Studies of electroosmotic flow and the effects of protein adsorption in plasma‐polymerized microchannel surfaces

This paper presents a study of EOF properties of plasma‐polymerized microchannel surfaces and the effects of protein (fibrinogen and lysozyme) adsorption on the EOF behavior of the surface‐modified microchannels. Three plasma polymer surfaces, i.e. tetraglyme, acrylic acid and allylamine, are tested. Results indicate EOF suppression in all plasma‐coated channels compared with the uncoated glass microchannel surfaces. The EOF behaviors of the modified microchannels after exposure to protein solutions are also investigated and show that even low levels of protein adsorption can significantly influence EOF behavior, and in some cases, result in the reversal of flow. The results also highlight that EOF measurement can be used as a method for detecting the presence of proteins within microchannels at low surface coverage (<1 ng/cm² on glass). Critically, the results illustrate that the non‐fouling tetraglyme plasma polymer is able to sustain EOF. Comparison of the plasma‐polymerized surfaces with conventionally grafted polyelectrolyte surfaces demonstrates the stabilities of the plasma polymer films, enabling multiple EOF runs over 3 days without deterioration in performance. The results of this study clearly demonstrate that plasma polymers enable the surface chemistry of microfluidic devices to be tailored for specific applications. Critically, the deposition of the non‐fouling tetraglyme coating enables stable EOF to be induced in the presence of protein.     

Radial basis function interpolation supplemented lattice Boltzmann method for electroosmotic flows in microchannel

Large gradients of physical variables near the channel walls are characteristic of EOF. The previous numerical simulations of EOFs with the lattice Boltzmann method (LBM) utilize uniform lattice and are not efficient, especially when the electric double layer (EDL) thickness is significantly smaller than the channel height. The efficient LBM simulation of EOF in microchannel calls for a nonuniform mesh which is dense in the EDL region and sparse in the bulk region. In this article, we formulate a radial basis function (RBF)-based interpolation supplemented LBM (ISLBM) to solve the governing equations of EOF, that is, the Poisson, Nernst–Planck, and Navier–Stokes equations, in a nonuniform mesh system. Unlike the conventional ISLBM, the RBF-ISLBM determines the prestreaming distribution functions by using the local RBF-based interpolation over circular supporting regions and is particularly suitable for nonuniform meshes. The RBF-ISLBM is validated by the EOFs in infinitely long and finitely long microchannels. The results show that the RBF-ISLBM possesses excellent robustness and accuracy. Finally, we use the RBF-ISLBM to simulate the EOFs with the hitherto highest electrokinetic parameter, κa, defined by the ratio of channel height a to EDL thickness κ⁻¹, in LBM simulations of EOF.     

Reversible covalent patterning of self-assembled monolayers on gold and silicon oxide surfaces

This paper describes the generation of reversible patterns of self-assembled monolayers (SAMs) on gold and silicon oxide surfaces via the formation of reversible covalent bonds. The reactions of (patterned) SAMs of 11-amino-1-undecanethiol (11-AUT) with propanal, pentanal, decanal, or terephthaldialdehyde result in dense imine monolayers. The regeneration of these imine monolayers to the 11-AUT monolayer is obtained by hydrolysis at pH 3. The (patterned) monolayers were characterized by Fourier transform infrared reflection absorption spectroscopy, X-ray photoelectron spectroscopy, contact angle and electrochemical measurements, and atomic force microscopy. Imines can also be formed by microcontact printing of amines on terephthaldialdehyde-terminated substrates. Lucifer Yellow ethylenediamine was employed as a fluorescent amine-containing marker to visualize the reversible covalent patterning on a terephthaldialdehyde-terminated glass surface by confocal microscopy. These experiments demonstrate that with reversible covalent chemistry it is possible to print and erase chemical patterns on surfaces repeatedly.     

