Tunable Ionic Transport Control inside a Bio‐Inspired Constructive Bi‐Channel Nanofluidic Device

Inspired by the cooperative functions of the asymmetrical ion channels in living cells, a constructive bi‐channel nanofluidic device that demonstrates the enhanced capability of multiple regulations over both the ion flux amount and the ionic rectification property is prepared. In this bi‐channel system, the construction routes of the two asymmetric conical nanochannels provide a way to efficiently transform the nanodevice into four different functional working modes. In addition, the variation of external pH conditions leads the nanodevice to the uncharged, semi‐charged and charged states, where the multistory ionic regulating function property is enhanced by the charged degree. This intelligent integration of the single functional nanochannels demonstrates a promising future for building more functional multi‐channel integrated nanodevices as well as expands the functionalities of the bio‐inspired smart nanochannels.     

Tunable Confinement for Bridging Single‐Cell Manipulation and Single‐Molecule DNA Linearization

DNA linearization by nanoconfinement has offered a new avenue toward large‐scale genome mapping. The ability to smoothly interface the widely different length scales from cell manipulation to DNA linearization is critical to the development of single‐cell genomic mapping or sequencing technologies. Conventional nanochannel technologies for DNA analysis suffer from complex fabrication procedures, DNA stacking at the nanochannel entrance, and inefficient solution exchange. In this work, a dynamic and tunable confinement strategy is developed to manipulate and linearize genomic‐length DNA molecules from a single cell. By leveraging pneumatic microvalve control and elastomeric collapse, an array of nanochannels with confining dimension down to 20 nm and length up to sub‐millimeter is created and can be dynamically tuned in size. The curved edges of the microvalve form gradual transitions from microscale to nanoscale confinement, smoothly facilitating DNA entry into the nanochannels. A unified micro/nanofluidic device that integrates single‐cell trapping and lysis, DNA extraction, purification, labeling, and linearization is developed based on dynamically controllable nanochannels. Mbp‐long DNA molecules are extracted directly from a single cell and in situ linearized in the nanochannels. The device provides a facile and promising platform to achieve the ultimate goal of single‐cell, single‐genome analysis.     

Assays of endogenous caspase activities: a comparison of mass spectrometry and fluorescence formats

This paper describes a label-free assay for measuring endogenous caspase protease activities in cell lysates. The assay format, termed SAMDI-MS (self-assembled monolayers for matrix assisted laser desorption ionization time-of-flight mass spectrometry), is based on the enzymatic modification of peptides immobilized to monolayer substrates, followed by direct detection of the products with mass spectrometry. Monolayers presenting peptide substrates for either caspase-3 or -8 were treated with lysates from Jurkat cells that were stimulated with staurosporine and SKW6.4 cells that were stimulated with LzCD95L. In both cases, the SAMDI assays reported on the activation of endogenous caspase enzymes with levels of detection that are similar to those observed using the commonly employed fluorogenic assays. The use of longer peptide substrates, which are not compatible with the fluorogenic assays, provided for a better resolution of the two caspase activities. This work is significant because it demonstrates that the SAMDI assay can be used to measure endogenous enzyme activities and because it avoids the loss of activity and specificity that often accompany label-dependent assay formats.     

Biomolecule‐Functionalized Solid‐State Ion Nanochannels/Nanopores: Features and Techniques

Solid‐state ion nanochannels/nanopores, the biomimetic products of biological ion channels, are promising materials in real‐world applications due to their robust mechanical and controllable chemical properties. Functionalizations of solid‐state ion nanochannels/nanopores by biomolecules pave a wide way for the introduction of varied properties from biomolecules to solid‐state ion nanochannels/nanopores, making them smart in response to analytes or external stimuli and regulating the transport of ions/molecules. In this review, two features for nanochannels/nanopores functionalized by biomolecules are abstracted, i.e., specificity and signal amplification. Both of the two features are demonstrated from three kinds of nanochannels/nanopores: nucleic acid–functionalized nanochannels/nanopores, protein‐functionalized nanochannels/nanopores, and small biomolecule‐functionalized nanochannels/nanopores, respectively. Meanwhile, the fundamental mechanisms of these combinations between biomolecules and nanochannels/nanopores are explored, providing reasonable constructs for applications in sensing, transport, and energy conversion. And then, the techniques of functionalizations and the basic principle about biomolecules onto the solid‐state ion nanochannels/nanopores are summarized. Finally, some views about the future developments of the biomolecule‐functionalized nanochannels/nanopores are proposed.     

