Characterization and manipulation of microscopic biochemically active regions by scanning electrochemical microscopy (SECM).

Preparation and characterization of microscopic biochemically active regions are important for the development of miniaturized bioanalytical systems with proteins, such as miniaturized enzyme electrode arrays. Scanning electrochemical microscopy (SECM) has emerged as an ideal tool for prototyping such systems. The technique is based on electrochemical conversions of dissolved species at a micrometer-sized probe electrode. It offers several mechanisms for local surface modifications under conditions compatible with conservation of protein functionality of enzymes and antibodies. The subsequent imaging of the immobilized activity provides direct information about local immobilized enzyme activities. The working modes of the techniques are illustrated by recent studies from this laboratory for the design and characterization of patterned enzyme layers covalently linked to gold surfaces via thiol self-assembly chemistry.     

Effects of self-assembly process of latex spheres on the final topology of macroporous silica

This paper surveys the topology of macroporous silica prepared using latex templates covering the submicrometric range (0.1-0.7 mum). The behavior of latex spheres in aqueous dispersion has been analyzed by dynamic light scattering (DLS) measurement indicating the most appropriate conditions to form well-defined cubic arrays. The optical behavior of latex spheres has been analyzed by transmittance and reflectance measurements in order to determine their diameter and filling factor when they were assembled in bidimensional arrays. Macroscopic templates have been obtained by a centrifugation process and their crystalline ordering has been confirmed by porosimetry and scanning electron microscopy. These self-assembled structures have been used to produce macroporous silica, whose final topology depends on the pore size distribution of the original template. It has been seen that latex spheres are ordered in a predominant fcc arrangement with slipping of tetragonal pores due to the action of attractive electrostatic interactions. The main effect is to change the spherical shape of voids in macroporous silica into a hexagonal configuration with possible applications to fabricate photonic devices with novel optical properties.     

Electrostatic and dispersion interactions during protein adsorption on topographic nanostructures

Recently, biomaterials research has focused on developing functional implant surfaces with well-defined topographic nanostructures in order to influence protein adsorption and cellular behavior. To enhance our understanding of how proteins interact with such surfaces, we analyze the adsorption of lysozyme on an oppositely charged nanostructure using a computer simulation. We present an algorithm that combines simulated Brownian dynamics with numerical field calculation methods to predict the preferred adsorption sites for arbitrarily shaped substrates. Either proteins can be immobilized at their initial adsorption sites or surface diffusion can be considered. Interactions are analyzed on the basis of Derjaguin-Landau-Verway-Overbeek (DLVO) theory, including electrostatic and London dispersion forces, and numerical solutions are derived using the Poisson-Boltzmann and Hamaker equations. Our calculations show that for a grooved nanostructure (i.e., groove and plateau width 8 nm, height 4 nm), proteins first contact the substrate primarily near convex edges because of better geometric accessibility and increased electric field strengths. Subsequently, molecules migrate by surface diffusion into grooves and concave corners, where short-range dispersion interactions are maximized. In equilibrium, this mechanism leads to an increased surface protein concentration in the grooves, demonstrating that the total amount of protein per surface area can be increased if substrates have concave nanostructures.     

Fabrication of large-area ferromagnetic arrays using etched nanosphere lithography

Nanosphere lithography (NSL) is a simple, cost-effective, and powerful technique capable of producing large-area arrays of ferromagnetic nanostructures with dimensions below 100 nm. These properties make NSL an attractive process for the fabrication of arrays of magnetic elements with applications in magnetic data storage. The main disadvantage with conventional NSL is that the monolayer of spheres always contains imperfections that are transferred to the resulting nanostructures. This can significantly affect the structural and magnetic properties of the fabricated array. In this paper we present a novel adaptation of NSL that reduces the effect of such defects on the resulting nanostructures. The technique also offers excellent control over the diameter, aspect ratio, and pitch of the fabricated elements. These properties are demonstrated through the fabrication of arrays of Ni elements of 210 nm diameter and arrays of Co elements with diameters between 200 and 320 nm.     

Nanoparticle-assisted surface immobilization of phospholipid liposomes

Phospholipid liposomes (100-200 nm diameter) are deposited onto solid substrates after stabilizing them against fusion with the solid by allowing charged nanoparticles to adsorb at approximately 25% surface coverage. The immobilized vesicles remain stable over a period of days. Epifluorescence imaging shows that they diffuse freely over surfaces with the same charge but adsorb tightly onto surfaces with opposite charge. Nanoparticle adsorption to surface patterns of opposite charge provides a facile method to create large-scale surface-supported arrays of intact liposomes. This surface attachment method is simple chemically and applies generally for solid surfaces that can be hydrophobic or hydrophilic. Offering routes to localize proteins and other vesicle-contained objects at surfaces in tailored spatial patterns, these immobilized liposome arrays may find diverse applications in the emerging field of nanobiotechnology.     

