Submicron streptavidin patterns for protein assembly

Micron and submicron-scale features of aldehyde functionality were fabricated in polymer films by photolithography to develop a platform for protein immobilization and assembly at a biologically relevant scale. Films containing the pH-reactive polymer poly(3,3'-diethoxypropyl methacrylate) and a photoacid generator (PAG) were patterned from 500 nm to 40 mum by exposure to 365 nm (i-line) light. Upon PAG activation and hydrolysis of acetals, aldehyde groups formed. After the films were incubated with a biotinylated aldehyde reactive probe, the X-ray photoelectron spectroscopy results were consistent with biotin being attached to the surface. The background was subsequently passivated by flood exposure and incubation with an aminooxy-terminated poly(ethylene glycol), resulting in a 98% reduction in nonspecific protein adsorption. Protein patterning and assembly was demonstrated using streptavidin, biotinylated anthrax toxin receptor-1, and the protective antigen moiety of anthrax toxin and confirmed by fluorescence microscopy and atomic force microscopy (AFM). AFM demonstrated that 500 nm protein features were achieved. Because of the abundance of biotinylated proteins, this methodology provides a platform for protein immobilization and assembly for various applications in biotechnology.     

A Versatile Approach towards the Compaction,Decompaction, and Immobilization of DNA at Interfaces by Using Cyclodextrins

We investigate the temperature dependence of interactions of β‐cyclodextrin (CD)/hexadecyltrimethylammonium bromide (CTAB) self‐assemblies with DNA during the decompaction of DNA/CTAB complexes. By combining direct imaging techniques with density and sound‐velocity measurements, we can explain the decompaction process and suggest a suitable model. The DNA‐decompaction process by using CDs is accompanied by interactions with surfaces, such as glass or mica. The mechanism of β‐CD/CTAB self‐assembly is elucidated and the immobilization of DNA onto negatively charged surfaces is explained. Differences between the fractal dimensions of DNA that is adsorbed onto the surfaces are related to strong and weak binding, which permit the partial relaxation of DNA on the surfaces. The β‐CD/CTAB self‐assembled monolayers are demonstrated to be a facile and efficient route for surface functionalization, which allows for the immobilization of biomacromolecules in close proximity without any intermediate binding or deprotection steps. Moreover, this route is expected to show several advantages that might contribute to improving the performance of future biosensors as gentle immobilization‐limiting alteration of the protein structure, oriented immobilization, thereby allowing homogeneous accessibility, reversible immobilization, thereby allowing reutilizations, and high compatibility with various types of biomacromolecules.     

Directed supramolecular surface assembly of SNAP-tag fusion proteins

Supramolecular assembly of proteins on surfaces and vesicles was investigated by site-selective incorporation of a supramolecular guest element on proteins. Fluorescent proteins were site-selectively labeled with bisadamantane by SNAP-tag technology. The assembly of the bisadamantane functionalized SNAP-fusion proteins on cyclodextrin-coated surfaces yielded stable monolayers. The binding of the fusion proteins is specific and occurs with an affinity in the order of 10(6) M(-1) as determined by surface plasmon resonance. Reversible micropatterns of the fusion proteins on micropatterned cyclodextrin surfaces were visualized by using fluorescence microscopy. Furthermore, the guest-functionalized proteins could be assembled out of solution specifically onto the surface of cyclodextrin vesicles. The SNAP-tag labeling of proteins thus allows for assembly of modified proteins through a host-guest interaction on different surfaces. This provides a new strategy in fabricating protein patterns on surfaces and takes advantage of the high labeling efficiency of the SNAP-tag with designed supramolecular elements.     

Highly selective directed assembly of functional actomyosin on Au surfaces

The capability of assembling biomotors onto specific locations of solid substrates is a key for development of biomotor-based nanomechanical systems. We developed a method to direct the assembly of the heavy meromyosin fragment from rabbit skeletal muscle myosin onto specific locations of Au substrates utilizing surface molecular patterns. In this strategy, chemically directed patterns of streptavidin were achieved to direct highly specific assembly of biotinylated heavy meromyosin on the substrates--a strategy applicable for patterning a variety of biotinylated molecules--while BSA was utilized to avoid nonspecific adsorption. In vitro motility assays of filament sliding were used to confirm functionality of assembled actomyosin.     

