Antibody Immobilization Techniques Research Articles

Antibody Immobilization Techniques in Mass Sensitive Immunosensor: Enhanced Sensitivity through Limited Mass Load

Background: Biosensors are analytical devices that include a sample-delivery approach between a biological recognition element and a transducer required to convert the physicochemical change produced from the interaction of biological molecules-receptor interaction into signal. The immunosensor is a special type of biosensors that includes an antibody as a biorecognition element to detect analyte as antigens. In mass-sensitive sensors, antigen-antibody interactions can be specified by measuring the frequency change and most commonly knowns are surface acoustic wave, bulk acoustic wave, quartz crystal microbalance and microcantilevers. Methods: Different methods for antibody immobilization including functionalization of the transducer surface with specific groups have been reported for antibody immobilization. This stage affects the limit of detection and overall performance. In this review, perspectives on immobilization strategies of mass sensitive immunosensors according to transducer types will be presented. The choice of immobilization methods and their impact on performance in terms of capture molecule loading, orientation and signal improvement is will also be discussed. Results: One of the most critical point during configuration of the biorecognition layer is to improve the sensitivity. Therefore, we initially focused on comparisons of the antibody immobilization strategies in the biorecognition layer in terms of mass load level and high sensitivity. Conclusion: The lack of significant data on the mass accumulations up to the functionalization and antibody immobilization steps, which are the basis of immusensor production, has been identified. However, mass sensitive immunosensors have the potential to become more common and effective analytical devices for many application areas.

Configurable Digital Virus Counter on Robust Universal DNA Chips.

Here, we demonstrate real-time multiplexed virus detection by applying a DNA-directed antibody immobilization technique in a single-particle interferometric reflectance imaging sensor (SP-IRIS). In this technique, the biosensor chip surface spotted with different DNA sequences is converted to a multiplexed antibody array by flowing antibody-DNA conjugates and allowing for specific DNA-DNA hybridization. The resulting antibody array is shown to detect three different recombinant vesicular stomatitis viruses (rVSVs), which are genetically engineered to express surface glycoproteins of Ebola, Marburg, and Lassa viruses in real time in a disposable microfluidic cartridge. We also show that this method can be modified to produce a single-step, homogeneous assay format by mixing the antibody-DNA conjugates with the virus sample in the solution phase prior to incubation in the microfluidic cartridge, eliminating the antibody immobilization step. This homogenous approach achieved detection of the model Ebola virus, rVSV-EBOV, at a concentration of 100 PFU/mL in 1 h. Finally, we demonstrate the feasibility of this homogeneous technique as a rapid test using a passive microfluidic cartridge. A concentration of 104 PFU/mL was detectable under 10 min for the rVSV-Ebola virus. Utilizing DNA microarrays for antibody-based diagnostics is an alternative approach to antibody microarrays and offers advantages such as configurable sensor surface, long-term storage ability, and decreased antibody use. We believe that these properties will make SP-IRIS a versatile and robust platform for point-of-care diagnostics applications.

Antibody immobilization technique using protein film for high stability and orientation control of the immobilized antibody

The development of antibody immobilization techniques is essential for creating antibody-based biomaterials. Although numerous methods for antibody immobilization have been demonstrated, low stability and disordered orientation of the immobilized antibody remain an important problem. In this work, an original antibody immobilization technique using a protein film, which achieved a high stability and orientation control of the immobilized antibody, has been described. In this method, an antibody-immobilized albumin film was prepared by adding the cross-linked albumin solution to the substrate, where antibodies were attached in uniform orientation, followed by subsequent drying, and detaching the formed film from the substrate by heating at 120 °C in a dry state. Antibodies in the film showed high antigen-binding capacity, at a level comparable to the oriented immobilized antibody using protein G. The stability of antibodies in the film was found to be significantly high; their antigen-binding capacity was completely retained even after storage at 40 °C in a dry state for one month. Thus, this approach provides useful information to immobilize the antibody on solid surfaces while controlling its orientation and increasing its stability.

Label-free detection of rheumatoid factor using YbYxOy electrolyte–insulator–semiconductor devices

