Specific Capture of Peptide‐Receptive Major Histocompatibility Complex Class I Molecules by Antibody Micropatterns Allows for a Novel Peptide‐Binding Assay in Live Cells

Binding assays with fluorescently labeled ligands and recombinant receptor proteins are commonly performed in 2D arrays. But many cell surface receptors only function in their native membrane environment and/or in a specific conformation, such as they appear on the surface of live cells. Thus, receptors on live cells should be used for ligand binding assays. Here, it is shown that antibodies preprinted on a glass surface can be used to specifically array a peptide receptor of the immune system, i.e., the major histocompatibility complex class I molecule H‐2K^b, into a defined pattern on the surface of live cells. Monoclonal antibodies make it feasible to capture a distinct subpopulation of H‐2K^b and hold it at the cell surface. This patterned receptor enables a novel peptide‐binding assay, in which the specific binding of a fluorescently labeled index peptide is visualized by microscopy. Measurements of ligand binding to captured cell surface receptors in defined confirmations apply to many problems in cell biology and thus represent a promising tool in the field of biosensors.     

Kinetics of antigen binding to arrays of antibodies in different sized spots

A fluorescence-based array biosensor has been developed which can measure the binding kinetics of an antigen to an immobilized antibody in real time. A patterned array of antibodies immobilized on the surface of a planar waveguide was used to capture a Cy5-labeled antigen present in a solution that was continuously flowed over the surface. The CCD image of the waveguide was monitored continuously for 25 min. The resulting exponential rise in fluorescence signal was determined by image analysis software and fitted to a reaction-limited kinetics model, giving a kf of 3.6 x 10(5) M(-1) s(-1). Different spot sizes were then patterned on the surface of the waveguide using either a PDMS flow cell or laser exposure, producing width sizes ranging from 80 to 1145 microm. It was demonstrated that under flow conditions, the reduction of spot size did not alter the association rate of the antigen with immobilized antibody; however, as the spot width decreased to < 200 nm, the signal intensity also decreased.     

A quantitative assessment of heterogeneity for surface-immobilized proteins

Many biotechnological applications use protein receptors immobilized on solid supports. Although, in solution, these receptors display homogeneous binding affinities and association/dissociation kinetics for their complementary ligand, they often display heterogeneous binding characteristics after immobilization. In this study, a fluorescence-based fiber-optic biosensor was used to quantify the heterogeneity associated with the binding of a soluble analyte, fluorescently labeled trinitrobenzene, to surface-immobilized monoclonal anti-TNT antibodies. The antibodies were immobilized on silica fiber-optic probes via five different immobilization strategies. We used the Sips isotherm to assesses and compare the heterogeneity in the antibody binding affinity and kinetic rate parameters for these different immobilization schemes. In addition, we globally analyzed kinetic data with a two-compartment transport-kinetic model to analyze the heterogeneity in the analyte-antibody kinetics. These analyses provide a quantitative tool by which to evaluate the relative homogeneity of different antibody preparations. Our results demonstrate that the more homogeneous protein preparations exhibit more uniform affinities and kinetic constants.     

Biomolecular Tuning of Electronic Transport Properties of Carbon Nanotubes via Antibody Functionalization

Carbon nanotubes (CNTs) are remarkable solid-state nanomaterials due to their unique electrical and mechanical properties. The electronic properties of nanotubes combined with biological molecules such as proteins could make miniature devices for biological sensing applications. In this paper, the noncovalent interaction of single-wall CNTs with antibodies is presented for its potential applications for detecting overexpressed cell surface receptors in breast cancer cells. The degree of binding of antibodies on CNTs was found to be more than 80% for an extended sampling area by confocal microscopy. The key to achieve such high degree of functionalization is due to the separation of CNTs using surfactants that leads to a high surface area to volume ratio and higher number of active sites for charge transfer that enhance binding. This paper also presents tuning of electronic transport properties of CNTs by monoclonal antibodies that are specific to insulin-like growth factor 1 receptor in breast cancer     

Densities and orientations of antibodies on nano-textured silicon surfaces

High surface densities of properly oriented antibodies are desired for enhancing the sensitivity of immunosensors. A systematic investigation of the densities and orientations of antibodies, immobilized without and with intermediate protein molecules (protein-A and streptavidin) on nano-textured silicon surfaces (RMS roughness < 100 nm) was performed and the results were compared to those obtained from non-textured surfaces. The primary antibody densities and orientations (through densities of secondary antibodies) were quantitatively measured for the different cases. Higher densities were obtained for all nano-textured surfaces compared to non-textured ones. It was observed that higher primary antibody densities were obtained when no intermediate proteins were used. The different nano-texturing conditions did not have a significant effect on the densities with no intermediate proteins and with protein-A, but had an effect with streptavidin. The densities of the properly oriented primary antibodies increased on most of the nano-textured surfaces used as compared to the non-textured samples. The effect of the texturing on the densities was observed for several of the surfaces studied. Design nano-texturing could be used to maximize as well as tune the densities of the properly oriented antibodies on the substrate surface.     

