Elucidation of protein adsorption behavior on polymeric surfaces: toward high-density, high-payload protein templates

The elucidation of protein adsorption behavior on polymeric surfaces is very important, since their use as arrays and carriers of biomolecules is ever growing for a wide variety of bioapplications. We evaluate protein adsorption characteristics on chemically homogeneous and heterogeneous polymeric surfaces by employing polystyrene-block-polymethylmethacrylate (PS-b-PMMA) diblock copolymer, PS homopolymer, PMMA homopolymer, and PS/PMMA blend as protein templates. We also investigate distance-dependent protein adsorption behavior on the interfacial region between PS and PMMA. We observe selective protein adsorption exclusively onto PS areas for the chemically heterogeneous PS-b-PMMA and PS/PMMA blend templates. On blend films, protein adsorption is highly favored on the PS regions located near the PS:PMMA interface over that on the PS areas situated away from the interface. Protein density on PS domains is inversely proportional to the separation distance between two neighboring PS:PMMA interfaces. We also observe a higher protein density on the PS-b-PMMA than on the PS or PMMA homopolymer templates. This effect is due to the fact that chemically heterogeneous PS-b-PMMA presents periodically spaced PS:PMMA interfaces on the nanometer scale, whereas no such interfaces are present on homopolymer films. The density of protein molecules on the heterogeneous PS-b-PMMA surface is approximately 3-4-fold higher than on the homogeneous PS surface for the identical experimental conditions. These results demonstrate that self-assembling, chemically heterogeneous, nanoscale domains in PS-b-PMMA diblock copolymers can be used as excellent, high-payload, high-density protein templates. The unique advantages of the diblock copolymer may prove the spontaneously constructed protein nanotemplates to be highly suitable as functional substrates in many proteomics applications.     

Nanoscale protein patterning using self-assembled diblock copolymers

Novel methods for immobilizing proteins on surfaces have the potential to impact basic biological research as well as various biochip applications. Here, we demonstrate a unique method to pattern proteins with a nanometer periodicity on silicon oxide substrates using microphase-separated diblock copolymer thin films. We developed a straightforward and effective protein immobilization technique using the microphase-separated domains of polystyrene-block-poly(methyl methacrylate) to localize various model protein molecules such as bovine immunoglobulin G, fluorescein isothiocyanate conjugated anti-bovine immunoglobulin G, and protein G. The self-organizing nature of the diblock copolymer was exploited to produce periodically alternating, nanometer-spaced polymeric domains exposing the two chemical compositions of the diblock to surface. We demonstrate that the model proteins selectively self-organize themselves on the microdomain regions of specific polymer components due to their preferential interactions with one of the two polymer segments. This diblock copolymer-based, self-assembly approach represents a step forward for facile, nanometer-spaced protein immobilization with high areal density and could provide a pathway to high-throughput proteomic arrays and biosensors.     

Fabrication of nanoporous templates from diblock copolymer thin films on alkylchlorosilane-neutralized surfaces

The fabrication of nanoporous templates from poly(styrene)-b-poly(methyl methacrylate) diblock copolymer thin films (PS-b-PMMA, volume ratio 70:30) on silicon requires precise control of interfacial energies to achieve a perpendicular orientation of the PMMA cylindrical microdomains relative to the substrate. To provide a simple, rapid, yet tunable approach for surface neutralization, we investigated the self-assembled ordering of PS-b-PMMA diblock copolymer thin films on silicon substrates modified with a partial monolayer of octadecyldimethyl chlorosilane (ODMS), i.e., a layer of ODMS with a grafting density less than the maximum possible monolayer surface coverage. We demonstrate herein the fabrication of nanoporous PS templates from annealed PS-b-PMMA diblock copolymer thin films on these partial ODMS SAMs.     

Two-dimensionally self-arranged protein nanoarrays on diblock copolymer templates

Novel methods for creating protein arrays with two-dimensional control can significantly enhance basic biological research as well as various bioarray applications. We demonstrate that the structural variety and chemical heterogeneity of self-assembled, hexagonal polystyrene-b-poly(vinylpyridine) micelles can be successfully exploited as templates for easy and rapid fabrication of functional protein arrays over a large scale. Spontaneous formation of such polymeric template-guided protein molecules yields high-density protein arrays that exhibit repeat spacings in a nanoscopic dimension. The ensuing self-assembled protein molecules in the array maintain their natural conformation and activity over a very long time period. By tuning the size of the underlying block copolymer templates, our amphiphilic diblock copolymer-based approach to create high-density protein patterns also permits spatial control over two-dimensional repeat spacings of protein nanoarrays. These unique advantages of polystyrene-b-poly(vinylpyridine) templates make the spontaneously constructed protein nanoarrays highly suitable as functional protein sensor substrates. Therefore, our novel two-dimensional protein assembly method can be greatly beneficial for high-throughput proteomic assays and multiplexed high-density protein sensing applications.     

