Functionalization of thioctic acid-capped gold nanoparticles for specific immobilization of histidine-tagged proteins

This paper presents an efficient strategy for the specific immobilization of fully functional proteins onto the surface of nanoparticles. Thioctic acid-derivatized gold clusters are used as a scaffold for further stepwise modification, leading to a cobalt(II)-terminated ligand shell. A histidine tag introduced by genetic engineering into a protein is coordinated to this transition metal ion. The specific immobilization has been demonstrated for the cases of a genetically engineered horseradish peroxidase and ferredoxin-NADP(+) reductase, confirming the attachment of the fully functional proteins to the Co(II)-terminated nanointerface. The absence of nonspecific protein adsorption and the specificity of the binding site have been verified using several analogues of the enzymes without the histidine tag.     

Macroporous silica in chromatography and immobilization of biopolymers

Macroporous silicas--silica gel (xerogels) and silochroms--are used for the chromatography of proteins and viruses and for the immobilization of several enzymes. The stabilization of silica by aluminum and zirconium salts was investigated. The hydrolytic stability of silica in alkaline media can be significantly increased by the substitution of aluminum ions for hydrogen atoms of the surface silanol groups. The aluminum modification also enhances the stability of glucose isomerase immobilized on silica. The maximum capacity for the immobilization of enzymes is found with silicas having average pore sizes of 5 to 10 times those of the protein globules. A thin layer of pyrolytic carbon deposited on silica enhances the retention of the adsorbed enzyme by the carrier. The original, aluminated and modified by γ-aminopropyltriethoxysilane and carbohydrates silica gels and silochroms are applicable in the liquid chromatography of acid and neutral proteins and in the purification of viruses. Adsorption and steric and ionic exclusion were observed in the chromatography of biopolymers.     

An infrared study of adsorbed metal ions on modified silica: Comparative chelation between mercury, cadmium, and lead divalent ions to silica functionalized with ortho- and para-aminothiophenoles

Chelating selectivity and capacity of silica functionalized 2- and 4-aminothiophenoles (2-ASP-[silica] and 4-ASP-[silica]) toward mercury, lead, and cadmium ions in aqueous medium are studied. In this comparative study, the three metal ions were allowed to interact individually and simultaneously with two aminothiophenol (ASP) derivatives namely, 2- and 4-ASP once as free chelates in solution and secondly as immobilized chelates on silica. Upon individually or simultaneously interacting the three metal ions with 4-ASP-[silica], Hg(II) ions are preferentially adsorbed where 100% of Hg(II) is removed compared to 83.0% of Pb(II) and 76% of Cd(II) ions. In solution, Hg(II) ions are found to be preferentially adsorbed by 2-ASP when compared to 4-ASP. Whereas, anchoring 4-ASP to a silica surface via amide linkage provides a significant enhancement in selectivity and extent of chelation toward Hg(II) over Cd(II) and Pb(II) ions. In the case of 4-ASP-[silica], the existence of a free SH group allows an easy-accessible and strain-free binding site for the incoming Hg(II) ions. Whereas, in 2-ASP-[silica], the SH group is sterically hindered due to proximity to the point of attachment with the surface. As a result, 2-ASP-[silica] showed less potential for Hg(II) binding compared to the modified analogue, 4-ASP-[silica] with less chelation extent observed in solution compared to that observed at the surface.     

Enzyme-encapsulated silica monolayers for rapid functionalization of a gold surface

We report a simple and rapid method for the deposition of amorphous silica onto a gold surface. The method is based on the ability of lysozyme to mediate the formation of silica nanoparticles. A monolayer of lysozyme is deposited via non-specific binding to gold. The lysozyme then mediates the self-assembled formation of a silica monolayer. The silica formation described herein occurs on a surface plasmon resonance (SPR) gold surface and is characterized by SPR spectroscopy. The silica layer significantly increases the surface area compared to the gold substrate and is directly compatible with a detection system. The maximum surface concentration of lysozyme was found to be a monolayer of 2.6 ng/mm(2) which allowed the deposition of a silica layer of a further 2 ng/mm(2). For additional surface functionalization, the silica was also demonstrated to be a suitable matrix for immobilization of biomolecules. The encapsulation of organophosphate hydrolase (OPH) was demonstrated as a model system. The silica forms at ambient conditions in a reaction that allows the encapsulation of enzymes directly during silica formation. OPH was successfully encapsulated within the silica particles and a detection limit for the substrate, paraoxon, using the surface-encapsulated enzyme was found to be 20 microM.     

