Living-DNA Nanogel Appendant Enables In Situ Modulation and Quantification of Regulation Effects on Membrane Proteins.

Screening appendants on membrane proteins to understand their varied regulation effects is desirable for finding the potential candidates of the membrane-protein-targeted drugs. However, most artificial appendants can hardly support in situ condition screening because they cannot evolve in situ, neither can they send out signals to reflect the modulation. Here, we designed living-DNA appendants to enable such screening. First, the living-cell rolling-circle amplification (LCRCA) strategy was developed to elongate the DNA appendants for self-tangled physical nanogels. The nanogels unify both the functions of membrane-protein modulation and quantification: their sizes increase with the increased time length of LCRCA, which change the regulation effect on the membrane proteins; their large number of repeating short sequences allow quantification of their sizes in the presence of the complementary fluorophore-tagged short DNA. Then, the performance of the living-DNA appendants was examined taking α6ß4 integrins as the target, where effective regulation over the distribution of actin filaments, cell viability, and chances of anoikis are all validated. The screening also clearly elucidates the interesting nonlinear relationships between the regulations and the effects. We hope this screening strategy based on living-DNA appendants can stand for a prototype for deeper understanding of natural behaviors of membrane proteins and help in the accurate designing of the membrane-protein-targeted drugs.

Abnormal Liquid Chasing Effect in Paper Capillary Enables Versatile Gradient Generation on Microfluidic Paper Analytical Devices.

Microfluidic paper analytical devices (µPADs) are smart and accessible substituents to traditional counterparts in point-of-care tests (POCT), which exploited delicate control over passive fluid in microscale for rich functions. In this work, we are extending such control by introducing novel ways to generate 1D and 2D gradients on µPADs. It is achieved by using paper-capillary-based serial sampling. The paper capillary is composed of a concaved paper channel sealed with tape, with test pads properly distributed aside. In such a structure, the liquid can not only quickly and automatically flow along the capillary but also be continuously consumed by the peripheral test pads. Thus, when we do serial sampling, an abnormal liquid chasing effect can be observed, where the later liquid sample chases and surpasses the earlier parts in the paper capillary, leading to reversely ordered sample distribution compared with that in a typical glass capillary. This specialty allows for fast, ordered, and tunable sequential sampling and enables efficient generation of 1D and 2D concentration gradient with one, two, and even three components on µPADs. Besides, we verified the applicability of this technique for arrayed assays, including 1D serial dilution-based metal ion colorimetry and a 2D bacterial antibiotic susceptibility test for synergic effect evaluations, which paves the way for high-throughput sample analysis and information-rich condition screening on µPADs.

Paper Capillary Enables Effective Sampling for Microfluidic Paper Analytical Devices.

The paper capillary is introduced to enable effective sampling on microfluidic paper analytical devices. By coupling the macroscale capillary force of paper capillary and the microscale capillary forces of native paper, fluid transport can be flexibly tailored with proper design. Subsequently, a hybrid-fluid-mode paper capillary device was proposed which enables fast and reliable sampling in an arrayed form with less surface adsorption and bias for different components. The resulting device thus supports high-throughput, quantitative, and repeatable assays by manual operation. With all these merits, multiplex analysis of ions, proteins, and microbes have all been realized on this platform, which has paved the way to higher analysis on µPADs.

Microfluidic liquid-air dual-gradient chip for synergic effect bio-evaluation of air pollutant.

In this paper, a novel prototype liquid-air dual gradient chip is introduced, which has paved the way for effective synergic effect bio-evaluation of air pollutant. The chip is composed of an array of the agarose liquid-air interfaces, top air gradient layer and bottom liquid gradient layer. The novel agarose liquid-air interface allows for non-biased exposure of cells to all the substances in the air and diffusive interactions with the liquid phase; while the dual liquid-air gradient provides powerful screening abilities, which well reduced errors, saved time and cost from repeated experiment. Coupling the two functions, the chip subsequently facilitates synergic effect evaluation of both liquid and air factors on cells. Here cigarette smoke was taken as the model air pollutant, and its strong synergic effects with inflammatory level of A549 lung cancer cells on their fate were successfully quantified for the first time. These results well testified that the proposed dual-gradient chip is powerful and indispensable for bio-evaluation of air pollutant.

Dual-Functional Carbon Dots Pattern on Paper Chips for Fe3+ and Ferritin Analysis in Whole Blood.