The use of surface tension to predict the formation of 2D arrays of latex spheres formed via the Langmuir-Blodgett-like technique

Highly ordered hexagonal arrays of latex spheres on highly ordered pyrolytic graphite (HOPG) have been prepared from a Langmuir-Blodgett-like (LB-like) technique using both polymers and surfactants as spreading agents. The role of spreading agent concentration in forming a well-ordered, stable monolayer at the air-liquid interface was studied by means of atomic force microscopy, scanning electron microscopy, optical microscopy, and surface tension measurements for three different systems: a nonionic surfactant, octylphenoxy poly(ethyleneoxy)ethanol (Igepal CO 630); an anionic surfactant, sodium dodecyl sulfate; and a low-molecular-weight, water-soluble polymer, polyacrylamide. For both the anionic surfactant and the water soluble polymer, a correlation was found between a unique feature in surface tension measurements of the latex-spreading agent mixture and the concentrations at which hexagonal arrays of latex spheres form on the surface of HOPG. For the nonionic surfactant, no ordered structures were found on HOPG for any surfactant concentration, consistent with no appearance of the unique feature in surface tension measurements. These results show that a tensiometer can be used to determine the conditions under which well-ordered latex films have the possibility of forming on a substrate using the LB-like technique; however, other factors, such as pulling speed and surface chemistry, play a role as well.     

Silver hierarchical bowl-like array: synthesis, superhydrophobicity, and optical properties

We present a facile synthetic route to a silver bowl-like array film with hierarchical structures on glass substrate using the colloidal monolayer as a template. In these special hierarchical structures, microstructures were provided by a colloidal template of polystyrene latex spheres and nanostructures resulting from the thermal decomposition of silver acetate. These structures were chemically modified with 1-hexadecanethiol, and a corresponding self-assembled monolayer (SAM) was formed on their surfaces. Due to the lotus leaf-like morphology with hierarchical micro/nanostructures, the film displayed an extraordinary superhydrophobicity after chemical modification. Water contact angle and sliding angle were 169 degrees and 3 degrees (the weight of water droplets: 3 mg), respectively. Additionally, its optical property has also been investigated. This structure could be used in microfluidic devices, optical devices, and biological science.     

Microfluidic fabrication of addressable tethered lipid bilayer arrays and optimization using SPR with silane-derivatized nanoglassy substrates

We report the microfluidic fabrication of robust and fluid tethered bilayer arrays within a poly(dimethylsiloxane) (PDMS) chip, and demonstrate its addressability and biosensing by incorporating the GM1 receptor into the bilayer framework for detection of cholera toxin. Rapid optimization of the experimental conditions is achieved by using nanoglassified surfaces in combination with surface plasmon resonance. The ultrathin glassy film on gold mimics glass surfaces employed in microfluidics, allowing real-time monitoring of multiple assembly steps and therefore permitting rapid prototyping of microfluidic arrays. The tethered bilayer array utilizes a covalently immobilized biotinylated protein for generation of well-defined capture zones where a streptavidin link is employed for the immobilization of biotinylated vesicles. Fusion of captured vesicles is accomplished using a concentrated PEG solution, and the lateral diffusion of the tethered bilayer membrane is characterized by fluorescence recovery after photobleaching methods. The tethered membrane arrays demonstrate marked stability and high mobility, which provide an ideal host environment for membrane-associated proteins and open new avenues for high-throughput analysis of these proteins.     

Biomimetic antireflective hierarchical arrays

We report a simple method for the fabrication of biomimetic antireflective hierarchical arrays based on the combination of self-assembled polymer spheres and nanoimprint lithography (NIL). The hierarchical structures are fabricated by creating nanopillars on the microscale round protrusion arrays, which are similar to natural mosquito eyes consisting of combined micro- and nanostructures. The hierarchical arrays dramatically suppress the surface reflection from visible to near-infrared regions with an angle of incidence of up to 70°.     