Application of structural analogs of dimercaptosuccinic acid-functionalized silica nanoparticles (DMSA-[silica]) to adsorption of mercury, cadmium, and lead

In order to gain additional insight into mercury selectivity with nano-sized DMSA-[silica], we investigated a series of ligands related to DMSA, these are: monomercaptosuccinic acid; MMSA, 2-mercapto-4-methyl-5-thiazoleacetic acid; MCT, ortho-thiosalicylic acid; o-TSA and para-thiosalicylic acid; p-TSA. The MMSA chelate is structurally similar to DMSA except that it has only one thiol group. The chelates o-TSA and p-TSA each have one thiol and one carboxylic acid group. MCT includes neutral S and N atoms in close proximity to the thiolate binding site. MCT, o-TSA and p-TSA resemble each other in having equal numbers of carboxylic acid and thiol groups and formation of amide bonds with the linker on silica is expected to eliminate the carboxylate binding sites, making thiolates the only binding sites for Hg(II), Cd(II), and Pb(II) metals ions in the nano-sized; MMSA-[silica], MCT-[silica], o-TSA-[silica], and p-TSA-[silica]. Each of the nano-sized MMSA-[silica], MCT-[silica], o-TSA-[silica], and p-TSA-[silica], show a higher preference for Hg(II) over Cd(II) and Pb(II) compared to the same free chelates in solution, respectively. In addition, there are differences in the level of metal ion chelation for each functionalized silica nanoparticle. These differences in the degree of metal chelation for each functionalized silica nanoparticles surface are explained by the difference in thiolate/carboxylate ratio upon attachment to the surface and on steric reasons based on the orientation of the thiol groups on the surface. When attached via amide bond formation, the thiolate site in o-TSA-[silica] will face towards the silica surface, while for p-TSA-[silica], the thiolate site is expected to be pointed outwards and away from the silica surface. In comparing MMSA-[silica] to DMSA-[silica], the thiolate/carboxylate ratio decreases from 2/1 in DMSA-[silica] to 1/1 in MMSA-[silica] (assuming attachment via one amide bond in each case). This effect of increasing the ratio of thiolate to carboxylate upon attachment to the surface is believed to play a role in the selectivity enhancement towards Hg(II) over Cd(II) and Pb(II).     

Fl-DFO molecules@mesoporous silica materials: highly sensitive and selective nanosensor for dosing with iron ions

Highly sensitive and selective nanosensor for labile iron pool (LIP) determination, has been designed and prepared by immobilization of Fluoresceine-Desferrioxamine (Fl-DFO), a bifunctional fluoro-siderophore probe molecule with great affinity for iron ions (pKf=30.7), into highly ordered mesoporous silica structure. Different immobilization methods of Fl-DFO molecules, such as their encapsulation in surfactant micelles used as templating agents for the synthesis of mesoporous silica, direct impregnation into the mesochannels of as-synthesized mesoporous silica and their surface anchoring by covalent binding with propylamine groups implanted by post-synthesis on the internal surface of mesochannels, have been explored. Each nanohybrid has been fully characterized by small angle XRD, TEM, SEM, solid state (29)Si and (13)C MAS NMR and N(2) adsorption-desorption. The fluorescence properties of nanohybrids obtained have been correlated with the immobilization methods, generating interesting information concerning the localization of Fl-DFO molecules in the channels of mesoporous silica. The leaching of Fl-DFO molecules from mesoporous materials has been investigated. The nanosensor prepared by surface anchoring of Fl-DFO at the internal surface of mesochannels showed high performances with no leaching effect and high sensitivity in regards to its responses to ferric ions. Its fluorescence intensity decreased as soon as first Fe(III) ions are in contact with this nanosensor. A linear relationship between the fluorescence intensity and the ferric ions concentration was observed in low micromolar range. The selectivity of this nanosensor towards other metal ions has also been tested and shown its high affinity to ferric ions. This study can allow the design of a stable, portable, simple, regenerable and cost-effective nanosensor highly sensitive and selective for iron ions with detection limits in the range of cellular LIP in cells, e.g. lower micromolar range.     

