Ultrasensitive Electrochemiluminescence Imaging Detection of Multiple miRNAs in Single Cells with a Closed Bipolar Electrode Array Chip.

In situ sensitive detection of multiple biomarkers in a single cell was highly necessary for understanding the pathogenesis mechanism and facilitating disease diagnosis. Herein, a bipolar electrode (BPE)-electrochemiluminescence (ECL) imaging chip was designed for ultrasensitive in situ detection of multiple miRNAs in single cells based on a dual-signal amplification strategy. A single cell was trapped and lysed within the microtrap of the cathode chamber and an HCR amplification process and nanoprobes (Fc/DNA/Fe3O4) were introduced, leading to a large number of electroactive molecules (Fc) being modified on the surface. Under a suitable potential, Fc+ in the cathodic chamber was reduced to Fc and L-012 was oxidized in the anodic chamber according to the electric neutrality principle of the bipolar electrode system, resulting in the ECL signal recorded by EMCCD. Ascribed to the dual-signal amplification, sensitive visual detection of miRNA-21 and miRNA-155 in single cells was achieved. For MCF-7 cells, miRNA-21 and miRNA-155 were calculated to be 4385 and 1932 copies/cell (median), respectively. For HeLa cells, miRNA-21 and miRNA-155 were calculated to be 1843 and 1012 copies/cell (median), respectively. The comprehensive evaluation of two kinds of miRNA could effectively eliminate error signals, and the detection precision was improved by 10%.

A Portable Device for in Situ Noninvasive Monitoring of Cell Secretions and Communications with Fluorescence and Nanochannel Electrochemistry.

In situ monitoring of cell secretions and communications plays a fundamental role in screening of disease diagnostic biomarkers and drugs. Quantitative detection of cell secretions and monitoring of intercellular communication have been separately reported, which often rely on target labeling or complex pretreatment steps, inevitably causing damage to the target. Simultaneous in situ noninvasive detection of cell secretions and monitoring of intercellular communication are challenging and have never been reported. Herein, we smartly developed a portable device for in situ label-free monitoring of cell secretions and communications with fluorescence and ion-transport-based nanochannel electrochemistry. Based on the dual signal mode, a series of nonelectroactive secretions were sensitively and accurately quantified. The detection limits for VEGF, MUC1, and ATP were 3.84 pg/mL, 32.7 pg/mL, and 47.4 fM (3σ/S), which were 1/3.9, 1/1.1, and 1/41 of those of commercial ELISA kits, respectively. More interestingly, under the released secretions, the gradual opening of the nanochannel connected the two cells in the left and right chambers of the device; thus, the secretion mediated intercellular communication can be monitored. The proposed platform may provide a promising tool for understanding the mechanism of intercellular communication and discovering new therapeutic targets.

Electrochemiluminescence Analysis of Multiple Glycans on Single Living Cell with a Closed Bipolar Electrode Array Chip.

The profiling of multiple glycans on a single cell is important for elucidating glycosylation mechanisms and accurately identifying disease states. Herein, we developed a closed bipolar electrode (BPE) array chip for live single-cell trapping and in situ galactose and sialic acid detection with the electrochemiluminescence (ECL) method. Methylene blue-DNA (MB-DNA) as well as biotin-DNA (Bio-DNA) codecorated AuNPs were prepared as nanoprobes, which were selectively labeled on the cell surface through chemoselective labeling techniques. The individual cell was captured and labeled in the microtrap of the cathodic chamber, under an appropriate potential, MB molecules on the cellular membrane underwent oxidation, triggering the reduction of [Ru(bpy)3]2+/TPA and consequently generating ECL signals in the anodic chamber. The abundance of MB groups on the single cell enabled selective monitoring of both sialic acid and galactosyl groups with high sensitivity using ECL. The sialic acid and galactosyl content per HepG2 cell were detected to be 0.66 and 0.82 fmol, respectively. Through comprehensive evaluation of these two types of glycans on a single cell, tumor cells, and normal cells could be effectively discriminated and the accuracy of single-cell heterogeneous analysis was improved. Additionally, dynamic monitoring of variations in galactosyl groups on the surface of the single cell was also achieved. This work introduced a straightforward and convenient approach for heterogeneity analysis among single cells.