Defect-free nanoporous thin films from ABC triblock copolymers

The self-assembly of triblock copolymers of poly(ethylene oxide-b-methyl methacrylate-b-styrene) (PEO-b-PMMA-b-PS), where PS is the major component and PMMA and PEO are minor components, provides a robust route to highly ordered, nanoporous arrays with cylindrical pores of 10-15 nm that show promise in block copolymer lithography. These ABC triblock copolymers were synthesized by controlled living radical polymerization, and after solvent annealing, thin films showing defect-free cylindrical microdomains were obtained. The key to the successful generation of highly regular, porous thin films is the use of PMMA as a photodegradable mid-block which leads to nanoporous structures with an unprecedented degree of lateral order. The power of using a triblock copolymer when compared to a traditional diblock copolymer is evidenced by the ability to exploit and combine the advantages of two separate diblock copolymer systems, the high degree of lateral ordering inherent in PS-b-PEO diblocks plus the facile degradability of PS-b-PMMA diblock copolymer systems, while negating the corresponding disadvantages, poor degradability in PS-b-PEO systems and no long-range order for PS-b-PMMA diblocks.     

Evaluation of enzymatic activity on nanoscale polystyrene-block-polymethylmethacrylate diblock copolymer domains

Understanding structural and functional changes of polymeric surface-bound proteins is extremely important as polymers play an increasingly significant role as arrays and substrates in proteomics applications. We carried out, for the first time, quantitative activity measurements of horseradish peroxidase (HRP) enzymes immobilized selectively on the polystyrene domains of microphase-separated polystyrene-block-polymethylmethacrylate ultrathin films. The specific enzymatic activity of HRP adsorbed on the diblock copolymer surface was evaluated and compared to that of HRP in free solution. We demonstrate that the polymeric surface-bound HRP molecules maintain approximately 85% of their activity in free solution. The unique advantages of diblock copolymer templates, involving nanoscale self-assembly and largely retained protein functionality, make the spontaneously constructed enzyme nanoarrays highly suitable as proteomics substrates. Our novel assembly method of providing functional enzymes on diblock copolymer thin films can be greatly beneficial for high-throughput and high-density protein assays.     

Controlling the size of nanostructures in thin films via blending of block copolymers and homopolymers

The effects of molecular weight and concentration of poly (methyl methacrylate) (PMMA) homopolymer or symmetric short polystyrene-block-poly (methyl methacrylate) (PS-b-PMMA) diblock copolymer on the size of the nanostructures of its blends with symmetric long PS-b-PMMA diblock copolymer have been investigated by atomic force microscopy. By careful controlling of the film thickness, solvent selectivity, and annealing time, PMMA cylindrical microdomains oriented normal to the film surface were obtained in all thin films. With the addition of both low- and high-molecular-weight PMMA homopolymers, the cylindrical domain sizes increased although it was less obvious for the lower molecular weight homopolymer. In contrast to the homopolymer, adding the short chain diblock copolymer resulted in a decrease in the cylindrical domain size, which was ascribed to the reduction of the interfacial tension and increase in the stretching energy.     

Deposition of DNA-functionalized gold nanospheres into nanoporous surfaces

We report the deposition of DNA-conjugated gold nanospheres into arrays of surface nanopores obtained from hexagonally ordered thin polystyrene-b-poly(methyl methacrylate) (PS-b-PMMA) diblock copolymer films on silicon. The deposition occurs spontaneously from aqueous solution and is driven by either electrostatic interactions or specific DNA hybridization events between the DNA nanospheres and the surface nanopores. To mitigate this spontaneous deposition, we have chemically modified the nanopores with either positively charged aminosilanes or oligonucleotide probe sequences. The deposition of DNA nanospheres into the surface nanopores was characterized by atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). We have observed preferential immobilization of individual DNA nanospheres within the nanopores, based on the size matching between the two entities. The inclusion density and selectivity of DNA nanosphere deposition into the surface nanopores was found to depend predominantly on the methods through which the nanoporous surfaces were prepared and chemically functionalized.     