Arginine- and lysine-specific polymers for protein recognition and immobilization

Free radical polymerization of methacrylamide-based bisphosphonates turns weak arginine binders into powerful polymeric protein receptors. Dansyl-labeled homo- and copolymers with excellent water solubility are accessible through a simple copolymerization protocol. Modeling studies point to a striking structural difference between the stiff rodlike densely packed homopolymer 1 and the flexible copolymer 2 with spatially separated bisphosphonate units. Fluorescence titrations in buffered aqueous solution (pH = 7.0) confirm the superior affinity of the homopolymer toward oligoarginine peptides reaching nanomolar K(D) values for the Tat peptide. Basic proteins are bound almost equally well by 1 and 2 with micromolar affinities, with the latter producing much more soluble complexes. The Arg selectivity of the monomer is transferred to the polymer, which binds Arg-rich proteins 1 order of magnitude tighter than lysine-rich pendants of comparable pI, size, and (Arg/Lys vs Glu/Asp) ratio. Noncovalent deposition of both polymers on glass substrates via polyethyleneimine layers results in new materials suitable for peptide and protein immobilization. RIfS measurements allow calculation of association constants K(a) as well as dissociation kinetics k(D). They generally confirm the trends already found in free solution. Close inspection of electrostatic potential surfaces suggest that basic domains favor protein binding on the flat surface. The high specificity of the bisphosphonate polymers toward basic proteins is demonstrated by comparison with polyvinyl sulfate, which has almost no effect in RIfS experiments. Thus, copolymerization of few different comonomer units without cross-linking enables surface recognition of basic proteins in free solution as well as their effective immobilization on surfaces.     

Calmodulin-mediated reversible immobilization of enzymes

This work demonstrates the use of the protein calmodulin, CaM, as an affinity tag for the reversible immobilization of enzymes on surfaces. Our strategy takes advantage of the of the reversible, calcium-mediated binding of CaM to its ligand phenothiazine and of the ability to produce fusion proteins between CaM and a variety of enzymes to reversibly immobilize enzymes in an oriented fashion to different surfaces. Specifically, we employed two different enzymes, organophosphorus hydrolase (OPH) and beta-lactamase and two different solid supports, a silica surface and cellulose membrane modified by covalently attaching a phenothiazine ligand, to demonstrate the versatility of our immobilization method. Fusion proteins between CaM-OPH and CaM-beta-lactamase were prepared by using genetic engineering strategies to introduce the calmodulin tail at the N-terminus of each of the two enzymes. In the presence of Ca(2+), CaM adopts a conformation that favors interaction between hydrophobic pockets in CaM and phenothiazine, while in the presence of a Ca(2+)-chelating agent such as EGTA, the interaction between CaM and phenothiazine is disrupted, thus allowing for removal of the CaM-fusion protein from the surface under mild conditions. CaM also acts as a spacer molecule, orienting the enzyme away from the surface and toward the solution, which minimizes enzyme interactions with the immobilization surface. Since the method is based on the highly selective binding of CaM to its phenothiazine ligand, and this is covalently immobilized on the surface, the method does not suffer from ligand leaching nor from interference from other proteins present in the cell extract. An additional advantage lies in that the support can be regenerated by passing through EGTA, and then reused for the immobilization of the same or, if desired, a different enzyme. Using a fusion protein approach for immobilization purposes avoids the use of harsh conditions in the immobilization and/or regeneration steps, which could cause inactivation of the immobilized enzyme. Moreover, we have demonstrated that the CaM affinity tag allows immobilization of enzymes on a variety of surfaces without compromising their enzymatic activity substantially; for example, the immobilized OPH retained more than 80% of the activity of the free enzyme. Our results with beta-lactamase showed the feasibility of using a phenothiazine surface in several consecutive loading and regeneration cycles. This can be advantageous when expensive and/or difficult to obtain immobilization surfaces have to be employed; the immobilization surface could be reused to immobilize the same or a different enzyme using the CaM affinity tail. We also determined that the phenothiazine-modified silica particles are stable for long periods of time, i.e., up to 2 years when stored at 4 degrees C. It is envisioned that this type of reversible immobilization may find applications in the development of reversible, reusable biosensors and bioreactors endowed with the additional advantage that the biological element at the surface of the sensor or bioreactor could be replaced under mild conditions when needed to sense or process a different target molecule.     

