Enhancing Antibodies’ Binding Capacity through Oriented Functionalization of Plasmonic Surfaces

Protein A has long been used in different research fields due to its ability to specifically recognize immunoglobulins (Ig). The protein derived from Staphylococcus aureus binds Ig through the Fc region of the antibody, showing its strongest binding in immunoglobulin G (IgG), making it the most used protein in its purification and detection. The research presented here integrates, for the first time, protein A to a silicon surface patterned with gold nanoparticles for the oriented binding of IgG. The signal detection is conveyed through a metal enhanced fluorescence (MEF) system. Orienting immunoglobulins allows the exposition of the fragment antigen-binding (Fab) region for the binding to its antigen, substantially increasing the binding capacity per antibody immobilized. Antibodies orientation is of crucial importance in many diagnostics devices, particularly when either component is in limited quantities.

Scanning Electrochemical Impedance Microscopy in Redox-Competition Mode for the Investigation of Antibodies Labelled with Horseradish Peroxidase

Scanning electrochemical microscopy enhanced by electrochemical impedance spectroscopy (SEIM) was applied to detect immobilized antibodies labelled with horseradish peroxidase (Ab-HRP). The localized HRP activity was investigated by the SEIM redox competition (RC-SEIM) mode using hydrogen peroxide as a substrate and hexacyanoferrate as a redox mediator. Electrochemical impedance shows to be related to the consumption of hydrogen peroxide at the ultramicroelectrode. For the evaluation of impedimetric results, an equivalent electric circuit was applied with solution resistance, double-layer capacitance, and charge-transfer resistance. These equivalent circuit characteristics depend on the distance between the sample and ultramicroelectrode, and the concentration of substrate. From the gathered data, the charge-transfer resistance appeared to be the parameter describing the behavior of HRP catalyzed reaction as it showed a linear dependence on H2O2 concentration. The RC-SEIM mode suitability for the studying of HRP catalyzed reactions and for the evaluation of Ab-HRP bound to the surface was demonstrated. Additionally, the applicability of RC-SEIM mode for the determination of Ab-HRP affinity bound to the target analyte was discussed.

Bioaffinity-based Surface Immobilization of Antibodies to Capture Endothelial Colony-forming Cells

Maximizing the re-endothelialization of vascular implants such as prostheses or stents has the potential to significantly improve their long-term performance. Endothelial progenitor cell capture stents with surface-immobilized antibodies show significantly improved endothelialization in the clinic. However, most current antibody-based stent surface modification strategies rely on antibody adsorption or direct conjugation via amino or carboxyl groups which leads to poor control over antibody surface concentration and/or molecular orientation, and ultimately bioavailability for cell capture. Here, we assess the utility of a bioaffinity-based surface modification strategy consisting of a surface-conjugated cysteine-tagged protein G molecules that immobilize Immunoglobulin G (IgG) antibodies via the Fc domain to capture circulating endothelial colony-forming cells (ECFCs). The cysteine-tagged protein G was grafted onto aminated substrates at different concentrations as detected by an enzyme-linked immunosorbent assay and fluorescence imaging. Different IgG antibodies were successfully immobilized on the protein G-modified surfaces and higher antibody surface concentrations were achieved compared to passive adsorption methods. Surfaces with immobilized antibodies targeting endothelial surface proteins, such as CD144, significantly enhanced the capture of circulating ECFCs in vitro compared to surfaces with non-endothelial specific antibodies such as anti-CD14. This work presents a potential avenue for enhancing the clinical performance of vascular implants by using covalent grafting of protein G to immobilize IgG antibodies more effectively.

Design and Analysis of Silicon Nanowire FET for the Detection of Cardiac Troponin I Biomarker

Cardiac arrests are one of the major health problems in present days. Cardiac Troponin-I (cTnI) is one of the important enzymes that causes cardiac arrest. Early diagnosis and proper medication of this saves human life. One of the prominent devices to diagnose troponin I is FET based bio-sensor. Normally, for these sensors’ higher sensitivities will be obtained as these biosensors structure consists of nanowire FETs. Proper selection of materials, dimensions, and doping concentrations of nanowire FET imply the perfection of a nanowire FET-based biosensor. In this work, Silicon Nanowire (SiNW) FET sensor is designed and simulated using COMSOL Multiphysics. Through this design, Identified the presence of different concentrations of cTnI present in human blood. The presence of different enzymes like cTnT, cTnI etc., bring changes in characteristics of SiNW FET sensor. With these changes in characteristics, we can identify the presence of these enzymes of a lower concentration also. The lower concentrations of these biomarkers will bring notable changes in the drain current. The characteristics were analysed with the SiNW FET which is equipped with immobilized antibodies on it. The considerable changes observed in these characteristics of FET sensor identifies the presence of cTnI biomarker and are attached to the monoclonal Antibodies (mAb). Our observations shown that the properties of designed SiNW FET changes with presence of these bio marker materials and a limit of detection is obtained the order of 2pg/mL. with further the design bio sensor with SiNW FET can be used for microfluidic and Lab-on-Chip applications also.