Multiple sampling in single-cell enzyme assays using CE-laser-induced fluorescence to monitor reaction progress

A novel method for assaying enzymes from a single cell or small cell populations is described. The key advantage of this method is the ability to repeatedly sample a single cell enzyme reaction. Whereas multiple sampling has been achieved for larger cell types with a diameter of 1 mm, we report a technique by which single cell enzyme assays of small cells (15 microm in diameter) can be repeatedly carried out. Individual cells were isolated using an in-house-built micromanipulator and placed in nanoliter-scale reaction vessels. The cells were lysed with solution containing substrate, and enzyme activity was assayed by removing 5-nL aliquots with a recently developed nanopipettor. The reaction aliquot was then analyzed using capillary electrophoresis with laser-induced fluorescence detection to quantitate enzyme activity. Sf9 cells were assayed at the single cell level and found to be highly heterogeneous with respect to alpha-glucosidase II activity. Since only 5 nL of the single cell reaction was removed, multiple sampling was possible, allowing triplicate analysis of enzyme activity for each individual cell. Multiple sampling also permitted a single cell reaction to be monitored over time. The sensitivity of this method was demonstrated in the analysis of a low-abundance enzyme, alpha1,3-N-acetylgalactosaminyltransferase, from single HT29 cells. Detecting the product of this enzyme reaction required minimizing the dilution of cellular contents. To demonstrate the potential applications of this methodology in small scale biochemical analyses, single Arabidopsis knf embryos lacking the alpha-glucosidase I encoding KNOPF gene were assayed. Mutant embryos demonstrated insignificant conversion of a triglucose substrate, as compared to wild type, confirming the deletion of alpha-glucosidase I. Embryos were simultaneously assayed for a second enzyme, beta-galactosidase, illustrating that the mutants were viable except for their lack of alpha-glucosidase I activity.     

Self-assembled monolayers for MALDI-TOF mass spectrometry for immunoassays of human protein antigens

This paper reports a method that combines self-assembled monolayers with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to perform immunoassays on clinical samples. The immunosensors are prepared by immobilizing His-tagged protein G (or A) to a monolayer presenting the Ni2+ chelates, followed by immobilization of IgG antibodies with specificity for the intended analyte. The SAMDI mass spectrometry technique confirms the presence of the two proteins on the immunosensor and additionally provides a label-free analysis of antigens that bind to the sensor. This paper reports examples of detecting several proteins from human serum, including multianalyte assays that resolve each analyte according to their mass-to-charge ratio in the SAMDI spectra. An example is described wherein SAMDI is used to identify a proteolytic fragment of cystatin C in cerebral spinal fluids from patients diagnosed with multiple sclerosis. The SAMDI-TOF immunoassay, which combines well-defined surface chemistries for the selective and reproducible localization of analytes with mass spectrometry for label-free detection of analytes, may offer an alternative methodology to address many of the issues associated with standardized clinical diagnostics.     

Dynamic Curvature Nanochannel‐Based Membrane with Anomalous Ionic Transport Behaviors and Reversible Rectification Switch

Biological nanochannels control the movements of different ions through cell membranes depending on not only those channels' static inherent configurations, structures, inner surface's physicochemical properties but also their dynamic shape changes, which are required in various essential functions of life processes. Inspired by ion channels, many artificial nanochannel‐based membranes for nanofluidics and biosensing applications have been developed to regulate ionic transport behaviors by using the functional molecular modifications at the inner surface of nanochannel to achieve a stimuli‐responsive layer. Here, the concept of a dynamic nanochannel system is further developed, which is a new way to regulate ion transport in nanochannels by using the dynamic change in the curvature of channels to adjust ionic rectification in real time. The dynamic curvature nanochannel‐based membrane displays the advanced features of the anomalous effect of voltage, concentration, and ionic size for applying simultaneous control over the curvature‐tunable asymmetric and reversible ionic rectification switching properties. This dynamic approach can be used to build smart nanochannel‐based systems, which have strong implications for flexible nanofluidics, ionic rectifiers, and power generators.     

