Micropatterning of polystyrene nanoparticles and its bioapplications

Micropatterning of biomolecules forms the basis of cell culture, biosensor and microarray technology. Currently, the most widely used techniques are photoresist lithography, soft lithography or using robots which all involve multi-step surface modification directly on a planar substrate. Here we report a method to pattern biomolecules through self-assembling polystyrene nanoparticles in arrayed microwells on a solid surface to form well-ordered patterning, followed by attaching biomolecules to the assembled nanoparticles. The formation of colloidal patterns depends on capillary force, surface wettability and physical confinement. This method can be used for micropatterning a variety of biomolecules such as protein and antibody.     

Micropatterning of proteins on nanospheres

Currently micropatterning of proteins is mainly carried out on a planar substrate, which involves multi-step surface modifications directly on the substrate. Efficiency of chemical reactions is usually low, resulting in low signal-to-noise (S/N) ratio and poor repeatability of results. Here we presented a micropatterning method using polystyrene nanospheres with non-planar surface as a solid support for attaching proteins, which introduces many advantages. The patterning of proteins was carried out in two approaches: one was to dispense polystyrene nanospheres into an array of microwells and then attach proteins onto the nanospheres, and another was to coat polystyrene nanospheres with proteins first and then deposit the spheres into the microwells. For both approaches, a uniform pattern of proteins was generated. The amount of proteins attached via nanospheres was much higher than that on planar surface.     

Duplexed sandwich immunoassays on a fiber-optic microarray

In this paper, we describe a duplexed imaging optical fiber array-based immunoassay for immunoglobulin A (IgA) and lactoferrin. To fabricate the individually addressable array, microspheres were functionalized with highly specific monoclonal antibodies. The microspheres were loaded in microwells etched into the distal face of an imaging optical fiber bundle. Two microsphere-based sandwich immunoassays were developed to simultaneously detect IgA and lactoferrin, two innate immune system proteins found in human saliva. Individual microspheres could be interrogated for the simultaneous measurement of both proteins. The working concentration range for IgA detection was between 700 pM and 100 nM, while the working concentration range for lactoferrin was between 385 pM and 10 nM. The cross-reactivity between detection antibodies and their non-specific targets was relatively low in comparison to the signal generated by the specific binding with their targets. These results suggest that the degree of multiplexing on this fiber-optic array platform can be increased beyond a duplex.     

Spatial geometric arrangements of disulfide-crosslinked loops in nonplanar proteins

The method presented earlier [T. Kikuchi, G. Némethy, and H.A. Scheraga, (1986) J. Comput. Chem. 7 , 67] for the classification of patterns of the three-dimensional folding of a covalently crosslinked polypeptide chain has been extended to nonplanar proteins. The procedure described earlier was applicable only to proteins termed planar, i.e., with a connexity of the crosslinks (e.g., disulfide bonds) that can be represented in a planar diagram. The procedure described in the present work is applicable to any (planar or nonplanar) pattern of crosslinking. The classification is based on a systematic and objective method of enumeration of spatial geometric arrangements of loops (SGAL) using no information other than the location of the disulfide bonds in the amino acid sequence. Various SGAL classes correspond to the presence of different ways of mutual penetration of loops, called thrustings and entanglements. Information on SGAL classes can be of use in structural predictions of folding patterns of proteins.     

Recent advances in the development of graphene-based surface plasmon resonance (SPR) interfaces

Surface plasmon resonance (SPR) is a powerful technique for measurement of biomolecular interactions in real-time in a label-free environment. One of the most common techniques for plasmon excitation is the Kretschmann configuration, and numerous studies of ligand–analyte interactions have been performed on surfaces functionalized with a variety of biomolecules, for example DNA, RNA, glycans, proteins, and peptides. A significant limitation of SPR is that the substrate must be a thin metal film. Post-coating of the metal thin film with a thin dielectric top layer has been reported to enhance the performance of the SPR sensor, but is highly dependent on the thickness of the upper layer and its dielectric constant. Graphene is a single-atom thin planar sheet of sp2 carbon atoms perfectly arranged in a honeycomb lattice. Graphene and graphene oxide are good supports for biomolecules because of their large surface area and rich π conjugation structure, making them suitable dielectric top layers for SPR sensing. In this paper, we review some of the key issues in the development of graphene-based SPR chips. The actual challenges of using these interfaces for studying biomolecular interactions will be discussed and the first examples of the use of graphene-on-metal SPR interfaces for biological sensing will be presented.     