Oriented immobilization of proteins

Immobilized enzymes have found numerous applications in analytical, clinical, environmental and industrial chemistry. However, in most cases, immobilization leads to partial or total loss of activity. It is widely believed that the loss in activity is due to attachment of proteins on the immobilization support through several amino acid residues. This results in a random orientation of the immobilized protein and in increased structural deformation due to multi-point attachment. Several researchers have explored ways to orient proteins on surfaces, such that orderly organization, single point attachment and accessibility of the active site (or binding site) are possible. This article reviews the various approaches available to achieve oriented immobilization of proteins and its applications in several disciplines.     

Conjugated polyelectrolyte amplified fluorescent assays with probe functionalized silica nanoparticles for chemical and biological sensing

Low-cost sensors with high sensitivity and selectivity for chemical and biological detection are of high scientific and economic importance. Silica nanoparticles (NPs) have shown vast promise in sensor applications by virtue of their controllable surface modification, good chemical stability, and biocompatibility. This mini-review summarizes our recent development of silica NP-based assays for chemical and biological detection, where silica NPs serve as the substrate for probe immobilization, target recognition, and separation. The assay performance is further improved through the introduction of conjugated polyelectrolyte to amplify the detection signal. The assays have been demonstrated to be successful for the detection of DNA, small molecules, and proteins. They could be generalized for other targets based on specific interactions, such as DNA hybridization, antibody-antigen recognition, and target-aptamer binding.     

Encapsulation of the ferritin protein in sol-gel derived silica glasses

A significant recent development in sol-gel science has been the encapsulation of biomolecules such as proteins and enzymes in optically transparent silica glasses. This paper reports on the encapsulation of an iron (Fe) storage protein, ferritin, to develop a magnetic silica glass. Native ferritin, which has a nanometer-sized microcrystalline Fe oxide core, was encapsulated in optically transparent silica glasses using the sol-gel process. Fe could be released from ferritin but could not be reconstituted into apoferritin when the protein was trapped in the pores of the glass. Transmission electron microscopy of ferritin-doped aged silica gels indicated that crystallinity of the Fe oxide core was retained upon sol-gel encapsulation. Magnetic measurements on ferritin-doped silica gels indicated the material to be paramagnetic, but not superparamagnetic.     

Immobilization of binding proteins on nonporous supports

Four different nonporous particulate materials, nylon, polystyrene, soda-lime silicate glass, and fused silica glass, have been evaluated for their appropriateness as immobilization supports for immunoglobulins. A method of protein quantitation that is usually applied to solutions, the bicinchoninic acid (BCA) assay, was used successfully to directly measure ng amounts of protein immobilized on the supports. Two proteins, a monoclonal antibody to theophylline and the biotin binding protein avidin, were studied. Radioactive theophylline and radioactive biotin were used to measure the activity of the immobilized protein. Ligand binding capacity per mm2 of support was measured as a function of amount of protein immobilized. By measuring both the amount of protein immobilized and its ligand binding capacity, we have determined that antitheophylline antibody adsorbed on polystyrene balls loses almost 90% of its binding activity after 65 h, although little protein is lost from the balls over this time. Avidin retains nearly full activity for biotin on polystyrene. The binding activity of biotinyl-antibody conjugate immobilized on avidin-adsorbed polystyrene is stable, even when stored for over 22 wk. Antibody covalently immobilized on soda-lime silicate glass beads retains its binding activity over long-term storage, although only 0.1 mol of 3H-theophylline bind per mol of immobilized antibody. Using fused silica glass particles as the solid support, the same antibody binds approx 0.6 mol of ligand per mol of immobilized antibody protein. The structural "softness" of the immunoglobulin requires that interaction with the surface be prevented in order to maintain activity.     