Immobilization of histidine-tagged proteins on electrodes

The development of new enzyme immobilization techniques that do not affect catalytic activity or conformation of a protein is an important research task in biotechnology including biosensor applications and heterogeneous reaction systems. One of the most promising approaches for controlled protein immobilization is based on the immobilized metal ion affinity chromatography (IMAC) principle originally developed for protein purification. Here we describe the current status and future perspectives of immobilization of His-tagged proteins on electrode surfaces. Recombinant proteins comprising histidine-tags or histidine rich native proteins have a strong affinity to transition metal ions. For metal ion immobilization at the electrode surface different matrices can be used such as self-assembled monolayers or conductive polymers. This specific technique allows a reversible immobilization of histidine-tagged proteins at electrodes in a defined orientation which is an important prerequisite for efficient electron transfer between the electrode and the biomolecule. Any application requiring immobilized biocatalysts on electrodes can make use of this immobilization approach, making future biosensors and biocatalytic technologies more sensitive, simpler, reusable and less expensive while only requiring mild enzyme modifications.     

Direct site-selective covalent protein immobilization catalyzed by a phosphopantetheinyl transferase

Immobilization of proteins onto solid supports is important in the preparation of functional protein microarrays and in the development of bead-based bioassays, biosensors, and industrial biocatalysts. In order to generate the stable, functional, and homogeneous materials required for these applications, attention has focused on methods that enable the efficient and site-specific covalent immobilization of recombinant proteins onto a wide range of platforms. To this end, the phosphopantetheinyl transferase Sfp was employed to catalyze the direct immobilization of recombinant proteins bearing the small, genetically encoded ybbR tag onto surfaces functionalized with CoA. Using mass spectrometry, it was shown that the Sfp catalyzes immobilization of a model acyl carrier protein (ACP) onto CoA-derivatized PEGA resin beads through specific covalent bond formation. Luciferase (Luc) and glutathione-S-transferase (GST) ybbR-fusion proteins were similarly immobilized onto PEGA resin retaining high levels of enzyme activity. This strategy was also successfully applied for the immobilization of the ACP, as well as ybbR-Luc, -GST, and -thioredoxin fusion proteins, on hydrogel microarray slides. Overall, the Sfp-catalyzed surface ligation is mild, quantitative, and rapid, occurring in a single step without prior chemical modification of the target protein. Immobilization of the target proteins directly from a cell lysate mixture was also demonstrated.     

Dendrimer-activated surfaces for high density and high activity protein chip applications

Highly functional Si and glass surfaces for protein immobilization have been prepared by a facile activation of native surface silanol groups. Poly(propyleneimine) dendrimers of generations 1-5 were immobilized onto the surface using a facile room-temperature coupling procedure that involved activation of native silanol groups of glass using 1,1'-carbonyldiimidazole under anhydrous conditions. The dendrimer-coated surfaces were used to immobilize proteins and were characterized with respect to surface loading and activity. A number of different chemical, physical, and biochemical techniques including contact angle measurement, ellipsometry, and fluorescence microscopy were used to characterize the resulting surfaces. Increasing the dendrimer generation past G-3 led to increased surface amine content, immobilized protein concentration, and the activity of immobilized alkaline phosphatase (used as a test system). Very high activity of the immobilized proteins in the case of higher generation (G-4 and G-5) dendrimers led us to conclude that such an approach has true potential for creating highly functional surfaces for protein chip applications.     

Solvent-induced novel morphologies in diblock copolymer blend thin films

We report the morphology and phase behaviors of blend thin films containing two polystyrene-b-poly(methyl methacrylate) (PS-b-PMMA) diblock copolymers with different blending compositions induced by a selective solvent for the PMMA block, which were studied by transmission electron microscopy (TEM). The neat asymmetric PS-b-PMMA diblock copolymers employed in this study, respectively coded as a1 and a2, have similar molecular weights but different volume fractions of PS block (fPS=0.273 and 0.722). Another symmetric PS-b-PMMA diblock copolymer, coded as s, which has a PS block length similar to that of a1, was also used. For the asymmetric a1/a2 blend thin films, circular multilayered structures were formed. For the asymmetric a1/symmetric s blend thin films, inverted phases with PMMA as the dispersed domains were observed, when the weight fraction of s was less than 50%. The origins of the morphology formation in the blend thin films via solvent treatment are discussed. Combined with the theoretical prediction by Birshtein et al. (Polymer 1992, 33, 2750), we interpret the formation of these special microstructures as due to the packing frustration induced by the difference in block lengths and the preferential interactions between the solvent and PMMA block. Results obtained here suggest that diblock copolymer blend thin films treated with a selective solvent offer an alternative and attractive approach to control the self-organization of polymers.     