Stationary phase structures enhancing electroosmotic flow in capillary electrochromatography

Several chemically bonded silicas with C18 groups were examined with respect to electroosmotic flow (EOF) velocities under CEC conditions. Stationary phases with low hydrophobic selectivity generally provided high EOFs. The stationary phases prepared by using octadecyltrichlorosilane showed greater EOF than those from octadecyldimethylchlorosilane. Restricted-access reversed-phase (RARP) packing materials having C18 groups inside the pores and silanols on the external surfaces showed higher EOF than monomeric C18 phases with similarly high hydrophobic selectivity. The RARP-type structure having silanols at the external surface seems to be effective for increasing EOF while maintaining the hydrophobic character of the solute binding sites.     

Cobalt(III)‐Mediated Permanent and Stable Immobilization of Histidine‐Tagged Proteins on NTA‐Functionalized Surfaces

We present the cobalt(III)‐mediated interaction between polyhistidine (His)‐tagged proteins and nitrilotriacetic acid (NTA)‐modified surfaces as a general approach for a permanent, oriented, and specific protein immobilization. In this approach, we first form the well‐established Co²⁺‐mediated interaction between NTA and His‐tagged proteins and subsequently oxidize the Co²⁺ center in the complex to Co³⁺. Unlike conventionally used Ni²⁺‐ or Co²⁺‐mediated immobilization, the resulting Co³⁺‐mediated immobilization is resistant toward strong ligands, such as imidazole and ethylenediaminetetraacetic acid (EDTA), and washing off over time because of the high thermodynamic and kinetic stability of the Co³⁺ complex. This immobilization method is compatible with a wide variety of surface coatings, including silane self‐assembled monolayers (SAMs) on glass, thiol SAMs on gold surfaces, and supported lipid bilayers. Furthermore, once the cobalt center has been oxidized, it becomes inert toward reducing agents, specific and unspecific interactions, so that it can be used to orthogonally functionalize surfaces with multiple proteins. Overall, the large number of available His‐tagged proteins and materials with NTA groups make the Co³⁺‐mediated interaction an attractive and widely applicable platform for protein immobilization.     

DNA-modified electrodes; part 4: optimization of covalent immobilization of DNA on self-assembled monolayers

The covalent immobilization of DNA onto self-assembled monolayer (SAM) modified gold electrodes (SAM/Au) was studied by X-ray photoelectron spectrometry and electrochemical method so as to optimize its covalent immobilization on SAMs. Three types of SAMs with hydroxyl, amino, and carboxyl terminal groups, respectively, were examined. Results obtained by both X-ray photoelectron spectrometry and cyclic voltammetry show that the largest covalent immobilization amount of dsDNA could be gained on hydroxyl-terminated SAM/Au. The ratio of amount of dsDNA immobilized on hydroxyl-terminated SAMs to that on carboxyl-terminated SAMs and to that on amino-terminated SAMs is (3-3.5): (1-1.5): 1. The dsDNA immobilized covalently on hydroxyl-terminated SAMs accounts for 82.8-87.6% of its total surface amount (including small amount of dsDNA adsorbed). So the hydroxyl-terminated SAM is a good substrate for the covalent immobilization of dsDNA on gold surfaces.     

Poly(oxyethylene) based surface coatings for poly(dimethylsiloxane) microchannels

Control of surface properties in microfluidic systems is an indispensable prerequisite for successful bioanalytical applications. Poly(dimethylsiloxane) (PDMS) microfluidic devices are hampered from unwanted adsorption of biomolecules and lack of methods to control electroosmotic flow (EOF). In this paper, we propose different strategies to coat PDMS surfaces with poly(oxyethylene) (POE) molecules of varying chain lengths. The native PDMS surface is pretreated by exposure to UV irradiation or to an oxygen plasma, and the covalent linkage of POE-silanes as well as physical adsorption of a triblock-copolymer (F108) are studied. Contact angle measurements and atomic force microscopy (AFM) imaging revealed homogeneous attachment of POE-silanes and F108 to the PDMS surfaces. In the case of F108, different adsorption mechanisms to hydrophilic and hydrophobic PDMS are discussed. Determination of the electroosmotic mobilities of these coatings in PDMS microchannels prove their use for electrokinetic applications in which EOF reduction is inevitable and protein adsorption has to be suppressed.     

