Plasma-based surface modification of polystyrene microtiter plates for covalent immobilization of biomolecules

In recent years, polymer surfaces have become increasingly popular for biomolecule attachment because of their relatively low cost and desirable bulk physicochemical characteristics. However, the chemical inertness of some polymer surfaces poses an obstacle to more expansive implementation of polymer materials in bioanalytical applications. We describe use of argon plasma to generate reactive hydroxyl moieties at the surface of polystyrene microtiter plates. The plates are then selectively functionalized with silanes and cross-linkers suitable for the covalent immobilization of biomolecules. This plasma-based method for microtiter plate functionalization was evaluated after each step by X-ray photoelectron spectroscopy, water contact angle analysis, atomic force microscopy, and bioimmobilization efficacy. We further demonstrate that the plasma treatment followed by silane derivatization supports direct, covalent immobilization of biomolecules on microtiter plates and thus overcomes challenging issues typically associated with simple physisorption. Importantly, biomolecules covalently immobilized onto microtiter plates using this plasma-based method retained functionality and demonstrated attachment efficiency comparable to commercial preactivated microtiter plates.     

Site‐Specific Surface Functionalization via Microchannel Cantilever Spotting (µCS): Comparison between Azide–Alkyne and Thiol–Alkyne Click Chemistry Reactions

Different types of click chemistry reactions are proposed and used for the functionalization of surfaces and materials, and covalent attachment of organic molecules. In the present work, two different catalyst‐free click approaches, namely azide–alkyne and thiol–alkyne click chemistry are studied and compared for the immobilization of microarrays of azide or thiol inks on functionalized glass surfaces. For this purpose, the surface of glass is first functionalized with dibenzocyclooctyne‐acid (DBCO‐acid), a cyclooctyne with a carboxyl group. Then, the DBCO‐terminated surfaces are functionalized via microchannel cantilever spotting with different fluorescent and nonfluorescent azide and thiol inks. Although both routes work reliably for surface functionalization, the protein binding experiments reveal that using a thiol–alkyne route will obtain the highest surface density of molecular immobilization in such spotting approaches. The obtained achievements and results from this work can be used for design and manufacturing of microscale patterns suitable for biomedical and biological applications.     

Patterned immobilization of biomolecules on a polymer surface functionalized by radiation grafting

Patterned graft polymerization of a functional monomer on a hydrophobic polymer surface was proposed for biomolecule patterning. A poly(vinylidene fluoride) (PVDF) film surface was selectively activated by ion implantation through a pattern mask and acrylic acid (AA) was then graft polymerized onto the activated regions of the PVDF surfaces. The peroxide concentration on the implanted surface depended on the fluence, which had a considerable effect on the grafting degree of AA. Afterwards, amine-functionalized biotin and probe DNA were immobilized on the poly(acrylic acid)-grafted regions of the PVDF surfaces. Specific binding of biotin with streptavidin and hybridization of probe DNA with complimentary DNA proved successful protein and DNA patterning and well-defined 50 microm dot-type patterns of the streptavidin and DNA were obtained. These results confirmed the potential of this strategy for patterning of various biomolecules.     

Microstructured bioreactive surfaces: covalent immobilization of proteins on Au(1 1 1)/silicon via aminoreactive alkanethiolate self-assembled monolayers

Micrometer-scale patterns of a defined surface chemistry and structure were produced on both ultraflat Au(1 1 1) and on gold-coated monocrystalline silicon surfaces by a method combining microcontact printing, wet chemical etching and the replacement of etch-resist self-assembled monolayers (SAMs) by functionalized or reactive SAMs. Key steps in this methodology were characterized by X-ray photoelectron spectroscopy (XPS), ellipsometry and contact angle measurements. The covalent immobilization of (functional) biological systems on these surfaces was tested using an N-hydroxysuccinimide ester -functionalized disulphide (DSU), which covalently binds primary amines without the need for further activation steps. Atomic force microscope images of native collagen V single molecules immobilized on these patterned surfaces revealed both high spatial resolution and strong attachment to the monolayer/gold surface. Microcontact printing of DSU is shown to be feasible on specially prepared, ultraflat Au(1 1 1) surfaces providing a valuable tool for scanning probe experiments with biomolecules. The retention of enzymatic activity upon immobilization of protein was demonstrated for the case of horseradish peroxidase. The described approach can thus be used to confine biological activity to predetermined sites on microstructured gold/silicon devices – an important capability in biomedical and biomolecular research. © 1999 Kluwer Academic Publishers     

