Selective Immobilization Of Proteins Research Articles

Selective biofunctionalization of 3D cell-imprinted PDMS with collagen immobilization for targeted cell attachment

Cell-imprinted polydimethylsiloxane substrates, in terms of their ability to mimic the physiological niche, low microfabrication cost, and excellent biocompatibility were widely used in tissue engineering. Cells inside the mature cells' cell-imprinted PDMS pattern have been shown in previous research to be capable of being differentiated into a specific mature cell line. On the other hand, the hydrophobicity of PDMS substrate leads to weak cell adhesion. Moreover, there was no guarantee that the cells would be exactly located in the cavities of the cells' pattern. In many studies, PDMS surface was modified by plasma treatment, chemical modification, and ECM coating. Hence, to increase the efficiency of cell-imprinting method, the concavity region created by the cell-imprinted pattern is conjugated with collagen. A simple and economical method of epoxy silane resin was applied for the selective protein immobilization on the desired regions of the PDMS substrate. This method could be paved to enhance the cell trapping into the cell-imprinted pattern, and it could be helpful for stem cell differentiation studies. The applied method for selective protein attachment, and as a consequence, selective cell integration was assessed on the aligned cell-imprinted PDMS. A microfluidic chip created the aligned cell pattern. After Ar+ plasma and APTES treatment of the PDMS substrate, collagen immobilization was performed. The immobilized collagen was removed by epoxy silane resin stamp from the ridge area where the substrate lacked cell pattern and leaving the collagen only within the patterned areas. Coomassie brilliant blue staining was evaluated for selective collagen immobilization, and the collagen-binding stability was assessed by BCA analysis. MTT assay for the evaluation of cell viability on the modified surface was further analyzed. Subsequently, the crystal violet staining has confirmed the selective cell integration to the collagen-immobilized site on the PDMS substrate. The results proved the successfully selective collagen immobilization on the cell-imprinted PDMS and showed that this method increased the affinity of cells to attach inside the cell pattern cavity.

Biotinylation of Membrane Proteins for Binder Selections.

The selective immobilization of proteins represents an essential step in the selection of binding proteins such as antibodies. The immobilization strategy determines how the target protein is presented to the binders and thereby directly affects the experimental outcome. This poses specific challenges for membrane proteins due to their inherent lack of stability and limited exposed hydrophilic surfaces. Here we detail methodologies for the selective immobilization of membrane proteins based on the strong biotin-avidin interaction and with a specific focus on its application for the selection of nanobodies and sybodies. We discuss the challenges in generating and benefits of obtaining an equimolar biotin to target-protein ratio.

Stress-specific aggregation of proteins in the amyloid bodies.

Physiological amyloid aggregation occurs within the nuclei of stress-treated cells. These structures, termed Amyloid bodies (A-bodies), assemble through the rapid accumulation of proteins into dense membrane-less organelles, which possess the same biophysical properties as plaques observed in many amyloid-based diseases. Here, we demonstrate that A-body proteomic compositions vary significantly between stimuli, as constituent proteins can be sequestered by one or more stressors. Stimulus exposure alone was insufficient to induce aggregation, demonstrating that this pathway is not regulated solely by stress-induced conformational changes of the A-body targets. We propose that different environmental conditions induce the formation of A-body subtypes containing distinct protein residents. This selective immobilization of proteins may have evolved as a finely tuned mechanism for surviving divergent stressors.

Bioinspired Pseudozwitterionic Hydrogels with Bioactive Enzyme Immobilization via pH-Responsive Regulation.

Hydrogels are hydrated networks of flexible polymers with versatile biomedical applications, and their resistance to nonspecific protein adsorption is critical. On the other hand, functionalization with other biomacromolecules would greatly enhance their biotechnological potential. The aim of this research is to prepare low fouling hydrogel polymers for selective protein immobilization. Initially, hydrogels were prepared by controlling the composition ratios of 2-carboxyethyl acrylate (CA) and 2-dimethylaminoethyl methacrylate (DMAEMA) monomers in an N, N-methylene-bis-acrylamide (NMBA) cross-linked free radical polymerization reaction. This series of hydrogels (C1D9 to C9D1) were then analyzed by X-ray photoelectron spectroscopy (XPS) and dynamic laser scattering to confirm the actual polymer ratios and surface charge. When the composition ratio was set at CA:6 vs DMEAMA:4 (C6D4), the hydrogel showed nearly neutral surface charge and an equivalent reaction ratio of CA vs DMAEMA in the hydrogel. Subsequent analysis showed excellent antifouling properties, low blood cell adhesion, hemocompatibility, and platelet deactivation. Moreover, this hydrogel exhibited pH responsiveness to protein adsorption and was then used to facilitate the immobilization of lipase as an indication of active protein functionalization while still maintaining a low fouling status. In summary, a mixed-charge nonfouling pseudozwitterionic hydrogel could be prepared, and its pH-responsive adsorption holds potential for designing a biocompatible tissue engineering matrix or membrane enzyme reactors.