Assembly of multilayer films incorporating a viral protein cage architecture

Protein cage architectures such as viral capsids, heat shock proteins, ferritins, and DNA-binding proteins are nanoscale modular subunits that can be used to expand the structural and functional range of composite materials. Here, layer-by-layer (LbL) assembly was used to incorporate cowpea chlorotic mottle virus (CCMV) into multilayer films. Three types of multilayer films were prepared. In the first type, ionic interactions were employed to assemble CCMV into triple layers. In the second type, complementary biological interactions (streptavidin/biotin) were used for this purpose. In a third variation of LbL assembly, complementary biological interactions were employed to produce nanotextured films that exhibit in-plane order over a micron scale without the need to adsorb onto a prepatterned template.     

Selective assembly and alignment of actin filaments with desired polarity on solid substrates

We report a new strategy to selectively assemble and align filamentous actin (F-actin) onto desired locations on a solid substrate with a specific structural polarity. In this strategy, biotinylated gelsolin caps the structural minus end of F-actin so that the F-actin binds onto a streptavidin pattern with a specific structural polarity. We also demonstrate that an electric field can be utilized to align bound F-actin along a desired direction. This can be one of the major technical breakthroughs toward the assembly of nanomechanical systems based on myosin biomotors.     

Selective immobilization of DNA and antibody probes on electrode arrays: simultaneous electrochemical detection of DNA and protein on a single platform

A proof of concept procedure for the electroaddressable covalent immobilization of DNA and protein on arrayed electrodes along with simultaneous detection of multiple bioagents in the same sample solution is described. Carboxyphenyldiazonium was selectively deposited onto five of nine individually addressable electrodes in an array via bias assisted assembly. Amine functionalized DNA probes were covalently coupled to the carboxyl surface via carbodiimide chemistry. This was followed by the covalent immobilization of diazonium-antibody conjugates into the remaining four electrodes via cyclic voltammetry. Simultaneous electrochemical detection of a DNA sequence related to the breast cancer BRCA1 gene and the human cytokine protein interleukin-12, which is a substantial component in the immune system response and attack of tumor cells, is reported. These results demonstrate the possibility of selective patterning of diverse biomolecules on a single device and may have significant implications for future development of microarrays and biosensors.     

Patterning biomolecules with a water-soluble release and protection interlayer

We report on a general lithography method for high-resolution biomolecule patterning with a bilayer resist system. Biomolecules are first immobilized on the surface of a substrate and covered by a release-and-protection interlayer of water-soluble polymer. Patterns can then be obtained by lithography with a spin-coated resist layer in a conventional way and transferred onto the substrate by reactive ion etching. Afterward, the resist layer is removed by dissolution in water. To demonstrate a high-resolution patterning, soft UV nanoimprint lithography has been used to produce high-density dot arrays of poly-(L-lysine) molecules on a glass substrate. Both fluorescence images and cell proliferation behaviors on such a patterned substrate have shown evidence of improved stability of biomolecule immobilization comparing to that obtained by microcontact printing techniques.     

SNAP-tag fluorogenic probes for wash free protein labeling

In this review, we described the design strategies of SNAP-tag fl uorogenic probes with turn-on fl uorescence responses, which minimized the fl uorescence background and allowed for direct imaging in living cells without wash-out steps. These probes can apply in real-time analysis of protein localization, dynamics, and protein– protein interactions in living cells. Furthermore, the excellent fl uorescent properties made it possible to apply some of the probes in super-resolution fl uorescence imaging.     

Dual functional, polymeric self-assembled monolayers as a facile platform for construction of patterns of biomolecules