Though microfluidic paper analytical devices (µPADs) have attracted paramounting attentions in recent years as promising devices for low cost point-of-care tests, their real applications for blood analysis are still challenged by integrating sample preparation with different detection modes on a same µPAD. Herein, we developed a novel µPAD, which well coupled automatic serum extraction with reliable dual mode iron health tests: fluorescent analysis for Fe3+ and colorimetric ELISA for ferritin. All these functions are made available by in situ carbon dots (CDs) and AuNPs sequential patterning techniques. For CDs immobilization, hydrothermal reaction was taken on paper, to which a patterned through-hole polydimethylsiloxane (PDMS) mask was applied. None fluorescence CDs (nF-CDs) were generated on exposed regions, while the fluorescent CDs (F-CDs) were generated simultaneously on covered regions. Sensitive serum iron quantification was realized on the F-CDs modified regions, where Fe3+ ion can selectively quench the fluorescence of F-CDs. For AuNPs immobilization, electroless plating was taken on nF-CDs modified regions. The resulting AuNPs on nF-CDs layer on one hand triggered the coagulation of blood cells and thus led to the longest ever wicking distance for serum separation, on the other hand facilitated colorimetric enzyme linked immunosorbent assay (ELISA) for detection of serum ferritin. Combining the two readings, the µPAD can provide reliable measurement for serum iron and serum ferritin in whole blood. Furthermore, as CDs and AuNPs modified µPAD has the features of easy handling, low-cost, lightweight, and disposability, it is accounting for a promising prototype for whole blood point-of-care analysis.

Microfluidic PDMS on paper (POP) devices.

In this paper, we propose a generalized concept of microfluidic polydimethylsiloxane (PDMS) on paper (POP) devices, which combines well the merits of paper chips and PDMS chips. First, we optimized the conditions for accurate PDMS spatial patterning on paper, based on screen printing and a high temperature enabled superfast curing technique, which enables PDMS patterning to an accuracy of tens of microns in less than ten seconds. This, in turn, makes it available for seamless, reversible and reliable integration of the resulting paper layer with other PDMS channel structures. The integrated POP devices allow for both porous paper and smooth channels to be spatially defined on the devices, greatly extending the flexibility for designers to be able to construct powerful functional structures. To demonstrate the versatility of this design, a prototype POP device for the colorimetric analysis of liver function markers, serum protein, alkaline phosphatase (ALP) and aspartate aminotransferase (AST), was constructed. On this POP device, quantitative sample loading, mixing and multiplex analysis have all been realized.

A microfluidic cigarette smoke collecting platform for simultaneous sample extraction and multiplex analysis.

In this work, we report a novel microfluidic gas collecting platform aiming at simultaneous sample extraction and multiplex mass spectrometry (MS) analysis. An alveolar-mimicking elastic polydimethylsiloxane (PDMS) structures was designed to move dynamically driven by external pressure. The movement was well tuned both by its amplitude and rhythm following the natural process of human respiration. By integrating the alveolar units into arrays and assembling them to gas channels, a cyclic contraction/expansion system for gas inhale and exhale was successfully constructed. Upon equipping this system with a droplet array on the alveolar array surface, we were able to get information of inhaled smoke in a new strategy. Here, with cigarette smoke as an example, analysis of accumulation for target molecules during passive smoking is taken. Relationships between the breathing times, distances away from smokers and inhaled content of nicotine are clarified. Further, by applying different types of extraction solvent droplets on different locations of the droplet array, simultaneous extraction of nicotine, formaldehyde and caproic acid in sidestream smoke (SS) are realized. Since the extract droplets are spatially separated, they can be directly analyzed by MS which is fast and can rid us of all complex sample separation and purification steps. Combining all these merits, this small, cheap and portable platform might find wide application in inhaled air pollutant analysis both in and outdoors.

Versatile microfluidic droplets array for bioanalysis.

We propose a novel method to obtain versatile droplets arrays on a regional hydrophilic chip that is fabricated by PDMS soft lithography and regional plasma treatment. It enables rapid liquid dispensation and droplets array formation just making the chip surface in contact with solution. By combining this chip with a special Christmas Tree structure, the droplets array with concentrations in gradient is generated. It possesses the greatly improved performance of convenience and versatility in bioscreening and biosensing. For example, high throughput condition screening of toxic tests of CdSe quantum dots on HL-60 cells are conducted and cell death rates are successfully counted quickly and efficiently. Furthermore, a rapid biosensing approach for cancer biomarkers carcinoma embryonic antigen (CEA) is developed via magnetic beads (MBs)-based sandwich immunoassay methods.

A novel microfluidic platform with stable concentration gradient for on chip cell culture and screening assays.