Highly sensitive pyrosequencing system with polymer-supported enzymes for high-throughput DNA analysis

A highly sensitive massively parallel pyrosequencing system employing a gel matrix to immobilize enzymes at high density in microreaction chambers is demonstrated. Reducing the size of microreaction chambers in a DNA analyzer is important to achieve a high throughput utilizing a commercially available detection device or camera. A high-performance system can be attained by detecting signals from one reaction chamber with one photopixel of around several micrometers by utilizing a 1:1 image magnification. However, the use of small beads immobilizing DNA has a disadvantage in detecting luminescence because only small amounts of DNA can be immobilized on the bead surfaces for sequencing. As luminescence intensity could be enhanced by increasing the luciferase density in the chambers, we overcame this difficulty by using a gel matrix to immobilize luciferase at a high concentration in the microreaction chambers. Luminescence 1 order of magnitude higher could be observed with the new method compared to the conventional method. Consequently, the chamber size and bead size immobilizing DNA could be reduced to as small as 6.5 and 4 μm, respectively. This can be successfully applied to achieving small, inexpensive, pyrosequencing systems with high throughput.     

Apparatus for precise drift time change measurements by means of photoelectrons emitted from the cathode

A drift chamber which measures the drift time of photoelectrons emitted from the cathode surface by means of a UV laser pulse has been built. The chamber is used for monitoring the drift time changes in the muon chambers of the L3 detector at CERN due to changes of gas parameters with the accuracy of 0.1%. The construction and experiments to determine the accuracy and stability of the device are described.     

Micromachined Fabry-Pérot interferometer with embedded nanochannels for nanoscale fluid dynamics

We describe a microfabricated Fabry-Pérot interferometer with nanochannels of various heights between 6 and 20 nm embedded in its cavity. By multiple beam interferometry, the device enables the study of liquid behavior in the nanochannels without using fluorescent substances. During filling studies of ethanol and water, an intriguing filling mode for partially wetting water was observed, tentatively attributed to the entrapment of a large amount of gas inside the channels.     

Electrical Scanning Probe Microscopy on Active Organic Electronic Devices

Polymer‐ and small‐molecule‐based organic electronic devices are being developed for applications including electroluminescent displays, transistors, and solar cells due to the promise of low‐cost manufacturing. It has become clear that these materials exhibit nanoscale heterogeneities in their optical and electrical properties that affect device performance, and that this nanoscale structure varies as a function of film processing and device‐fabrication conditions. Thus, there is a need for high‐resolution measurements that directly correlate both electronic and optical properties with local film structure in organic semiconductor films. In this article, we highlight the use of electrical scanning probe microscopy techniques, such as conductive atomic force microscopy (c‐AFM), electrostatic force microscopy (EFM), scanning Kelvin probe microscopy (SKPM), and similar variants to elucidate charge injection/extraction, transport, trapping, and generation/recombination in organic devices. We discuss the use of these tools to probe device structures ranging from light‐emitting diodes (LEDs) and thin‐film transistors (TFT), to light‐emitting electrochemical cells (LECs) and organic photovoltaics.     

Entrapment of protein in nanotubes formed by a nanochannel and ion-channel hybrid structure of anodic alumina

The nanochannel (in a porous layer) and ion-channel (in a barrier layer) hybrid structure of anodic alumina is used as a protein-trapping device. The transmembrane potential drives the electromigration of the charged proteins (FITC-labeled) into the nanochannels, but electromigration across the barrier layer is impossible due to the size-exclusion effect. As a result, the proteins can be continuously trapped in the nanochannels.     