Engineering camel single-domain antibodies and immobilization chemistry for human prostate-specific antigen sensing

The specificity and affinity characteristics of antibodies make them excellent probes in biosensor applications. Unfortunately, their large size, unstable behavior, and random immobilization properties create numerous problems. The single-domain antigen-binding fragment derived from heavy-chain antibodies of camelids (termed VHH) offers special advantages in terms of size, stability, and ease of generating different antibody constructs. In this study, we show the potential of those VHHs in sensing human prostate-specific antigen (hPSA) by SPR technology. Different VHH constructs were immobilized onto commercial and custom-built sensor surfaces by metal chelation, biotin-streptavidin interaction, or covalent coupling. The detection of subnanogram per milliliter hPSA concentrations could be attained on a covalently coupled three-dimensional dextran surface. Moreover, the ratio of different hPSA isoform concentrations could be assessed via a sandwich assay and resulted in the detection of clinically significant antigen concentrations within 15 min. In addition, for the first time, the intrinsic protein stability is presented as an important probe design factor, since our results reveal that higher intrinsic stability offers higher resistance to harsh regeneration conditions. In conclusion, we present VHHs as a novel class of biosensor probes rivaling conventional antibodies and their derived antibody fragments.     

Gold surface functionalization and patterning for specific immobilization of olfactory receptors carried by nanosomes

There is substantial interest in engineering solid supports to achieve functional immobilization of membrane receptors both for investigation of their biological function and for the development of novel biosensors. Three simple and practical strategies for immobilization of a human olfactory receptor carried by nanosomes are presented. The basis of the functionalization of solid gold surfaces is a self-assembled monolayer (SAM) containing biotinyl groups. Biotinyl groups are subsequently used to attach neutravidin and then biotinylated monoclonal antibody directed against the receptor to allow its specific grafting. Surface plasmon resonance technique is implemented for real-time monitoring of step-by-step surface functionalization and, in addition, for testing the functional response of immobilized olfactory receptors. We show that OR1740 is functional when immobilized via a tag attached to its C-terminus, but not via its N-terminus. Finally, we demonstrate that gold surfaces can be patterned by the SAMs tested using microcontact printing. AFM images of immobilized nanosomes onto a patterned surface suggest that small nanosomes flatten and fuse into larger vesicles but do not merge into a continuous layer. The whole study emphasizes the outstanding performances of the BAT/PEGAT SAM, which could be useful for developing on-a-chip sensor formats for membrane receptor investigations and use.     

Significance of antibody orientation unraveled: well-oriented antibodies recorded high binding affinity

To investigate the effect of antibody orientation on its immunological activities, we developed a novel and versatile platform consisting of a well-defined phospholipid polymer surface on which staphylococcal protein A (SpA) was site-selectively immobilized. The application of a biocompatible phospholipid-based platform ensured minimal denaturation of immobilized antibodies, and the site-selective immobilization of SpA clarified the effect of antibody orientation on immunological activities. The phospholipid polymer platform was prepared on silicon substrates using the surface-initiated atom transfer radical polymerization (SI-ATRP) technique. An enzymatic reaction was performed for orientation-selective coupling of SpA molecules to the polymer brush surface. Orientation-controlled antibodies were achieved using enzymatic reactions, and these antibodies captured 1.8 ± 0.1 antigens on average, implying that at least 80% of immobilized antibodies reacted with two antigens. Theoretical multivalent binding analysis further revealed that orientation-controlled antibodies had antigen-antibody reaction equilibrium dissociation constants (K(d)) as low as 8.6 × 10(-10) mol/L, whereas randomly oriented and partially oriented antibodies showed K(d) values of 2.0 × 10(-7) and 1.2 × 10(-7) mol/L, respectively. Strict control of antibody orientation not only formed an approximately 100-fold stronger antigen-antibody complex than the controls but also sustained the native antibody K(d) (10(-10)-10(-9) mol/L). These findings support the significance of antibody orientation because controlling the orientation resulted in high reactivity and theoretical binding capacity.     

