Bifunctional oligo(ethylene glycol) decorated surfaces which permit covalent protein immobilization and resist protein adsorption

In this article, surface coatings derived from homo-bifunctional tri(ethylene glycol) (EG₃) and hexa(ethylene glycol) (EG₆) molecules which have two terminal aldehyde groups are reported. These homo-bifunctional molecules can be used to functionalize amine-terminated surfaces through crosslinking one aldehyde group to surface amine groups, while leaving the other aldehyde group available for covalent immobilization of proteins. Best of all, after reducing remaining aldehyde groups on the surface with a reducing agent, sodium borohydride, the surface becomes oligo(ethylene glycol) (OEG)-terminated. The OEG-terminated surface can resist nonspecific protein adsorption, a feature that is often required for many biosensors and biomedical devices. Although some mixed self-assembled monolayers formed from two different organothiols also permit covalent protein immobilization and resist nonspecific protein adsorption, the procedure reported herein requires only one type of homo-bifunctional molecule and can be applied to both silicon and gold surfaces.     

RGD-grafted poly-L-lysine-graft-(polyethylene glycol) copolymers block non-specific protein adsorption while promoting cell adhesion

A novel class of surface-active copolymers is described, designed to protect surfaces from nonspecific protein adsorption while still inducing specific cell attachment and spreading. A graft copolymer was synthesized, containing poly-(L-lysine) (PLL) as the backbone and substrate binding and poly(ethylene glycol) (PEG) as protein adsorption-resistant pendant side chains. A fraction of the grafted PEG was pendantly functionalized by covalent conjugation to the peptide motif RGD to induce cell binding. The graft copolymer spontaneously adsorbs from dilute aqueous solution onto negatively charged surfaces. The performance of RGD-modified PLL-g-PEG copolymers was analyzed in protein adsorption and cell culture assays. These coatings efficiently blocked the adsorption of serum proteins to Nb(2)O(5) and tissue culture polystyrene while specifically supporting attachment and spreading of human dermal fibroblasts. This surface functionalization technology is expected to be valuable in both the biomaterial and biosensor fields, because different signals can easily be combined, and sterilization and application are straightforward and cost-effective.     

Multifunctional CMOS microchip coatings for protein and peptide arrays

Complementary metal oxide semiconductor (CMOS) microelectronic chips fulfill important functions in the field of biomedical research, ranging from the generation of high complexity DNA and protein arrays to the detection of specific interactions thereupon. Nevertheless, the issue of merging pure CMOS technology with a chemically stable surface modification which further resists interfering nonspecific protein adsorption has not been addressed yet. We present a novel surface coating for CMOS microchips based on poly(ethylene glycol)methacrylate graft polymer films, which in addition provides high loadings of functional groups for the linkage of probe molecules. The coated microchips were compatible with the harshest conditions emerging in microarray generating methods, thoroughly retaining structural integrity and microelectronic functionality. Nonspecific adsorption of proteins on the chip's surface was completely obviated even with complex serum protein mixtures. We could demonstrate the background-free antibody staining of immobilized probe molecules without using any blocking agents, encouraging further integration of CMOS technology in proteome research.     

Use of thiol-terminal silanes and heterobifunctional crosslinkers for immobilization of antibodies on silica surfaces

A procedure for covalent immobilization of functional proteins on silica substrates was developed using thiol-terminal silanes and heterobifunctional cross-linkers. Using this procedure, a high density of functional antibodies was bound to glass cover slips and silica fibers. The amount of anti-IgG antibody immobilized was determined to be in the range of 0.66 to 0.96 ng/mm2 using radiolabeled antibody. The relative amount of IgG antigen bound by the immobilized antibody (0.37 to 0.55 mol antigen/mol antibody) was three to five times greater than other investigators have reported. In addition, the amount of protein nonspecifically adsorbed to the antibody-coated surface was further reduced by the addition of blocking agents so that nonspecific adsorption of protein antigens represented only 2-6% of the total antigen binding. With this low background, IgG antigen binding could be measured at levels as low as 150 fmol when an antigen concentration of 3 pmol/ml was applied. The process for antibody immobilization is straightforward, easy to perform, and adaptable for modifying mass quantities of biosensor components.     