Soft-landing of peptide ions onto self-assembled monolayer surfaces: an overview

This review is focused on what has been learned in recent research studies concerned with fundamental aspects of soft-landing and reactive landing of peptide ions on self-assembled monolayer surfaces (SAMs). Peptide ions are particularly attractive model systems that provide important insights on the behavior of soft landed proteins, while SAMs provide a convenient and flexible platform for tailoring the interfacial properties of metals and semiconductor surfaces. Deposition of mass-selected ions on surfaces is accompanied by a number of processes including charge reduction, neutralization, covalent and non-covalent binding, and thermal desorption of ions and molecules from the substrate. Factors that affect the competition between these processes are discussed.     

Fabrication of monodisperse asymmetric colloidal clusters by using contact area lithography (CAL)

We report a new fabrication method of asymmetric colloidal clusters by using contact area lithography with site-selective growth. Nanometric surface patterns (approximately 44, 60, and 81 nm in diameter) were prepared by coating surfaces with self-assembled monolayers (SAMs; octadecyltrichlorosilane (OTS) in this study) except the contact area either between colloidal particles or between colloids and substrate. Nanoscale site-specific heterogeneous nucleation and growth of oxide materials of titanium were studied using the patterns of OTS-SAMs onto the either flat or curved surfaces of SiO2. Experimental results suggest that a combination of the large difference in the surface energy between the growing and surrounding surfaces and the diffusion-controlled growth leads to complete nanoscale site specificity. We also fabricated superstructrures of silica spheres with hemispheres of titania (<20 nm in dimension) on their surfaces and discussed the optical properties of colloidal films consisting of the monodisperse asymmetric colloidal clusters in terms of photonic band gap.     

Cationic poly(methacryl oxyethyl trimethylammonium) and its poly(ethylene glycol)‐grafted analogue as capillary coating materials in electrophoresis

Cationic polyelectrolytes were synthesized and used as semipermanent coating materials for capillaries in electrophoresis. The polyelectrolytes used were a homopolymer of poly(methacryl oxyethyl trimethylammonium chloride) (PMOTAC) and its poly(ethylene glycol) (PEG)‐grafted analogue. Two PMOTAC polyelectrolytes, with molar masses of 85,000 and 300,000 g/mol, and PEG‐grafted PMOTAC with a molar mass of 280,000 g/mol were synthesized and then characterized by size exclusion chromatography (SEC) and nuclear magnetic resonance (NMR) spectroscopy. Attachment of the polyelectrolytes to the wall of the fused silica capillary for electrophoresis caused the electroosmotic flow (EOF) to reverse. The polyelectrolyte coatings were tested over the pH range 2–11 at different buffer ionic strengths, and the most stable and strongest anodic EOFs were obtained at acidic pH values with low ionic strength buffers. Between runs the capillary is merely rinsed for 2 or 3 min with the background electrolyte solution. With the PMOTAC coatings at pH values ≤5, the RSDs of the EOFs were less than 2.9% after 60 injections. The effects of the molar mass of the polycation and of PEGylation of PMOTAC on the interactions between the polycations and basic proteins were studied at acidic pH values. The differences in the effective electrophoretic mobilities, resolution values, and plate numbers of the proteins with the different coatings were due to the EOF, as demonstrated through calculations of reduced mobilities, relative resolution values, and relative plate numbers. © 2007 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 45: 2655–2663, 2007     

Semicarbazide-functionalized Si(111) surfaces for the site-specific immobilization of peptides

The covalent attachment of semicarbazide-functionalized layers to hydrogen-terminated Si(111) surfaces is reported. The surface modification, based on the photoinduced hydrosilylation of a Si(111) surface with protected semicarbazide-functionalized alkenes, was investigated by means of X-ray photoelectron spectroscopy (XPS), contact angle measurements, and atomic force microscopy (AFM). The removal of the protecting group yielded a semicarbazide-terminated monolayer which was reacted with peptides bearing a glyoxylyl group for site-specific alpha-oxo semicarbazone ligation.     

