Integrated Distance-Based Origami Paper Analytical Device for One-Step Visualized Analysis

An integrated distance-based origami paper analytical device (ID-<i>o</i>PAD) is developed for simple, user friendly and visual detection of targets of interest. The platform enables complete integration of target recognition, signal amplification, and visual signal output based on aptamer/invertase-functionalized sepharose beads, cascaded enzymatic reactions, and a 3D microfluidic paper-based analytical device with distance-based readout, respectively. The invertase–DNA conjugate is released upon target addition, after which it permeates through the cellulose and flows down into the bottom detection zone, whereas sepharose beads with larger size are excluded and stay in the upper zone. Finally, the released conjugate initiates cascaded enzymatic reactions and translates the target signal into a brown bar chart reading. By simply closing the device, the ID-<i>o</i>PAD enables a sample-in-answer-out assay within 30 min with visual and quantitative readout. Importantly, bound/free probe separation is achieved by taking advantage of the size difference between sepharose beads and cellulose pores, and the downstream enzymatic amplification is realized based on the compatibility of multiple enzymes with corresponding substrates. Overall, with the advantages of low-cost, disposability, simple operation, and visual quantitative readout, the ID-<i>o</i>PAD offers an ideal platform for point-of-care testing, especially in resource-limited areas

Legumain expression at the mRNA and protein levels.

<p>(a) Western blotting analysis of Legumain in normal gastric cell lines (GES-1) and gastric cancer cell lines (MKN28, AGS, SGC-7901, MGC-803, BGC-823). BGC-823, MGC-803 cell (poorly differentiated) and AGS, SGC-7901 (moderately differentiated) showed lower expression of Legumain in comparison with GES-1 (normal gastric cell lines) and MKN28 (well differentiated), (b) β-actin is the loading control. Legumain expression was confirmed in all the cell lines by realtime PCR.</p

Facile and Rapid Generation of Large-Scale Microcollagen Gel Array for Long-Term Single-Cell 3D Culture and Cell Proliferation Heterogeneity Analysis

Microfabricated devices are suitable for single-cell analysis due to their high throughput, compatible dimensions and controllable microenvironment. However, existing devices for single-cell culture and analysis encounter some limitations, such as nutrient depletion, random cell migration and complicated fluid shear influence. Moreover, most of the single-cell culture and analysis devices are based on 2D cell culture conditions, even though 3D cell culture methods have been demonstrated to better mimic the real cell microenvironment in vivo. To solve these problems, herein we develop a microcollagen gel array (μCGA) based approach for high-throughput long-term single-cell culture and single-cell analysis under 3D culture conditions. Type-I collagen, a well-established 3D cell culture medium, was used as the scaffold for 3D cell growth. A 2 × 2 cm PDMS chip with 10 000 μCGA units was fabricated to encapsulate thousands of single cells in less than 15 min. Single cells were able to be confined and survive in μCGA units for more than 1 month. The capability of large-scale and long-term single-cell 3D culture under open culture conditions allows us to study cellular proliferation heterogeneity and drug cytotoxicity at the single-cell level. Compared with existing devices for single-cell analysis, μCGA solves the problems of nutrient depletion and random cellular migration, avoids the influence of complicated fluid shear, and mimics the real 3D growth environment in vivo, thereby providing a feasible 3D long-term single-cell culture method for single-cell analysis and drug screening

Massively Parallel Single-Molecule and Single-Cell Emulsion Reverse Transcription Polymerase Chain Reaction Using Agarose Droplet Microfluidics

A microfluidic device for performing single copy, emulsion Reverse Transcription Polymerase Chain Reaction (RT-PCR) within agarose droplets is presented. A two-aqueous-inlet emulsion droplet generator was designed and fabricated to produce highly uniform monodisperse picoliter agarose emulsion droplets with RT-PCR reagents in carrier oil. Template RNA or cells were delivered from one inlet with RT-PCR reagents/cell lysis buffer delivered separately from the other. Efficient RNA/cell encapsulation and RT-PCR at the single copy level was achieved in agarose-in-oil droplets, which, after amplification, can be solidified into agarose beads for further analysis. A simple and efficient method to graft primer to the polymer matrix using 5′-acrydite primer was developed to ensure highly efficient trapping of RT-PCR products in agarose. High-throughput single RNA molecule/cell RT-PCR was demonstrated in stochastically diluted solutions. Our results indicate that single-molecule RT-PCR can be efficiently carried out in agarose matrix. Single-cell RT-PCR was successfully performed which showed a clear difference in gene expression level of EpCAM, a cancer biomarker gene, at the single-cell level between different types of cancer cells. This work clearly demonstrates for the first time, single-copy RT-PCR in agarose droplets. We believe this will open up new possibilities for viral RNA detection and single-cell transcription analysis

