Enzyme solid-state support assays: a surface plasmon resonance and mass spectrometry coupled study of immobilized insulin degrading enzyme

Solid-support based assays offer several advantages that are not normally available in solution. Enzymes that are anchored on gold surfaces can interact with several different molecules, opening the way to high throughput array format based assays. In this scenario, surface plasmon resonance (SPR) and mass spectrometry (MS) investigations have often been applied to analyze the interaction between immobilized enzyme and its substrate molecules in a tag-free environment. Here, we propose a SPR-MS combined experimental approach aimed at studying insulin degrading enzyme (IDE) immobilized onto gold surfaces and its ability to interact with insulin. The latter is delivered by a microfluidic system to the IDE functionalized surface and the activity of the immobilized enzyme is verified by atmospheric pressure/matrix assisted laser desorption ionization (AP/MALDI) MS analysis. The SPR experiments allow the calculation of the kinetic constants involved for the interaction between immobilized IDE and insulin molecules and evidence of IDE conformational change upon insulin binding is also obtained.     

Nature of immobilized antibody layers linked to thioctic acid treated gold surfaces

Utilization of 125I-labeled IgG enables an investigation of protein immobilized to gold electrodes sputter deposited on microporous nylon membranes, including the precise nature of the surface-protein bond (i.e. covalent or non-specific adsorption), physical location of the immobilized protein (i.e. on the surface of the gold electrode or within the pores of the membrane), and the amount of protein immobilized. This is accomplished by comparing the mass of protein immobilized to gold surfaces that have been treated in several different fashions, as well as, deposition of the gold on nylon membranes that have been treated differently. It is shown that these microporous gold electrodes, proposed previously for conducting novel non-separation electrochemical enzyme immunoassays, consist of multiple protein layers non-specifically adsorbed. Approximately, half of the total adsorbed protein is immobilized to the gold surface with the remaining protein bound within the pores on the nylon membrane.     

Development of a whole-cell biosensor by cell surface display of a gold-binding polypeptide on the gold surface

Cell surface display was used as a strategy to display the gold-binding polypeptide (GBP) fusion protein on the surface of Escherichia coli , and consequently to immobilize the cells on the gold surface. The DNA encoding the GBP was fused to the truncated fadL gene and was expressed by the tac promoter. For the display of the core streptavidin (cSA) of Streptomyces avidinii , the cSA gene was fused to the truncated oprF gene. After the dual display of FadL–GBP and OprF–cSA on the surface of E. coli , binding of cells on the gold surface and the interaction of OprF–cSA with the biotin–horseradish peroxidase (HRP) were studied by surface plasmon resonance (SPR) analysis. Cells displaying the FadL–GBP fusion protein could be immobilized on the SPR sensor chip as shown by the SPR angle shift of 0.5°, which was stably bound at least for 60 h with a washing solution. When the FadL–GBP and OprF–cSA fusion proteins were displayed on the same cell surface, the former was used to immobilize the cells on the gold surface and the latter was used for the interaction studies with the biotin–HRP, which demonstrates that the strategy should be useful for developing whole-cell biosensor chips.     

Enhancing the reusability of endoglucanase-gold nanoparticle bioconjugates by tethering to polyurethane microspheres

The synthesis of polyurethane microsphere-gold nanoparticle "core-shell" structures and their use in the immobilization of the enzyme endoglucanase are described. Assembly of gold nanoparticles on the surface of polymer microspheres occurs through interaction of the nitrogens in the polymer with the nanoparticles, thereby precluding the need for modifying the polymer microspheres to enable such nanoparticle binding. Endoglucanse could thereafter be bound to the gold nanoparticles decorating the polyurethane microspheres, leading to a highly stable biocatalyst with excellent reuse characteristics. The immobilized enzyme retains its biocatalytic activity and exhibits improved thermal stability relative to free enzyme in solution. The high surface area of the host gold nanoparticles renders the immobilized enzyme "quasi free", while at the same time retaining advantages of immobilization such as ease of reuse, enhanced temporal and thermal stability, etc.     