Fabrication and Visualization of Metal‐Ion Patterns on Glass by Dip‐Pen Nanolithography

Fluorescent self‐assembled monolayers (SAMs) are used as dip‐pen nanolithography (DPN) substrates for the fabrication of patterns of Ca²⁺ and Cu²⁺ ions. The driving force for the transfer of these ions from an atomic force microscopy (AFM) tip to the surface is their complexation to organic ligands on the monolayer. By means of fluorescent surfaces, the patterns can be visualized under a fluorescence microscope. We use a custom‐built atomic force fluorescence microscope (AFFM), a combination of atomic force and confocal fluorescence microscopes, to deposit the metal ions onto the sensing SAMs by DPN and to subsequently visualize modulations of fluorescence intensity in a sequential write–read mode.     

Protein interactions with self-assembled monolayers presenting multimodal ligands: a surface plasmon resonance study

This paper describes the use of surface plasmon resonance (SPR) spectroscopy and self-assembled monolayers (SAMs) to understand the characteristics of surfaces that promote the adsorption of proteins at high ionic strengths (high-salt conditions). We synthesized SAMs presenting different multimodal ligands and determined the influence of surface composition, solution composition, and the nature of the protein on the extent of protein adsorption onto the SAMs. Our results confirm that hydrophobic interactions can contribute significantly to protein adsorption under high-salt conditions. In particular, the extent of protein adsorption under high-salt conditions increased with increasing surface hydrophobicity. The extent of protein adsorption was also influenced by the solution composition and decreased with an increase in the chaotropicity of the anion. The combination of SPR and SAMs is well-suited for studying the interaction of proteins with complex surfaces of relevance to chromatography.     

Fabrication of biomolecular nanostructures by scanning near-field photolithography of oligo(ethylene glycol)-terminated self-assembled monolayers

The UV photo-oxidation of oligo(ethylene glycol) (OEG)-terminated self-assembled monolayers (SAMs) has been studied using static secondary ion mass spectrometry, X-ray photoelectron spectroscopy, contact angle measurement, and friction force microscopy. OEG-terminated SAMs are oxidized to yield sulfonates, but photodegradation of the OEG chain also occurs on a more rapid time scale, yielding degradation products that remain bound to the surface via gold-sulfur bonds. The oxidation of these degradation products is the rate-limiting step in the process. Photopatterning of OEG-terminated SAMs may be accomplished by using a mask and suitable light source or by using scanning near-field photolithography (SNP) in which the mask is replaced by a scanning near-field optical microscope coupled to a UV laser. Using SNP, it is possible to fabricate patterns in SAMs with a full width at half-maximum height (fwhm) as small as 9 nm, which is approximately 15 times smaller than the conventional diffraction limit. SNP-patterned OEG-terminated SAMs may be used to fabricate protein nanopatterns. By adsorbing carboxylic acid-terminated thiols into oxidized regions and converting these to active ester intermediates, it has been possible to fabricate lines of protein molecules with widths of only a few tens of nanometers.     

Physisorbed surface coatings for poly(dimethylsiloxane) and quartz microfluidic devices

Surface modifications of microfluidic devices are of essential importance for successful bioanalytical applications. Here, we investigate three different coatings for quartz and poly(dimethylsiloxane) (PDMS) surfaces. We employed a triblock copolymer with trade name F₁₀₈, poly(l-lysine)-g-poly(ethylene glycol) (PLL-PEG), as well as the hybrid coating n-dodecyl-β-d-maltoside and methyl cellulose (DDM/MC). The impact of these coatings was characterized by measuring the electroosmotic flow (EOF), contact angle, and prevention of protein adsorption. Furthermore, we investigated the influence of static coatings, i.e., the incubation with the coating agent prior to measurements, and dynamic coatings, where the coating agent was present during the measurement. We found that all coatings on PDMS as well as quartz reduced EOF, increased reproducibility of EOF, reduced protein adsorption, and improved the wettability of the surfaces. Among the coating strategies tested, the dynamic coatings with DDM/MC and F₁₀₈ demonstrated maximal reduction of EOF and protein adsorption and simultaneously best long-term stability concerning EOF. For PLL-PEG, a reversal in the EOF direction was observed. Interestingly, the static surface coating strategy with F₁₀₈ proved to be as effective to prevent protein adsorption as dynamic coating with this block copolymer. These findings will allow optimized parameter choices for coating strategies on PDMS and quartz microfluidic devices in which control of EOF and reduced biofouling are indispensable.     

