Cell culture arrays using magnetic force-based cell patterning for dynamic single cell analysis

In order to understand the behavior of individual cells, single cell analyses have attracted attention since most cell-based assays provide data with values averaged across a large number of cells. Techniques for the manipulation and analysis of single cells are crucial for understanding the behavior of individual cells. In the present study, we have developed single cell culture arrays using magnetic force and a pin holder, which enables the allocation of the magnetically labeled cells on arrays, and have analyzed their dynamics. The pin holder was made from magnetic soft iron and contained more than 6000 pillars on its surface. The pin holder was placed on a magnet to concentrate the magnetic flux density above the pillars. NIH/3T3 fibroblasts that were labeled with magnetite cationic liposomes (MCLs) were seeded into a culture dish, and the dish was placed over the pin holder with the magnet. The magnetically labeled cells were guided on the surface where the pillars were positioned and allocated on the arrays with a high resolution. Single-cell patterning was achieved by adjusting the number of cells seeded, and the target cell was collected by a micromanipulator after removing the pin holder with the magnet. Furthermore, change in the morphology of magnetically patterned cells was analyzed by microscopic observation, and cell spreading on the array was observed with time duration. Magnetic force-based cell patterning on cell culture arrays would be a suitable technique for the analysis of cell behavior in studies of cell-cell variation and cell-cell interactions.     

Inhibition assay of yeast cell walls by plasmon resonance Rayleigh scattering and surface-enhanced Raman scattering imaging

We report on plasmon resonance Rayleigh scattering (PRRS) and surface enhanced Raman scattering (SERS) imaging for inhibition assay of yeast cell walls. This assay reveals that the proteins having alkali sensitive linkage bound to β1,3 glucan frameworks in cell walls are involved in SERS activity. The result is further confirmed by comparison of genetically modified cells and wild type cells. Finally, we find that PRRS and SERS spots do not appear on cell walls when daughter cells are enough smaller than parent ones, but appear when size of daughter cells are comparable to parent cells. This finding indicates the relationship between expression of the proteins that generate SERS spots and cell division. These results demonstrate that PRRS and SERS imaging can be a convenient and sensitive method for analysis of cell walls.     

Photoactivated release of cargo from the cavity of polyelectrolyte capsules to the cytosol of cells

Polyelectrolyte capsules with metal nanoparticles in their walls and fluorescently labeled polymers as cargo inside their cavity were prepared. Capsules were ingested by living cells with no uncontrolled release of the cargo upon the incorporation process. Photoinduced heating of the metal nanoparticles in the capsule walls lead to rupture of the capsule walls, and the polymeric cargo was released to the whole cytosol. Viability tests demonstrate that opening of capsules at moderate light intensities does not impair the cellular metabolism, whereas capsule opening at high light intensities ultimately leads to cell death.     

Micropallet arrays with poly(ethylene glycol) walls

Arrays of releasable micropallets with surrounding walls of poly(ethylene glycol) (PEG) were fabricated for the patterning and sorting of adherent cells. PEG walls were fabricated between the SU-8 pallets using a simple, mask-free strategy. By utilizing the difference in UV-transmittance of glass and SU-8, PEG monomer was selectively photopolymerized in the space surrounding the pallets. Since the PEG walls are composed of a cross-linked structure, the stability of the walls is independent of the pallet array geometry and the properties of the overlying solution. Even though surrounded with PEG walls, the individual pallets were detached from the array by the mechanical force generated by a focused laser pulse, with a release threshold of 6 microJ. Since the PEG hydrogels are repellent to protein adsorption and cell attachment, the walls localized cell growth to the pallet top surface. Cells grown in the microwells formed by the PEG walls were released by detaching the underlying pallet. The released cells/pallets were collected, cultured and clonally expanded. The micropallet arrays with PEG walls provide a platform for performing single cell analysis and sorting on chip.     