Controlling the reactivity of ampiphilic quantum dots in biological assays through hydrophobic assembly of custom PEG derivatives

Modifications of the quantum dot (QD) surface are routinely performed via covalent biomolecule attachment, and poly(ethylene glycol) (PEG) derivatization has previously been shown to limit nonspecific cellular interactions of QD probes. Attempts to functionalize ampiphilic QDs (AMP-QDs) with custom PEG derivatives having a hydrophobic terminus resulted in self-assembly of these PEG ligands to the AMP-QD surface in the absence of covalent coupling reagents. We demonstrate, via electrophoretic characterization techniques, that these self-assembled PEG-QDs exhibit improved passivation in biological environments and are less susceptible to unwanted protein adsorption to the QD surface. We highlight the artifactual fluorescent response protein adsorption can cause in biological assays, and discuss considerations for improved small molecule presentation to facilitate specific QD interactions.     

Composite surface for blocking bacterial adsorption on protein biochips

The design and fabrication of protein biochips requires characterization of blocking agents that minimize nonspecific binding of proteins or organisms. Nonspecific adsorption of Escherichia coli, Listeria innocua, and Listeria monocytogenes is prevented by bovine serum albumin (BSA) or biotinylated BSA adsorbed on SiO(2) surfaces of a biochip that had been modified with a C(18) coating. Biotinylated BSA forms a protein-based surface that in turn binds streptavidin. Because streptavidin has multiple binding sites for biotin, it in turn anchors other biotinylated proteins, including antibodies. Hence, biotinylated BSA simultaneously serves as a blocking agent and a foundation for binding an interfacing protein, avidin or streptavidin, which in turns anchors biotinylated antibody. In our case, the antibody is C11E9, an IgG-type antibody that binds Listeria spp. Nonspecific adsorption of another bacterium, Escherichia coli, is also minimized due to the blocking action of the BSA. The blocking characteristics of BSA adsorbed on C(18)-derivatized SiO(2) surfaces for construction of a protein biochip for electronic detection of pathogenic organisms is investigated.     

Protein-liposome conjugates using cysteine-lipids and native chemical ligation

Liposomes have become popular drug delivery vehicles and have more recently also been applied as contrast agents for molecular imaging. Most current methods for functionalization of liposomes with targeting proteins rely on reactions of amine or thiol groups at the protein exterior, which generally result in nonspecific conjugation at multiple sites on the protein. In this study, we present native chemical ligation (NCL) as a general method to covalently couple recombinant proteins in a highly specific and chemoselective way to liposomes containing cysteine-functionalized phospholipids. A cysteine-functionalized phospholipid (Cys-PEG-DSPE) was prepared and shown to readily react with the MESNA thioester of EYFP, which was used as a model protein. Characterization of the EYFP-liposomes using fluorescence spectroscopy showed full retention of the fluorescent properties of conjugated EYFP and provides a lower limit of 120 proteins per liposome. The general applicability of NCL was further tested using CNA35, a collagen-binding protein recently applied in fluorescent imaging of collagen. NCL of CNA35 thioester yielded liposomes containing approximately 100 copies of CNA35 per liposome. The CNA35-liposomes were shown to be fully functional and bind collagen with a 150-fold higher affinity compared to CNA35. Our results show that NCL is an attractive addition to existing conjugation methods that allows direct, covalent, and highly specific coupling of recombinant proteins to liposomes and other lipid-based assemblies.     

Nanoneedle Surface Modification with 2-Methacryloyloxyethyl Phosphorylcholine Polymer to Reduce Nonspecific Protein Adsorption in a Living Cell

Previously, we developed a new molecular delivery system to target single living cells by using atomic force microscope and ultrathin needle referred to as nanoneedle. This system delivers molecules into the cell by attaching them to the surface of nanoneedle. However, nonspecific protein adsorption on the nanoneedle surface inside the living cells limits the range of application of this system. In the present study, we focused on nonspecific protein adsorption onto the nanoneedle surface inside the cells and examined whether this protein adsorption was reduced by modifying the nanoneedle surface with a biocompatible phospholipid polymer containing 2-methacryloyloxyethyl phosphorylcholine (MPC) unit. MPC polymer coating of the surface of silicon wafer reduced nonspecific adsorption of proteins from liver extracts and prevented the formation of clot-like protein aggregates. MPC polymer also decreased nonspecific adsorption of cytosolic protein onto the nanoneedle surface inside the living cell. On the other hand, MPC polymer showed no effect on nonspecific mechanical interaction between nanoneedle and the cell components. Surface modification with MPC polymer is a useful technique to modify the surface properties of nanoneedle.     