Electrospun gold nanofiber electrodes for biosensors

A new form of high surface area bioelectrode, based on nanofibers of electrospun gold with immobilized fructose dehydrogenase, was developed. The gold fibers were prepared by electroless deposition of gold nanoparticles on an electrospun poly(acrylonitrile)-HAuCl(4) fiber. The material was characterized using electron microscopy, XRD and BET, as well as cyclic voltammetry and biochemical assay of the immobilized enzyme. The electrochemical surface area of the gold microfibers was 0.32 ± 0.04 m(2)/g. Fructose dehydrogenase was covalently coupled to the gold surface through glutaraldehyde crosslinks to a cystamine monolayer. The enzyme exhibited mediated electron transfer directly to the gold electrode and catalytic currents characteristic of fructose oxidation in the presence of a ferrocene methanol mediator were observed. The limit of detection of fructose was 11.7 μM and the K(M) of the immobilized enzyme was 5mM. The microfiber electrode was stable over 20 cycles with a 3.05% standard deviation. The response time of the sensor was less than 2.2s and reached half maximum value within 3.6s. The sensor was proven to be accurate and precise in both serum and popular beverages sweetened with high fructose corn syrup. The addition of glucose isomerase enabled the sensor to perform with glucose, thus expanding the available analyte selection for the sensor.     

A surface plasmon resonance probe with a novel integrated reference sensor surface

A surface plasmon resonance (SPR) sensor probe with integrated reference surface is described. In order to fabricate the integrated reference surface, two dielectric layers with different thickness were deposited on the single gold SPR sensor surface via plasma polymerization of hexamethyldisiloxane. The working sensor surface was a 34 nm dielectric layer with immobilized bovine serum albumin (BSA) antigen and an adjacent thin 1 nm dielectric layer without BSA provided reference surface. A specific immunoreaction of anti-BSA antibody was detected after immersion of the SPR probe into sample solution. Simultaneous observation of reference and working surface response enabled determination of the immunoreaction without the need for the baseline measurement. Moreover, compensation of nonspecific adsorption could be confirmed using anti-human serum albumin antibody.     

Characterization and application of a surface modification designed for QCM-D studies of biotinylated biomolecules

The rapid development of surface sensitive biosensor technologies, especially towards nanoscale devices, requires increasing control of surface chemistry to provide reliable and reproducible results, but also to take full advantage of the sensing opportunities. Here, we present a surface modification strategy to allow biotinylated biomolecules to be immobilized to gold coated sensor crystals for quartz crystal microbalance with dissipation monitoring (QCM-D) sensing. The unique feature of QCM-D is its sensitivity to nanomechanical (viscoelastic) properties at the sensing interface. The surface modification was based on mixed monolayers of oligo(ethylene glycol) (OEG) disulfides, with terminal -OH or biotin groups, on gold. Mixtures containing 1% of the biotin disulfide were concluded to be the most appropriate based on the performance when streptavidin was immobilized to biotinylated sensors and the subsequent biotinylated bovine serum albumin (BSA) interaction was studied. The OEG background kept the unspecific protein binding to a minimum, even when subjected to serum solutions with a high protein concentration. Based on characterization by contact angle goniometry, ellipsometry, and infrared spectroscopy, the monolayers were shown to be well-ordered, with the OEG chains predominantly adopting a helical conformation but also partly an amorphous structure. Storage stability was concluded to depend mainly on light exposure while almost all streptavidin binding activity was retained when storing the sensors cold and dark for 8 weeks. The surface modification was also tested for repeated antibody-antigen interactions between BSA and anti-BSA (immobilized to biotinylated protein A) in QCM-D measurements lasting for >10h with intermediate basic regeneration. This proved an excellent stability of the coating and good reproducibility was obtained for 5 interaction cycles. With this kind of generic surface modification QCM-D can be used in a variety of biosensing applications to provide not only mass but also relevant information of the structural properties of adlayers.     

Assembling of engineered IgG-binding protein on gold surface for highly oriented antibody immobilization

The B-domain, which is one of IgG-binding domains of staphylococcal protein A, was repeated five times and a cysteine residue was introduced at its C-terminus by a genetic engineering technique. The resulting protein, designated B5C1, retained the same IgG-binding activity as native protein A. The B5C1 was assembled on a gold plate surface by utilizing a strong affinity between thiol of cysteine and a gold surface. IgG-binding activity of B5C1 on a gold surface was much higher than that of physically adsorbed B5, which lacks cysteine residue. Furthermore, antigen-binding activity of immobilized antibody molecules through the use of assembled B5C1 on a gold surface was about 4.3 times higher than that of physically adsorbed antibody molecules. Immobilization of highly oriented antibody molecules was realized with the engineered IgG-binding protein.     