Immobilization of epidermal growth factor on titanium and stainless steel surfaces via dopamine treatment

Titanium and stainless steel were modified with dopamine for the immobilization of biomolecules, epidermal growth factor (EGF). First, the treatment of metal surfaces with a dopamine solution under different pH conditions was investigated. At higher pH, the dopamine solution turned brown and formed precipitates. Treatment of the metals with dopamine at pH 8.5 also resulted in the development of brown color at the surface of the metals. The hydrophobicity of the surfaces increased after treatment with dopamine, independently of pH. X-ray photoelectron spectroscopy revealed the formation of a significant amount of an organic layer on both surfaces at pH 8.5. According to ellipsometry measurements, the organic layer formed at pH 8.5 was about 1000 times as thick as that formed at pH 4.5. The amount of amino groups in the layer formed at pH 8.5 was also higher than that observed in the layer formed at pH 4.5. EGF molecules were immobilized onto the dopamine-treated surfaces via a coupling reaction using carbodiimide. A greater amount of EGF was immobilized on surfaces treated at pH 8.5 compared with pH 4.5. Significantly higher growth of rat fibroblast cells was observed on the two EGF-immobilized surfaces compared with non-immobilized surfaces in the presence of EGF. The present study demonstrated that metals can become bioactive via the surface immobilization of a growth factor and that the effect of the immobilized growth factor on metals was greater than that of soluble growth factor.     

Quantitative photochemical immobilization of biomolecules on planar and corrugated substrates: a versatile strategy for creating functional biointerfaces

Methods for the generation of substratespresenting biomolecules in a spatially controlled manner are enabling tools for applications in biosensor systems, microarray technologies, fundamental biological studies and biointerface science. We have implemented a method to create biomolecular patterns by using light to control the direct covalent immobilization of biomolecules onto benzophenone-modified glass substrates. We have generated substrates presenting up to three different biomolecules patterned in sequence, and demonstrate biomolecular photopatterning on corrugated substrates. The chemistry of the underlying monolayer was optimized to incorporate poly(ethylene glycol) to enable adhesive cell adhesion onto patterned extracellular matrix proteins. Substrates were characterized with contact angle goniometry, AFM, and immunofluorescence microscopy. Importantly, radioimmunoassays were performed to quantify the site density of immobilized biomolecules on photopatterned substrates. Retained function of photopatterned proteins was demonstrated both by native ligand recognition and cell adhesion to photopatterned substrates, revealing that substrates generated with this method are suitable for probing specific cell receptor-ligand interactions. This molecularly general photochemical patterning method is an enabling tool for the creation of substrates presenting both biochemical and topographical variation, which is an important feature of many native biointerfaces.     

Quantitative Fourier analysis of schlieren masks: the transition from shadowgraph to schlieren

In a schlieren setup, a lens system forms an image of the refractive index fluctuations of a transparent sample onto a matrix detector while an intensity mask is positioned in the Fourier plane of a collecting lens to perform the required spatial filtering. In the absence of the mask, the resulting technique is that of a shadowgraph. The two methods provide different information about the refractive index of transparent fluids and can be used both for visualization purposes and scattering measurements. Here, we describe the effect of the intensity mask on the technique transfer function, i.e., its ability to detect different spatial frequencies and show how the special cases of shadowgraph, schlieren, and the transition between the two can be derived. We also present experimental data that agree well with our predictions.     

Fabrication of histidine-tagged fusion protein arrays for surface plasmon resonance imaging studies of protein-protein and protein-DNA interactions