Protein immobilization at gold–thiol surfaces and potential for biosensing

Self-assembled monolayers (SAMs) provide a convenient, flexible and simple system to tailor the interfacial properties of metals, metal oxides and semiconductors. Monomolecular films prepared by self-assembly are attractive for several exciting applications because of the unique possibility of making the selection of different types of terminal functional groups and as emerging tools for nanoscale observation of biological interactions. The tenability of SAMs as platforms for preparing biosurfaces is reviewed and critically discussed. The different immobilization approaches used for anchoring proteins to SAMs are considered as well as the nature of SAMs; particular emphasis is placed on the chemical specificity of protein attachment in view of preserving protein native structure necessary for its functionality. Regarding this aspect, particular attention is devoted to the relation between the immobilization process and the electrochemical response (i.e. electron transfer) of redox proteins, a field where SAMs have attracted remarkable attention as model systems for the design of electronic devices. Strategies for creating protein patterns on SAMs are also outlined, with an outlook on promising and challenging future directions for protein biochip research and applications.     

THE EFFECTS OF PATTERNED SURFACES ON THE PHASE SEPARATION FOR DIBLOCK COPOLYMERS

The phase behaviors of symmetric diblock copolymer thin films confined between two hard,parallel and diversified patterned surfaces are investigated by three-dimensional dissipative particle dynamics(DPD)simulations.The induction of diversified patterned surfaces on phase separation of symmetric diblock copolymer films in snapshots,density profiles and concentration diagrams of the simulated systems are presented.The phase separations can be controlled by the patterned surfaces.In the meantime,the mean-s...     

A general approach to controlling the surface composition of poly(ethylene oxide)-based block copolymers for antifouling coatings

To control the surface properties of a polystyrene-block-poly(ethylene oxide) diblock copolymer, perfluorinated chemical moieties were specifically incorporated into the block copolymer backbone. A polystyrene-block-poly[(ethylene oxide)-stat-(allyl glycidyl ether)] [PS-b-P(EO-stat-AGE)] statistical diblock terpolymer was synthesized with varying incorporations of allyl glycidyl ether (AGE) in the poly(ethylene oxide) block from 0 to 17 mol %. The pendant alkenes of the AGE repeat units were subsequently functionalized by thiol-ene chemistry with 1H,1H,2H,2H-perfluorooctanethiol, yielding fluorocarbon-functionalized AGE (fAGE) repeat units. (1)H NMR spectroscopy and size-exclusion chromatography indicated well-defined structures with complete functionalization of the pendant alkenes. The surfaces of the polymer films were characterized after spray coating by X-ray photoelectron spectroscopy (XPS) and near-edge X-ray absorption fine structure spectroscopy (NEXAFS), showing that the P(EO-stat-fAGE) block starts to compete with polystyrene to populate the surface after only 1 mol % incorporation of fAGE. Increasing the incorporation of fAGE led to an increased amount of perfluorocarbons on the surface and a decrease in the concentration of PS. At a fAGE incorporation of 8 mol %, PS was not detected at the surface, as measured by NEXAFS spectroscopy. Water contact angles measured by the captive-air-bubble technique showed the underwater surfaces to be dynamic, with advancing and receding contact angles varying by >20°. Protein adsorption studies demonstrated that the fluorinated surfaces effectively prevent nonspecific binding of proteins relative to an unmodified PS-b-PEO diblock copolymer. In biological systems, settlement of spores of the green macroalga Ulva was significantly lower for the fAGE-incorporated polymers compared to the unmodified diblock and a polydimethylsiloxane elastomer standard. Furthermore, the attachment strength of sporelings (young plants) of Ulva was also reduced for the fAGE-containing polymers, affirming their potential as fouling-release coatings.     

Low temperature splinting activity and catalytic behavior of nano Ag doped sulphamicacid bridged diblock copolymer

A novel silver nanoparticle doped diblock copolymer was synthesized by a 3‐step process via bulk polymerization process under nitrogen atmosphere. The above prepared polymer is characterized by FTIR spectroscopy, fluorescence emission spectroscopy, circular dichroism (CD), HRTEM, and FESEM. The sulphamicacid end capped poly(ε‐caprolactone) (P1) system exhibited higher tensile strength than the sulphamicacid bridged diblock copolymer (P2) and nano Ag doped sulphamicacid bridged diblock copolymer (P3) systems. The splinting activity of the diblock copolymers was tested and confirmed the low temperature splinting activity of the diblock copolymer. The Ag nanoparticle catalyzed catalytic reduction of p‐nitrophenol (NiP) was tested, and the apparent rate constant (k_app) was determined as 7.36 × 10⁻³ sec⁻¹. The thermal studies were carried out by DSC and TGA methods. The TGA study declared that the P1 system has higher degradation temperature than the P2 and P3 systems. The P1 system has higher melting temperature (T_m) (75.5°C) than the P2 and P3 systems. The CD study indicated that the conformation of sulphamicacid was not changed even after the formation of nano Ag doped sulphamicacid bridged diblock copolymer.     