Planar or nonplanar: what is the structure of urea in aqueous solution?

A combined quantum chemical statistical mechanical method has been used to study the solvation of urea in water, with emphasis on the structure of urea. The model system consists of three parts: a Hartree-Fock quantum chemical core, 99 water molecules described with a polarizable force-field, and a dielectric continuum. A free-energy profile along the transition of urea from planar to a nonplanar structure is calculated. This mode in aqueous solution is found to be floppy. That is, the structure of urea in water is not well-defined because the planar to nonplanar transition requires an energy of the order of the thermal energy at room temperature. We discuss the implications of this finding for simulation studies of urea in polar environments like water and proteins.     

Programmable assembly of colloidal particles using magnetic microwell templates

A substrate of thin micromagnets covered by a template of microwells is used to direct the assembly of superparamagnetic colloidal beads into two-dimensional arrays. It is confirmed that the magnetization of the micromagnets can direct beads to programmed locations on the substrate with assistance of externally applied magnetic fields. Empirical investigations on this topic were guided by mathematical models with the intent to elucidate the conditions that promote a single bead to be assembled in the desired microwells. To demonstrate that this technique is programmable, heterogeneous arrays of colored beads are produced.     

Preparation and pore-size control of hydrophilic monodispersed polymer microspheres for size-exclusive separation of biomolecules by the SPG membrane emulsification technique

This paper describes an experiment directed toward the preparation of monodispersed porous polymer microspheres with a diameter of ca. 50 m, which is applicable to the chromatographic separation of biomolecules such as proteins and peptides by size exclusion. Fairly monodispersed polymer microspheres were successfully prepared by suspension copolymerization of polyethylene glycol monomethacrylate and ethylene glycol dimethacrylate as monomer and cross-linker, respectively. Monodispersed O/W emulsion was prepared by the SPG membrane emulsification technique, and was used in the subsequent droplet-swelling process in which monodispersed seed droplets were swollen by adsorbing the secondary emulsion droplets. The effects of the organic diluent in suspension polymerization and comonomer on the porous structure of the polymer microspheres were investigated by scanning electron microscopy and mercury-intrusion porosimetry, and the separation performances of polystyrene, polyethylene glycol, and various biomolecules by size-exclusion chromatography. As a result, it was evident that benzene, 1-butanol, and butyl acetate worked as nonsolvents for the polymer prepared in this study, and that polymer microspheres prepared with these solvents had larger pores. Size-exclusion chromatography revealed that the exclusive limiting molecular weight was 1.9×10⁵ when polystyrene was used as a standard polymer, and 3.5×10⁴ when polyethylene glycol was used as a standard polymer. Furthermore, we confirmed that the monodispersed polymer microspheres with defined pores clearly separated the six representative kinds of biomolecules with molecular weights ranging from 75 to 6.4×10⁵.     

Automated sample preparation method for suspension arrays using renewable surface separations with multiplexed flow cytometry fluorescence detection

