Ab Immobilization Research Articles

Antibody immobilization on a nanoporous aluminum surface for immunosensor development

A method of antibody (Ab) immobilization on a nanoporous aluminum surface for an electrochemical immunosensor is presented. To achieve good attachment and stability of Ab on an aluminum surface, aluminum was silanized with 3-aminopropyltryethoxysilane (APTES), and then covalently cross-linked to self-assembled layers (SALs) of APTES. Both the APTES concentration and the silanization time affected the formation of APTES-SALs as Ab immobilization. The formation of APTES-SALs was confirmed using the water contact angle on the APTES-SALs surface. The reactivity of APTES-SALs with Ab was investigated by measuring the fluorescence intensity of fluorescein isothiocyanate-labeled Ab-immobilized on the aluminum surface. Silanization of aluminum in 2% APTES for 4h resulted in higher water contact angles and greater amounts of immobilized Ab than other APTES concentrations or silanization times. More Ab was immobilized on the nanoporous surface than on a planar aluminum surface. Electrochemical immunosensors developed on the nanoporous aluminum via the Ab immobilization method established in this study responded functionally to the antigen concentration in the diagnostic solution.

Highly Sensitive Photoelectrochemical Immunoassay with Enhanced Amplification Using Horseradish Peroxidase Induced Biocatalytic Precipitation on a CdS Quantum Dots Multilayer Electrode

Herein we demonstrate the protocol of a biocatalytic precipitation (BCP)-based sandwich photoelectrochemical (PEC) horseradish peroxidase (HRP)-linked immunoassay on the basis of their synergy effect for the ultrasensitive detection of mouse IgG (antigen, Ag) as a model protein. The hybrid film consisting of oppositely charged polyelectrolytes and CdS quantum dots (QDs) is developed by the classic layer by layer (LbL) method and then employed as the photoactive antibody (Ab) immobilization matrix for the subsequent sandwich-type Ab-Ag affinity interactions. Improved sensitivity is achieved through using the bioconjugates of HRP-secondary antibodies (Ab(2)). In addition to the much enhanced steric hindrance compared with the original one, the presence of HRP would further stimulate the BCP onto the electrode surface for signal amplification, concomitant to a competitive nonproductive absorption that lowers the photocurrent intensity. As a result of the multisignal amplification in this HRP catalyzed BCP-based PEC immunoassay, it possesses excellent analytical performance. The antigen could be detected from 0.5 pg/mL to 5.0 ng/mL with a detection limit of 0.5 pg/mL.

Current Advances in Antibody Immobilization on Different Surfaces and Beads

Antibody immobilization is of considerable interest for miscellaneous fields of interest, e.g. detecting biomarkers in cancer diagnostics, e.g. prostate specific antigen (PSA) for prostate cancer and pathogens like Escherichia coli, a foodborne pathogen. For this purpose, specific antibody captures are required and all of them should guarantee highest reproducibility and capacity possible. Especially in clinical applications very complex and specific immunoassays are needed. However, the complexity of pinning antibodies necessitates particular methods concerning the immobilization technique itself- because of their structure and specificity- and of course the analyzing methods. Glass, polymers, gold and even fullerene beads have been used as carriers, treated with various spacers and activation steps within the last few years. Furthermore, considerable attention is drawn to the regeneration of matrices and their capabilities for further antibody attachment. Commonly applied verification tools for the detection of the trapped antibodies include impedance spectroscopy (IS), atomic fluorescence microscopy (AFM), electrophoresis (EP), enzyme linked immunosorbent assays (ELISA), quartz crystal microbalance (QCM) and infrared spectroscopy (IR). Matrix assisted laser desorption/ionization-time-offlight mass spectrometry (MALDI-TOF/MS) is also a useful tool for proving the success of new antibody fixing procedures. Immobilization techniques have been improved concerning their reproducibility, binding capacity and high specificity. In this review, recent developments of antibody (Ab) immobilization are summarized, the individual applications are mentioned, advantages and disadvantages are discussed in detail. Keywords: Immunoassay, IgG, immobilization, antibody, beads, surface, gold, glass, polysaccharose, polymer

Systematic comparison of surface coatings for protein microarrays

To process large numbers of samples in parallel is one potential of protein microarrays for research and diagnostics. However, the application of protein arrays is currently hampered by the lack of comprehensive technological knowledge about the suitability of 2-D and 3-D slide surface coatings. We have performed a systematic study to analyze how both surface types perform in combination with different fluorescent dyes to generate significant and reproducible data. In total, we analyzed more than 100 slides containing 1152 spots each. Slides were probed against different monoclonal antibodies (mAbs) and recombinant fusion proteins. We found two surface coatings to be most suitable for protein and antibody (Ab) immobilization. These were further subjected to quantitative analyses by evaluating intraslide and slide-to-slide reproducibilities, and the linear range of target detection. In summary, we demonstrate that only suitable combinations of surface and fluorescent dyes allow the generation of highly reproducible data.

Probing direct binding affinity in electrochemical antibody-based sensors

Antibodies (Ab) are commonly used in the development of biosensors for large molecules. Their effectiveness in the direct analysis of small analyte at low concentrations can be compromised by the mode of Ab immobilization to solid substrates. The association of atrazine with anti-atrazine IgG monoclonal antibodies bound to gold-deposited quartz-crystal (GQC) and indium-doped tin oxide (ITO) electrode surfaces was characterized by electrochemical impedance spectroscopy (EIS) as a function of immobilization chemistry. Relative differences in the capacitance of unmodified and antibody modified gold electrode surfaces varied inversely with the degree of surface roughness as determined by atomic force microscopy (AFM). In addition, the surface resistance and capacitance elements measured at antibody modified GQC and ITO-electrodes were shown to reflect antibody–antigen binding.