Influence of chemical nature of surface and geometrical characteristics of silica matrices on immobilization of proteins

The immobilization patterns of enzymes on the surface of dispersed silicas were studied in order to obtain active heterogeneous preparations. The chemical nature of the activated silica matrices used have practically no influence on the optimal pH value of the immobilization and time of completion of this process, but determines mainly the degree of retention of activity of the grafted biocatalysts. The geometrical characteristics of the carriers influence to a great extent the rate of binding of the enzymes with the carriers and their capacity with respect to the protein.Translated from Teoreticheskaya i Eksperimental'naya Khimiya, Vol. 26, No. 2, pp. 201–209, March–April, 1990.     

Aspects of surface modification, structure characterization, thermal stability and metal selectivity properties of silica gel phases-immobilized-amine derivatives

Four silica gel phases-bound-amine derivatives (I-IV) were prepared based on chemical immobilization technique. The surface modification was identified by determination of the coverage values in mmol g⁻¹ via thermal desorption method (1.463-1.807) and elemental analysis of nitrogen and carbon contents (1.089-2.456). Structure characterization related to immobilization of the amine derivatives was accomplished and evaluated by means of infrared (IR) and secondary ion mass spectrometric (SIMS) technique. The modified silica gel phases (I-IV) along with their interaction products with copper(II) were also examined by electron impact mass spectrometric analysis (EI-MS) as a method for evaluation of their thermal stability and structure elucidation. Potentiometric titration as a method of characterization was applied for the modified silica gel phases (II-IV) and their copper(II)-adduct. A series of bi- and trivalent metal ions were selected to focus more aspects of the selectivity properties incorporated into the modified silica gel phases for binding and interaction with these metals based on determination of the distribution coefficient and separation factor. The results of these evaluation processes were found to prove higher selectivity and preference of these four phases for binding with lead(II) and cadmium(II) compared to other metal ions.     

Enzyme-functionalized mesoporous silica for bioanalytical applications

The unique properties of mesoporous silica materials (MPs) have attracted substantial interest for use as enzyme-immobilization matrices. These features include high surface area, chemical, thermal, and mechanical stability, highly uniform pore distribution and tunable pore size, high adsorption capacity, and an ordered porous network for free diffusion of substrates and reaction products. Research demonstrated that enzymes encapsulated or entrapped in MPs retain their biocatalytic activity and are more stable than enzymes in solution. This review discusses recent advances in the study and use of mesoporous silica for enzyme immobilization and application in biosensor technology. Different types of MPs, their morphological and structural characteristics, and strategies used for their functionalization with enzymes are discussed. Finally, prospective and potential benefits of these materials for bioanalytical applications and biosensor technology are also presented. Figure Enzyme-functionalized mesoporous silica fibers and their integration in a biosensor design. The immobilization process takes place essentially in the silica micropores.     

Biochemically functionalized silica nanoparticles

In this report, we demonstrate the biochemical modification of silica based nanoparticles. Both pure and dye-doped silica nanoparticles were prepared, and their surfaces were modified with enzymes and biocompatible chemical reagents that allow them to function as biosensors and biomarkers. The nanoparticles produced in this work are uniform in size with a 1.6% relative standard deviation. They have a pure silica surface and can thus be modified easily with many biomolecules for added biochemical functionality. Specifically, we have modified the nanoparticle surfaces with enzyme molecules (glutamate dehydrogenase (GDH) and lactate dehydrogenase (LDH)) and a biocompatible reagent for cell membrane staining. Experimental results show that the silica nanoparticles are a good biocompatible solid support for enzyme immobilization. The immobilized enzyme molecules on the nanoparticle surface have shown excellent enzymatic activity in their respective enzymatic reactions. The nanoparticle surface biochemical functionalization demonstrates the feasibility of using nanoparticles for biosensing and biomarking applications.     