The creation and characterization of histidine-tagged fusion protein arrays using nitrilotriacetic acid (NTA) capture probes on gold thin films for the study of protein-protein and protein-DNA interactions is described. Self-assembled monolayers of 11-mercaptoundecylamine were reacted with the heterobifunctional linker N-succinimidyl S-acetylthiopropionate (SATP) to create reactive sulfhydryl-terminated surfaces. NTA capture agents were immobilized by reacting maleimide-NTA molecules with the sulfhydryl surface. The SATP and NTA attachment chemistry was confirmed with Fourier transform infrared reflection absorption spectroscopy. Oriented protein arrays were fabricated using a two-step process: (i) patterned NTA monolayers were first formed through a single serpentine poly(dimethylsiloxane) microchannel; (ii) a second set of parallel microchannels was then used to immobilize multiple His-tagged proteins onto this pattern at discrete locations. SPR imaging measurements were employed to characterize the immobilization and specificity of His-tagged fusion proteins to the NTA surface. SPR imaging measurements were also used with the His-tagged fusion protein arrays to study multiple antibody-antigen binding interactions and to monitor the sequence-specific interaction of double-stranded DNA with TATA box-binding protein. In addition, His-tagged fusion protein arrays created on gold surfaces were also used to monitor antibody binding with fluorescence microscopy in a sandwich assay format.     

A strategy for the generation of surfaces presenting ligands for studies of binding based on an active ester as a common reactive intermediate: a surface plasmon resonance study

This paper describes the immobilization of ten proteins and two low-molecular-weight ligands on mixed self-assembled monolayers (SAMs) of alkanethiolates on gold generated from the tri(ethylene glycol)-terminated thiol 1 (HS(CH2)11(OCH2CH2)3OH) (chi(1) = 1.0-0.0) and the longer, carboxylic acid-terminated thiol2(HS(CH2)11(OCH2-CH2)6OCH2CO2H) (chi(2) = 0.0-1.0). The immobilization was achieved by a two-step procedure: generation of reactive N-hydroxysuccinimidyl esters from the carboxylic acid groups of 2 in the SAM and coupling of these groups with amines on the protein or ligand. Because this method involves a common reactive intermediate that is easily prepared, it provides a convenient method for attaching ligands to SAMs for studies using surface plasmon resonance spectroscopy (and, in principle, other bioanalytical methods that use derivatized SAMs on gold, silver, and other surfaces). These SAMs were resistant to nonspecific adsorption of proteins having a wide range of molecular weights and isoelectric points. The pH of the coupling buffer, the concentration of protein, the ionic strength of the solution of protein, and the molecular weight of the protein all influenced the amount of the protein that was immobilized. For the proteins investigated in detail--carbonic anhydrase and lysozyme--the highest quantities of immobilized proteins were obtained when using a low ionic strength solution at a value of pH approximately one unit below the isoelectric point (pI) of the protein, at a concentration of approximately 0.5 mg mL-1. Comparisons of the kinetic and thermodynamic constants describing binding of carbonic anhydrase and vancomycin to immobilized benzenesulfonamide and N-alpha-Ac-Lys-D-Ala-D-Ala groups, respectively, on mixed SAMs (by methods described in this paper) and in the carboxymethyl dextran matrix of commercially available substrates yielded (for these systems) essentially indistinguishable values of Kd, koff, and kon.     

Quantitative methods based on twisted nematic liquid crystals for mapping surfaces patterned with bio/chemical functionality relevant to bioanalytical assays

We report methods for the acquisition and analysis of optical images formed by thin films of twisted nematic liquid crystals (LCs) placed into contact with surfaces patterned with bio/chemical functionality relevant to surface-based assays. The methods are simple to implement and are shown to provide easily interpreted maps of chemical transformations on surfaces that are widely exploited in the preparation of analytic devices. The methods involve acquisition of multiple images of the LC as a function of the orientation of a polarizer; data analysis condenses the information present in the stack of images into a spatial map of the twist angle of the LC on the analytic surface. The potential utility of the methods is illustrated by mapping (i) the displacement of a monolayer formed from one alkanethiol on a gold film by a second thiol in solution, (ii) coadsorption of mixtures of amine-terminated and ethylene glycol-terminated alkanethiols on gold films, which leads to a type of mixed monolayer that is widely exploited for immobilization of proteins on analytic surfaces, and (iii) patterns of antibodies printed onto surfaces. These results show that maps of the twist angle of the LC constructed from families of optical images can be used to reveal surface features that are not apparent in a single image of the LC film. Furthermore, the twist angles of the LC can be used to quantify the energy of interaction of the LC with the surface with a spatial resolution of <10 microm. When combined, the results described in this paper suggest nondestructive methods to monitor and validate chemical transformations on surfaces of the type that are routinely employed in the preparation of surface-based analytic technologies.     