A computational reaction-diffusion model for the analysis of transport-limited kinetics

Optical, evanescent wave biosensors have become popular tools for quantitatively characterizing the kinetic properties of biomolecular interactions. Analyzing data from biosensor experiments, however, is often complicated when mass-transfer influences the detection kinetics. We present a computational, transport-kinetic model that can be used to analyze transport-limited biosensor data. This model describes a typical biosensor experiment in which a soluble analyte diffuses through a flow chamber and binds to a receptor immobilized on the transducer surface. Analyte transport in the flow chamber is described by the diffusion equation while the kinetics of analyte-surface association and dissociation are captured by a reactive boundary condition at the sensor surface. Numerical integration of the model equations and nonlinear least-squares fitting are used to compare model kinetic data to experimental results and generate estimates for the rate constants that describe analyte detection. To demonstrate the feasibility of this model, we use it to analyze data collected for the binding of fluorescently labeled trinitrobenzene to immobilized monoclonal anti-TNT antibodies. A successful analysis of this antigen-antibody interaction is presented for data collected with a fluorescence-based fiber-optic immunoassay. The results of this analysis are compared with the results obtained with existing methods for analyzing diffusion-limited kinetic data.     

Fibronectin immobilized by covalent conjugation or physical adsorption shows different bioactivity on aminated-PET

To manipulate the cellular response to synthetic surfaces, extracellular matrix (ECM) proteins such as fibronectin (FN) and collagen are often immobilized on the surface to promote interaction between these ligands and the cell receptors. In this study we compared the biological properties of FN-decorated polyethylene terephthalate (PET) produced by two widely used immobilization techniques: adsorption and conjugation. As revealed by the micro-bicinchoninic acid (micro-BCA) assay and AFM, the modified surface topography was dependent on the immobilization methods. Adsorption method preserved the compact conformation of FN, reaching saturation when a monolayer of FN was formed. Covalent conjugation induced FN unfolding and fibrillogenesis, forming multiple layers of FN. Biological characterization by adhesion of baby hamster kidney 21 (BHK21) cells and enzyme-linked immunosorbent assay (ELISA) for active Arg-Gly-Asp (RGD) domains suggested that the difference in conformation of FN led to different bioactivities. Adsorption maintained a more active RGD domain, thereby promoting cell adhesion, whereas conjugation induced fibrillogenesis and blocked the access of RGD, consequently suppressing cell adhesion as the surface density of FN increased. This study suggests that in addition to choosing the nature of the adhesion molecule, the mode of immobilization may also significantly influence the bioactivity of the surface.     

Stepwise functionalization of SiNx surfaces for covalent immobilization of antibodies

A stepwise functionalization of silicon nitride surfaces is followed by X-ray photoelectron spectroscopy (XPS). The first step involves a silanization reaction leading to the formation of a silane film with a thickness estimated by XPS of one or two molecular layers. A monoprotected homobifunctionalized linker is then used to avoid the formation of bridge structures on the surface. The linker reacts quantitatively with the amino groups of the surface as outlined by the absence of residual unreacted CNH₂/CNH₃⁺ groups in XPS analyses. Deprotection of the ester groups of the immobilized linker and subsequent reaction with N-hydroxysuccinimid lead to N-hydroxysuccinimid activated surfaces able to react with biological species. These surfaces were then incubated with anti-transferrin antibodies. As seen by XPS and atomic force microscopy analyses, the concentration and incubation conditions of antibodies are important to obtain a compact layer of antibodies on the surface. All chemical steps of the procedure are compatible with microelectronic process on silicon. Moreover, antibodies introduced under native conditions at physiological pH, in the last step of the immobilization process, recognized specifically antigens, as shown by fluorescence competitive assay.     

Antibody arrays prepared by cutinase-mediated immobilization on self-assembled monolayers

Antibody arrays hold considerable potential in a variety of applications including proteomics research, drug discovery, and diagnostics. Many of the schemes used to fabricate the arrays fail to immobilize the antibodies at a uniform density or in a single orientation; consequently, the immobilized antibodies recognize their antigens with variable efficiency. This paper describes a strategy to immobilize antibodies in a single orientation, with a controlled density, using the covalent interaction between cutinase and its suicide substrate. Protein fusions between cutinase and five antibodies of three different types (scFv, V(HH), and FN3) were prepared and immobilized upon self-assembled monolayers (SAMs) presenting a phosphonate capture ligand. The immobilized antibodies exhibit high affinity and selectivity for their target antigens, as monitored by surface plasmon resonance and fluorescence scanning. Furthermore, by changing the density of capture ligand on the SAM the density of the immobilized antibody could be controlled. The monolayers, which also present a tri(ethylene glycol) group, are inert to nonspecific adsorption of proteins and allow the detection of a specific antigen in a complex mixture. The demonstration of cutinase-directed antibody immobilization with insert SAMs provides a straightforward and robust method for preparing antibody chips.     