Intercalation Effect of Attapulgite in g‑C₃N₄ Modified with Fe₃O₄ Quantum Dots To Enhance Photocatalytic Activity for Removing 2‑Mercapto­benzo­thiazole under Visible Light

A novel magnetic intercalation Fe₃O₄-QDs@g-C₃N₄/ATP photocatalyst was first prepared by a combined eutectic method with deposition technology; it shows superior degradation efficiency for removing 2-mercapto­benzo­thiazole (MBT) under visible light. The improved photocatalytic performance is mainly attributed to the intercalation effect of attapulgite (ATP) in g-C₃N₄ together with the quantum effect of Fe₃O₄ quantum dots (QDs) and the better conductivity between ATP and g-C₃N₄ resulting in the enhanced separation efficiency of photogenerated electron–hole pairs in the light absorption range. Moreover, insight into this mechanism indicates that the holes and superoxide radicals are the major active species in the MBT removal procedure. This work provides an efficient and promising approach to construct new high-performance g-C₃N₄-based photocatalytic materials for wastewater treatment

Denaturation of dsDNA Induced by Specific Major Groove Binding of Cadmium Ion to Thymine

The toxicity of cadmium causes varying degrees of risk to organisms. The underlying mechanism has been conventionally attributed to Cd²⁺-ion-induced oxidative stress. Here, we propose that the Cd²⁺ ion directly and stably binds with the thymine specifically in the major groove and causes denaturation of dsDNA. Using molecular dynamics simulations, it was found that the Cd²⁺ ion preferred to bind to the thymine exposed in the major groove. This then destroyed the hydrogen bonds between adenine and thymine, resulting in a mismatched structure of dsDNA. Our findings are expected to promote the understanding of cadmium-induced direct destruction of genomic stability and may also be helpful for the facilitation of the experimental detection of the binding sites

Detection of T4 Polynucleotide Kinase via Allosteric Aptamer Probe Platform

As a vital enzyme in DNA phosphorylation and restoration, T4 polynucleotide kinase (T4 PNK) has aroused great interest in recent years. Therefore, numerous strategies have been established for highly sensitive detection of T4 PNK based on diverse signal amplification techniques. However, they often need sophisticated design, a variety of auxiliary reagents and enzymes, or cumbersome manipulations. We have designed a new kind of allosteric aptamer probe (AAP) consisting of streptavidin (SA) aptamer and the complementary DNA (cDNA) for simple detection of T4 PNK without signal amplification and with minimized interference in complex biological samples. When the 5′-terminus of the cDNA is phosphorylated by T4 PNK, the cDNA is degraded by lambda exonuclease to release the fluorescein amidite (FAM)-labeled SA aptamer, which subsequently binds to streptavidin beads. The enhancement of the fluorescence signal on SA beads can be detected precisely and easily by a microscope or flow cytometer. Our method performs well in complex biological samples as a result of the enrichment of the signaling molecules on beads, as well as simple manipulations to discard the background interference and nonbinding molecules. Without signal amplification techniques, our AAP method not only avoids complicated manipulations but also decreases the time required. With the advantages of ease of operation, reliability, and robustness for T4 PNK detection in buffer as well as real biological samples, the AAP has great potential for clinical diagnostics, inhibitor screening, and drug discovery

Univariate analysis of prognostic variables in overall survival (OS) for patients.

*<p>Significant difference.</p

Highly Sensitive and Automated Surface Enhanced Raman Scattering-based Immunoassay for H5N1 Detection with Digital Microfluidics

Digital microfluidics (DMF) is a powerful platform for a broad range of applications, especially immunoassays having multiple steps, due to the advantages of low reagent consumption and high automatization. Surface enhanced Raman scattering (SERS) has been proven as an attractive method for highly sensitive and multiplex detection, because of its remarkable signal amplification and excellent spatial resolution. Here we propose a SERS-based immunoassay with DMF for rapid, automated, and sensitive detection of disease biomarkers. SERS tags labeled with Raman reporter 4-mercaptobenzoic acid (4-MBA) were synthesized with a core@shell nanostructure and showed strong signals, good uniformity, and high stability. A sandwich immunoassay was designed, in which magnetic beads coated with antibodies were used as solid support to capture antigens from samples to form a beads–antibody–antigen immunocomplex. By labeling the immunocomplex with a detection antibody-functionalized SERS tag, antigen can be sensitively detected through the strong SERS signal. The automation capability of DMF can greatly simplify the assay procedure while reducing the risk of exposure to hazardous samples. Quantitative detection of avian influenza virus H5N1 in buffer and human serum was implemented to demonstrate the utility of the DMF-SERS method. The DMF-SERS method shows excellent sensitivity (LOD of 74 pg/mL) and selectivity for H5N1 detection with less assay time (<1 h) and lower reagent consumption (∼30 μL) compared to the standard ELISA method. Therefore, this DMF-SERS method holds great potentials for automated and sensitive detection of a variety of infectious diseases