In this paper, we describe a new method of automated sample preparation for multiplexed biological analysis systems that use flow cytometry fluorescence detection. In this approach, color-encoded microspheres derivatized to capture particular biomolecules are temporarily trapped in a renewable surface separation column to enable perfusion with sample and reagents prior to delivery to the detector. This method provides for separation of the biomolecules of interest from other sample matrix components as well as from labeling solutions. After sample preparation, the beads can be released from the renewable surface column and delivered to a flow cytometer for direct on-bead analysis one bead at a time. Using mixtures of color-encoded beads derivatized for various analytes yields suspension arrays for multiplexed analysis. Development of this approach required a new technique for automated capture and release of the color-encoded microspheres within a fluidic system. We developed a method for forming a renewable filter and demonstrate its use for capturing microspheres that are too small to be easily captured in previous flow cells for renewable separation columns. The renewable filter is created by first trapping larger beads in the flow cell, and then smaller beads are captured either within or on top of the bed of larger beads. Both the selective microspheres and filter bed are automatically emplaced and discarded for each sample. A renewable filter created with 19.9 μm beads was used to trap 5.6 μm optically encoded beads with trapping efficiencies of 99%. The larger beads forming the renewable filter did not interfere with the detection of color-encoded 5.6 μm beads by the flow cytometer fluorescence detector. The use of this method was demonstrated with model reactions for a variety of bioanalytical assay types including a one-step capture of a biotinylated label on Lumavidin beads, a two-step sandwich immunoassay, and a one-step DNA binding assay. A preliminary demonstration of multiplexed detection of two analytes using color-encoded beads was also demonstrated. The renewable filter for creating separation columns containing optically encoded beads provides a general platform for coupling renewable surface methods for sample preparation and analyte labeling with flow cytometry detectors for suspension array multiplexed analyses.     

Encapsulation and release of biomolecules from Ca-P/PHBV nanocomposite microspheres and three-dimensional scaffolds fabricated by selective laser sintering

This study focused on the fabrication of calcium phosphate (Ca-P)/poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) nanocomposite scaffolds loaded with biomolecules using the selective laser sintering (SLS) technique and their evaluation. Ca-P/PHBV nanocomposite microspheres loaded with bovine serum albumin (BSA) as the model protein were fabricated using the double emulsion solvent evaporation method. The encapsulation efficiency of BSA in PHBV polymer microspheres and Ca-P/PHBV nanocomposite microspheres were 18.06 ± 0.86% and 24.51 ± 0.60%, respectively. The BSA loaded Ca-P/PHBV nanocomposite microspheres were successfully produced into three-dimensional porous scaffolds with good dimensional accuracy using the SLS technique. The nanocomposite microspheres served as protective carriers and maintained the bioactivity of BSA during SLS. The effects of SLS parameters such as laser power and scan spacing on the encapsulation efficiency of BSA in the scaffolds and in vitro BSA release were studied. An initial burst release was observed, which was followed by a slow release of BSA. After 28-day release, The PHBV matrix was slightly degraded after 28-day in vitro release study. It was shown that nanocomposite scaffolds with controlled architecture obtained via SLS could be incorporated with biomolecules, enhancing them with more functions for bone tissue engineering application or making them suitable for localized delivery of therapeutics.     

Aspects of prewetting at nonplanar surfaces

We employ Monte Carlo simulations in the grand canonical ensemble (GCEMC) to investigate the impact of nonplanarity of a solid substrate on the locus of the prewetting phase transition. The substrate is modelled as a periodic sequence of furrows of depth D and periodicity sx in the x direction; the furrows are infinitely long in the y direction. Our results indicate that a necessary prerequisite for a prewetting transition is the formation of a(n approximately) planar interface between molecularly thin films and an adjacent (bulk) gas. Thus, in general the prewetting transition is shifted to larger chemical potentials because the formation of a planar film-gas interface is more difficult next to a nonplanar compared with a planar solid surface. However, this shift turns out to be nonmonotonic depending on D on account of subtle packing effects manifested in the deviation of the local density Deltarho(x,Deltaz;D) at the nonplanar solid surface from that at a planar substrate. If D becomes sufficiently large prewetting as a discontinuous phase transition is suppressed because inside the furrow a highly ordered film forms that prevents a planar film-gas interface from forming.     

自组装超薄膜: 从纳米层状构筑到功能组装

总结了一种新型的超薄膜自组装技术－交替沉积组装技术的发展现状,着重对成膜推动力、生物分子的层状组装、无机／有机杂化结构、有机小分子化合物的层状组装、超薄膜化学修饰电极、层间化学反应及非平衡基底上的层状构筑等几个方面的问题进行了讨论。     

Scanning tunneling microscopy and scanning tunneling spectroscopy studies of planar and nonplanar naphthalocyanines on graphite (0001). Part 1: effect of nonplanarity on the adlayer structure and voltage-induced flipping of nonplanar tin-naphthalocyanine