Modification of ISFETs with a monolayer of latex beads for specific detection of proteins

The so-called ion-step method is a novel potentiometric approach that can detect protein adsorbed onto the gate area of modified ion-sensitive field-effect transistors (ISFETs). In this report, a generic technology is described for immobilization of peptides and proteins to the ISFET gate in order to confer specific binding properties to the ISFET. For this, the surface of the ISFET was covered with a monolayer of Amino beads (diameter, 0.9 microm) followed by immobilization of protein ligands onto these beads. Amino beads are latex spheres that contain primary amino groups at the outer surface. Preactivation of the latex-bound amino groups with glutaraldehyde, and consecutive incubation with polylysine resulted in covalent immobilization of this polyamine, as revealed by ion stepping measurements. For ImmunoFET applications, human serum albumin (HSA) was immobilized onto the Amino bead-covered ISFETs, by passive adsorption but also by covalent coupling. Resulting devices were used for qualitative detection of alpha-HSA antibodies by means of the ion step method. The binding of antibody was very specific and fast (most of the binding was accomplished in 15 min) with signal yields up to 17 mV. Efforts to increase the antibody-binding capacity of the solid phase on the ISFET exploiting amino group activation (with glutaraldehyde or other homobifunctional cross linkers) before HSA coupling, did not improve signal yield. The bead technology described in this report is an easy, generic method for coating the ISFET with a solid phase that, using the ion-step method, can be applied to immunosensing.     

Photonic immobilization of BSA for nanobiomedical applications: creation of high density microarrays and superparamagnetic bioconjugates

Light assisted molecular immobilization has been used for the first time to engineer covalent bioconjugates of superparamagnetic nanoparticles and proteins. The technology involves disulfide bridge disruption upon UV excitation of nearby aromatic residues. The close spatial proximity of aromatic residues and disulfide bridges is a conserved structural feature in proteins. The created thiol groups bind thiol reactive surfaces leading to oriented covalent protein immobilization. We have immobilized a model carrier protein, bovine serum albumin, onto Fe(3)O(4)@Au core-shell nanoparticles as well as arrayed it onto optically flat thiol reactive surfaces. This new immobilization technology allows for ultra high dense packing of different bio-molecules on a surface, allowing the creation of multi-potent functionalized active new biosensor materials, biomarkers identification and the development of nanoparticles based novel drug delivery system.     

Patterned biofunctional poly(acrylic acid) brushes on silicon surfaces

Protein patterning was carried out using a simple procedure based on photolithography wherein the protein was not subjected to UV irradiation and high temperatures or contacted with denaturing solvents or strongly acidic or basic solutions. Self-assembled monolayers of poly(ethylene glycol) (PEG) on silicon surfaces were exposed to oxygen plasma through a patterned photoresist. The etched regions were back-filled with an initiator for surface-initiated atom transfer radical polymerization (ATRP). ATRP of sodium acrylate was readily achieved at room temperature in an aqueous medium. Protonation of the polymer resulted in patterned poly(acrylic acid) (PAA) brushes. A variety of biomolecules containing amino groups could be covalently tethered to the dense carboxyl groups of the brush, under relatively mild conditions. The PEG regions surrounding the PAA brush greatly reduced nonspecific adsorption. Avidin was covalently attached to PAA brushes, and biotin-tagged proteins could be immobilized through avidin-biotin interaction. Such an immobilization method, which is based on specific interactions, is expected to better retain protein functionality than direct covalent binding. Using biotin-tagged bovine serum albumin (BSA) as a model, a simple strategy was developed for immobilization of small biological molecules using BSA as linkages, while BSA can simultaneously block nonspecific interactions.     

Immobilization of hydrophobic peptidic ligands to hydrophilic chromatographic matrix: a preconcentration approach

This study presents a methodology for covalent attachment of hydrophobic peptidic ligands to hydrophilic chromatographic matrices with improved coupling efficiency. Preconcentration was introduced through the use of polyethylene glycol (PEG)-based crosslinkers. Immobilization of model hydrophobic peptide pep12 (ITLISSEGYVSS) to hydrophilic silica-amine matrix was investigated in the absence/presence of PEG-based linker. The effect of linker densities 14.2, 27.6, and 56.4 μmol/g beads on coupling efficiency was investigated. Whereas a ligand coupling efficiency of 67% was obtained in the absence of the linker, incorporating PEG-based linker at low densities allowed a 30% increase in the coupling efficiency. Although the heterobifunctional crosslinker, maleimide-PEG-NHS (N-hydroxysuccinimide) ester, can be used to couple thiol-bearing ligands to amine-functionalized matrices, no method is available for quenching free amine moieties on the matrix after ligand immobilization. The efficacy of acylating agents, acetyl chloride and oxalyl chloride, in blocking free amine groups when immobilizing the model peptide pep14 (CITLISSEGYVSSK) to silica-amine matrix using maleimide-PEG-NHS ester crosslinker was investigated. Because oxalyl chloride was nonreactive to maleimides, it allowed successful coupling of pep14 to the maleimide termini of the linkers. Adsorption studies between pep14-immobilized microspheres and human immunoglobulin M (hIgM) suggested retention of ligand activity and a 95% decrease in nonspecific binding of proteins to the matrix.     

