a great enhancement in the electrochemical stripping signal due to the accumulation of metallic silver by enzyme-generated product. Compared with the method using chemical deposition of silver, the electrochemical deposition of silver on a separate detection electrode apartment avoids the possible influence of silver deposition on the enzyme activity.

A sensitive immunosensor using colloidal gold as electrochemical label.

A sensitive immunosensor using colloidal gold as electrochemical label is described. In this method, the capture protein was first immobilized on a carbon paste electrode surface through passive adsorption to bind quantitatively with corresponding antigen and colloidal gold labeled antibody to perform a sandwich assay. To detect the amount of the colloidal gold captured on the electrode surface, the colloid was first oxidized electrochemically to produce AuCl(4)(-) ions which were adsorbed strongly on the electrode surface. Adsorptive voltammetry was then employed for the determination of the adsorbed AuCl(4)(-) ions. A linear relationship between reduction wave peak current and the antigen concentration (human IgG) from 10 to 500 ng/ml is obtained with a detection limit of 4.0 ng/ml.