Journal: ACS Sensors
Article Title: Sandwich Immunosensor Based on Particle Motion: How Do Reactant Concentrations and Reaction Pathways Determine the Time-Dependent Response of the Sensor?
doi: 10.1021/acssensors.3c01549
Figure Lengend Snippet: Sandwich immunosensing using Biosensing by Particle Motion. (A) Schematic representation of Biosensing by Particle Motion. Particles are tracked over time using video microscopy with particle identification and tracking software. In the presence of the analyte, the antibodies on the substrate and on the particles bind to the analyte and form a sandwich bond, resulting in a bound state of the particles. The sketch is not to scale; the particles are much larger than the molecules. (B) Close-up of the molecular components of the BPM immunosensor, with bovine lactoferrin as the analyte molecule. Antibodies are physisorbed on a polystyrene substrate, and open areas are blocked with bovine serum albumin (BSA). Streptavidin-coated particles (1 μm diameter) are functionalized with biotinylated antibodies and blocked with biotin-PEG. (C) The bound fraction was measured for varying analyte concentrations using endpoint measurements in 96-well plates. (D) The signal response over time was measured by using flow cells. Upon addition of the analyte, the bound fraction increases over time until a plateau is reached. In this study, the factors are investigated that contribute to the characteristic response time (τ), maximal signal ( S end ), and initial slope. (E) The molecular sandwich complex between particle and substrate can be formed via two pathways. Pathway AP: an analyte molecule is first captured by an antibody on a particle and subsequently by an antibody on the substrate. Pathway AS: an analyte molecule is first captured by an antibody on the substrate and, subsequently, by an antibody on a particle. Analyte reaction-diffusion plays a role in the first processes (A → AP and A → AS) and particle reaction-diffusion plays a role in the second processes (AP → SAP and AS → SAP).
Article Snippet: Transparent 96-well plates (Nunc MaxiSorb flat-bottom), Dynabeads MyOne Streptavidin C1, and bovine lactoferrin polyclonal antibody (A10–126A) were purchased from Thermo Fisher Scientific.
Techniques: Microscopy, Software, Diffusion-based Assay
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![Influence of the analyte concentration and the substrate binder concentration on the sensor response. (A) The signal of the BPM sensor as a function of time was studied in a static flow cell for different lactoferrin concentrations. The polystyrene substrates were functionalized by physisorption of antibodies using concentrations of 5 nM (green), 50 nM (orange), and 500 nM (red). The data were fitted with single-exponential curves according to eq . (B) The maximal signal S end was extracted from the fits in (A). (C) The characteristic response time τ extracted from the fits in (A) plotted on log–log scales. The τ values show a slope of roughly minus 1/3, which means that τ scales roughly as τ ∝ [A] −1/3 . (D) The initial slope (bound fraction per second) extracted from linear fits of the first 2000 s (Supporting Information Figure S4 ). The solid lines in (B–D) are a guide to the eye and the error bars (not always visible) indicate the 95% confidence interval of the values extracted from the fits.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_3507/pmc10683507/pmc10683507__se3c01549_0003.jpg)