Autoantibody detection by direct counting of antigen-displayed yeast cells

In this study, we report a new immunoassay platform based on yeast surface display technology for detection of autoantibodies involved in autoimmune diseases, e.g., systemic lupus erythematosus (SLE) and Sj?gren's syndrome (SS). The autoantigens of Ro52/SSA epitope and SmD were chosen to be displayed on the yeast surface with their respective antibodies as the analytes. By using magnetic beads modified with protein G, yeast cells bound with specific target antibody can be captured. The amount of analytes could be determined by counting the number of fluorescent yeast cells captured in a magnetic field. The platform showed promising results in the detection of SLE autoantibodies with high sensitivity and multiplex detection capability over the traditional approaches.     

Directed positioning of single cells in microwells fabricated by scanning probe lithography and wet etching methods

Scanning probe microscopy has emerged as a powerful technique for mapping the surface morphology of biological specimens, including proteins and cells. In addition to providing measurements of topographic images, it enables the fabrication of micro-/nanostructures with a high spatial resolution. Herein, we demonstrate a simple and reliable method for the preparation of single Escherichia coli bacterial cell arrays using pre-fabricated microwell structures. Using a <100>-oriented silicon substrate, microwell arrays with inclined sidewalls were fabricated by scanning probe lithography and sequential chemical wet etching. The trapping efficiency of single cells was optimized by controlling the geometries of the microwells. These data suggest that single-cell arrays may be applicable in a variety of areas, including drug testing and toxicology, as well as basic cell biology.     

Atomic force microscopy demonstrates that disulfide bridges are required for clustering of the yeast cell wall integrity sensor Wsc1

In yeasts, cell surface stresses are detected by a family of plasma membrane sensors. Among these, Wsc1 contains an extracellular cysteine-rich domain (CRD), which mediates sensor clustering and is believed to anchor the sensor in the cell wall. Although the formation of Wsc1 clusters and their interaction with the intracellular pathway components are important for proper stress signaling, the molecular mechanisms underlying clustering remain poorly understood. Here, we used the combination of single-molecule atomic force microscopy (AFM) with genetic manipulations to demonstrate that Wsc1 clustering involves disulfide bridges of the CRD. Using AFM tips carrying nitrilotriacetate groups, we mapped the distribution of individual His-tagged sensors on living yeast cells. While Wsc1 formed nanoscale clusters on native cells, clustering was no longer observed after treatment with the reducing agent dithiothreitol (DTT), indicating that intra- or intermolecular disulfide bridges are required for clustering. Moreover, DTT treatment resulted in a significant increase in cell surface roughness, suggesting that disulfide bridges between other cell-wall proteins are crucial for proper cell surface topology. The remarkable sensor properties unravelled here may well apply to other sensors and receptors with cysteine-rich domains throughout biology. Our combined method of AFM with genetic manipulations offers great prospects to explore the mechanisms underlying the clustering of cell surface proteins.     

Electromagnetophoretic force measurement of a single binding interaction between lectin and yeast cell surfaces.

A novel measurement method of the binding force between a micrometer-sized particle and a solid surface in an electrolyte solution has been established by using the electromagnetophoretic buoyancy on the particle. By this method, we investigated the binding force between a yeast cell surface and an oligosaccharide-binding protein, concanavalin A (Con A), fixed on a silica capillary wall. The force measurement was carried out up to 60 pN. In a lower surface concentration of Con A, yeast cells could be desorbed by a force less than 60 pN. However, in a higher surface concentration after treated by 1 mg ml(-1) solution, yeast cells were adsorbed with a force stronger than 60 pN. In this case, the addition of 10 mg ml(-1) D-mannose solution to the medium reduced the binding force to less than 60 pN. The observed adsorption force of yeast cells ranged within 30 - 40 pN, regardless of the interfacial amount of Con A. This force was thought to be the single binding force between a mannose group of the cell surface and an active site of Con A. Moreover, the dissociation rate constant of the single binding of yeast cell and Con A complex was determined as 4.6 x 10(-3) s(-1) and the increment of the binding distance at the transition state as 0.33 nm from the desorption kinetic experiments of yeast cell under the constant pulling conditions of 10, 20 and 30 pN. Such satisfactory results demonstrate the novel advantages of the present method.     