Hydrolysis of cellobiose by immobilized β-glucosidase entrapped in maintenance-free gel spheres

A crude preparation of Aspergillus niger β-glucosidase (27.5 cello-biase U/mg protein at 40°C, pH 5.0) was immobilized on concanavalin A-Sepharose (CAS). The cellobiase activity of the immobilized enzyme was 1334 U/mg dried CAS or 108 U/mL CAS gel. The β-glucosidase-CAS complex was entrapped within crosslinked propylene glycol alginate/bone-geletin gel spheres that possessed between 0.67 and 2.35 cellobiase U/mL spheres, depending on their size. The effect of cellobiose concentration (10–300 mM) on the activity of native, immobilized, and gel-entrapped enzyme was determined. It was shown that concentrations of cellobiose between 10 and 180 mM were not inhibitory to the entrapped enzyme, although inhibition was found to occur with the native and immobilized enzyme. Exogenous ion addition was not necessary to maintain the structural integrity of the spheres, which were stable for 4 d at 40°C.     

Monitoring the transformation of colloidal crystals by styrene vapor using atomic force microscopy

The stages of transformation of a colloidal crystalline film of latex spheres to a new periodic structure were imaged by atomic force microscopy. Colloidal crystalline films were prepared with 320 nm diameter poly(styrene-co-2-hydroxyethyl methacrylate) (PSt/HEMA) spheres. The hexagonally ordered surfaces of the colloidal crystalline films were transformed with styrene vapor at room temperature to a new morphology having holes in the surface and the same periodicity as the original films. The surfaces of colloidal crystals and the transformed films have a raspberry-like texture superposed on the 320 nm hexagonal periodicity. Both height images and phase images reveal that the latex spheres shrink and the transformation proceeds by an order-disorder-order sequence. The final structure is an interconnected colloidal array with smaller polystyrene particles dispersed in a continuous PSt/HEMA matrix.     

Imaging the binding ability of proteins immobilized on surfaces with different orientations by using liquid crystals

We report an investigation of the binding ability of a protein immobilized on surfaces with different orientations but in identical interfacial microenvironments. The surfaces present mixed self-assembled monolayers (SAMs) of 11-[19-carboxymethylhexa(ethylene glycol)]undecyl-1-thiol, 1, and 11-tetra(ethylene glycol) undecyl-1-thiol, 2. Whereas 2 is used to define an interfacial microenvironment that prevents nonspecific adsorption of proteins, 1 was activated by two different schemes to immobilize ribonuclease A (RNase A) in either a preferred orientation or random orientations. The binding of the ribonuclease inhibitor protein (RI) to RNase A on these surfaces was characterized by using ellipsometry and the orientational behavior of liquid crystals. Ellipsometric measurements indicate identical extents of immobilization of RNase A via the two schemes. Following incubation of both surfaces with RI, however, ellipsometric measurements indicate a 4-fold higher binding ability of the RNase A immobilized with a preferred orientation over RNase A immobilized with a random orientation. The higher binding ability of the oriented RNase A over the randomly oriented RNase A was also apparent in the orientational behavior of nematic liquid crystals of 4-cyano-4'-pentylcyanobiphenyl (5CB) overlayed on these surfaces. These results demonstrate that the orientations of proteins covalently immobilized in controlled interfacial microenvironments can influence the binding activities of the immobilized proteins. Results reported in this article also demonstrate that the orientational states of proteins immobilized at surfaces can be distinguished by examining the optical appearances of liquid crystals.     

Modification and characterization of artificially patterned enzymatically active surfaces by scanning electrochemical microscopy

This review summarizes the characterization of localized enzymatic activity by scanning electrochemical microscopy (SECM). After introducing the concepts of feedback imaging and generator-collector experiments with enzyme-modified solid surfaces, a comparison of the merits and limitations of both approaches is given and further illustrated by selected applications. They include enzyme-modified patterned monolayers, enzyme-modified polymer microstructures and enzyme-modified metal microstructures. Such configurations are important for the development of miniaturized bioanalytical systems with proteins, such as miniaturized enzyme electrode arrays. SECM has emerged as an ideal tool for prototyping of such systems. It also offers several mechanisms for local surface modifications under conditions compatible with conservation of protein functionality of enzymes and antibodies. The subsequent imaging of the immobilized activity provides direct information about local immobilized enzyme activity. The range of biotechnological applications can be expanded by labeling other biomolecules, such as monoclonal antibodies, with appropriate enzymes. Miniaturized electrochemical enzyme immunoassays that apply the sandwich format and SECM as the detection method are reviewed. They have been performed on microstructured supports after reagent spotting or on agglomerates of surface-modified magnetic microbeads. Finally, current challenges are listed with indications of ongoing research to overcome current limitations by means of instrumental improvements.     