The adsorption of base-free naphthalocyanine (Nc), a planar molecule, and tin-naphthalocyanine (SnNc), a nonplanar molecule, on a freshly cleaved highly oriented pyrolytic graphite (HOPG) surface at low sample temperature (50 K) has been studied using a variable-temperature scanning tunneling microscope in ultra-high vacuum conditions. The planar molecules form large areas of defect-free ordered monolayer with high molecular packing density while the nonplanar molecules show different phases of adsorption with lower molecular packing density. The SnNc adlayers follow the same geometry as the graphite substrate and form pure phases of adsorption with either all molecules in a Sn(2+) up or Sn(2+) down geometry. Moreover, a one-dimensional selectivity is observed in still another phase of Sn(2+) down geometry. Multilayers show a completely different kind of adsorption in each case. Nc molecules show columnar pi-stacking whereas the SnNc molecules exhibit noncolumnar stacking. Distinctly, a voltage-induced flipping of nonplanar tin-naphthalocyanine in the monolayer has been observed which can possibly be applied to single-molecular information storage.     

Synthesis and Reactions of Hydrophilic Functional Microspheres for Immunological Studies

There is a need for hydrophilic polymeric microspheres with functional groups on their surface which can be reacted efficiently with proteins. These microspheres with antibodies (immunoglobulins) covalently bound to their surfaces constitute valuable immunoreagents capable of marking specific receptors (antigens) on cell surface membranes. The main requirements of the microspheres for the above applications are: insolubility in aqueous or organic media, absence of aggregation and of nonspecific interaction with cells and presence of suitable functional groups for covalent binding with antibodies. Hydrophobic polystyrene or poly(methyl methacrylate) latices do not meet these requirements. Copolymerization of hydrophilic monomers under suitable experimental conditions yielded microspheres with the required characteristics. Emulsion polymerization and ionizing radiation were found to constitute convenient techniques for the synthesis of hydrophilic and crosslinked (and therefore insoluble) functional microspheres ranging in diameter from 0.01 to 8 μm. By choosing suitable comonomers, it was possible to incorporate hydroxyl, carboxyl, amido, and dimethylamino functional groups into the particles. Copolymerization with isomeric vinylpyridines or dimethylamino methacrylate yielded weakly or strongly basic groups, respectively, capable of binding with acids. The experimental conditions suitable for obtaining desired particle sizes, in a relatively narrow distribution, were determined. It was found that the particle size depended to a large extent on the water solubility of the monomers, the presence or absence of stabilizer, the concentration of a surfactant, and the monomer concentration. The preferred technique to bind antibodies to the microsphere surface consisted of reacting amino groups with glutaraldehyde followed by the reaction with proteins. The use of polyglutaraldehyde instead of glutaraldehyde was also investigated. For this purpose the rate of polymerization of glutaraldehyde as a function of concentration and pH was first studied, followed by a study of the reactivity of polyglutaraldehyde micro-spheres with immunoglobulins. A recent new development of importance for cell separation is the synthesis of functional microspheres containing magnetic iron oxide. Preliminary investigations show that red blood cells and lymphocytes labeled with magnetic immunomicrospheres can be efficiently separated by means of permanent magnet. Separation of labeled from unlabeled human red blood cells was also achieved by means of a free-flow electrophoretic instrument.     

Morphological characteristics of modified freeze-dried poly(<Emphasis Type="Italic">N</Emphasis>-isopropylacrylamide) microspheres studied by optical microscopy,SEM, and DLS