Magnetization of individual yeast cells by in situ formation of iron oxide on cell surfaces

Magnetic functionalization of living cells has intensively been investigated with the aim of various bioapplications such as selective separation, targeting, and localization of the cells by using an external magnetic field. However, the magnetism has not been introduced to individual living cells through the in situ chemical reactions because of harsh conditions required for synthesis of magnetic materials. In this work, magnetic iron oxide was formed on the surface of living cells by optimizing reactions conditions to be mild sufficiently enough to sustain cell viability. Specifically, the reactive LbL strategy led to formation of magnetically responsive yeast cells with iron oxide shells. This facile and direct post-magnetization method would be a useful tool for remote manipulation of living cells with magnetic interactions, which is an important technique for the integration of cell-based circuits and the isolation of cell in microfluidic devices.     

Probing the Intracellular Glutathione Redox Potential by In‐Cell NMR Spectroscopy

Non‐invasive and real‐time analysis of cellular redox processes has been greatly hampered by lack of suitable measurement techniques. Here we describe an in‐cell nuclear magnetic resonance (NMR) based method for measuring the intracellular glutathione redox potential by direct and quantitative measurement of isotopically labeled glutathione introduced exogenously into living yeast. By using this approach, perturbations in the cellular glutathione redox homeostasis were also monitored as yeast cells were subjected to oxidative stress.     

Copper benzene tricarboxylate metal-organic framework with wide permanent mesopores stabilized by Keggin polyoxometallate ions

Porous solids with organized multiple porosity are of scientific and technological importance for broadening the application range from traditional areas of catalysis and adsorption/separation to drug release and biomedical imaging. Synthesis of crystalline porous materials offering a network of uniform micro- and mesopores remains a major scientific challenge. One strategy is based on variation of synthesis parameters of microporous networks, such as, for example, zeolites or metal-organic frameworks (MOFs). Here, we show the rational development of an hierarchical variant of the microporous cubic Cu(3)(BTC)(2) (BTC = 1,3,5-benzenetricarboxylate) HKUST-1 MOF having strictly repetitive 5 nm wide mesopores separated by uniform microporous walls in a single crystal structure. This new material coined COK-15 (COK = Centrum voor Oppervlaktechemie en Katalyse) was synthesized via a dual-templating approach. Stability was enhanced by Keggin type phosphotungstate (HPW) systematically occluded in the cavities constituting the walls between the mesopores.     

磁性微纳米材料的功能化及其在食物样品前处理中的应用进展

磁性固相萃取是当前对复杂样品中痕量目标物进行有效分离富集的热门技术,功能化磁性微纳米粒子是该技术应用中的关键材料。本文综述了各种已报道的功能化磁性微纳米材料,总结了包括表面嫁接有机小分子、表面包覆碳或无机氧化物、表面嫁接或包覆聚合物、载体表面或孔道内负载磁性纳米粒子、载体骨架内掺入磁性纳米粒子、物理共混法制备磁性功能材料在内的6种功能化方法,并对功能化磁性微纳米材料在食物样品前处理中的应用进行了简要评述。     

Visualized investigation of yeast transformation induced with Li+ and polyethylene glycol

The effects of Li⁺ and polyethylene glycol (PEG) on the genetic transformation of Saccharomyces cerevisiae were investigated by using fluorescence microscopy (FM) to visualize the binding of plasmid DNA labeled with YOYO-1 to the surface of yeast cells, scanning electron microscopy (SEM) and atomic force microscopy (AFM) to image the change in surface topography of yeast cells, coupled with transformation frequency experiments. The results showed that under the same conditions, the transformation frequencies of yeast protoplasts were much higher than those of intact yeast cells. PEG was absolutely required for the binding of DNA to the surface of intact yeast cells or yeast protoplasts, and had no effect on the surface topography of intact yeast cells or yeast protoplasts. In the presence of PEG, Li⁺ could greatly enhance the binding of plasmid DNA to the surface of intact yeast cells, increase their transformation frequency, and affect their surface topography. On the other hand, no effect on the DNA binding to the surface of protoplasts and no increase in the number of transformants and no surface topography changes were found upon the treatment with Li⁺ to protoplasts. In the present work, the effects of Li⁺ and PEG on yeast genetic transformation were directly visualized, rather than those deduced from the results of transformation frequencies. These results indicate that cell wall might be a barrier for the uptake of plasmid DNA. Li⁺ could increase the permeability of yeast cell wall, then increase the exposed sites of DNA binding on intact yeast cells. The main role of PEG was to induce DNA binding to cell surface.     