Surface tailoring for controlled protein adsorption: effect of topography at the nanometer scale and chemistry

Protein adsorption behavior is at the heart of many of today's research fields including biotechnology and materials science. With understanding of protein-surface interactions, control over the conformation and orientation of immobilized species may ultimately allow tailor-made surfaces to be generated. In this contribution protein-surface interactions have been examined with particular focus on surface curvature with and without surface chemistry effects. Silica spheres with diameters in the range 15-165 nm with both hydrophilic and hydrophobic surface chemistries have been used as model substrates. Two proteins differing in size and shape, bovine serum albumin (BSA) and bovine fibrinogen (Fg), have been used in model studies of protein binding with detailed secondary structure analysis being performed using infrared spectroscopy (IR) on surface-bound proteins. Although trends in binding affinity and saturation values were similar for both proteins, albumin is increasingly less ordered on larger substrates, while fibrinogen, in contrast, loses secondary structure to a greater extent when adsorbing onto particles with high surface curvature. These effects are compounded by surface chemistry, with both proteins becoming more denatured on hydrophobic surfaces. Both surface chemistry and topography play key roles in determining the structure of the bound proteins. A model of the binding characteristics of these two proteins onto surfaces having differing curvature and chemistry is presented. We propose that properties of an adsorbed protein layer may be guided through careful consideration of surface structure, allowing the fabrication of materials/surface coatings with tailored bioactivity.     

Aqueous particulate foams stabilized solely with polymer latex particles

In this article, a wide range of latexes are evaluated as possible foam stabilizers. These include near-monodisperse, poly(N-vinyl pyrrolidone)-stabilized polystyrene [PNVP-PS] latexes with diameters ranging from 170 nm to 1.62 microm, submicrometer-sized poly(ethylene glycol)-stabilized polystyrene [PEGMA-PS] latex particles, a PNVP-stabilized poly(4-bromostyrene) [PNVP-PBrS] latex with a mean diameter of 870 nm, two PNVP-stabilized poly(methyl methacrylate) [PNVP-PMMA] latexes with mean diameters of 730 nm and 1.20 microm, a PNVP-stabilized poly(2-hydroxypropyl methacrylate) [PNVP-PHPMA] latex with a mean diameter of 630 nm, and a charge-stabilized anionic PS latex of 220 nm diameter. The effect of varying the particle size, latex concentration, and latex surface composition on foam stability were studied in detail. The larger PNVP-PS latexes, the PNVP-PBrS, and the two PNVP-PMMA latexes gave highly stable foams, whereas PEGMA-PS, PNVP-PHPMA, and the charge-stabilized PS latex produced either no foams or foams with inferior long-term stabilities. Scanning electron microscopy studies revealed hexagonally close-packed latex arrays in the walls of the dried foam, which leads to localized moiré patterns being observed by optical microscopy. Moreover, these dried foams are highly iridescent in bright transmitted light.     

Synthesis of nanometer-scale polymeric structures on surfaces from template assisted admicellar polymerization: a comparative study with protein adsorption

A novel method for the formation of nanometer-scale polymer structures via template assisted admicellar polymerization (TAAP) is described. Admicellar polymerization uses a surfactant layer adsorbed on a surface to localize monomer to the surface prior to polymerization of the monomer. Nanostructures are formed by restricting adsorption to the uncovered sites of an already-templated surface, in this case to the interstitial sites between adsorbed latex spheres. Unlike most other process that form polymer nanostructures, polymer dimensions can be significantly smaller than the interstitial size because of sphere-surfactant interactions. Protein adsorption in the interstitial sites of colloidal arrays was also studied for three different proteins, and the results were compared with those obtained via admicellar polymerization.     

Controlling the shape and alignment of mesopores by confinement in colloidal crystals: designer pathways to silica monoliths with hierarchical porosity