We report a facile approach to the construction of patterns of biomolecules based on polymeric self-assembled monolayers (pSAMs) that possess dual functions: "bio-reactive (post-functionalizable)" and "bio-inert (anti-biofouling)" properties. To prepare pSAMs on Si/SiO2 wafers were synthesized new random copolymers by radical polymerization of poly(ethylene glycol) methyl ether methacrylate (PEGMA), 3-(trimethoxysilyl)propyl methacrylate (TMSMA), and N-acryloxysuccinimide (NAS), and referred to as poly(TMSMA-r-PEGMA-r-NAS). Poly(TMSMA-r-PEGMA-r-NAS) was designed to play triple roles: "surface-reactive", "bio-reactive", and "bio-inert" ones. The pSAMs of poly(TMSMA-r-PEGMA-r-NAS) were formed on Si/SiO2 wafers with 1 h incubation of the substrates in the polymer solution, which showed approximately a 1 nm-thick film as measured by ellipsometry. After the formation of the pSAMs, the feasibility of the pSAMs as a dual functional surface (bio-inert and bio-reactive properties) was examined. The ability of the pSAMs to block nonspecific adsorption of proteins was evaluated against bovine serum albumin as a model protein. High-resolution N(1s) X-ray photoelectron spectroscopy (XPS) analysis on the protein adsorption revealed that significant reduction up to approximately 97% was observed compared to the unmodified Si/SiO2 wafer. In addition, micropatterns of streptavidin with high signal-to-noise ratios were achieved using microcontact printing (microCP) of NH2-bearing biotin onto the pSAMs of poly(TMSMA-r-PEGMA-r-NAS) on glass slides, which suggests that other biomolecules could also be efficiently immobilized onto the pSAMs with high specificity while minimizing nonspecific adsorption. On the other hand, the surface density of both bio-reactive and anti-biofouling functionality could be tailored by simply changing initial feed ratios of each monomer in the polymer synthesis: different molar ratios of the bio-reactive group (NAS: 33%, 20%, and 10%, respectively) were employed. When micropatterns of streptavidin were constructed, the pSAMs with 33% NAS moiety showed the highest immobilization of the protein. Taken together, the present dual functional, random copolymers may have warrant applications in the field of biosensors and biochips.     

Surface plasmon resonance spectroscopy and quartz crystal microbalance study of streptavidin film structure effects on biotinylated DNA assembly and target DNA hybridization

Surface plasmon resonance (SPR) spectroscopy is employed for the study of biotinylated DNA assembly on streptavidin modified gold surfaces for target DNA hybridization. Two immobilization strategies are involved for constructing streptavidin films, namely, (1) physical adsorption on biotin-containing thiol treated surfaces through biotin-streptavidin links and (2) covalent attachment to 11-mercaptoundecanoic acid (MUA) treated surfaces through amine coupling. To understand the structural properties of the streptavidin films, a quartz crystal microbalance with energy dissipation monitoring (QCM-D) is used to monitor the streptavidin immobilization procedures. The simultaneously measured frequency (Deltaf) and dissipation factor (DeltaD) changes, together with the SPR angle shifts (Deltatheta), suggest that the streptavidin film assembled on the biotin-containing surface is highly rigid with a well-ordered structure while the streptavidin film formed through amine coupling is highly dissipative and less structured. The subsequent biotinylated DNA (biotin-DNA) assembly and target hybridization results show that the streptavidin film structure has distinct effects on the biotin-DNA binding amount. On the streptavidin matrix, not only the probe DNA density but also the strand orientation mediated by the streptavidin films has distinct effects on hybridization efficiency. Particularly, the molecularly ordered streptavidin films formed on the biotin-containing surfaces ensure a well-ordered DNA assembly, which in turn allows for a higher efficiency in target DNA capture and for a higher sensitivity in the hybridization analysis when compared to the biotin-DNA assembled on the less structured streptavidin films formed through amine coupling.     

Positive microcontact printing with mercaptoalkyloligo(ethylene glycol)s

The soft lithographic replication of patterns with a low filling ratio by microcontact printing (microCP) is problematic due to the poor mechanical stability of common elastomeric stamps. A recently described strategy to avoid this problem employs a modified patterning method, positive microcontact printing ((+)microCP), in which a stamp with a mechanically more stable inverted relief pattern is used. In contrast to conventional negative microCP ((-)microCP), in the contact areas a self-assembled monolayer (SAM) is printed of a "positive ink", which provides only minor etch protection, whereas the noncontacted areas are subsequently covered with a different, etch-resistant SAM, prior to development by chemical etching. With the aim to identify novel, highly versatile positive inks, the patterning of gold by (+)microCP with mercaptoalkyloligo(ethylene glycol)s (MAOEGs), the subsequent adsorption of octadecanethiol (ODT), and the final development by wet chemical etching have now been studied. A polydisperse mixture of mercaptoundecylocta(ethylene glycol) derivatives was found to provide the best patterning results. The surface spreading of the positive ink during stamping, the exchange of printed MAOEGs with ODT, and the choice of the right etching bath were identified as key parameters that influence the achievable pattern resolution and contrast. Due to the modular composition of functionalized alkyloligo(ethylene glycol) derivatives, (+)microCP with these positive inks has the potential for easy adaptation to a variety of materials and development conditions.     