Influence of the initiator and additional hydrophobic copolymer on the morphology of thermosensitive poly(N-isopropylacrylamide) (pNIPAM) microspheres, and their presumed application for the stabilization of biologically active molecules were evaluated in this study. Three different types of pNIPAM were synthesized, applying various components: PN1 is a polymer with terminal anionic groups resulting from potassium persulfate initiator; PN2 was synthesized with a 2,2′-azobis(2-methylpropionamidine) dihydrochloride initiator introducing cationic amidine terminal groups; in the PN3 polymer, anionic terminals were implemented, however, increased hydrophobicity was maintained using N-tert-butyl functional groups. Turbidity measurements of the obtained dispersions confirmed specific thermosensitivity of synthesized microspheres in the range of 32–33°C. The polymerization course was proved by infrared spectroscopy and ¹H NMR assessments, whereas the size of the synthesized microspheres, expressed as planar area, was evaluated by dynamic light scattering (DLS), scanning electron microscopy (SEM) and optical microscopy (OM). The respective surface patterns of the freeze-dried microspheres were evaluated by SEM. Planar area of the synthesized macromolecules was in the range between 0.41–3.22 μm, depending on the substrates composition and the method applied for the measurements. The assessments performed in the dry stage gave higher values of the diameter and planar area of the observed microspheres. The measured diameter and planar area increased in the following order for the PN3 microspheres: DLS, OM, SEM. In the case of PN1 and PN2, the observed diameters were positioned as: DLS, SEM, OM. These differences were assigned both to varied intramolecular hydrophobic-hydrophilic interactions of the polymer chains and to the environment, i.e. low pressure in the SEM conditions and aqueous solvent in the DLS measurements. The observed gaps in the freeze-dried PN2 polymer resulted in an attempt to evaluate the application of this polymer for mechanical stabilization of certain macromolecules or nanocrystals in the size range between 10 nm and 20 nm.     

Synthesis of Fe3O4@poly(methylmethacrylate-co-divinylbenzene) magnetic porous microspheres and their application in the separation of phenol from aqueous solutions

A simple strategy to fabricate magnetic porous microspheres of Fe(3)O(4)@poly(methylmethacrylate-co-divinylbenzene) was demonstrated. The magnetic microspheres, consisting of polymer-coated iron oxide nanoparticles, were synthesized by the modified suspension polymerization of methacrylate and divinylbenzene in the presence of a magnetic fluid. The morphology and the properties of the magnetic porous microspheres were examined by scanning electron microscopy, transmission electron microscopy, superconducting quantum interference device, Fourier transform infrared spectroscopy, thermogravimetry, and X-ray powder diffraction. The pore size distribution and the specific surface area of the microspheres were measured by nitrogen sorption and mercury porosimetry technique. As predicted from the previous knowledge, the magnetic porous microspheres possessed a high specific surface area using n-hexane as a porogen. It was further found that the amounts of divinylbenzene and methacrylate, the ratio of porogens, and the dosage of ferrofluids affect the specific surface area of the microspheres. Furthermore, the microspheres were applied to remove phenol from aqueous solutions. The results showed that the microspheres had a high adsorption capacity for phenol and a high separation efficiency due to their porous structure, polar groups, and superparamagnetic characteristic.     

Efficient incorporation of quantum dots into porous microspheres through a solvent-evaporation approach

Quantum dot (QD)-encoded microspheres play an important role in suspension arrays by acting as supports for various reactions between biomolecules. With regard to QD-encoded microspheres utilized in suspension arrays, three key requirements are controllable size, abundant surface functional groups, and especially excellent fluorescence properties. In this paper, narrowly dispersed poly(styrene-co-divinylbenzene-co-methylacrylic acid) (PSDM) microspheres with specific size, surface carboxyl groups, and porous structures were synthesized by seeded copolymerization. In order to improve the incorporation efficiency of QDs within microspheres, we developed a swelling-evaporation approach in which the swelling process was combined with gradual evaporation of the solvent and thus gradual concentration of QDs in the dispersion solution. This approach was demonstrated to be an efficient method for improving the fluorescence intensity of resultant microspheres compared with the use of swelling alone. Moreover, the porous structure was shown to aid the penetration of QDs into the interiors of the microspheres. Through this approach, microspheres encoded with either single or multiple wavelength-emitting QDs were fabricated effectively. The suspension immunoassays were then founded based on the QD-encoded microspheres, by coating mouse antihuman chorionic gonadotropin as the probe for goat antimouse IgG detection. The positive results determined by Luminex 100 and the low cytotoxicity of the QD-encoded microspheres demonstrated their great potential in suspension arrays.     