Spatial-phase code-division multiple-access system with multiplexed Fourier holography switching for reconfigurable optical interconnection

A new, to our knowledge, space-variant optical interconnection system based on a spatial-phase code-division multiple-access technique with multiplexed Fourier holography is described. In this technique a signal beam is spread over wide spatial frequencies by an M-sequence pseudorandom phase code. At a receiver side a selected signal beam is properly decoded, and at the same time its spatial pattern is shaped with a Fourier hologram, which is recorded by light that is encoded with the same M-sequence phase mask as the desired signal beam and by light whose spatial beam pattern is shaped to a signal routing pattern. Using the multiplexed holography, we can simultaneously route multisignal flows into individually specified receiver elements. The routing pattern can also be varied by means of switching the encoding phase code or replacing the hologram. We demonstrated a proof-of-principle experiment with a doubly multiplexed hologram that enables simultaneous routing of two signal beams. Using a numerical model, we showed that the proposed scheme can manage more than 250 routing patterns for one signal flow with one multiplexed hologram at a signal-to-noise ratio of ~5.     

Oriented immobilization of antibodies onto the gold surfaces via their native thiol groups

The general approach for site-oriented immobilization of antibodies onto gold supports is reported. The immobilization is carried out using the native sulfide groups of immunoglobulin (IgG). To liberate the thiol groups, the intact IgG was split into two half-IgG fragments without destruction of the binding site of the antibody. The immobilization of half-IgG fragments on the gold surface was carried out by simple adsorption. The antigen binding capacity of the half-IgG modified gold supports is similar to that of the gold surfaces with the traditionally linked antibodies and is much higher than for nonspecifically adsorbed intact IgGs. The immobilized antibodies, according to the proposed approach, maintain high antigen binding constants. The immobilization procedure provides orientation of IgG fragments in terms of the similar distance between the binding site of the antibody and the surface of the gold support, which does not cause the distribution of the apparent affinity constants. The high operational stability of half-IgG modified gold electrodes makes them applicable for analytical applications.     

Patterning of electrically conductive poly(aniline-co-aniline sulfonic acid) and its application in the immobilization of cytochrome c

We have synthesized poly(aniline-co-aniline sulfonic acid) and then investigated the feasibility of application as a specific and electrically conductive binding template for biomolecules. Poly(aniline-co-aniline sulfonic acid)s were prepared by oxidation polymerization of aniline and aniline sulfonic acid under various ratios. A fine pattern of the conducting copolyaniline was obtained by using a deep UV lithographic technique. Cytochrome c was immobilized onto the photochemically patterned conducting copolyaniline with a self-assembly method. Physical and electrochemical properties of the self-assembled cytochrome c monolayer were studied from atomic force microscopy and cyclic voltammetry. The self-assembled cytochrome c monolayer immobilized onto the copolyaniline with a high electrical conductivity showed a high electrochemical activity.     

Electrochemical detection of biomolecule with mixed self-assembled monolayers of ferrocene-undecanethiol

Thiol self-assembled monolayers (SAMs) have been widely used as a modification method to immobilize biomolecules to gold surfaces. However, the additional layers of SAM and biomolecules make electron transfer difficult, leading to a large overpotential in electrical signal. Electron transfer mediation is the most popular solution to overcome the problem of the overpotential for an electrochemical biosensor. We introduced mixed SAMs of mercapto-dodecanoic acid (MDA) and ferrocene-undecanthiol. Ferrocene-undecanthiol acts as an electron transfer mediator and MDA is used for immobilization of biomolecule. The electron transferability of mixed SAMs is affected by pH, kinds of electrolytes, and the composition of the thiol molecule. We optimized the carboxyl acid and ferrocene molecule ratio which is a crucial factor in the performance of mixed SAMs and electrochemically detected the avidin. The detection limit was 2.0 microg/mL of avidin concentration.     