Spatially addressable protein array: ssDNA-directed assembly for antibody microarray

Protein microarray offers a means for high-throughput profiling of cellular proteins to provide insights into the mechanisms of biological processes. This study describes the design and fabrication of a robust platform, spatially addressable protein array (SAPA), by exploring the specificity of ssDNA hybridization for self-assembly of semi-synthetic ssDNA-antibody conjugates which capture antigens from complex biological samples. This approach does not involve the direct immobilization of antibodies nor antigen, but instead captures the target antigens in the solution phase followed by self-directed assembly of the complex onto the surface. In an effort to optimize the platform, the effects of surface chemistry, nonspecific protein adsorption, facile preparation, and purification of ssDNA-conjugated antibody and capture of the antigen from a complex biological sample such as cell lysate were examined. This platform allowed antigen detection in cell lysate with high sensitivity (1 pM). The method described herein can be extended to the high-throughput detection of other interacting molecules in solution phase and their subsequent assembly onto any substrate.     

Patterning polymerized lipid vesicles with soft lithography

The applications of soft lithography in patterning polymerized lipid vesicles of 1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine on glass substrates are reported. We demonstrate that the polymerized vesicles can be used as a high molecular weight ink to be transferred from a PDMS stamp onto a glass substrate to form two-dimensional stripes with a controlled separation. By combining channel flow with dewetting within microfluidic networks, we assemble the polymerized vesicle into three-dimensional stripes and one-dimension lines on glass substrates. Atomic force microscopy shows that these patterned vesicle structures are stable on glass substrates. The simple, stable, and precise immobilization of lipid vesicles on solid substrates will open up the possibility of integrating them in biosensors and microelectronic devices.     

Well-oriented ZZ–PS-tag with high Fc-binding onto polystyrene surface for controlled immobilization of capture antibodies

The site specificity and bioactivity retention of antibodies immobilized on a solid substrate are crucial requirements for solid phase immunoassays. A fusion protein between an immunoglobulin G (IgG)-binding protein (ZZ protein) and a polystyrene-binding peptide (PS-tag) was constructed, and then used to develop a simple method for the oriented immobilization of the ZZ protein onto a PS support by the specific attachment of the PS-tag onto a hydrophilic PS. The orientation of intact IgG was achieved via the interaction of the ZZ protein and the constant fragment (Fc), thereby displayed the Fab fragment for binding antigen. The interaction between rabbit IgG anti-horseradish peroxidase (anti-HRP) and its binding partner HRP was analyzed. Results showed that the oriented ZZ–PS-tag yielded an IgG-binding activity that is fivefold higher than that produced by the passive immobilization of the ZZ protein. The advantage of the proposed immunoassay strategy was demonstrated through an enzyme-linked immunosorbent assay, in which monoclonal mouse anti-goat IgG and HRP-conjugated rabbit F(ab′)₂ anti-goat IgG were used to detect goat IgG. The ZZ–PS-tag presented a tenfold higher sensitivity and a wider linear range than did the passively immobilized ZZ protein. The proposed approach may be an attractive strategy for a broad range of applications involving the oriented immobilization of intact IgGs onto PS supports, in which only one type of phi-PS (ZZ–PS-tag) surface is used.     

Immobilized streptavidin gradients as bioconjugation platforms

Surface density gradients of streptavidin (SAV) were created on solid surfaces and demonstrated functionality as a bioconjugation platform. The surface density of immobilized streptavidin steadily increased in one dimension from 0 to 235 ng cm(-2) over a distance of 10 mm. The density of coupled protein was controlled by its immobilization onto a polymer surface bearing a gradient of aldehyde group density, onto which SAV was covalently linked using spontaneous imine bond formation between surface aldehyde functional groups and primary amine groups on the protein. As a control, human serum albumin was immobilized in the same manner. The gradient density of aldehyde groups was created using a method of simultaneous plasma copolymerization of ethanol and propionaldehyde. Control over the surface density of aldehyde groups was achieved by manipulating the flow rates of these vapors while moving a mask across substrates during plasma discharge. Immobilized SAV was able to bind biotinylated probes, indicating that the protein retained its functionality after being immobilized. This plasma polymerization technique conveniently allows virtually any substrate to be equipped with tunable protein gradients and provides a widely applicable method for bioconjugation to study effects arising from controllable surface densities of proteins.     