Synergistic ROS generation and directional overloading of endogenous calcium induce mitochondrial dysfunction in living cells.

Taking advantage of endogenous Ca2+ to upregulate intramitochondrial Ca2+ level has become a powerful mean for mitochondrial dysfunction-mediated tumor therapy. However, the Ca2+ entered into mitochondria is limited ascribing to the uncontrollability and non-selectivity of endogenous Ca2+ transport. It remains a great challenge to make the maximum use of endogenous Ca2+ to ensure sufficient Ca2+ overloading in mitochondria. Herein, we smartly fabricate an intracellular Ca2+ directional transport channel to selectively transport endogenous Ca2+ from endoplasmic reticulum (ER) to mitochondria based on cascade release nanoplatform ABT-199@liposomes/doxorubicin@FeIII-tannic acid (ABT@Lip/DOX@Fe-TA). In tumor acidic microenvironment, Fe3+ ions are firstly released and reduced by tannic acid (TA) to Fe2+ for ROS generation. Subsequently, under the NIR light irradiation, the released ABT-199 molecules combine with ROS contribute to the formation of IP3R-Grp75-VDAC1 channel between ER and mitochondria, thus Ca2+ ions are directionally delivered and intramitochondrial Ca2+ level is significantly upregulated. The synergetic ROS generation and mitochondrial Ca2+ overloading effectively intensifies mitochondrial dysfunction, thereby achieving efficient tumor inhibition. This work presents a new insight and promising avenue for endogenous Ca2+-involved tumor therapies.

Gold Nanowires Array-Based Closed Bipolar Nanoelectrode System for Electrochemiluminescence Detection of α-Fetoprotein on Cell Surface.

Inspired by the promising applications of a closed bipolar electrodes (c-BPEs) system in electrochemiluminescence (ECL) detection of cell adhesion and disease-related biomarkers, here, a gold nanowires array-based c-BPEs system was constructed for cell surface protein detection. Regular and uniform gold nanowires array were prepared by intermittent potentiostatic deposition. Then, two poly(dimethylsiloxane) (PDMS) chips with a hole diameter of 2 mm as a reservoir were placed at both sides of Au nanowires array to construct c-BPEs system. Thionine-functionalized silicon dioxide nanoparticles conjugated to antibody (Ab2-Th@SiO2) were used as the electrochemical probe, while [Ru(bpy)3]2+-wrapped SiO2 nanoparticles (Ru(II)@SiO2) were employed as the ECL signal readout. Taking α-fetoprotein (AFP) as model, the gold nanowires array-based c-BPEs system allowed sensitive detection of AFP at a linear range from 0.002 to 50.0 ng/mL and at least 6 living cells ascribing to the synergetic amplification effect at both sensing and reporting chambers. Besides, the amount of AFP expressed by HepG2 cells was calculated to be 6.71 pg/cell. The presented strategy with high sensitivity provided a promising and universal platform for the detection of other cancer cells and disease-related biomarkers (such as proteins, glycan, miRNA).

Biomimetic nanoreactor for targeted cancer starvation therapy and cascade amplificated chemotherapy.