Reversible protein adsorption and bioadhesion on monolayers terminated with mixtures of oligo(ethylene glycol) and methyl groups

Surface-grafted, environmentally responsive polymers have shown great promise for controlling adsorption and desorption of macromolecules and cells on solid surfaces. In the paper, we demonstrate that certain mixed self-assembled monolayers (SAMs) of oligo(ethylene glycol) (OEG) and methyl-terminated alkanethiolates on gold form surfaces with switchable hydrophobicity and tendency for protein adsorption and cellular attachment. At temperatures above 32 degrees C, SAMs with a surface density of approximately 50% OEG adsorbed significant amounts of pyruvate kinase and lysozyme, whereas below this temperature, these same SAMs were resistant to the adsorption of these proteins. Furthermore, protein layers adsorbed to these SAMs above 32 degrees C were removed upon rinsing with water below this temperature. Similar results were seen for attachment and release of the marine bacterium, Cobetia marina. The change from nonresistance to adsorptive state of the SAMs was concomitant with a change in advancing water contact angle. Vibrational sum frequency generation spectroscopy suggests that the temperature-induced changes coincide with a disorder-to-partial order transition of the hydrated methylene chains of the OEG moieties within the SAMs. Mixed OEG-methyl SAMs represent both a convenient means of controlling macromolecular and cellular adsorption within the laboratory and a useful tool for relating adsorption properties to molecular structures within the SAMs.     

Attachment of Amine‐ and Maleimide‐Containing Ferrocene Derivatives onto Self‐Assembled Alkanethiol and Alkanedithiol Monolayers: Voltammetric Evaluation of Cross‐Linking Efficiencies and Surface Coverage of Electroactive Groups

Ferrocene derivatives containing primary amines and maleimide groups were attached covalently onto N‐hydrosuccinimidyl (NHS)‐terminated alkanethiol self‐assembled monolayers (SAMs) and SAMs of alkanedithiol. The surface coverage and efficiencies of the two cross‐linking reactions were evaluated with cyclic voltammetry. All the ferrocene derivatives attached onto the alkanethiol or alkanedithiol SAMs exhibit reversible redox waves. The surface coverage of the aminated ferrocene groups was compared to that of N‐hydrosuccinimidyl (NHS)‐terminated alkanethiol SAM. The covalent attachment of β‐ferrocenylethylamine onto a 11,11′‐dithio‐bis(succinimidylundecanoate) SAM yielded an efficiency as high as 63.1%. The cross‐linking efficiency of this reaction was found to increase with the nucleophilicity of the amino groups. SAMs of longer alkyl chains favor the attachment of a greater number of ferrocene derivatives. As for the Michael‐type electrophilic addition between the sulfhydryl groups of the alkanedithiol SAMs and the ferrocenyl maleimide, the cross‐linking efficiencies were found to range from 6.5% to 25.7%, depending on the alkanedithiol chain length. The difference in the efficiencies between the two types of cross‐linking reactions might be partially attributable to the steric hindrance imposed by the SAMs and the relative sizes of the functional groups.     