Reversible Immobilization of Proteins in Sensors and Solid-State Nanopores.

The controlled functionalization of surfaces with proteins is crucial for many analytical methods in life science research and biomedical applications. Here, a coating for silica-based surfaces is established which enables stable and selective immobilization of proteins with controlled orientation and tunable surface density. The coating is reusable, retains functionality upon long-term storage in air, and is applicable to surfaces of complex geometry. The protein anchoring method is validated on planar surfaces, and then a method is developed to measure the anchoring process in real time using silicon nitride solid-state nanopores. For surface attachment, polyhistidine tags that are site specifically introduced into recombinant proteins are exploited, and the yeast nucleoporin Nsp1 is used as model protein. Contrary to the commonly used covalent thiol chemistry, the anchoring of proteins via polyhistidine tag is reversible, permitting to take proteins off and replace them by other ones. Such switching in real time in experiments on individual nanopores is monitored using ion conductivity. Finally, it is demonstrated that silica and gold surfaces can be orthogonally functionalized to accommodate polyhistidine-tagged proteins on silica but prevent protein binding to gold, which extends the applicability of this surface functionalization method to even more complex sensor devices.

Microfluidic Devices: DNA-SMART: Biopatterned Polymer Film Microchannels for Selective Immobilization of Proteins and Cells (Small 17/2017)

Microfluidic Devices: DNA-SMART: Biopatterned Polymer Film Microchannels for Selective Immobilization of Proteins and Cells (Small 17/2017)

DNA-SMART: Biopatterned Polymer Film Microchannels for Selective Immobilization of Proteins and Cells.

A novel SMART module, dubbed "DNA-SMART" (DNA substrate modification and replication by thermoforming) is reported, where polymer films are premodified with single-stranded DNA capture strands, microthermoformed into 3D structures, and postmodified with complementary DNA-protein conjugates to realize complex biologically active surfaces within microfluidic devices. As a proof of feasibility, it is demonstrated that microchannels presenting three different proteins on their inner curvilinear surface can be used for selective capture of cells under flow conditions.

Coating morphology and surface composition of acrylic terpolymers with pendant catechol, OEG and perfluoroalkyl groups in varying ratio and the effect on protein adsorption

This work aims at developing versatile low-biofouling polymeric coatings by using acrylic terpolymers (DOFs) that bear pendant catechol (D), oligo(ethylene glycol) (O), and perfluoroalkyl (F) groups in varying ratios. The polymers were endowed with the ability to form firmly coatings on virtually any surfaces and undergo surface microphase separation and self-assembly, as revealed by the surface enrichment of F pendants and the morphology variation from irregular solid domains to discrete crater-type aggregates of different size. The effect on protein adsorption was investigated using bovine serum albumin (BSA) and adhesive fibrinogen (Fib) as model proteins. The coating of DOF164 (low F content), which has morphology of discrete crater-type aggregates of ∼400nm in size, adsorbed a least amount of protein but with a highest protein unit activity as determined by SPR and immunosorbent assay; whereas the coating of DOF1612 (high F content) showed a 12.3-fold higher adsorption capacity toward Fib. Interestingly, a 2.2-fold lower adsorption amount but with a 1.8-fold higher unit activity was found for Fib adsorbed on the DOF164 surface than on DOF250 (without F fraction), whose OEG segments being a widely recognized protein compatible material. The features of the DOF164 terpolymer presenting a robust coating ability and a minimal protein adsorption capacity while with a high protein unit activity suggest its potential application as a non-fouling surface-modifier for medical antifouling coatings and as a matrix material for selective protein immobilization and activity preservation in biosensor construction.

Fluorescent Biotin Analogues for Microstructure Patterning and Selective Protein Immobilization.