Consuming glucose by glucose oxidase (GOx) has attracted great interest in cancer starvation therapy, but the therapeutic effect is severely limited by the tumor hypoxia environment. Herein, to overcome such limitation, cancer cell membranes disguised biomimetic nanoreactors were elaborately established for synergetic cancer starvation therapy and cascade amplificated hypoxia activated chemotherapy. Via a metallothionein-like self-assembly and infiltration approach, GOx and hypoxia activated prodrug banoxantrone (AQ4N) were efficiently loaded into metal-organic framework ZIF-8 nanocarriers to yield nanoreactor AQ4N/GOx@ZIF-8. Subsequently, the biomimetic nanoreactor (AQ4N/GOx@ZIF-8@CM) was obtained by camouflaging the nanoreactor with cancer cell membrane, which endowed the biomimetic nanoreactor homotypic targeting, immune escape and prolonged blood circulation features. Once targeted accumulating into tumor sites, the acid environment triggered the decomposition of ZIF-8, then encapsulated GOx and AQ4N were released. GOx would rapidly exhaust endogenous glucose and O2 to shut off the energy supply of tumor cells for starvation treatment. Furthermore, the aggravated tumor intracellular hypoxia environment would activate the cytotoxicity of AQ4N for chemotherapy. In vitro and in vivo results demonstrated that the designed biomimetic nanoreactor exhibited negligible systemic toxicity, besides, the combination of starvation therapy and cascade amplified hypoxia activated chemotherapy significantly inhibited the tumor growth and improved the therapeutic efficacy.

Electrofluorochromic Imaging Analysis of Glycan Expression on Living Single Cell with Bipolar Electrode Arrays.

The in situ glycan profiling of a single tumor cell plays an important role in personalized cancer treatment. Herein, an integrated microfluidic system was designed for living single-cell trapping and real-time monitoring of galactosyl expression on the surface, combining closed bipolar electrode (BPE) arrays and electrofluorochromic (EFC) imaging. Galactosyl groups on human liver cancer HepG2 cells were used as the model analysts, galactose oxidase (GAO) could selectively oxidize hydroxyl sites of galactosyl groups on the cell surface to aldehydes, and then biotin hydrazide (BH) was used to label the aldehydes by aniline-catalyzed hydrazone ligation. With the biotin-avidin system, nanoprobes were finally introduced to the galactosyl groups on the cell surface with avidin as a bridge, which was prepared by simultaneously assembling ferrocene-DNA (Fc-DNA) and biotin-DNA (Bio-DNA) on gold nanoparticles (AuNPs) due to their large surface area and excellent electrical conductivity. After a labeled single cell was captured in the anodic microchannel, the Fc groups attached on the cell surface were oxidized under suitable potential, and the nonfluorescent resazurin on the cathode was correspondingly reduced to produce highly fluorescent resorufin, collected by fluorescence confocal microscope. The combination of EFC imaging and BPE realized monitoring galactosyl group expression of 5.0 × 108 molecules per cell. Furthermore, the proposed platform had the ability to distinguish a single cancer cell from a normal cell according to the expression level of galactosyl groups and to dynamically monitor the galactosyl group variation on the cell surface, providing a simple and accessible method for the single-cell analysis.

A single-liquid miniature biofuel cell with boosting power density via gas diffusion bioelectrodes.

The low solubility of gas molecules in aqueous solutions has limited the power density output of enzymatic biofuel cells. Herein, a single-liquid miniature glucose-O2 fuel cell was constructed by using gas diffusion electrodes, which were prepared by immobilizing glucose oxidase (GOx) or laccase (Lac) modified on a porous structured carbon paper (CP). Due to the fast and direct O2 diffusion from air to the active sites of the immobilized enzyme through the pores of the CP anode/cathode with controlled wettability, the maximum power output densities dramatically increased to 9.64 µW cm-2 at 0.43 V and 53.0 µW cm-2 at 0.45 V for the cell in 5 mM glucose and after exposing the cell to air or O2 atmosphere, respectively. Interestingly, the resulting single-liquid cell could harvest power from human serum operating at a maximum power density of 49.0 µW cm-2 at 0.2 V. The biofuel cell fabricated by the gas diffusion electrodes displayed advantages such as high output power density, low cost and high 'on-chip' integrability and miniaturization, which suggest its great potential for implantable self-powered sensors and for many future applications.

Promoting Photodegradation Efficiency via a Heterojunction Photocatalyst Combining with Oxygen Direct and Fast Diffusion from the Gas Phase to Active Catalytic Sites.