Microfluidic enzyme immunoassay using silicon microchip with immobilized antibodies and chemiluminescence detection

Silicon microchips with immobilized antibodies were used to develop microfluidic enzyme immunoassays using chemiluminescence detection and horseradish peroxidase (HRP) as the enzyme label. Polyclonal anti-atrazine antibodies were coupled to the silicon microchip surface with an overall dimension of 13.1 x 3.2 mm, comprising 42 porous flow channels of 235-microm depth and 25-microm width. Different immobilization protocols based on covalent or noncovalent modification of the silica surface with 3-aminopropyltriethoxysilane (APTES) or 3-glycidoxypropyltrimethoxysilane (GOPS), linear polyethylenimine (LPEI, MW 750,000), or branched polyethylenimine (BPEI, MW 25,000), followed by adsorption or covalent attachment of the antibody, were evaluated to reach the best reusability, stability, and sensitivity of the microfluidic enzyme immunoassay (microFEIA). Adsorption of antibodies on a LPEI-modified silica surface and covalent attachment to physically adsorbed BPEI lead to unstable antibody coatings. Covalent coupling of antibodies via glutaraldehyde (GA) to three different functionalized silica surfaces (APTES-GA, LPEI-GA, and GOPS-BPEI-GA) resulted in antibody coatings that could be completely regenerated using 0.4 M glycine/HCl, pH 2.2. The buffer composition was shown to have a dramatic effect on the assay stability, where the commonly used phosphate buffer saline was proved to be the least suitable choice. The best long-term stability was obtained for the LPEI-GA surface with no loss of antibody activity during one month. The detection limits in the microFEIA for the three different immuno surfaces were 45, 3.8, and 0.80 ng/L (209, 17.7, and 3.7 pM) for APTES-GA, LPEI-GA, and GOPS-BPEI-GA, respectively.     

Bone cell grafts in bioreactor: a study of feasibility of bone cell autograft in large defects

Cancellous bone cells were isolated from adult dogs, introduced into cell culture, subcultured and grown on hydroxylapatite granules. Cells immobilized on these granules were used to make bioreactors which were implanted in dog ulna diaphyse to fill osseous defects. The bioreactor implantation constituted a bone cell autograft and showed bone formation in a reactor containing cultured cells but not in the control reactor containing hydroxylapatite granules without cells. These results indicate that hydroxylapatite material can be used in bioartificial organs. The properties of hydroxylapatite used in bone reconstruction are due to the cells and extra-cellular matrix immobilized on its surface.     

Immobilization of antibodies on polyaniline films and its application in a piezoelectric immunosensor

Conducting polymers, especially polyaniline (PAni), have been extensively used in biosensor applications. A protocol for covalent immobilization of human IgG on polyaniline using glutaraldehyde as the cross-linker is described in this report and utilized in development of a piezoelectric immunosensor. Here, PAni was used as the substrate for immobilization. The electropolymerization parameters were optimized to get suitable thickness and surface morphology of the PAni for obtaining high density and uniformity of immobilized antibodies on the surface of our films. Possible reaction between PAni thin films and glutaraldehyde was explored using FT-IR characterization in grazing angle mode and XPS. The protocol has been characterized with the help of quartz crystal microbalance analysis. An antibody surface density of 4.86 ng/mm2 was obtained. A piezoelectric biosensor developed for detection of IgG with the proposed protocol was capable of differentiating the target analyte concentrations between 500 ng/mL and 25 microg/mL with nonspecific binding of approximately 10%.     

Formation of amine functionalized films by chemical vapour deposition

Thermally Activated Chemical Vapour Deposition (TA-CVD) has been used for the biofunctionalization of silicon substrates, among others. This technique uses 3-aminopropyltriethoxysilane as organometallic precursor. The deposited films show biofunctional properties, with reactive amines on the surface, as it was shown by FTIR and confocal microscopy. In this work, the influence of the deposition parameters in the microstructure and functionality of the films was investigated. Antibodies were immobilized on the films that had higher and more homogeneous distribution of amines. The confocal microscopy images show that the amines react with the antibodies and that these biomolecules keep their biological functionality.     