In this study, we investigated the effect of yttrium content on the structural properties and sensing characteristics of YbYxOy sensing membranes for electrolyte–insulator–semiconductor (EIS) sensors to detect the rheumatoid factor (RF). The YbYxOy EIS device prepared at the 60 W plasma condition exhibited a higher sensitivity of 65.77 mV/pH, a lower hysteresis voltage of ∼1 mV, and a smaller drift rate of 0.14 mV/h than did those prepared at the other conditions. We attribute this behavior to the optimal yttrium content in the YbYxOy film forming a smooth surface. Furthermore, we used a novel YbTixOy EIS biosensor to measure the RF antigen in human serum because of its rapid and label-free detection. Two different techniques were used for the immobilization of RF antibody onto the surface of an YbTixOy EIS sensor. The RF antibody was directly immobilized on the EIS surface modified with 3-aminopropyltriethoxysilane (APTES) followed by glutaraldehyde (GA). In contrast, a mixture of 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) solution was used to functionalize the carboxyl groups at the tail of RF antibodies. RF antibodies functionalized with the active NHS esters were covalently immobilized on the APTES-modified YbTixOy surface. The immobilized RF antibodies on the EIS that are functionalized with the EDC and NHS exhibit higher (41.11mV/pCRF) for detection of serum RF antigen in the range 10−7 to 10−3 M, compared to traditional antibody immobilization technique via APTES and GA linkage. The YbTixOy EIS biosensor is a promising analytical tool for RF antigen monitoring due to its good sensitivity, stability and repeatability.

Immobilization of Fab' fragments onto substrate surfaces: A survey of methods and applications.

Antibody immobilization onto surfaces has widespread applications in many different fields. It is desirable to bind antibodies such that their fragment-antigen-binding (Fab) units are oriented away from the surface in order to maximize analyte binding. The immobilization of only Fab' fragments yields benefits over the more traditional whole antibody immobilization technique. Bound Fab' fragments display higher surface densities, yielding a higher binding capacity for the analyte. The nucleophilic sulfide of the Fab' fragments allows for specific orientations to be achieved. For biosensors, this indicates a higher sensitivity and lower detection limit for a target analyte. The last thirty years have shown tremendous progress in the immobilization of Fab' fragments onto gold, Si-based, polysaccharide-based, plastic-based, magnetic, and inorganic surfaces. This review will show the current scope of Fab' immobilization techniques available and illustrate methods employed to minimize non-specific adsorption of undesirables. Furthermore, a variety of examples will be given to show the versatility of immobilized Fab' fragments in different applications and future directions of the field will be addressed, especially regarding biosensors.

Bio-nanomechanical Detection of Diabetic Marker HbA1c

We report the development of an ultrasensitive immunosensor system for the detection of specific diabetic biomarker glycated hemoglobin (HbA1c) using specific antibody functionalized micromechanical cantilever chips. The biomolecules-induced deflection of the cantilever beam reflects the interplay between the strain energy increase of the cantilever and the free energy reduction of the interaction, providing a unique system for investigating the connection between the nanomechanics and the chemistry of antibody–antigen interaction at very low concentration. Cantilevers were functionalized with specific antibodies using site-directed antibody immobilization technique which was adopted to attach antibody on to the gold substrate. The antibody immobilized cantilevers were used to detect the level of the expressed biomarker in standard samples by adopting direct immunoassay format without using any labels. The assay exhibited an excellent sensitivity for HbA1c in the dynamic range from 0.147 to 1.47 pM. This label-free detection method could be used for fast, high-throughput screening of specific biomarkers for the therapeutic management of diabetes mellitus in clinical diagnostic at a very low cost.

Comparison of Oriented and Random Antibody Immobilization Techniques on the Efficiency of Immunosensor

An immunosensor for bovine leukemia virus antigen (gp51) determination was developed using surface plasmon resonance (SPR) technique. During this work the SPR-chip coated with gold was used for immunosensor design. Different strategies of antibodies immobilization - oriented immobilization of specific antibodies fragments (frag-anti-gp51) on gold surface obtained after chemical reduction and random immobilization of intact antibodies (anti-gp51) on gold surface, were investigated. The immobilization based on the application of frag-anti-gp51 was found to be the most suitable for the design of SPR-immunosensor for gp51 detection. SPR sensor created using frag-anti-gp51 was tested in a range from 0.01 to 0.5 mg/mL of gp51 antigen concentration. Developed immunosensor exhibited sufficient antigen binding capacity, simplicity and low cost in respect to the currently evaluated techniques.

Comparative Study of Random and Oriented Antibody Immobilization Techniques on the Binding Capacity of Immunosensor