Application of response surface methodology to optimization of the fixation of ribonuclease a on glutaraldehyde activated amine silica

Summary We have used response surface methodology to study the interactions between various parameters (pH, temperature, enzyme concentration) controlling the immobilization of ribonuclease A on to glutaraldehyde activated-amine Spherosil beads. The optimum activity of immobilized ribonuclease A was observed at pH=9.1. The specific activity of the immobilized enzyme was affected by the pH and by the enzyme concentration.     

High-throughput analysis of GST-fusion protein expression and activity-dependent protein interactions on GST-fusion protein arrays with a spectral surface plasmon resonance biosensor

We modified gold arrays with a glutathione (GSH) surface, and investigated high-throughput protein interactions with a spectral surface plasmon resonance (SPR) biosensor. We fabricated the GSH exterior on gold surfaces by successive modification with aminoethanethiol, 4-maleimidobutyric acid N-hydroxysuccinimide ester and GSH. We immobilized GST-Rac1, GST-RhoA, the GST-Rho-binding domain of rhotekin and the GST-p21-binding domain of PAK1 onto the GSH surface, and observed specific antigen-antibody interactions on the GST-fusion protein arrays. We determined the expression of GST-fusion proteins in Escherichia coli on the GSH surface with the SPR biosensor. We then analyzed the interactions of tissue transglutaminase (tTGase), a Ca2+-dependent enzyme, with RhoA and Rac1 on the GST-fusion protein arrays with the SPR biosensor. We found that tTGase interacted with RhoA and Rac1 in a Ca2+-dependent manner, indicating that the interactions were dependent on tTGase activity. In addition, transamidation of Rac1 by tTGase was dependent on Ca2+ concentration. We obtained similar results with GST pull-down assays. Thus, protein arrays prepared on the GSH surface provide a useful system for the high-throughput analysis of GST-fusion protein expression and activity-dependent protein interactions with the spectral SPR biosensors.     

Immobilization of lipase by ultrafiltration and cross-linking onto the polysulfone membrane surface

In this study, a biphasic enzymatic membrane reactor was made by immobilizing Candida Rugosa lipase onto the dense surface of polysulfone ultrafiltration membrane by filtration and then cross-linking with glutaraldehyde solution. The reactor was further applied for the hydrolysis of olive oil, the performance of which was evaluated in respect of apparent reaction rate based on the amount of fatty acids extracted into the aqueous phase per minute and per membrane surface. It was found that the ultrafiltration and cross-linking process greatly improved the reaction rate per unit membrane area and the enzyme lifetime. The highest reaction rate reached 0.089 micromol FFA/min cm2 when the enzyme loading density was 0.098 mg/cm2. The results also indicated that the performance of lipase immobilized on the membrane surface was superior to that immobilized in the pores, and the apparent reaction rate and stability of immobilized lipases were improved greatly after cross-linking. It suggested that immobilization of enzymes by filtration and then cross-linking the enzymes onto the membrane surface is a simple and convenient way to prepare a high-activity immobilized enzyme membrane.     

Purification and characterization of a glycoprotein from the surface of Ascaridia galli

Employing papain as the enzyme and agarose bound Ricinus communis agglutinin as the affinity gel, a glycoprotein has been isolated and purified from the surface of Ascaridia galli. The glycoprotein shows an apparent molecular weight of 68 kilo daltons and contains fucose, galactose, rhamnose and glucosamine as sugar moieties. Only 2% of its entire molecule has been found to possess alpha-helical configuration.     