Ultrathin functional star PEG coatings for DNA microarrays

In this study, star PEG coatings on glass substrates have been used as support material for oligonucleotide microarrays. These coatings are prepared from solutions of six armed star shaped prepolymers that carry reactive isocyanate endgroups. As described earlier, such films prevent the adsorption of proteins and the adhesion of cells but can easily be functionalized for specific biological recognition. Here we used the high functionality of these coatings for the covalent immobilization of amino terminated 20mer oligonucleotides, both by microcontact printing and spotting techniques. The permanent immobilization of fluorescently labeled DNA as well as hybridization of 20mer oligonucleotides have been monitored by fluorescence microscopy. The hybridization efficiency as determined by fluorescence intensity varied from 30% to 80% depending on the way of layer preparation. The direct spotting without additional activation and blocking steps of the surface demonstrates the potential of star PEG coatings as ultrathin surface modification for microarrays.     

Genetically encoding latent bioreactive amino acids and the development of covalent protein drugs

As natural proteins generally do not bind targets in a covalent mode, the therapeutic potential of covalent protein drugs remains largely unexplored. Recently, latent bioreactive amino acids have been incorporated into proteins through genetic code expansion, which selectively react with nearby natural residues via proximity-enabled reactivity, generating diverse covalent linkages for proteins in vitro and in cells. These new covalent linkages provide novel avenues for protein research and engineering. In addition, a general platform technology, proximity-enabled reactive therapeutics (PERx), has been established for the development of covalent protein drugs. The first covalent protein drug demonstrates advantageous features in cancer immunotherapy in mice. Selective introduction of covalent bonds into proteins will advance biological studies, synthetic biology, and biotherapeutics with the power of biocompatible covalent chemistries.     

A method for the comprehensive proteomic analysis of membrane proteins

We describe a method that allows for the concurrent proteomic analysis of both membrane and soluble proteins from complex membrane-containing samples. When coupled with multidimensional protein identification technology (MudPIT), this method results in (i) the identification of soluble and membrane proteins, (ii) the identification of post-translational modification sites on soluble and membrane proteins, and (iii) the characterization of membrane protein topology and relative localization of soluble proteins. Overlapping peptides produced from digestion with the robust nonspecific protease proteinase K facilitates the identification of covalent modifications (phosphorylation and methylation). High-pH treatment disrupts sealed membrane compartments without solubilizing or denaturing the lipid bilayer to allow mapping of the soluble domains of integral membrane proteins. Furthermore, coupling protease protection strategies to this method permits characterization of the relative sidedness of the hydrophilic domains of membrane proteins.     

Multifunctional poly(ethylene glycol) semi-interpenetrating polymer networks as highly selective adhesive substrates for bioadhesive peptide grafting

Novel artificial extracellular matrices were synthesized in the form of semi-interpenetrating polymer networks containing copolymers of poly(ethylene glycol) and acrylic acid (PEG-co-AA) grafted with synthetic bioadhesive peptides onto exposed carboxylic acid moieties. These substrates were very resistant to cell adhesion, but when they were grafted with adhesive peptides they were highly biospecific in their ability to support cell adhesion. Extensive preadsorption of adhesive proteins or peptides did not render these materials cell adhesive; yet covalent grafting of adhesive peptides did render these materials highly cell adhesive even in the absence of serum proteins. Polymer networks containing immobilized PEG-co-AA were grafted with peptides at densities of 475 +/- 40 pmol/cm(2). Polymer networks containing immobilized PEG-co-AA N-terminally grafted with GRGDS supported cell adhesion efficiencies of 42 +/- 4% 4 h after seeding and became confluent after 12 h. These cells displayed cell spreading and cytoskeletal grafted with inactive control peptides (GRDGS, GRGES, or no peptide) supported cell adhesion efficiencies of 0 +/- 0%, even when challenged with high seeding densities (to 100,000 cell/cm(2)) over 14 days. These polymer networks are suitable substrates to investigate in vitro cell-surface interactions in the presence of serum proteins without nonspecific protein adsorption adhesion signals other than those immobilized for study.     