Chitosan biotinylation and electrodeposition for selective protein assembly

An alternative route to protein assembly at surfaces based on using the unique capabilities of biological materials for the spatially selective assembly of proteins is described. Specifically, the stimuli-responsive properties of aminopolysaccharide chitosan are combined with the molecular-recognition capabilities of biotin-streptavidin binding. Biotinylated chitosan retains its stimuli-responsive properties and is capable of electrodepositing at specific electrode addresses. Once deposited, it is capable of binding streptavidin, which can mediate the subsequent assembly of biotinylated proteins. Spatially selective protein assembly using biotinylated Protein A and fluorescently-labeled antibodies is demonstrated.     

Surface modification of elastomeric stamps for microcontact printing of polar inks

Chemical modification of the surface of a stamp used for microcontact printing (microCP) is interesting for controling the surface properties, such as the hydrophilicity. To print polar inks, plasma polymerization of allylamine (PPAA) was employed to render the surface of poly(dimethylsiloxane) (PDMS), polyolefin plastomers (POP), and Kraton elatomeric stamps hydrophilic for long periods of time. A thin PPAA film of about 5 nm was deposited on the stamps, which increased the hydrophilicity, and which remained stable for at least several months. These surface-modified stamps were used to transfer polar inks by microCP. The employed microCP schemes are as follows: (a) a second generation of dendritic ink having eight dialkyl sulfide end groups to fabricate patterns on gold substrates by positive microCP, (b) fluorescent guest molecules on beta-cyclodextrin (beta-CD) printboards on glass employing host-guest recognition, and (c) Lucifer Yellow ethylenediamine resulting in covalent patterning on an aldehyde-terminated glass surface. All experiments resulted in an excellent performance of all three PPAA-coated stamp materials to transfer the polar inks from the stamp surface to gold and glass substrates by microCP, even from aqueous solutions.     

A native, affinity-based protein blot for the analysis of streptavidin heterogeneity: consequences for the specificity of streptavidin mediated binding assays

Commercial preparations of streptavidin, a bacterial biotin-binding protein, were analyzed by isoelectric focusing combined with an affinity-based protein blot using biotinylated, protein-saturated nitrocellulose. The colorimetrical detection of streptavidin with biotinylated alkaline phosphatase allows the selective visualization of streptavidin molecules with at least two active biotin-binding sites. Dependent on the preparation, seven to sixteen streptavidin forms were found with isoelectric points ranging from 5 to 8. Molecular weight analysis of the subunits of streptavidin showed that the observed heterogeneity was mainly due to limited proteolysis, which does not destroy the biotin-binding activity. The preparations differed also in the nonspecific reactivity of streptavidin with single-stranded DNA, bovine serum albumin and Tween 20. No relationship was observed between heterogeneity and non-specific binding activity. Data obtained from protein blots onto nitrocellulose saturated with single-stranded DNA showed that it cannot be excluded that streptavidin with only a single active biotin-binding site is mainly responsible for the nonspecific reactivity of some streptavidin preparations.     

Reactivity of acid fluoride-terminated self-assembled monolayers on gold

This report describes the reactivity of acid fluoride (AF)-terminated self-assembled monolayers (SAMs) on gold toward amine and alcohol compounds and the potentiality of AF as a reactive intermediate for surface functionalizations. The AF group was generated in situ on a gold surface by reacting the terminal carboxylic acid group in the SAM of 16-mercaptohexadecanoic acid with cyanuric fluoride and pyridine under the optimized conditions. AF was found to be highly reactive toward various amine groups, such as primary and secondary amines, but it did not react effectively with alcohol. In addition, the amide coupling reaction by microcontact printing (microCP) was compared with the solution-based reaction: when amine-derivatized ferrocene compound was used for 1-min microCP on the AF-activated surface, the surface coverage of the reaction product was about 83% of 3.45 x 1014 cm-2, the coverage obtained in the solution-based reaction. On the basis of the high reaction efficiency of microCP, the AF-activated surface was also used as a platform for patterning a biological ligand, biotin.