Cellular Assays with a Molecular Endpoint Measured by SAMDI Mass Spectrometry

Cell‐based, high‐throughput screening (HTS) assays are increasingly important tools used in drug discovery, but frequently rely on readouts of gene expression or phenotypic changes and require development of specialized, labeled reporters. Here a cell‐based, label‐free assay compatible with HTS is introduced that can report quantitatively on enzyme activities by measuring mass changes of substrates with matrix‐assisted laser desorption/ionization mass spectrometry. The assay uses self‐assembled monolayers to culture cells on arrays presenting substrates, which serve as reporters for a desired enzyme activity. Each spot of cells is treated with a compound, cultured and lysed, enabling endogenous enzymes to act on the immobilized peptide substrate. It is demonstrated that the assay can measure protein tyrosine phosphatase (PTP) activity from as few as five cells and a screen is described that identifies a compound that reduces PTP activity in cell lysates. This approach offers a valuable addition to the methods available for cell‐based screening.     

Solving initial value problems by differential quadrature method—part 1: first‐order equations

In this paper, the differential quadrature method is used to solve first‐order initial value problems. The initial condition is given at the beginning of a time interval. The time derivative at a sampling grid point within the time interval can be expressed as a weighted linear sum of the given initial condition and the function values at the sampling grid points within the time interval. The order of accuracy and the stability property of the quadrature solutions depend on the locations of the sampling grid points. It is shown that the order of accuracy of the quadrature solutions at the end of a time interval can be improved to 2n–1 or 2n if the n sampling grid points are chosen carefully. In fact, the approximate solutions are equivalent to the generalized Padé approximations. The resultant algorithms are therefore unconditionally stable with controllable numerical dissipation. The corresponding sampling grid points are found to be given by the roots of the modified shifted Legendre polynomials. From the numerical examples, the accuracy of the quadrature solutions obtained by using the proposed sampling grid points is found to be better than those obtained by the commonly used uniformly spaced or Chebyshev–Gauss–Lobatto sampling grid points. Copyright © 2001 John Wiley & Sons, Ltd.     

High Virus Removal by Self‐Organized Nanostructured 2D Liquid‐Crystalline Smectic Membranes for Water Treatment

To obtain high quality of drinking water free from biocontaminants is especially important issue. A new strategy employing smectic liquid‐crystalline ionic membranes exhibiting 2D structures of layered nanochannels for water treatment is proposed for efficient virus removal and sufficient water flux. The smectic A (SmA) liquid‐crystalline membranes obtained by in situ polymerization of an ionic mesogenic monomer are examined for removal of three distinct viruses with small size: Qβ bacteriophage, MS2 bacteriophage, and Aichi virus. The semi‐bilayer structure of the SmA significantly obstructs the virus penetration with an average log reduction value of 7.3 log₁₀ or the equivalent of reducing 18 million viruses down to 1. Furthermore, the layered nanochannels of the SmA liquid crystal allow efficient water permeation compared to other types of liquid‐crystalline membrane consisting of nanopores.     

Biocatalytic Polymer Coatings: On‐Demand Drug Synthesis and Localized Therapeutic Effect under Dynamic Cell Culture Conditions

Biocatalytic surface coatings are prepared herein for localized synthesis of drugs and their on‐demand, site‐specific delivery to adhering cells. This novel approach is based on the incorporation of an enzyme into multilayered polymer coatings to accomplish enzyme‐prodrug therapy (EPT). The build‐up of enzyme‐containing multilayered coatings is characterized and correlations are drawn between the multilayer film assembly conditions and the enzymatic activity of the resulting coatings. Therapeutic effect elicited by the substrate mediated EPT (SMEPT) strategy is investigated using a prodrug for an anticancer agent, SN‐38. The performance of biocatalytic coatings under flow conditions is investigated and it is demonstrated that EPT allows synthesizing the drugs on‐demand, at the time desired and in a controllable amount to suit particular applications. Finally, using cells cultured in sequentially connected flow chambers, it is demonstrated that SMEPT affords a site‐specific drug delivery, that is, exerts a higher therapeutic effect in cells adhering directly to the biocatalytic coatings than in the cells cultured “downstream”. Taken together, these data illustrate biomedical opportunities made possible by engineering tools of EPT into multilayered polymer coatings and present a novel, highly versatile tool for surface mediated drug delivery.     