Cytometry on a chip: cellular phenotypic and functional analysis using grating-coupled surface plasmon resonance

Grating-coupled surface plasmon resonance imaging (GCSPRI) is a method for the accurate assessment of both cell phenotype and function. In GCSPRI, cells and/or proteins of interest are flowed across antibodies immobilized on a gold-coated sensor chip. The surface of the chip is illuminated with monochromatic light that couples with surface plasmons in the gold. At a specific angle of incidence, the GCSPR angle, the maximum amount of coupling occurs. Shifts in the GCSPR angle can be correlated with refractive index changes following cell or analyte capture by the immobilized antibodies. In addition, GCSPRI can image the cells as they are being captured. GCSPRI's multiplexed format allows for the parallel assessment of up to 400 individual antibody regions. In this paper, we demonstrate GCSPRI's ability to identify cells and proteins of interest and compare results to a traditional flow cytometry system. This technology represents a fast and powerful method for the simultaneous assessment of cell phenotype and function.     

Immobilization of glucoamylase on ceramic membrane surfaces modified with a new method of treatment utilizing SPCP-CVD

Glucoamylase, as a model enzyme, was immobilized on a ceramic membrane modified by surface corona discharge induced plasma chemical process-chemical vapor deposition (SPCP-CVD). Characterizations of the immobilized enzyme were then discussed. Three kinds of ceramic membranes with different amounts of amino groups on the surface were prepared utilizing the SPCP-CVD method. Each with 1-time, 3-times and 5-times surface modification treatments and used for supports in glucoamylase immobilization. The amount of immobilized glucoamylase increased with the increase in the number of surface modification treatments and saturated to a certain maximum value estimated by a two-dimensional random packing. The operational stability of the immobilized glucoamylase also increased with the increase in the number of the surface treatment. It was almost the same as the conventional method, while the activity of immobilized enzyme was higher. The results indicated the possibility of designing the performance of the immobilized enzyme by controlling the amount of amino groups. The above results showed that the completely new surface modification method using SPCP was effective in modifying ceramic membranes for enzyme immobilization.     

Three-dimensional immobilization of beta-galactosidase on a silicon surface

Many alternative strategies to immobilize and stabilize enzymes have been investigated in recent years for applications in biosensors. The entrapment of enzymes within silica-based nanospheres formed through silicification reactions provides high loading capacities for enzyme immobilization, resulting in high volumetric activity and enhanced mechanical stability. Here we report a strategy for chemically associating silica nanospheres containing entrapped enzyme to a silicon support. beta-galactosidase from E. coli was used as a model enzyme due to its versatility as a biosensor for lactose. The immobilization strategy resulted in a three-dimensional network of silica attached directly at the silicon surface, providing a significant increase in surface area and a corresponding 3.5-fold increase in enzyme loading compared to enzyme attached directly at the surface. The maximum activity recovered for a silicon square sample of 0.5 x 0.5 cm was 0.045 IU using the direct attachment of the enzyme through glutaraldehyde and 0.16 IU when using silica nanospheres. The immobilized beta-galactosidase prepared by silica deposition was stable and retained more than 80% of its initial activity after 10 days at 24 degrees C. The ability to generate three-dimensional structures with enhanced loading capacity for biosensing molecules offers the potential to substantially amplify biosensor sensitivity.     

A real-time PCR-based method for determining the surface coverage of thiol-capped oligonucleotides bound onto gold nanoparticles

Here we report a real-time PCR-based method for determining the surface coverage of dithiol-capped oligonucleotides bound onto gold nanoparticles alone and in tandem with antibody. The detection of gold nanoparticle-bound DNA is accomplished by targeting the oligonucleotide with primer and probe binding sites, amplification of the oligonucleotide by PCR, and real-time measurement of the fluorescence emitted during the reaction. This method offers a wide dynamic range and is not dependant on the dissociation of the oligonucleotide strands from the gold nanoparticle surface; the fluorophore is not highly quenched by the gold nanoparticles in solution during fluorescence measurements. We show that this method and a fluorescence-based method give equivalent results for determining the surface coverage of oligonucleotides bound onto 13 or 30 nm gold nanoparticles alone and in tandem with antibody. Quantifying the surface coverage of immobilized oligonucleotides on metallic nanoparticle surfaces is important for optimizing the sensitivity of gold nanoparticle-based detection methods and for better understanding the interactions between thiol-functionalized oligonucleotides and gold nanoparticles.     