Immobilized Antibodies on Mercaptophenylboronic Acid Monolayers for Dual-Strategy Detection of 20S Proteasome

A dual strategy for the electrochemical detection for 20S proteasome (20S) is proposed, based on the oriented immobilization of a capture monoclonal antibody (Abβ) on a self-assembled monolayer of 4-mercaptophenylboronic acid (4-MPBA) on gold electrodes, which led to the Au/4-MPBA/Abβ immunosensor. The methodology comprises the correlation of 20S concentration with (i) its proteolytic activity toward the Z-LLE-AMC substrate, using the Au/4-MPBA/Abβ/20S, and (ii) the enzymatic activity of an alkaline phosphatase (AlkP) from the AlkP-labeled secondary antibody (Abcore-AlkP), which involves the conversion of aminophenylphosphate to the electroactive aminophenol using Au/4-MPBA/Abβ/20S/Abcore-AlkP. The step-by-step construction of the immunosensor and the interactions at its surface were evaluated by surface plasmon resonance and gravimetric analysis with quartz crystal microbalance, showing a high affinity between both antibodies and 20S. Morphological analysis by scanning electron microscopy demonstrated a pattern of parallel lines upon immobilization of Abβ on 4-MPBA and morphological changes to a well-organized granular structure upon binding of 20S. A voltametric and impedimetric characterization was performed after each step in the immunosensor construction. The two detection strategies were evaluated. It was shown that the immunosensor responds linearly with 20S concentration in the range between 5 and 100 µg mL−1, which corresponds to proteasome levels in serum in the case of diverse pathological situations, and LoD values of 1.4 and 0.2 µg mL−1 were calculated for the detection strategies. The immunosensor was applied to the detection of 20S in serum samples with recovery values ranging from 101 to 103%.

Highly Sensitive Detection of CA 125 Protein with the Use of an n-Type Nanowire Biosensor

The detection of CA 125 protein in a solution using a silicon-on-insulator (SOI)-nanowire biosensor with n-type chip has been experimentally demonstrated. The surface of nanowires was modified by covalent immobilization of antibodies against CA 125 in order to provide the biospecificity of the target protein detection. We have demonstrated that the biosensor signal, which results from the biospecific interaction between CA 125 and the covalently immobilized antibodies, increases with the increase in the protein concentration. At that, the minimum concentration, at which the target protein was detectable with the SOI-nanowire biosensor, amounted to 1.5 × 10−16 M.

Immunomagnetic Separation of Microorganisms with Iron Oxide Nanoparticles

The early detection of Legionella in water reservoirs, and the prevention of their often fatal diseases, requires the development of rapid and reliable detection processes. A method for the magnetic separation (MS) of Legionella pneumophila by superparamagnetic iron oxide nanoparticles is developed, which represents the basis for future bacteria detection kits. The focus lies on the separation process and the simplicity of using magnetic nanomaterials. Iron oxide nanoparticles are functionalized with epoxy groups and Legionella-specific antibodies are immobilized. The resulting complexes are characterized with infrared spectroscopy and tested for the specific separation and enrichment of the selected microorganisms. The cell-particle complexes can be isolated in a magnetic field and detected with conventional methods such as fluorescence detection. A nonspecific enrichment of bacteria is also possible by using bare iron oxide nanoparticles (BIONs), which we used as a reference to the nanoparticles with immobilized antibodies. Furthermore, the immunomagnetic separation can be applied for the detection of multiple other microorganisms and thus might pave the way for simpler bacterial diagnosis.

Label-Free Multi-Microfluidic Immunoassays with Liquid Crystals on Polydimethylsiloxane Biosensing Chips

We developed a new format for liquid crystal (LC)-based multi-microfluidic immunoassays, hosted on a polydimethylsiloxane substrate. In this design, the orientations of the LCs were strongly affected by the interface between the four microchannel walls and surrounding LCs. When the alignment layer was coated inside a microchannel, the LCs oriented homeotropically and appeared dark under crossed polarizers. After antigens bound to the immobilized antibodies on the alignment layer were coated onto the channel walls, the light intensity of the LC molecules changed from dark to bright because of disruption of the LCs. By employing pressure-driven flow, binding of the antigen/antibody could be detected by optical signals in a sequential order. The multi-microfluidic LC biosensor was tested by detecting bovine serum albumin (BSA) and an immunocomplex of BSA antigen/antibody pairs, a protein standard commonly used in labs. We show that this multi-microfluidic immunoassay was able to detect BSA and antigen/antibody BSA pairs with a naked-eye detection limitation of −0.01 µg/mL. Based on this new immunoassay design, a simple and robust device for LC-based label-free microfluidic immunodetection was demonstrated.