Benzyl substitution on ureido nitrogens of biotin led to manifestation of aggregation-induced emission, which was studied by steady-state fluorescence, microscopy, and TD-DFT, providing a rationale into the observed photophysical behavior. Besides exhibiting solvatochromism, the biotin derivatives revealed emission peaks centered at ∼430 and 545 nm, which has been attributed to the π-π stacking interactions. Our TD-DFT results also correlate the spectroscopic data and quantify the nature of transitions involved. The isothermal titration calorimetry data substantiates that the binding of the biotin derivatives with avidin are pretty strong. These derivatives on lithographic patterning present a platform for site specific strept(avidin) immobilization, thus opening avenues for potential applications exploiting these interactions. The fluorescent biotin derivatives can thus find applications in cellular biology and imaging.

Formation of highly stable self-assembled alkyl phosphonic acid monolayers for the functionalization of titanium surfaces and protein patterning.

A protocol for the preparation of improved phosphonate monolayers on a titanium (Ti) substrate is presented. Zirconium ions were used to enhance the bonding between the phosphonate headgroup and the pretreated Ti surface. Contact angle and X-ray photoelectron spectroscopy were used to characterize self-assembled monolayers (SAMs) of alkylphosphonic acid that formed spontaneously on Zr-mediated Ti (Zr/Ti) surfaces. The surfaces that were treated with an aqueous solution of zirconium oxychloride showed significantly enhanced stability in buffer compared with those formed directly on the native oxidized Ti. A bifunctional molecule, 10-mercaptodecanyl phosphonic acid (MDPA), was also used to form SAMs on Zr/Ti surfaces using an identical method, which enabled us to regulate the surface functionality through the terminal functional group. Protein patterning on the surface was carried out using UV irradiation through a mask to selectively degrade regions of the MDPA molecules. The surface was then backfilled with a protein-resistant molecule in the exposed regions followed by selective immobilization of proteins to the unexposed areas using a heterobifunctional linker molecule. The presented strategy significantly improved the stability of the phosphonate SAMs on oxidized Ti surfaces, which provided an ideal approach foundation for biomolecular immobilization and patterning onto the Ti surfaces. Thus, this method provided a versatile platform to activate the surfaces of biomedical Ti implants.

Nickel nanoparticle-doped paper as a bioactive scaffold for targeted and robust immobilization of functional proteins.

Cellulose-based materials are widely used in analytical chemistry as platforms for chromatographic and immunodiagnostic techniques. Due to its countless advantages (e.g., mechanical properties, three-dimensional structure, large surface to volume area, biocompatibility and biodegradability, and high industrial availability), paper has been rediscovered as a valuable substrate for sensors. Polymeric materials such as cellulosic paper present high protein capture ability, resulting in a large increase of detection signal and improved assay sensitivity. However, cellulose is a rather nonreactive material for direct chemical coupling. Aiming at developing an efficient method for controlled conjugation of cellulose-based materials with proteins, we devised and fabricated a hybrid scaffold based on the adsorption and in situ self-assembly of surface-oxidized Ni nanoparticles on filter paper, which serve as "docking sites" for the selective immobilization of proteins containing polyhistidine tags (His-tag). We demonstrate that the interaction between the nickel substrate and the His-tagged protein G is remarkably resilient toward chemicals at concentrations that quickly disrupt standard Ni-NTA and Ni-IDA complexes, so that this system can be used for applications in which a robust attachment is desired. The bioconjugation with His-tagged protein G allowed the binding of anti-Salmonella antibodies that mediated the immuno-capture of live and motile Salmonella bacteria. The versatility and biocompatibility of the nickel substrate were further demonstrated by enzymatic reactions.

Head group-functionalized poly(ethyleneglycol)–lipid (PEG–lipid) surface modification for highly selective analyte extractions on capillary-channeled polymer (C-CP) fibers

Polypropylene (PP) capillary-channeled polymer (C-CP) fibers were modified by adsorption of a head group-functionalized lipid to generate analyte-specific surfaces for application as a stationary phase in high performance liquid chromatography (HPLC) or solid phase extraction (SPE). The aliphatic moiety of the lipid adsorbs strongly to the hydrophobic PP surface, with the hydrophilic active head groups orienting themselves toward the more polar mobile phase, thus allowing for interactions with the desired solutes. Initial proof-of-concept was achieved by adsorbing a biotin-poly(ethylene glycol)-functionalized lipid to the surface of the PP C-CP fibers. Surface modification and uniformity was evaluated by binding streptavidin labeled with Texas Red (SAv-TR) to the biotin moiety. Isolation of SAv-TR from a mixture in neat buffer and in cleared lysate demonstrated the capability of the modified fibers to extract an analyte of interest from a complex viscous mixture. It is believed that this surface modification approach is generally applicable to a diversity of selective protein immobilization applications, including clinical diagnostics and preparative scale HPLC on C-CP fibers as well as to other hydrophobic supports.