A comparative study of four different antibody immobilization techniques that are suitable for modification of surface plasmon resonance (SPR) chip (SPR-chip) is reported. Antibodies against human growth hormone (anti-HGH) were used as the model system. The evaluated SPR-chip modification techniques were (i) random immobilization of intact anti-HGH (intact-anti-HGH) via self-assembled monolayer (SAM) based on 11-mercaptoundecanoic acid (MUA); (ii) random immobilization of intact-anti-HGH within carboxymethyl dextran (CMD) hydrogel by direct covalent amine coupling technique; (iii) oriented coupling of intact-anti-HGH via Fc-fragment to protein-G layer assembled on SAM consisting of MUA (MUA/pG); (iv) oriented immobilization of fragmented anti-HGH antibodies (frag-anti-HGH) via their native thiol-groups directly coupled to the gold. To liberate these thiol groups, the intact-anti-HGH was chemically "divided" into two frag-anti-HGH fragments by chemical reduction with 2-mercaptoethylamine (2-MEA). Optimal concentration of 2-MEA for preparation of anti-HGH was 15 mM. The surface concentration of immobilized antibodies and the antigen binding capacity for all four differently modified SPR-chips was evaluated and compared. The maximum surface concentration of immobilized intact-anti-HGH was obtained by immobilizing the antibody within CMD-hydrogel. The maximal antigen binding capacity was obtained by SPR-chip based on intact-anti-HGH immobilized via MUA/pG. The immobilization based on application of frag-anti-HGH was found to be the most suitable for design of SPR-immunosensor for HGH detection, due to its sufficient antigen binding capacity, simplicity, and low cost in respect to the currently evaluated techniques.

A new antibody immobilization technique based on organic polymers protected Prussian blue nanoparticles and gold colloidal nanoparticles for amperometric immunosensors

Abstract A new antibody immobilization strategy based on organic polymers protected Prussian blue (PB) nanoparticles and gold colloidal nanoparticles (GNPs) was used to develop a highly sensitive amperometric immunosensor for the detection of carcinoma antigen 125 (CA125). PB nanoparticles, as an electron transfer mediator, have been synthesized using chitosan (CS) and poly(diallyldimethylammonium chloride) (PDDA) as protective matrix, which were cast onto a glass carbon electrode surface directly. Then, GNPs were assembled onto the electrode through strong binding interactions between GNPs and amino groups of CS and electrostatic interaction between oppositely charged GNPs and PDDA. Finally, CA125 antibody (anti-CA125), used as a model antibody, was assembled onto the surface of GNPs. UV–vis absorption spectroscopy, transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy were employed to characterize CS and PDDA protected PB nanoparticles (CS–PDDA–PB). The assembly process of the modified electrode was characterized with cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and atomic force microscopy (AFM). The proposed immunosensor showed a high sensitivity, broad linear range and low detection limit for CA125 determination. Moreover, it possessed good reproducibility and stability, and would be applicable to clinical immunoassay of CA125. The use of the CS–PDDA–PB nanoparticles would open new horizons for fabrication of biosensors and biocatalysts.

An investigation of antibody immobilization methods employing organosilanes on planar ZnO surfaces for biosensor applications

One critical aspect for the development of label-free immunosensors is the employment of highly uniform and repeatable antibody immobilization techniques. In this study, we investigated the use of two different silane molecules (3-glycidyloxypropyl)trimethoxysilane (GPS), and (3-mercaptopropyl)trimethoxysilane (MTS) for the immobilization of fluorescently labeled IgG antibodies on planar ZnO surfaces. The chemical modification of the surfaces was investigated using water contact angle measurements, AFM, and fluorescence microscopy. The results of the water contact angle measurements indicate increased surface hydrophobicity after treatment with GPS and MTS as compared to the control. Surface modification was further verified through AFM measurements which demonstrate an increased surface roughness and particle height after treatment with antibodies. The results of the fluorescence studies indicate that the immobilization protocol employing MTS produced 21% higher fluorescence on average with greater uniformity than the GPS-based protocol, which indicates a higher overall density in antibody coverage on the surface of the ZnO. Acoustic sensor tests were employed to confirm the functionality of sensors treated with the MTS protocol. The results indicate that the immobilization protocol imparts sensitivity and specificity to the ZnO-based devices.

Detection of Antigens in Biologically Complex Fluids with Photografted Whole Antibodies

A highly sensitive (pM), efficient (t < 20 min) detection assay was developed by designing surfaces with grafted antibodies. Through this approach, a short half-life antigen, glucagon, was rapidly detected in a biologically complex plasma/blood environment. Tailoring of graft composition eliminates the need for time-consuming blocking steps, significantly reducing antigen-antibody incubation times, while maintaining antibody specificity and activity toward target antigen. Grafted antibodies were bound through solvated, mobile polymer chains, thereby circumventing problems associated with antibody accessibility, analyte diffusion, and steric limitations. The efficiency of this assay is provided through grafting synthesized, acrylated antibodies in the presence of PEG monoacrylate. This procedure eliminates the need for blocking steps, due to a decrease in nonspecific protein interactions. These polymerizable antibodies were tethered with a range of densities while retaining biological activity. Moreover, biological activity of acrylated antibodies was compared to that of unmodified antibodies and remained comparable. The acrylated antibodies were grafted from substrate surfaces using controlled radical photopolymerization, maintaining the advantages of classical antibody immobilization techniques while providing improved detection. Through integrating this antibody conjugation chemistry and immunoassay approach with photolithographic techniques, construction of spatial patterns on a microfluidic device was demonstrated for efficient, parallel screening of multiple antigens.