Functional artificial free-standing yeast biofilms

Here we report fabrication of artificial free-standing yeast biofilms built using sacrificial calcium carbonate-coated templates and layer-by-layer assembly of extracellular matrix-mimicking polyelectrolyte multilayers. The free-standing biofilms are freely floating multilayered films of oppositely charged polyelectrolytes and live cells incorporated in the polyelectrolyte layers. Such biofilms were initially formed on glass substrates of circular and ribbon-like shapes coated with thin layers of calcium carbonate microparticles. The templates were then coated with cationic and anionic polyelectrolytes to produce a supporting multilayered thin film. Then the yeast alone or mixed with various micro- and nanoparticle inclusions was deposited onto the multilayer composite films and further coated with outer polyelectrolyte multilayers. To detach the biofilms from the glass substrates the calcium carbonate layer was chemically dissolved yielding free-standing composite biofilms. These artificial biofilms to a certain degree mimic the primitive multicellular and colonial species. We have demonstrated the added functionality of the free-standing artificial biofilms containing magnetic, latex and silver micro- and nanoparticles. We have also developed "symbiotic" multicellular biofilms containing yeast and bacteria. This approach for fabrication of free-standing artificial biofilms can be potentially helpful in development of artificial colonial microorganisms composed of several different unicellular species and an important tool for growing cell cultures free of supporting substrates.     

In vitro and in vivo evaluation of microparticulate drug delivery systems composed of macromolecular prodrugs

Macromolecular prodrugs are very useful systems for achieving controlled drug release and drug targeting. In particular, various macromolecule-antitumor drug conjugates enhance the effectiveness and improve the toxic side effects. Also, polymeric micro- and nanoparticles have been actively examined and their in vivo behaviors elucidated, and it has been realized that their particle characteristics are very useful to control drug behavior. Recently, researches based on the combination of the concepts of macromolecular prodrugs and micro- or nanoparticles have been reported, although they are limited. Macromolecular prodrugs enable drugs to be released at a certain controlled release rate based on the features of the macromolecule-drug linkage. Micro- and nanoparticles can control in vivo behavior based on their size, surface charge and surface structure. These merits are expected for systems produced by the combination of each concept. In this review, several micro- or nanoparticles composed of macromolecule-drug conjugates are described for their preparation, in vitro properties and/or in vivo behavior.     

Chemically programmed cell adhesion with membrane-anchored oligonucleotides

Cell adhesion organizes the structures of tissues and mediates their mechanical, chemical, and electrical integration with their surroundings. Here, we describe a strategy for chemically controlling cell adhesion using membrane-anchored single-stranded DNA oligonucleotides. The reagents are pure chemical species prepared from phosphoramidites synthesized in a single chemical step from commercially available starting materials. The approach enables rapid, efficient, and tunable cell adhesion, independent of proteins or glycans, by facilitating interactions with complementary labeled surfaces or other cells. We demonstrate the utility of this approach by imaging drug-induced changes in the membrane dynamics of non-adherent human cells that are chemically immobilized on a passivated glass surface.     