Mesoporous silica nanoreactors for highly efficient proteolysis

Protein digestion inside the nanoreactor channels of mesoporous silica (SBA-15) is reported, and evaluated by using peptide-mass mapping. Both proteases and substrates were efficiently captured within these biocompatible nanoreactors. After 10 minutes, the mass spectrum of the protein digests released from the mesoporous-silica-based nanoreactors revealed the presence of eight peptides covering 58% of the protein sequence with an intense signal (signal/noise ratio > 70). In comparison, the conventional overnight in-solution digestion of proteins under otherwise identical conditions generated only three peptides (27% sequence coverage). We propose that this order-of-magnitude increase in the proteolytic reaction rate is mainly attributed to two factors: substrate enrichment within mesoporous silica channels and enzyme immobilization. The surface properties and macrostructure of the mesoporous silica were studied to reveal their significant influence on proteolytic reactions.     

A spatially resolved nucleic acid biochip based on a gradient of density of immobilized probe oligonucleotide

The potential for a new biochip design based on a continuous gradient of density of immobilized single-stranded DNA oligonucleotide probes (ssDNA) is explored. This gradient resolved information platform (GRIP) can provide sequence identification based on the spatial location and extent of hybridization by a target sequence. Surfaces based on indium-tin oxide (ITO) on glass were first functionalized by 3-aminopropyltriethoxysilane (APTES) followed by attachment of glutaraldehyde, prior to immobilization of oligonucleotide probe that was terminated with amine. The use of Cy₃ and Cy₅ dye-labelled ssDNA probes and targets allowed estimation of density and correlation of the location of binding of labelled targets. Probe molecules of 20 mer lengths were loaded to produce density gradients in the range of 1.0-200 ng/mm². The biochips could resolve a mixture of fully complementary five base-pair mismatched targets by the location of binding on the surface. Thermal control provided additional selectivity. Thermal cycling and washing provided for regeneration of the surface, and the fluorescence intensities showed no deterioration in at least five cycles of hybridization reactions.     

Immobilization of superoxide dismutase onto ordered mesoporous silica nanoparticles and improvement of its stability

In recent years, greater attention has been given to the application of mesoporous materials to the immobilization of enzymes. In this study, the immobilization of superoxide dismutase (SOD) onto the amine-functionalized cubic Ia3d mesoporous silica nanoparticles ([n-PrNH₂-KIT-6]) with an average pore diameter of around 6.5?nm was investigated. This organo-functionalized mesoporous silica was prepared using a non-ionic surfactant and was fully characterized by SEM, XRD, nitrogen adsorption?Cdesorption isotherm and FT-IR spectroscopy. UV?Cvisible spectroscopy studies demonstrated that the immobilized SOD was less prone to thermally induced aggregation than the free moiety. Further investigations using far-circular dichroism measurement intensity indicated that the structure of SOD before and after immobilization onto the [n-PrNH₂-KIT-6] nanoparticles was almost identical and the immobilized enzyme was more stable against GdHCl denaturation relative to the free one.     

Organic–inorganic hybrid silica as supporting matrices for selective recognition of bovine hemoglobin via covalent immobilization

The synthesis of poly‐aminophenylboronic acid (APBA) imprinted hybrid silica‐based polymers for selective recognition of bovine hemoglobin (BHb) was described, where the mesoporous hybrid silica supporting matrices were prepared by a mild sol–gel process with tetraethoxysilane and 3‐aminopropyltriethoxysilane as two precursors. Covalent immobilization of BHb was adopted in order to create homogeneous recognition sites. After removal of the template, the resulting imprinted polymers showed high binding affinity toward BHb and the imprinting factor (α) reached 2.12. The specificity of the BHb recognition was evaluated with competitive experiments, indicating the imprinted polymers have a higher selectivity for the template BHb. The easy preparation protocol and good protein recognition properties made the approach an attractive solution to depletion of high‐abundance protein from bovine blood.