DNA Directed Protein Immobilization on Mixed ssDNA/Oligo(ethylene glycol) Self-Assembled Monolayers for Sensitive Biosensors

A stable and versatile biosensor surface is prepared by site-directed immobilization of protein-DNA conjugates onto a mixed self-assembled monolayer (SAM) composed of ssDNA thiols and oligo(ethylene glycol) (OEG) terminated thiols. The protein conjugates consist of an antibody chemically linked to a ssDNA target with a sequence complementary to the surface-bound ssDNA probes and are immobilized on the surface via sequence-specific hybridization. Compared to standard antibody immobilization techniques, this approach offers many advantages. The exceptional specificity of DNA hybridization combined with the diversity of potential sequences makes this platform perfect for multichannel sensors. Once a surface is patterned with the appropriate probe sequences, sequence-specific hybridization will sort out the target conjugates and direct them to the appropriate spots on the surface. In addition, the DNA SAMs are very stable and well suited to recycling by dehybdridization of the conjugates from the surface-bound probes. In this work, we demonstrate the specificity, sensitivity, and convenience of using protein-DNA conjugates to convert a DNA/OEG SAM surface into a biosensor surface and apply this platform to the detection of human chorionic gonadotropin using surface plasmon resonance.

Simple method for plant virus detection: effect of antibody immobilization technique

The possibility has been demonstrated for applying a surface plasmon resonance for detecting plant viruses in real samples. An optimal mode for antiviral immunoglobulin immobilization on sensor surfaces is described. Out of three proposed techniques for sensor surface treatment, namely, unmodified gold surface, gold surface treated with (a) thiocyanate and (b) thiocyanate and protein A ( Staphylococcus aureus), the latter was chosen as most suited for retention of the formed native immunoglobulin layer.

Quantification of antibody immobilization using peroxidase enzyme–substrate reaction

Due to the multitude of antigens and antibodies that are currently being studied by immunoscientists and researchers, numerous and diverse antibody immobilization techniques were developed. Newer and improved methods of detection and immobilization are constantly being developed. As a result, especially when no prior research exists, scientists and researchers are faced with the challenge of choosing the best immobilization technique. Assessment of antibody immobilization based on the final antibody–antigen reaction may not always be accurate, as certain unknown processes or reactions may account for a part (or whole) of the transducer response. The present paper describes an alternative method for scientific quantification of antibody attachment on the biosensing surface or transducer using the peroxidase enzyme–substrate (TMB) colorimetric reaction. The proposed method can be used to pre-screen various immobilization techniques or pre-coatings, or can be used as a secondary quantification method. Four different pre-coatings on piezoelectric crystals and three affinity membranes were tested to evaluate the degree of antibody attachment. Results indicate that polystyrene-coated crystals had an average immobilization of 10.55% and the Immobilon membrane had 18.22% immobilization. Although the precision of the proposed technique varied significantly between different transducer surfaces (CV=1.7–21.7%), results clearly demonstrate that polystyrene coating on crystal is approximately 450% superior to glutaraldehyde coating. Similarly, the Immobilon membrane had 720% higher antibody immobilization than Ultrabind membrane. The sensitivity of the overall technique is so high that each test requires only 3 μl of diluted, labeled antibody solution (1 μg/ml).

Development and evaluation of a dipstick immunoassay format for the determination of atrazine residues on-site.

On the basis of a semi-quantitative dipstick immunoassay (IA) for atrazine with visual detection (Giersch, T., J. Agric. Food Chem., 1993, 41, 1006), a quantitative format suitable as a field assay for the analysis of pesticide residues in water and liquid food samples on-site is described. For antibody immobilization, different membranes and immobilization techniques were investigated. The measuring range for atrazine was 0.3-10 micrograms l-1 using reflectance detection. The total assay time was about 25 min with dipsticks previously coated with antibody. Atrazine-spiked water and liquid food samples were selected for assay evaluation. The samples could be measured directly without the need for any prior enrichment or clean-up steps. A satisfactory agreement was found between the results of the dipstick IA and HPLC or GC measurements of both the original and spiked samples.

A comparative study of protein immobilization techniques for optical immunosensors

This paper presents the results of a study of a number of antibody immobilization techniques for application to optical immunosensors. In particular, well-known methods such as covalent binding and physical adsorption have been extended to the Langmuir-Blodgett method in an attempt to improve the density and possibly the uniformity of orientation of monoclonal antibodies on an optical surface. The surface density of active immobilized antibodies was determined from enzyme immunoassay and their thickness and refractive index were deduced from ellipsometry. It is shown that, although high surface densities (500ng/cm 2) of antibody can be obtained, the major obstacle to the detection of low concentrations of antigens or haptens is the non-specific binding of foreign molecules to the sensing surface.