Imposition of boundary conditions by modifying the weighting coefficient matrices in the differential quadrature method

One of the important issues in the implementation of the differential quadrature method is the imposition of the given boundary conditions. There may be multiple boundary conditions involving higher‐order derivatives at the boundary points. The boundary conditions can be imposed by modifying the weighting coefficient matrices directly. However, the existing method is not robust and is known to have many limitations. In this paper, a systematic procedure is proposed to construct the modified weighting coefficient matrices to overcome these limitations. The given boundary conditions are imposed exactly. Furthermore, it is found that the numerical results depend only on those sampling grid points where the differential quadrature analogous equations of the governing differential equations are established. The other sampling grid points with no associated boundary conditions are not essential. Copyright © 2002 John Wiley & Sons, Ltd.     

Combinatorial Immunophenotyping of Cell Populations with an Electronic Antibody Microarray

Immunophenotyping is widely used to characterize cell populations in basic research and to diagnose diseases from surface biomarkers in the clinic. This process usually requires complex instruments such as flow cytometers or fluorescence microscopes, which are typically housed in centralized laboratories. Microfluidics are combined with an integrated electrical sensor network to create an antibody microarray for label‐free cell immunophenotyping against multiple antigens. The device works by fractionating the sample via capturing target subpopulations in an array of microfluidic chambers functionalized against different antigens and by electrically quantifying the cell capture statistics through a network of code‐multiplexed electrical sensors. Through a combinatorial arrangement of antibody sequences along different microfluidic paths, the device can measure the prevalence of different cell subpopulations in a sample from computational analysis of the electrical output signal. The device performance is characterized by analyzing heterogeneous samples of mixed tumor cell populations and then the technique is applied to determine leukocyte subpopulations in blood samples and the results are validated against complete blood cell count and flow cytometry results. Label‐free immunophenotyping of cell populations against multiple targets on a disposable electronic chip presents opportunities in global health and telemedicine applications for cell‐based diagnostics and health monitoring.     

Generation of Femtoliter Reactor Arrays within a Microfluidic Channel for Biochemical Analysis

We present a simple microfluidic method to generate high-density femotoliter-sized microreactor arrays within microfluidic channels. In general, we designed a main channel with many small chambers built into its walls. After sequentially infusing aqueous solution and organic solvent from a single tube into the device, aqueous droplets are confined in the chambers by the solvent flow. The generated reactors are small and stable enough for carrying out ultrasensitive biochemical assays at single molecule levels. As a demonstration, in this paper, we optically observed hydrolysis activity of β-galactosidase enzymatic molecules in the reactor arrays at single molecule levels. Further, this method has the following advantages: (1) the droplets are observable immediately after formation and (2) its simple procedure is sufficiently robust such that even handy infusion of the preloaded solutions is reproducible. We believe our method provides a platform attractive to a variety of single molecule studies and sensing applications such as clinical diagnostics.     

Shaping Giant Membrane Vesicles in 3D‐Printed Protein Hydrogel Cages

Giant unilamellar phospholipid vesicles are attractive starting points for constructing minimal living cells from the bottom‐up. Their membranes are compatible with many physiologically functional modules and act as selective barriers, while retaining a high morphological flexibility. However, their spherical shape renders them rather inappropriate to study phenomena that are based on distinct cell shape and polarity, such as cell division. Here, a microscale device based on 3D printed protein hydrogel is introduced to induce pH‐stimulated reversible shape changes in trapped vesicles without compromising their free‐standing membranes. Deformations of spheres to at least twice their aspect ratio, but also toward unusual quadratic or triangular shapes can be accomplished. Mechanical force induced by the cages to phase‐separated membrane vesicles can lead to spontaneous shape deformations, from the recurrent formation of dumbbells with curved necks between domains to full budding of membrane domains as separate vesicles. Moreover, shape‐tunable vesicles are particularly desirable when reconstituting geometry‐sensitive protein networks, such as reaction‐diffusion systems. In particular, vesicle shape changes allow to switch between different modes of self‐organized protein oscillations within, and thus, to influence reaction networks directly by external mechanical cues.