In this work a novel microfluidic platform for cell culture and assay is developed. On the chip a static cell culture region is coupled with dynamic fluidic nutrition supply structures. The cell culture unit has a sandwich structure with liquid channels on the top, the cell culture reservoir in the middle and gas channels on the bottom. Samples can be easily loaded into the reservoir and exchange constantly with the external liquid environment by diffusion. Since the flow direction is perpendicular to the liquid channel on the top of the reservoir, the cells in the reservoir are shielded from shear-force. By assembling the basic units into an array, a steady concentration gradient can be generated. Cell culture models both for continuous perfusion and one-off perfusion were established on the chip. Both adherent and suspended cells were successfully cultured on the chip in 2D and 3D culture modes. After culturing, the trapped cells were recovered for use in a later assay. As a competitive candidate for a standard cell culture and assay platform, this chip is also adaptable for cytotoxicity and cell growth assays.

Simultaneous electrochemical immunoassay using CdS/DNA and PbS/DNA nanochains as labels.

An electrochemical method for the simultaneous detection of two different tumor markers, carcinoembryonic antigen (CEA) and α-fetoprotein (AFP), in one-pot, using CdS/DNA and PbS/DNA nanochains as labels was developed. Herein, magnetic beads (MBs) as bimolecule immobilizing carriers, were used for co-immobilization of primary anti-CEA and anti-AFP antibodies. The distinguishable signal labels were synthesized by in situ growth of CdS and PbS nanoparticles on DNA chains, respectively, which were further employed to label the corresponding secondary antibodies. A sandwich-type immunoassay format was formed by the biorecognition of the antigens and corresponding antibodies. The assay was based on the peak currents of Cd(2+) and Pb(2+) dissolved from CdS and PbS nanoparticles by HNO(3) using square wave stripping voltammetry. Experimental results show that the multiplexed electrochemical immunoassay has enabled the simultaneous monitoring of CEA and AFP in a single run with wide working ranges of 0.1-100ng mL(-1) for CEA and 0.5-200ng mL(-1) for AFP. The detection limits reach to 3.3pg mL(-1) for CEA and 7.8pg mL(-1) for AFP.

A branched electrode based electrochemical platform: towards new label-free and reagentless simultaneous detection of two biomarkers.

A branched electrode platform was proposed for label-free and reagentless simultaneous tumor markers detection based on different redox substrates.

Liquid gradient in two-dimensional matrix for high throughput screening.

Based on the ingenious combination of two different gradient generation mechanisms, this work reports a novel approach for a high throughput linear liquid gradient in a two-dimensional (2D) matrix. Specifically, a typical Christmas Tree structure with two inlets was designed as the first mixture gradient generator, upon which the second diffusion gradient generator was coupled to produce the desired concentration series on the basis of the distance difference. Rather than a simple 1D line, the integration of the two generators would result in an innovative 2D matrix of reservoirs, which was then characterized both theoretically and experimentally. Theoretically, calculation of fluid field demonstrated the formation of a concentration gradient, which was then confirmed by the dye solution visualization analysis. For high throughput screening application, doxorubicin (Dox) was then selected as model medicine to treat the acute myeloblastic leukemia (HL-60) cells. Cell viability displayed that cell death rate enhanced with the increase of drug concentration, and this result was higher than that on a 96-well plate, and the corresponding mechanism was properly discussed. Subsequently, Dox and quercetin were employed simultaneously to generate an overlapping gradient and its effect on HL-60 cells was investigated. Due to the automatic formation of concentration gradient that could improve the work efficiency, this work provides a promising tool for future high throughput drug screening.

Liquid-gas dual phase microfluidic system for biocompatible CaCO3 hollow nanoparticles generation and simultaneous molecule doping.

Continuous CaCO(3) hollow nanoparticle generation at room temperature with simultaneous molecule doping is realized on a liquid-gas dual phase microfluidic system.

One step high quality poly(dimethylsiloxane)-hydrocarbon plastics bonding.

In this paper, one-step air plasma treatment is successfully used for poly(dimethylsiloxane)(PDMS)-plastic chip bonding. The technique is green, cheap, and requires no other reagent other than air. Hydrocarbon plastics: polystyrene (PS), cyclic olefin copolymer (COC), and polypropylene (PP) have all been successfully bonded to PDMS irreversibly. The corresponding compressed air resistances are measured to be around 500 kPa for PDMS-PS, PDMS-COC, and PDMS-PP hybrid chips. The bondings are also of good quality even after storage under different temperatures and subject to solutions from acid to base.

On chip steady liquid-gas phase separation for flexible generation of dissolved gas concentration gradient.

In this study, steady liquid-gas phase separation is realized by applying a hydrophobic small microchannel array (SMA) to bridge two large microchannels, one for liquid phase and one for gas phase. In this structure, a capillary pressure difference between that in the SMA and the larger channel results in a steady liquid-gas interface. The generated liquid-gas interface allows for fast gas dissolving speed. By coupling the liquid-gas interface with a one directional fluidic field, a steady dissolved gas concentration gradient (DgCG) is generated. The DgCG distribution is easily designable for linear or exponential modes, providing improved flexibility for gas participated processes on chip. To demonstrate its applicability, a CO(2) DgCG chip is fabricated and applied for screening CaCO(3) crystal growth conditions in the DgCG chip. Crystals with transitional structures are successfully fabricated, which is consistent with the CO(2) DgCG distribution.