Chemical assays of end-groups displayed on the surface of poly(ethylene terephthalate) (PET) films and membranes by radiolabeling

Poly(ethylene terephthalate) (PET) films and track-etched microporous membranes naturally display, on their surfaces, reactive chain-ends, i.e. carboxyl and hydroxyl functions. These were assayed by suitable activation (reaction with carbodiimide and tosyl chloride, respectively), followed by coupling with ³H-lysine and liquid scintillation counting of the sample-associated radioactivity. Values ranging between 5 and 30 pmol/cm² (open surface) of labeled end-groups were obtained, depending on the physico-chemical nature of the samples. Basic hydrolysis enriched the PET films with both types of endings (15–25 pmol/cm²). Reduction of films with the NaBH₄-catechol complex in tetrahydrofuran enriched their surfaces with hydroxyl groups. However, this procedure was not readily applicable to the surface modification of membranes; we observed an erosion effect that was confirmed by scanning electron microscope analyses. In contrast with the reduction process, the oxidation with KMnO₄ in 1.2N H₂SO₄ could be easily applied to the modification of either films or membranes; their surfaces were significantly enriched with carboxyl groups (15–50 pmol/cm²). This surface modification strategy has been used for the covalent coupling of adhesive proteins on PET membranes developed as supports for cell cultivation.     

Immobilization of peroxidase glycoprotein on gold electrodes modified with mixed epoxy-boronic Acid monolayers

The development of bioelectronic enzyme applications requires the immobilization of active proteins onto solid or colloidal substrates such as gold. Coverage of the gold surface with alkanethiol self-assembled monolayers (SAMs) reduces nonspecific adsorption of proteins and also allows the incorporation onto the surface of ligands with affinity for complementary binding sites on native proteins. We present in this work a strategy for the covalent immobilization of glycosylated proteins previously adsorbed through weak, reversible interactions, on tailored SAMs. Boronic acids, which form cyclic esters with saccharides, are incorporated into SAMs to weakly adsorb the glycoprotein onto the electrode surface through their carbohydrate moiety. To prevent protein release from the electrode surface, we combine the affinity motif of boronates with the reactivity of epoxy groups to covalently link the protein to heterofunctional boronate-epoxy SAMs. The principle underlying our strategy is the increased immobilization rate achieved by the weak interaction-induced proximity effect between slow reacting oxyrane groups in the SAM and nucleophilic residues from adsorbed proteins, which allows the formation of very stable covalent bonds. This approach is exemplified by the use of phenylboronates-oxyrane mixed monolayers as a reactive support and redox-enzyme horseradish peroxidase as glycoprotein for the preparation of peroxidase electrodes. Quartz crystal microbalance, atomic force microscopy, and electrochemical measurements are used to characterize these enzymatic electrodes. These epoxy-boronate functional monolayers are versatile, stable interfaces, ready to incorporate glycoproteins by incubation under mild conditions.     

Interfacial supramolecular self-assembled monolayers of C(60) by thiolated beta-cyclodextrin on gold surfaces via monoanionic C(60)

The supramolecular self-assembled monolayers (SAMs) of C(60) by thiolated beta-cyclodextrin (CD) on gold surfaces were constructed for the first time using C(60) monoanion. The results indicate that monoanionic C(60) plays a crucial role in the formation of the C(60)-containing self-assembled monolayers. The generation of C(60) monoanion and the formation process of C(60) SAMs were monitored in-situ by UV-visible and near-IR spectroscopy. The resulting C(60) SAMs were fully characterized by spectroscopic ellipsometry (SE), cyclic voltammetry, X-ray photoelectron spectroscopy (XPS), and water contact angle measurements. After the immobilization of C(60) by the SAMs of thiolated beta-CD, the film thickness increased by approximately 1 nm from 0.8 to 1.8 nm as determined by SE, demonstrating the formation of the supramolecular self-assembled monolayers of thiolated beta-CD/C(60). The new C(60) SAMs exhibited one quasi-reversible redox couple at half wave potential of -0.57 V vs SCE in aqueous solution containing 0.1 M KCl. The surface coverage of C(60) on the gold surfaces was estimated to be 1.1 x 10(-10) mol cm(-2). The XPS showed the assembly of C(60) over the thiolated beta-CD SAMs. The surface hydrophobicity increased greatly upon the formation of the C(60)-containing SAMs as analyzed by water contact angle measurements. The results are in agreement with the formation of 1:1 complex of C(60) and cyclodextrin on gold surfaces, though it also reveals some non-homogeneous features of the monolayers.