Validation of Arrayed Imaging Reflectometry Biosensor Response for Protein–Antibody Interactions: Cross-Correlation of Theory, Experiment, and Complementary Techniques

Abstract

One of the critical steps in the development of an analytical technique is to confirm that its experimental response correlates with predictions derived from the theoretical framework on which it is based. This validates the technique quantitatively, and, in the case of a biosensor, facilitates a correlation of the sensor’s output signal to the concentration of the analyte being tested. Herein we report studies demonstrating that the quantitative response of Arrayed Imaging Reflectometry (AIR), a highly sensitive label-free biosensing method, is a predictable function of probe and analyte properties. We first incorporated a standard one-site Langmuir binding model describing probe-analyte interactions at the surface into the theoretical model for thickness-dependent reflectance in AIR. This established a hypothetical correlation between analyte concentration and the AIR response. Spectroscopic ellipsometry, surface plasmon resonance (SPR) and AIR were then used to validate this model for two biomedically important proteins, fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF). While our studies demonstrated that the 1:1 one-site Langmuir model accurately described the observed response of macro spot AIR arrays, either a two-site Langmuir model or a Sips isotherm better described the behavior of AIR microarrays. These studies confirmed the quantitative performance of AIR across a range of probe-analyte affinities. Furthermore, the methodology developed here can be extended to other label-free biosensing platforms, thus facilitating a more accurate and quantitative interpretation of the sensor response.

A label-free optical biosensor for serotyping "unknown" influenza viruses

“…6 The technique has also been validated via comparisons to reference methods including surface plasmon resonance (SPR) and spectroscopic ellipsometry. 7 With regard to influenza-targeted applications, we have demonstrated that multiplex hemagglutinin arrays have utility for rapidly assessing human serum samples for the presence of anti-influenza antibodies in a vaccine trial, 8 and provide a simple method for multiplex serology on avian surveillance samples. 9 Here, we provide a preliminary description of an AIR array intended to simplify the task of classifying newly observed strains of influenza, by enabling patterns of reactivity with the chip to be compared with those generated by known strains of the virus.…”

Evaluation of Medicine Effects on the Interaction of Myoglobin and Its Aptamer or Antibody Using Atomic Force Microscopy

“…AFM silicon nitride (Si 3 N 4 ) tips (Benyuan Nano Instrument Co., China) were covalently attached by the aptamer (or antibody) according to previously reported procedures. 20−22 In detail, the tips were first cleaned and hydroxized through the treatment with chloroform, HF acid, alkaline solution (NH 4 The cleaned tips were incubated in 1.0% (v/v) MPTMS/ toluene solution for 2 h and then rinsed thoroughly with toluene to be modified with −SH groups. Then, the silanized tips were activated by incubation with NHS-PEG-MAL (1 mg mL −1 ) in 50% dimethyl sulfoxide for 3 h at room temperature.…”

Biomedical and Biochemical Tools of Förster Resonance Energy Transfer Enabled by Colloidal Quantum Dot Nanocrystals for Life Sciences

“…Many applications of biosensors in the literature heavily depend on the utilization of proteins as the recognition elements of the biosensor of interest [73]. They have also been utilized in many different FRET applications, and dye-conjugated proteins served as the acceptor or donor molecules.…”