Antibody immobilization on magnetic particles

Magnetic particles (MNPs) offer attractive possibilities in biotechnology. MNPs can get close to a target biological entity, as their controllable sizes range from a few nanometres up to tens of nanometres, and their surface can be modified to add affinity and specificity towards desired molecules. Additionally, they can be manipulated by an external magnetic field gradient. In this work, the study of ferric oxide (Fe3O4) MNPs with different coating agents was conducted, particularly in terms of strategies for antibody attachment at the surfaces (covalent and physical adsorption) and the effects of blocking buffer composition and incubation times on the specific and non-specific interactions observed. The considered biological model system consisted of a coating antibody (goat IgG), bovine serum albumin (BSA) as blocking agent, and a complementary antibody labelled with FITC (anti-goat IgG). The detection of antibody binding was followed by fluorescence microscopy and the intensity of the signals quantified. The ratio between the mean grey values of negative and positive controls, as well as the maximum intensity attainable in positive controls, were considered in the evaluation of the assays efficiency. The covalent immobilization of the coating antibody was more successful as opposed to protein adsorption. For covalent immobilization, silica-coated MNPs, a 5% (w/v) concentration of BSA in the blocking buffer and incubation times of 1 h produced the best results in terms of assay sensitivity. However, when conducting the assay for incubation periods of 10 min, the fluorescence signal was reduced by 44% but the assay specificity was maintained.     

Directed covalent immobilization of aminated DNA probes on aminated plates

A new protocol that enables the immobilization of DNA probes on aminated micro-titer plates activated with aldehyde-dextran via an amino group artificially introduced in the 3' end of the oligonucleotide probe is reported in this work. The method is based on the use of hetero-functional-dextran as a long and multifunctional spacer arm covalently attached to an aminated surface capable of immobilizing DNA oligonucleotides. The immobilization occurred only via the amino introduced in the 3' end of the probe, with no implication of the DNA bases in the immobilization, ensuring that the full length of the probe is available for hybridization. These plates having immobilized oligonucleotide probes are able to hybridize complementary DNA target molecules. The tailor-made hetero-functional aldehyde-aspartic-dextran together with the chemical blocking of the remaining primary amino groups on the support using acetic anhydride avoid the nonspecific adsorption of DNA on the surface of the plates. Using these activated plates, (studying the effect of the probe concentration, temperature, and time of the plate activation on the achieved signal), thus, the covalent immobilization of the aminated DNA probe was optimized, and the sensitivity obtained was similar to that achieved using commercial biotin-streptavidin systems. The new DNA plates are stable under very drastic experimental conditions (90% formamide, at 100 degrees C for 30 min or in 100 mM NaOH).     

Lyotropic salt effects in hydrophobic chromatography.

Pure hydrophobic chromatography can be observed with agarose gels containing caprylyl hydrazide. These nonionic gels show increased avidity in protein adsorption with higher content of caprylyl groups. Lyotropic salt effects can be used to control chromatographic behavior of proteins. Salting-out agents enhance binding of protein to sorbent, while salting-in agents diminish this binding. Based on these findings, the hydrophobic separation of β-lactoglobulin, ovalbumin, and bovine serum albumin is demonstrated.     

Protein immobilization on the surface of liposomes via carbodiimide activation in the presence of N-hydroxysulfosuccinimide

A method of the covalent immobilization of proteins on the surface of liposomes, containing 10% (by mol) of N-glutaryl phosphatidylethanolamine, is described. Carboxylic groups of liposomal N-glutaryl phosphatidylethanolamine were activated in the presence of water-soluble carbodiimide and N-hydroxysulfosuccinimide and reacted subsequently with protein amino groups. The liposome-protein conjugates formed contained up to 5 x 10(-4) mol protein/mol lipid. Lectins (RCA1 and WGA) upon immobilization on liposomes retained saccharide specificity and the ability to agglutinate red blood cells. The immobilization of mouse monoclonal IgG in a ratio of 3.5 x 10(-4) mol IgG/mol lipid was achieved. The liposome activation in the absence of N-hydroxysulfosuccinimide resulted in a 2-fold decrease of protein coupling yields.