Ionic electrets: electrostatic charging of surfaces by transferring mobile ions upon contact

This paper describes the fabrication and characterization of ionic electrets-materials that bear a long-lived electrostatic charge because of an imbalance between the number of cationic and anionic charges in the material. Crosslinked polystyrene microspheres that contain covalently bound ions and mobile counterions transfer some of their mobile ions in air, in the absence of bulk liquid, to another material upon contact. According to the ion-transfer model of contact electrification, this selective transfer of mobile ions yields microspheres that have a net electrostatic charge. A tool that operates on the principle of electrostatic induction measures the charge on individual microspheres (50-450 microm in diameter). Microspheres with a variety of covalently bound ionic functional groups (tetraalkylammonium, alkyltriphenylphosphonium, alkylsulfonate, and arylsulfonate) acquire charges consistent with this ion-transfer mechanism. The charge on a microsphere is proportional to its surface area (ca. 1 elementary charge per 2000 nm2) and close to the theoretical limit imposed by the dielectric breakdown of air. The charge density in an atmosphere of SF6 is more than twice that in an atmosphere of N2. These observations suggest that the charge density of these ionic electret microspheres is limited by the dielectric breakdown of the surrounding gas. Functionalizing the surfaces of glass or silicon with covalently bound ions and mobile counterions generates ionic electrets from these inorganic substrates. Soft lithography can pattern charge on a planar silicon surface (with oxide) and on the surface of 250-mum glass microspheres.     

Micro-well arrays for 3D shape control and high resolution analysis of single cells

In addition to rigidity, matrix composition, and cell shape, dimensionality is now considered an important property of the cell microenvironment which directs cell behavior. However, available tools for cell culture in two-dimensional (2D) versus three-dimensional (3D) environments are difficult to compare, and no tools exist which provide 3D shape control of single cells. We developed polydimethylsiloxane (PDMS) substrates for the culture of single cells in 3D arrays which are compatible with high-resolution microscopy. Cell adhesion was limited to within microwells by passivation of the flat upper surface through 'wet-printing' of a non-fouling polymer and backfilling of the wells with specific adhesive proteins or lipid bilayers. Endothelial cells constrained within microwells were viable, and intracellular features could be imaged with high resolution objectives. Finally, phalloidin staining of actin stress fibers showed that the cytoskeleton of cells in microwells was 3D and not limited to the cell-substrate interface. Thus, microwells can be used to produce microenvironments for large numbers of single cells with 3D shape control and can be added to a repertoire of tools which are ever more sought after for both fundamental biological studies as well as high throughput cell screening assays.     

Circulating IgSF Proteins Inhibit Adhesion of Antibody Targeted Microspheres to Endothelial Inflammatory Ligands

Proposed methods for detecting circulatory system disease include targeting ultrasound contrast agents to inflammatory markers on vascular endothelial cells. For antibody-based therapies, soluble forms of the targeted adhesion proteins of the immunoglobulin superfamily (IgSF) reduce adhesion yet were left unaccounted in prior reports. Microspheres labeled simply with a maximum level of antibodies can reduce the diagnostic sensitivity by adhering to proteins expressed normally at a low level, while sparsely coated particles may be rendered ineffective by circulating soluble forms of the targeted proteins. A new microdevice technique is applied to simultaneously measure the adhesion profile to a series of IgSF-protein-coated surfaces. In this investigation, we quantify the in vitro binding characteristics of 5-μm microspheres to oriented intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) protein-coated surfaces in the presence of human serum at physiological concentrations. Defined regions of a slide were coated with recombinant chimeric Fc-human ICAM-1 and VCAM-1 in variable ratios but constant total concentration. Monoclonal human anti-ICAM-1 or anti-VCAM-1 antibodies in competition with non-binding mouse anti-rabbit antibodies coat the microsphere surface at a constant surface density with variable yet controlled surface activities. Using multiple slide surface IgSF protein and microsphere antibody concentrations, an adhesion profile was developed for the microspheres with and without IgSF proteins from human serum, which demonstrated that exposure to serum reduced microsphere binding, on average, more than 50% compared to the no-serum condition.. The serum effects were limited to antibodies on the microsphere, since binding inhibition was reversed after rinsing serum from the system and fresh antibody-coated microspheres were introduced. This analysis quantifies the binding effects of soluble IgSF proteins from human serum on antibody-based targeted ultrasound detection and drug delivery methods.