Positive influence of aminosilanes on anti-EpCAM antibody immobilization on a glass surface

Immobilization of antibodies has a number of promising applications, including detection of biomolecules and cells. Well-oriented antibodies are required to bind them effectively. To eliminate the problem of random antibodies’ orientation, the surface of the device can be modified with silanes. This study aimed at elucidating if selected aminosilanes were able to bind antibodies in the appropriate orientation and thus retain their binding activity. Silanization of glass slides was performed using three amino-functional trialkoxysilanes – A, AE, and AEE. The immunofluorescent reaction was used to evaluate the potential of the silanized glass surface to bind anti-EpCAM antibodies. The affinity of selected anti-EpCAM HEA125 antibodies labeled with fluorochrome to tested silanized surfaces was evaluated by measuring the mean fluorescence intensity (MFI) in each analyzed area. The presented silanes effectively bound antibodies. Higher fluorescence intensity was noticed in the case of silane-coated glass slides in comparison to unmodified ones. The differences in the contact angles also confirmed this result. In the case of silane A, the fluorescence intensity reflected the amount of bound antibodies. However, there was no such a relation in the case of the silanes AE and AEE. Although our research gave promising results, the usefulness of selected silanes needs to be confirmed by further studies using cancer cells. Running title: Aminosilanes as enhancers of antibody immobilization

Design and Development of Antibody Functionalized Gold Nanoparticles for Biomedical Applications

Antibody-functionalized gold nanoparticle constitutes a powerful interface biosystem for biomedical applications where the properties of gold nanoparticles and the specificity of antibody–antigen interactions are combined. This study provides insight into the key factors for the development of antibody functionalized gold nanoparticles focusing on the immobilization of the antibody. Here, we address an oriented antibody immobilization procedure on gold nanoparticles. It comprises chelatemodified gold nanoparticles that are designed for oriented immobilization of IgG antibodies (end on spatial orientation) through the metal-chelation to histidine-rich metal binding site in the heavy chain (Fc) of the antibody.

Antibody Immobilization in ZnO-Thin Film Transistors for Low-Cost Biosensors Applications

The antibody immobilization with low-cost materials and label-free methods are a challenge for the fabrication of biosensor devices. In this work, it was developed a strategy for antibody immobilization on ZnO TFTs over polyethylene terephthalate (PET) as a recyclable plastic substrate. Antibodies were biofunctionalized using a label-free strategy for E. coli detection. The use of a recyclable plastic substrate PET enables the compatibility with flexible electronics that could contribute for a low-cost biosensor useful in rural communities that do not have the necessary infrastructure and trained personnel for pathogenic bacterial detection in food or water.

Immunomodulating nano-adaptors potentiate antibody-based cancer immunotherapy

Modulating effector immune cells via monoclonal antibodies (mAbs) and facilitating the co-engagement of T cells and tumor cells via chimeric antigen receptor- T cells or bispecific T cell-engaging antibodies are two typical cancer immunotherapy approaches. We speculated that immobilizing two types of mAbs against effector cells and tumor cells on a single nanoparticle could integrate the functions of these two approaches, as the engineered formulation (immunomodulating nano-adaptor, imNA) could potentially associate with both cells and bridge them together like an ‘adaptor’ while maintaining the immunomodulatory properties of the parental mAbs. However, existing mAbs-immobilization strategies mainly rely on a chemical reaction, a process that is rough and difficult to control. Here, we build up a versatile antibody immobilization platform by conjugating anti-IgG (Fc specific) antibody (αFc) onto the nanoparticle surface (αFc-NP), and confirm that αFc-NP could conveniently and efficiently immobilize two types of mAbs through Fc-specific noncovalent interactions to form imNAs. Finally, we validate the superiority of imNAs over the mixture of parental mAbs in T cell-, natural killer cell- and macrophage-mediated antitumor immune responses in multiple murine tumor models.

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

ABSTRACTHere, we demonstrate real-time multiplexed virus detection by applying DNA-directed antibody immobilization technique to 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 specific DNA-DNA hybridization. The resulting antibody array is shown to detect three different recombinant Vesicular Stomatitis Viruses (rVSVs) 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 solution phase prior to flowing 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 hour. 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 minutes 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 these properties will make SP-IRIS a versatile and robust platform for point-of-care diagnostics applications.