Field-flow fractionation of magnetic particles in a cyclic magnetic field

Although magnetic field-flow fractionation (MgFFF) is emerging as a promising technique for characterizing magnetic particles, it still suffers from limitations such as low separation efficiency due to irreversible adsorption of magnetic particles on separation channel. Here we report a novel approach based on the use of a cyclic magnetic field to overcome the particle entrapment in MgFFF. This cyclic field is generated by rotating a magnet on the top of the spiral separation channel so that magnetic and opposing gravitational forces alternately act on the magnetic particles suspended in the fluid flow. As a result, the particles migrate transversely between the channel walls and their adsorption at internal channel surface is prevented due to short residence time which is controlled by the rotation frequency. With recycling of the catch-release process, the particles follow saw-tooth-like downstream migration trajectories and exit the separation channel at velocities corresponding to their sedimentation coefficients. A retention model has been developed on the basis of the combined effects of magnetic, gravitational fields and hydrodynamic flow on particle migration. Two types of core-shell structured magnetic microspheres with diameters of 6.04- and 9.40-μm were synthesized and used as standard particles to test the proposed retention theory under varying conditions. The retention ratios of these two types of particles were measured as a function of magnet rotation frequency, the gap between the magnet and separation channel, carrier flow rate, and sample loading. The data obtained confirm that optimum separation of magnetic particles with improved separation efficiency can be achieved by tuning rotation frequency, magnetic field gradient, and carrier flow rate. In view of the widespread applications of magnetic microspheres in separation of biological molecules, virus, and cells, this new method might be extended to separate magnetically labeled proteins or organisms for multiplex analyte identification and purification.     

In situ study of electrochemical processes of metal nano-nuclei formation,growth, and stability on single carbon microfiber for surface multifunctionalization

With better understanding and control of metal layer formation on carbon surface, the electrical, magnetic, thermal, interfacial, and catalytic characteristics of carbon-based micro- and nanomaterials can be further improved for large-scale engineering applications. Experiments demonstrated that controlled metal electrodeposition on micro- and nanocarbon fibers can be realized in a cost-effective and reproducible fashion. Microbeam synchrotron X-ray diffraction and fluorescence techniques have been developed to provide in situ characterization capabilities to reveal the nuclei formation and growth processes on individual carbon microfibers with size, distribution, and microstructural information. The nuclei stability of the metal deposit is found to have strong dependence on its size as well as the deposition condition.     

Curvature driven transport of mouse macrophages in a pulsating magnetic garnet film ratchet

Magnetic fields varying on the colloidal length scale are used for the directed transport of magnetically labeled biological cells. The transport is achieved by using the ratchet effect which relies on an asymmetric, symmetry broken periodic potential where nonequilibrium fluctuations or oscillations generate a net cell current. Ferrofluid ingested mouse macrophages were placed on a magnetic garnet film with alternating stripe domain patterns, and a pulsating magnetic potential is provided by superposing an oscillating magnetic field normal to the film. The symmetry of the resulting periodic stripe potential is broken locally by the curvature of the stripes. We show, both experimentally and theoretically, the curvature of such stripes required for inducing directed transport of the macrophages in the ratchet. This may be useful for microfluidic devices such as a digital colloidal shift register for magnetically labeled biological cells.     

Magnetic field dependence of the diffusion of single dextran molecules within a hydrogel containing magnetite nanoparticles

We consider the effect of applied magnetic fields on the diffusion of single dextran molecules labeled with fluorescein isothiocyanate within a ferrogel [a composite of magnetite nanoparticles in a poly(methacrylic acid) hydrogel] using fluorescence correlation spectroscopy. We show that the mesh size of the ferrogel is controlled by the applied magnetic field, B, and scales as exp(-(4)√ξ(3)B(2)/2μ(0)k(B)T), where ξ is a correlation length, μ(0) the magnetic constant, k(B) the Boltzmann constant, and T is the absolute temperature. The diffusion coefficient of the dextran can be modeled with a simple Stokes-Einstein law, containing the same scaling behavior with magnetic field as the swelling of the hydrogel. Furthermore, the magnetic field-dependent release of dextran from the hydrogel is also controlled by the same relationship. The samples were characterized by small angle x-ray scattering (SAXS) and magnetometry experiments. Magnetic hysteresis loops from these ferrogels and zero field cooled∕field cooled measurements reveal single domain ferromagnetic behavior at room temperature with a similar coercivity for both as-prepared and fully swollen ferrogels, and for increasing magnetic nanoparticle concentration. SAXS experiments, such as the hysteresis loops, show that magnetite does not aggregate in these gels.