Glass etching to bridge micro- and nanofluidics.

In this study, a simple and economical fabrication technique bridging micro- and nanostructures is proposed. Glass molds with micro-nanostructures are fabricated by glass microlithography. The microlithography provides flexibility for structure design, and the glass etching contributes to transform the micro glass ridge to the nanoscale. Glass ridge structures with triangular cross sections are generated by undercutting, which coupled the isotropic character of glass and the shield effect of the top Cr layer upon HF etching. Further etching induced the height of the glass ridges to shrink from micro- to nanometres due to the edge effects. At the late etching stage, the geometrical change of the glass greatly slows down, which gives better control over the size of the glass ridge. By glass structure mold-copy, well repeatable, mechanically stable and tunable polydimethylsiloxane (PDMS) channels and cones are fabricated. Scanning electron microscopy (SEM) and laser interferometry (LI) are carried out to characterize the micro-nanostructures. To demonstrate their workability, sample preconcentration to a single nanochannel level is carried out.

Electrochemiluminescence analysis of folate receptors on cell membrane with on-chip bipolar electrode.

In this paper we report a transparent bipolar electrode based microfluidic chip-electrochemiluminescence (ECL) system for sensitive detection of folate receptors (FR) on cell membranes. This integrated system consists of a poly(dimethylsiloxane) (PDMS) layer containing a microchannel and a glass bottom sheet with indium tin oxide (ITO) strips as bipolar detectors. The ITO strips are fabricated using a PDMS micromold with carbon ink as a protective layer in place of traditional photoresist. The configuration of the bipolar electrode has great influence on the ECL intensity of Ru(bpy)(3)(2+)/tripropylamine(TPA) system. Further studies show that folic acid (FA) can strongly inhibit the ECL of the Ru(bpy)(3)(2+)/TPA system. Based on specific recognition between FA and FR on cell membrane, this microfluidic chip-ECL system is successfully applied for detecting the level of FR on human cervical tumor (HL-60) cells and MEF cells. It is found that the ECL intensity increases with the number of HL-60 cells in the range of 21 to 3.28 × 10(4) cells/mL. The average level of FR on HL-60 cells is calculated to be 8.05 ± 0.75 × 10(-18) mol/cell. While for MEF cells, it shows a much slower ECL increment than HL-60 cells due to the much lower FR level on MEF cells (5.30 ± 0.61 × 10(-19) mol/cell). Moreover, exocytosis of FA after FR mediated endocytosis was observed according to the change of the ECL signal with the incubation time of HL-60 cells in the FA- Ru(bpy)(3)(2+)/TPA system.

Large scale lithography-free nano channel array on polystyrene.

This paper reports a new fabrication method of lithography-free nanochannel array. It is based on the cracking process on the surface of a polystyrene (PS) Petri-dish, one type of thermoplastic that is composed of uni-axial macromolecular chains. Under proper conditions, parallel nanochannels with equal interspaces are obtained. Control over the channel depth from 20 nm to 200 nm is achieved, with the channel length reaching tens of millimetres. The PDMS replication based on PS nanochannel array has been successfully carried out. In combination with the microstructure, both an ion enrichment device and a current rectification device are fabricated, and their quantified characters manifested the applicability of the channel array structure in nanofluidics.

Bis(µ-4-nitro-phthalato)bis-[diaqua-(1,10-phenanthroline)manganese(II)].

In the title compound, [Mn(2)(C(8)H(3)NO(6))(2)(C(12)H(8)N(2))(2)(H(2)O)(4)], the Mn(II) atom in the centrosymmetric binuclear unit has a distorted octa-hedral geometry and is coordinated by a chelating 1,10-phenanthroline ligand, two monodentate carboxyl-ate anions from two 4-nitro-phthalates and two coordinated water mol-ecules. The two Mn(II) ions in the mol-ecule are bridged by two 4-nitro-phthalate anions, both in a bis-monodentate mode, which finally leads to the formation of the binuclear unit. Intra-molecular O-H⋯O hydrogen bonds between the coordinated and uncoordinated O atoms of one monodentate carboxyl-ate group and the corresponding coordinated water mol-ecules result in an eight-membered and two six-membered rings. In the crystal structure, inter-molecular O-H⋯O hydrogen bonds link the dinuclear mol-ecules into supra-molecular chains propagating parallel to [100].