Photoactivatable copolymers of vinylbenzyl thiocyanate as immobilization matrix for biochips

Biochip surfaces for immobilization of DNA and proteins require reactive polymers with high immobilization capacity and low nonspecific binding. Most reactive surfaces consist of matrixes that provide epoxy, aldehyde, or amino functions for biomolecule binding. The most widely used oligonucleotide modification is a C6-amino link. The high reactivity of isothiocyanate groups (NCS) toward amines was therefore the motivation to employ photogenerated NCS groups as binding sites for NH(2)-terminated oligonucleotides. Photosensitive poly(styrene-co-4-vinylbenzyl thiocyanate) (PST-co-VBT) was synthesized and applied as novel material for DNA and protein immobilization. The immobilization capacity of PST-co-VBT was a function of UV energy density used for photoactivation and was approximately 80% at 450 mJ cm(-)(2) (lambda(ex) = 254 nm). This surface was superior to tested commercial chip surfaces in signal-to-noise-ratio and reproducibility. Print buffer and spacer length were optimized for maximum fluorescence signal with DNA and proteins. UV exposure conditions and oligonucleotide modification were correlated, showing that this photochemical approach can be successfully applied for surface patterning of biochips.     

Rapid fabrication of diffractive optical elements by use of image-based excimer laser ablation

We describe a method of fabricating multilevel diffractive optics by excimer laser ablation. A portion of a chrome mask containing many patterns is illuminated by 193-nm laser light and imaged by an objective lens onto a poly(imide) substrate. Ablation of an entire single pattern is achieved in a single laser pulse. Multiple pulses are used to vary the ablation depth, and multiple patterns are used to create a variety of multilevel optics. We have successfully fabricated arrays of eight-level diffractive microlenses with varying focal lengths and decenters. The optics performed with diffraction-limited focusing and near-theoretical diffraction efficiency (92%).     

锗表面二维亚波长结构的反应离子刻蚀制备

为了获得具有金字塔结构的二维亚波长结构表面,提高其高宽比,用掩模曝光光刻及反应离子刻蚀技术,以SF6和O2为反应气体,在Ge衬底上制备了二维亚波长结构.用扫描电镜对刻蚀图形的形貌进行了观察,研究了功率、气压、气体流量及掩模图形对刻蚀图形的影响.结果表明:刻蚀图形腰部被优先刻蚀,形成凹陷的侧壁轮廓;O2流量增大有利于在侧壁形成保护层,从而减小腰部刻蚀、增大顶部及根部刻蚀;功率及气压过大或过小均会使侧壁刻蚀较大;方形图案比圆形图案掩模更有利于刻蚀出深度较大的亚波长结构.     

Gold surface functionalization and patterning for specific immobilization of olfactory receptors carried by nanosomes

There is substantial interest in engineering solid supports to achieve functional immobilization of membrane receptors both for investigation of their biological function and for the development of novel biosensors. Three simple and practical strategies for immobilization of a human olfactory receptor carried by nanosomes are presented. The basis of the functionalization of solid gold surfaces is a self-assembled monolayer (SAM) containing biotinyl groups. Biotinyl groups are subsequently used to attach neutravidin and then biotinylated monoclonal antibody directed against the receptor to allow its specific grafting. Surface plasmon resonance technique is implemented for real-time monitoring of step-by-step surface functionalization and, in addition, for testing the functional response of immobilized olfactory receptors. We show that OR1740 is functional when immobilized via a tag attached to its C-terminus, but not via its N-terminus. Finally, we demonstrate that gold surfaces can be patterned by the SAMs tested using microcontact printing. AFM images of immobilized nanosomes onto a patterned surface suggest that small nanosomes flatten and fuse into larger vesicles but do not merge into a continuous layer. The whole study emphasizes the outstanding performances of the BAT/PEGAT SAM, which could be useful for developing on-a-chip sensor formats for membrane receptor investigations and use.     

Solid‐Supported Biomolecules on Modified Silica Surfaces—A Tool for Fast Physicochemical Characterization and High‐Throughput Screening

Biofunctionalization for a wide variety of applications can be achieved by coating silica surfaces with biomolecules such as lipids or proteins. However, specific surface optimization of the inorganic SiO₂ is necessary to achieve biocompatible surfaces. Surface shielded porous silica beads can be non‐covalently coated with a single lipid bilayer. The lipids retain their fluidity in this handy solid‐supported system, perfectly mimicking the soft‐surface properties of cellular membranes. A supramolecular architecture can also be used for functional immobilization of membrane proteins: An artificial cytosolic compartment can be created with the aid of polymers; coating by lipid membranes and integration of membrane proteins results in a solid‐supported biofunctional cellular surface. Another surface modification enables a direct immobilization of human serum albumin (HSA) molecules onto silica surfaces. The HSA on this otherwise passivated surface provides a convenient material for the investigation of unspecific protein binding of pharmaceuticals on a high‐throughput scale.