Selective Protein Immobilization onto Gold Nanoparticles Deposited under Vacuum on a Protein-Repellent Self-Assembled Monolayer

The immobilization of proteins on flat substrates plays an important role for a wide spectrum of applications in the fields of biology, medicine, and biochemistry, among others. An essential prerequisite for the use of proteins (e.g., in biosensors) is the conservation of their biological activity. Losses in activity upon protein immobilization can largely be attributed to a random attachment of the proteins to the surface. In this study, we present an approach for the immobilization of proteins onto a chemically heterogeneous surface, namely a surface consisting of protein-permissive and protein-repellent areas, which allows for significant reduction of random protein attachment. As protein-permissive, i.e., as protein-binding sites, ultra pure metallic nanoparticles are deposited under vacuum onto a protein-repellent PEG-silane polymer layer. Using complementary surface characterization techniques (atomic force microscopy, quartz crystal microbalance, and X-ray photoelectron spectroscopy) we demonstrate that the Au nanoparticles remain accessible for protein attachment without compromising the protein-repellency of the PEG-silane background. Moreover, we show that the amount of immobilized protein can be controlled by tuning the Au nanoparticle coverage. This method shows potential for applications requiring the control of protein immobilization down to the single molecule level.

Plasma-Assisted Nanoscale Protein Patterning on Si Substrates via Colloidal Lithography

Selective immobilization of proteins in well-defined patterns on substrates has recently attracted considerable attention as an enabling technology for applications ranging from biosensors and BioMEMS to tissue engineering. In this work, a method is reported for low-cost, large scale and high throughput, selective immobilization of proteins on nanopatterned Si, based on colloidal lithography and plasma processing to define the areas (<300 nm) where proteins are selectively immobilized. A close-packed monolayer of PS microparticles is deposited on oxidized Si and, either after microparticle size reduction or alternatively after metal deposition through the PS close-packed monolayer, is used as etching mask to define SiO2 nanoislands (on Si). C4F8 plasma was used to selectively etch and modify the SiO2 nanoislands while depositing a fluorocarbon layer on the Si surface. The plasma-treated surfaces were chemically characterized in terms of functional group identification through XPS analysis and reaction with specific molecules. Highly selective protein immobilization mainly through physical adsorption on SiO2 nanoislands and not on surrounding Si was observed after C4F8 plasma-induced chemical modification of the substrate. The thickness of the immobilized protein monolayer was estimated by means of AFM image analysis. The method reported herein constitutes a cost-efficient route toward rapid, large surface, and high-density patterning of biomolecules on solid supports that can be easily applied in BioMEMS or microanalytical systems.

Protein patterning utilizing region-specific control of wettability by surface modification under atmospheric pressure

Wettability control can be crucial in improving the uniformity of selective protein immobilization in high-density microarrays. In this study, we propose an atmospheric-pressure plasma-enhanced chemical vapor deposition (AP-PECVD)-based method in conjunction with photolithography to implement region-specific control of wettability on Si substrate. The proposed PECVD method under atmospheric pressure condition would be a useful alternative of conventional reactive plasma-based treatments methods requiring vacuum condition for uniform protein patterning. Layers with dissimilar wettability and roughness prepared by AP-PECVD process using tetraethoxysilane (TEOS) or TEOS-O2 as precursors could realize uniform protein patterning in a micrometer-scale.

Photo selective protein immobilization using bovine serum albumin

A simple and selective technique which immobilizes protein onto a solid substrate by using UV illumination has been developed. In protein immobilization, a Bovine serum albumin (BSA) performed bifunctional role as a cross-linker between substrate and proteins and as a blocker inhibiting a nonspecific protein adsorption. A new photo-induced protein immobilization process has been investigated at each step by fluorescence microscopy, ellipsometry, and Fourier transform infrared (FT-IR) spectroscopy. A UV photomask has been used to induce selective protein immobilization on target regions of the surface of the SiO2 substrates under UV illumination with negligible nonspecific binding. The UV illumination also showed improved photostability than the conventional methods which employed bifunctional photo-crosslinker molecules of photo-reactive diazirine. This new UV illumination-based photo-addressable protein immobilization provides a new approach for developing novel protein microarrays for multiplexed sensing as well as other types of bio-immobilization in biomedical devices and biotechnologies.