Oriented Antibody Immobilization and Immunoassay Based on Boronic Acid-containing Polymer Brush

High sensitive immunoassay platforms have gained intense attention due to their vital roles in early-stage disease diagnosis and therapeutic information feedback.Although random covalent-binding of antibody has been widely adopted in immunoassays due to its simplicity and effectiveness,it readily loses its activity and fails to exhibit high antigen-binding capacity.In this work,copolymer of zwitterionic sulfobetaine methacrylate (SBMA) and glycidyl methacrylate (GMA) brushes were immobilized onto inert polypropylene (PP) via surface-initiated atom transfer radical polymerization (ATRP) based on biomimetic dopamine pretreatment.Subsequently,boronic acid (BA) groups were covalently bonded via active GMA units,followed by the introduction of oriented immobilization of antibody.Owing to the oriented immobilization of antibody facilitated by BA groups in polymer brush,the bioactivity of antibody is well preserved,which endows the surface with significantly enhanced antigen-binding capacity.Moreover,the existence of SBMA segments in polymer brushes renders the surface high resistance to nonspecific protein adsorption,significantly alleviating the signal interference of antigen recognition.This strategy could find potential applications in developing high sensitive immunoassay platforms based on the different substrates.     

Oriented immobilization of proteins

Immobilized enzymes have found numerous applications in analytical, clinical, environmental and industrial chemistry. However, in most cases, immobilization leads to partial or total loss of activity. It is widely believed that the loss in activity is due to attachment of proteins on the immobilization support through several amino acid residues. This results in a random orientation of the immobilized protein and in increased structural deformation due to multi-point attachment. Several researchers have explored ways to orient proteins on surfaces, such that orderly organization, single point attachment and accessibility of the active site (or binding site) are possible. This article reviews the various approaches available to achieve oriented immobilization of proteins and its applications in several disciplines.     

Surface segregation of polypeptide-based block copolymer micelles: An approach to engineer nanostructured and stimuli responsive surfaces

We describe the surface segregation of polypeptide-based block copolymer micelles to produce stimuli-responsive nanostructures at the polymer blend/air interface. Such structures were obtained by simultaneous surface migration and self assembly at the surface of diblock copolymer/homopolymer blends. We employed blends composed of homopolymer (PS) and an amphiphilic block copolymer polystyrene-b-poly(l-glutamic acid) (PS-b-PGA). The surface was functionalized based on the preferential segregation to the polymer blend/air interface of the hydrophilic PGA block of the diblock copolymer upon annealing to water vapor. The surface migration of the diblock copolymer to the interface was demonstrated both by XPS and contact angle measurements. As a consequence, the PGA interfacial attraction leads to a large surface excess on diblock copolymer which in turn, through macrophase and microphase separation, produced separated domains at the surface with regions composed either of homo or block copolymer. Herein we demonstrate that the use of asymmetric diblock copolymers with a higher content in PS lead to spherical micellar assemblies randomly distributed at the surface. As observed by AFM imaging the blend composition, i.e. the amount of block copolymer within the blend influences the density of micelles at the surface. Finally, when exposed to water, the pH affects the surface morphology. The PGA segments are collapsed at low pH values and extended at pH values above 4.8, thus inducing variations on the topography of the films at the nanometer scale.     

Morphologies in solvent-annealed thin films of symmetric diblock copolymer

We have systematically studied the thin film morphologies of symmetric poly(styrene)-block-poly(methyl methacrylate) (PS-b-PMMA) diblock copolymer after annealing to solvents with varying selectivity. Upon neutral solvent vapor annealing, terraced morphology is observed without any lateral structures on the surfaces. When using PS-selective solvent annealing, the film exhibits macroscopically flat with a disordered micellar structure. While PMMA-selective solvent annealing leads to the dewetting of the film with fractal-like holes, with highly ordered nanoscale depressions in the region of undewetted films. In addition, when decreasing the swelling degree of the film in the case of PMMA-selective solvent annealing, hills and valleys are observed with the coexistence of highly ordered nanoscale spheres and stripes on the surface, in contrast to the case of higher swelling degree. The differences are explained qualitatively on the basis of polymer-solvent interaction parameters of the different components.