Immobilization of immunoglobulin-G-binding domain of Protein A on a gold surface modified with biotin ligase

Protein A from Staphylococcus aureus specifically binds to the Fc region of immunoglobulin G (IgG) and is widely used as a scaffold for the immobilization of IgG antibodies on solid supports. It is known that the oriented immobilization of Protein A on solid supports enhances its antibody-binding capability in comparison with immobilization in a random manner. In the current work, we developed a novel method for the oriented immobilization of the IgG-binding domain of Protein A based on the biotinylation reaction from archaeon Sulfolobus tokodaii. Biotinylation from S. tokodaii has a unique property in that the enzyme, biotin protein ligase (BPL), forms a stable complex with its biotinylated substrate protein, biotin carboxyl carrier protein (BCCP). Here, BCCP was fused to the IgG-binding domain of Protein A, and the resulting fusion protein was immobilized on the BPL-modified gold surface of the sensor chip for quartz crystal microbalance through complexation between BCCP and BPL. The layer of the IgG-binding domain prepared in this way successfully captured the antibody, and the captured antibody retained high antigen-binding capability.     

alpha-D-galactose-bearing glycoproteins on the surface of stimulated murine peritoneal macrophages. Biochemical and immunochemical characterization of purified glycoproteins.

Two glycoproteins were isolated from lysates of thioglycollate-stimulated, murine peritoneal macrophages by affinity chromatography on immobilized Griffonia simplicifolia I lectin and by preparative SDS/PAGE. The glycoproteins were readily labeled on the surface of intact macrophages with 3H and 125I. The labeled glycoproteins migrated as broad bands of molecular mass 92-109 kDa and 115-125 kDa. The mobility of the glycoproteins decreased only slightly after reduction with dithiothreitol, indicating the absence of intersubunit disulfide bridges. The 92-kDa and 115-kDa glycoproteins had pI 5.2-5.4 and pI less than or equal to 4, respectively. Digestion of both glycoproteins with alpha-galactosidase released 23% of their 3H content and abolished their ability to bind to the G. simplicifolia I lectin, showing that they contain terminal alpha-D-galactosyl groups. After reduction with 2-mercaptoethanol, each glycoprotein fraction was sensitive to N-glycanase; the 115-kDa glycoproteins produced a smear with the front at approximately 67 kDa, whereas the 92-kDa glycoprotein gave two bands of 61 kDa and 75 kDa. Unreduced glycoproteins were insensitive to N-glycanase, suggesting the presence of intramolecular disulfide bonds. Although each glycoprotein fraction was sensitive to endoglycosidase H, this enzyme produced only slight changes in molecular mass when compared with N-glycanase. From these results as well as from the specificity of the enzymes involved, it is concluded that each glycoprotein fraction contains complex-type oligosaccharides and a small amount of high-mannose and/or hybrid-type oligosaccharides. While each glycoprotein fraction was bound to Datura stramonium lectin, they failed to react with anti-[i-(Den)] serum and their digestion with endo-beta-galactosidase did not cause a band shift in SDS/PAGE. Taken together, these results suggest the presence of N-acetyllactosamine units which are not arrayed in linear form but occur as single units, bound either to C2 and C6, or to C2 and C4, or both, of outer mannosyl residues on complex-type oligosaccharides. The glycoprotein(s) fraction precipitated with anti-[I (Step)] serum, suggesting the presence of branched lactosaminoglycans. Digestion of both glycoprotein fractions with a mixture of sialidase and O-glycanase did not alter their mobility in SDS/PAGE, suggesting a lack or low content of O-linked trisaccharides and tetrasaccharides. Each glycoprotein fraction was bound specifically to Sambucus nigra and Maackia amurensis immobilized lectins, indicating the presence of sialic acid linked alpha 2,6 to subterminal D-galactose or N-acetylgalactosamine residues, and alpha 2,3 to N-acetyllactosamine residues, respectively.     

Chemical force microscopy with active enzymes

The adhesion forces have been measured between an atomic force microscope tip derivatized with an active enzyme, shikimate kinase, and an ATP mimic immobilized on a gold surface. Experiments with competitive binding of other ligands in solution show that the observed adhesion forces arise predominantly from specific interactions between the immobilized enzyme and surface-bound adenine derivative. These experiments represent a step in the development of a screening methodology based upon chemical force microscopy.