The low solubility of oxygen in solution is the main obstacle for the biodegradation of organic pollutants in wastewater. To address this problem, inspired by the degradation mechanism of aerobic bacteria toward organic pollutants, a novel photodegradation system was presented and operated by a heterojunction photocatalyst combining with a hydrophobic triphase interface, allowing oxygen to directly diffuse from the gas phase to active catalytic sites submersed in polluted solutions. Especially, the heterojunction photocatalyst was fabricated by graphitic carbon nitride nanosheets (C3N4 NS) sensitized with 5,10,15,20-tetrakis(4-carboxylphenyl)porphyrin (TCPP). The resulting photocatalyst was coated on a certain part of the commercial superhydrophobic carbon paper (CP) and submersed in the polluted wastewater, while the other part of hydrophobic CP (without coating with C3N4-TCPP nanocomposite) was exposed to air to form a gas-liquid-solid tri-phase photodegradation system. With this system, the photodegradation rate was 10-fold higher than that of a conventional liquid/solid diphase system in oxygen-saturated solutions. This was, on one hand, due to the abundant oxygen on the surface of a photocatalyst coming from the fast and direct diffusion from the gas phase through the superhydrophobic nanoporous part of CP. On the other hand, the hybrid C3N4-TCPP nanocomposite enhanced the light absorption efficiency under simulated sunlight irradiation and restrained the recombination of photogenerated electron-hole. Moreover, the triphase photodegradation system was stable in aqueous solutions for a long time and can be reused almost without attenuation for five cycles, which provided a great potential to be utilized for practical wastewater treatment.

Visual Electrofluorochromic Detection of Cancer Cell Surface Glycoprotein on a Closed Bipolar Electrode Chip.

This work reports an electrofluorochromic strategy on the basis of electric field control of fluorescent signal generation on bipolar electrodes (BPEs) for visualizing cancer cell surface glycoprotein (mucin 1). The device included two separate cells: anodic sensing cell and cathodic reporting cell, which were connected by a screen-printing electrode patterned on poly(ethylene terephthalate) (PET) membrane. In the sensing cell, anti-MUC1 antibody immobilized on a chitosan-multiwalled carbon nanotube (CS-MWCNT)-modified anodic BPE channel was used for capturing mucin-1 (MUC1) or MCF-7 cancer cells. Then ferrocene (Fc)-labeled mucin 1 aptamers were introduced through hybridization. Under an applied voltage, the ferrocene was oxidized and the electroactive molecules of 1,4-benzoquinone (BQ) in the cathodic reporting cell were reduced according to electroneutrality. This produced a strongly basic 1,4-benzoquinone anion radical (BQ•-), which turned on the fluorescence of pH-responsive fluorescent molecules of (2-(2-(4-hydroxystyryl)-6-methyl-4 H-pyran-4-ylidene)malononitrile) (SPM) coexisting in the cathode reporting cell for both spectrophotometric detection and imaging. This strategy allowed sensitive detection of MUC1 at a concentration down to 10 fM and was capable of detecting a minimum of three MCF-7 cells. Furthermore, the amount of MUC1 on MCF-7 cells was calculated to be 6.02 × 104 molecules/cell. Our strategy also had the advantages of high temporal and spatial resolution, short response time, and high luminous contrast and is of great significance for human health and the promotion of life science development.

A multi-state fluorescent switch based on a diarylethene with an acridine unit.

A new asymmetrical fluorescent diarylethene derivative with an acridine unit was synthesized by Schiff base condensation. The derivative was sensitive to lights and special metal ions. Stimulated by UV/vis lights and Zn2+, distinct changes were observed in UV-vis and fluorescent spectra. Upon addition of Zn2+, the derivative emission peak was blue-shifted by 34nm and the emission intensity was enhanced by 16 fold, accompanied by the fluorescent color changed from red to light yellow, due to the formation of a 1:1 metal/ligand complex. The complex exhibited excellent fluorescence switching upon irradiation with UV light. Taking advantage of the lights and Zn2+ stimuli (inputs), and fluorescence intensity at 580nm (output), a molecular logic gate was constructed. Moreover, a new absorption band centered at 420-450nm emerged upon exposure to Zn2+. The dramatic color change of the solution made the 'naked-eyes' detection of Zn2+ possible.