Monolithic pieces of hierarchically structured silica, containing both periodic macropores and mesopores with well-controlled architecture, are synthesized by dual templating methods. Colloidal crystal templating with close-packed arrays of poly(methyl methacrylate) spheres yields regular, highly interconnected macropores a few hundred nanometers in diameter, and templating with nonionic surfactants produces mesoporosity (2.5-5.1 nm pore diameters) in the macropore walls. Several distinct mesostructures can be achieved within the silica skeleton, depending on the choice of surfactant, co-surfactant, and processing conditions. In the three-dimensional (3D) confinement of the colloidal crystal template, wormlike channels, cubic (Pm3n), or two-dimensional (2D) hexagonal (P6mm) mesostructures are produced with the surfactant Brij 56 (C16H33(OCH2CH2)nOH (n approximately 10) and dodecane as cosurfactant. In the 2D hexagonal structure, channels are oriented perpendicular to the polymer spheres, thereby connecting adjacent macropores through the silica walls. This orientation contrasts with channel alignment parallel to latex spheres when the polymeric surfactant Pluronic P123 (EO20PO70EO20) is used. On the basis of high-resolution 3D transmission electron microscopy, scanning electron microscopy, small-angle X-ray scattering, and nitrogen sorption measurements, structural and textural properties of the monoliths are described in detail as a function of the synthesis parameters. The control over the mesoarchitecture of these silica-surfactant systems in 3D confinement is explained by considering the relative dimensions of the mesostructures with respect to the interstitial space in the latex template, interfacial interactions, entropic effects, and structural frustration.     

Fabrication of Au hybrid protein chips and its application to SERS-based bioassay

Hybrid micro/nanostructures composed with alternative Au nanoparticle (NP) arrays and protein dots were fabricated via layer-by-layer self-assembly and the microsphere lithography technique. These micro/nanostructures were novel protein chips which had applications in the surface-enhanced Raman spectroscopy (SERS) based immunoassay. The synthetic processes were to fabricate Au nanowell arrays initially by using the templates of ordered monolayers of polystyrene (PS) microsphere arrays. Then, the proteins of antibody (avidin) were imbedded in the Au nanowells. Lastly, the immune reaction was implemented by adding atto 610-biotin. SERS spectra were recorded as the immunoassay readout, which showed the lowest detective concentration of 100 pg/mL. These new kind of SERS-based protein chips were easy to fabricate, inexpensive and supersensitive, and exhibit the potential application in bioassays, forensics and biosensors.     

Modification and characterization of artificially patterned enzymatically active surfaces by scanning electrochemical microscopy

This review summarizes the characterization of localized enzymatic activity by scanning electrochemical microscopy (SECM). After introducing the concepts of feedback imaging and generator-collector experiments with enzyme-modified solid surfaces, a comparison of the merits and limitations of both approaches is given and further illustrated by selected applications. They include enzyme-modified patterned monolayers, enzyme-modified polymer microstructures and enzyme-modified metal microstructures. Such configurations are important for the development of miniaturized bioanalytical systems with proteins, such as miniaturized enzyme electrode arrays. SECM has emerged as an ideal tool for prototyping of such systems. It also offers several mechanisms for local surface modifications under conditions compatible with conservation of protein functionality of enzymes and antibodies. The subsequent imaging of the immobilized activity provides direct information about local immobilized enzyme activity. The range of biotechnological applications can be expanded by labeling other biomolecules, such as monoclonal antibodies, with appropriate enzymes. Miniaturized electrochemical enzyme immunoassays that apply the sandwich format and SECM as the detection method are reviewed. They have been performed on microstructured supports after reagent spotting or on agglomerates of surface-modified magnetic microbeads. Finally, current challenges are listed with indications of ongoing research to overcome current limitations by means of instrumental improvements. Received: 19 December 2000 / Revised: 26 February 2001 / Accepted: 2 March 2001     

Rapid characterization of protein chips using microwave-assisted protein tryptic digestion and MALDI mass spectrometry

We demonstrate that the microwave-assisted protein enzymatic digestion (MAPED) method can be successfully applied to the mass spectrometric characterization of proteins captured on the affinity surfaces of protein chips. The microwave-assisted on-chip tryptic digestion method was developed using a domestic microwave, completing the on-chip proteolysis reaction in minutes, whereas the previous on-chip digestion methods by incubation took hours of incubation time. For the model protein chips, antibody-presenting surfaces were prepared, where anti-α-tubulin1 and antibovine serum albumin (BSA) were immobilized on self-assembled monolayers. The resulting digestion efficiency, displaying sequence coverages of 30 and 14% for α-tubulin1 and BSA, respectively, was comparable to the previous time-consuming incubation studies. It allowed the characterization of immunosensed proteins by MASCOT search using peptide mass fingerprinting. In an example of this method for protein chip applications, BSA naturally involved in fetal bovine serum was unambiguously identified on a model protein chip by imaging mass spectrometry. This work shows that biomass spectrometry techniques can be implemented for surface mass spectrometry and biochip applications. Along with recent advances in imaging mass spectrometry, this technique will provide a new opportunity for high-speed, and thus high-throughput in the future, label-free mass spectrometric assays using protein arrays.