Vinyl functionalized silica hybrid monolith-based trypsin microreactor for on line digestion and separation via thiol-ene “click” strategy

A novel thiol-ene “click” strategy for the preparation of monolithic trypsin microreactor was proposed. The hybrid organic–inorganic monolithic capillary column with ene-functionality was fabricated by sol–gel process using tetramethoxysilane (TMOS) and γ-methacryloxypropyltrimethoxysilane (γ-MAPS) as precursors. The disulfide bonds of trypsin were reduced to form free thiol groups. Then the trypsin containing free thiol groups was attached on the γ-MAPS hybrid monolithic column with ene-functionality via thiol-ene click chemistry to form a trypsin microreactor. The activity of the trypsin microreactor was characterized by detecting the substrate (Nα- p-tosyl- l-arginine methyl ester hydrochloride, TAME) and the product (Nα- p-tosyl- l-arginine, TA) with on-line capillary zone electrophoresis. After investigating various synthesizing conditions, it was found that the microreactor with poly(N,N′-methylenebisacrylamide) as spacer can deliver the highest activity, yielding a rapid reaction rate. After repeatedly sampling and analyzing for 100 times, the monolithic trypsin microreactor still remained 87.5% of its initial activity. It was demonstrated that thiol-ene “click” strategy for the construction of enzyme microreactor is a promising method for the highly selective immobilization of proteins under mild conditions, especially enzymes with free thiol radicals.

Nanoscale Protein Patterning on Si Substrates using Colloidal Lithography and Plasma Processing

Selective immobilization of proteins in well-defined patterns on substrates has recently attracted considerable attention as an enabling technology for applications ranging from basic research to biosensors and bioMEMS. In this work we demonstrate a low-cost and high throughput process for nanoscale, selective immobilization of proteins on patterned Si substrates, based on colloidal lithography and plasma processing in order to define the spots (< 300nm) where proteins are adsorbed. A close- packed monolayer of PS microparticles is deposited on oxidized Si substrates and is used as etching mask to define SiO2 spots (on Si), which after plasma-induced chemical modification are appropriate for selective protein immobilization. The immobilized proteins are detected by means of confocal microscopy and evaluated by means of atomic force microscopy. Such nanoscale immobilized proteins can be successfully integrated into BioMEMS and microanalytical systems.

Selective immobilization of proteins guided by photo-patterned poly(vinyl alcohol) structures

The development of “protein resistant” materials is challenging since protein hysisorption takes place on most surfaces due to van der Waals interactions, hydrogen bonding and entropy effects. In this work a new process for converting a surface resistant to protein adsorption is presented by using a photo-patternable poly(vinyl alcohol) (PVA) based film. This material minimizes effectively protein physisorption and it can be patterned through photolithography on top of any substrate. Herein the PVA-based film is patterned on top of a poly(styrene) (PS) film, in order to achieve selective protein patterning on the PS film and demonstrate the resistance of the PVA-based material to protein physisorption. The proposed methodology is expected to facilitate the fabrication of sensors and bioelectronic devices since it provides a patterning route with alignment capabilities for protein resistant-surfaces and it is based on an easy to implement process.

Protein patterning on polycrystalline silicon–germanium via standard UV lithography for bioMEMS applications

Polycrystalline silicon–germanium (poly-SiGe) is a promising structural material for the post-processing of micro electro-mechanical systems (MEMS) on top of complementary metal-oxide-semiconductor (CMOS) substrates. Combining MEMS and CMOS allows for the development of high-performance devices. We present for the first time selective protein immobilization on top of poly-SiGe surfaces, an enabling technique for the development of novel poly-SiGe based MEMS biosensors. Active regions made of 3-aminopropyl-triethoxysilane (APTES) were defined using silane deposition onto photoresist patterns followed by lift-off in organic solvents. Subsequently, proteins were covalently bound on the created APTES patterns. Fluorescein-labeled human serum albumin (HSA) was used to verify the immobilization procedure while the binding capability of the protein layer was tested by an antigen-labeled antibody pair. Inspection by fluorescence microscopy showed protein immobilization inside the desired bioactive areas and low non-specific adsorption outside the APTES pattern. Furthermore, the quality of the silane patches was investigated by treatment with 30 nm-diameter gold nanoparticles and scanning electron microscope observation. The developed technique is therefore a promising first step towards the realization of poly-SiGe based biosensors.