Single chain fragment variable recombinant antibody as a template for Fc sensors

A label free immunosensor for detection of Fc receptors expressed on cell surface was developed and characterized using a Quartz Crystal Microbalance (QCM) transducer. Taking advantage of the characteristics of single chain fragment variable (scFv) recombinant antibody and the multivalency of an antibody, the engineered recombinant scFv was immobilized onto preformed functionalized self-assembled monolayers (SAMs) template surface. The monomeric scFv can bind with the CH1 region of any rabbit IgG to form a highly oriented IgG layer with its Fc portion pointing toward a solution phase. This results in a highly oriented Fc sensor that can be used to study the thermodynamics and kinetics of binding between the Fc portion of immunoglobulin and the cell surface Fc receptor (FcR), an important area of the immune system. The Fc sensor was used to study the binding between Staphylococcus aureus and the Fc receptor on macrophage. Parallel characterization of cell surface Fc receptors in the same samples by ELISA was also performed.     

Quantification of antibodies to human growth hormone by high-performance protein G affinity chromatography with fluorescence detection

The technique of high-performance affinity chromatography (HPAC) is applied to the quantitative determination of antibodies to human growth hormone (hGH) in serum from patients. An affinity column consisting of covalently immobilized protein G on a rigid support is used to capture the antibodies. Texas Red labeled hGH (hGH-TR) is used as a fluorescence probe for detecting the anti-hGH antibodies. Calibration curves are established by using a well-characterized monoclonal antibody to hGH (GHC101). The minimum detectable concentration (MDC) of anti-hGH antibody in serum is 250 ng/mL (this represents 10 ng of anti-hGH injected onto the protein G column). Analytical recoveries are 92-110% for seven replicates with 250-4000 ng/mL of GHC101. A precision of 15% relative standard deviation (RSD) can be achieved at the MDC. The precision is better above the detection limit. The linear dynamic range of the method is approximately 2 orders of magnitude. The total fluorescence recovery from the affinity column is greater than or equal to 96%. Sample analysis times are on the order of 20 min. The HPAC technique gives results in absolute units of concentration that correlate well with binding capacity values determined by radioimmunoassay.     

Response to cardiac markers in human serum analyzed by guided-mode resonance biosensor

Cardiac markers in human serum with concentrations less than 0.1 ng/mL were analyzed by use of a guided-mode resonance (GMR) biosensor. Cardiac troponin I (cTnI), creatine kinase MB (CK-MB), and myoglobin (MYO) were monitored in the serum of both patients and healthy controls. Dose-response curves ranging from 0.05 to 10 ng/mL for cTnI, from 0.1 to 10 ng/mL for CK-MB, and from 0.03 to 1.7 μg/mL for MYO were obtained. The limits of detection (LOD) for cTnI, CK-MB, and MYO were less than 0.05, 0.1, and 35 ng/mL, respectively. Analysis time was 30 min, which is short enough to meet clinical requirements. Antibody immobilization and the hydrophilic properties of the guided-mode resonance filter (GMRF) surface were investigated by X-ray photoelectron spectroscopy (XPS) and by monitoring the peak wavelength shift and water contact angle (CA). Both assays used to evaluate the surface density of the immobilized antibodies, a sandwich enzyme-linked immunosorbent assay (ELISA) and a sandwich immunogold assay, showed that the antibodies were successfully immobilized and sufficiently aligned to detect the low concentration of biomarkers. Our results show that the GMR biosensor will be very useful in developing low-cost portable biosensors that can screen for cardiac diseases.     

Detection and Classification of Related Lipopolysaccharides via a Small Array of Immobilized Antimicrobial Peptides

A small array of antimicrobial peptides comprising three cysteine-terminated natural sequences covalently immobilized to pendant surface maleimide groups are used to bind and successfully discriminate five types of lipopolysaccharide (LPS) molecules. Using surface plasmon resonance, LPSs isolated from four strains of Escherichia coli and one strain of Pseudomonas aeruginosa yield distinct binding profiles to the three immobilized peptides. Linear discriminant analysis generated 100% training set and 80% validation set classification success for the 40 samples evaluated. This work demonstrates the discriminatory binding capabilities of immobilized antimicrobial peptides toward LPS molecules and alludes to their use as probes in pathogen sensing devices potentially superior to the current state-of-the-art.