Autonomous magnetically actuated continuous flow microimmunofluorocytometry assay

Abstract

This article presents a microfluidic device which integrates autonomous serial immunofluorocytometry binding reactions of cytometric beads with fluorescence detection and quantification in a continuous flow environment. The microdevice assay is intended to alleviate the extensive benchwork and large sample volumes used when conducting traditional immunoassays, without requiring complex external controls. The technology is based on the miniaturization and automation of the serial processing steps of an antigen sandwich immunoassay, with integrated fluorescence detection using paramagnetic microbeads. The continuous flow design may enable temporal tracking of time-varying protein concentrations in a continuously infused sample for clinical applications, specifically for monitoring inflammation marker proteins in blood produced during cardiac surgeries involving cardiopulmonary bypass (CPB) procedures. The device operation was first validated via a single incubation device which measured the concentration of a fluorescently labeled biotin molecule using streptavidin-coated paramagnetic cytometric beads. Subsequently, a dual incubation device was tested with samples of the anaphylatoxin complement protein C3a, and was shown to be capable of differentiating between samples at typical systemic concentrations of the protein (1–5 μg/ml), with very low sample usage (<6 μl/h). It is believed that this continuous flow, automated microimmunosensor technology will be a platform for high sample rate immunoassays capable of tracking and more thoroughly characterizing the systemic inflammation process, and may aid in the development of better treatment options for systemic inflammation during and after CPB.

Development and validation of a microfluidic immunoassay capable of multiplexing parallel samples in microliter volumes

“…Lab-on-a-disc bead based approaches have also been applied, first developed for single protein assays using fluorescence detection 49 , and then expanded to multiplexing by performing assays for different analytes in parallel in conjunction with absorbance detection 12 . Moreover, some devices have performed the immunoassay with beads flowing in solution and utilized off-chip flow cytometry 25, 50 . While the overall assay principle in each of these technologies is similar, the major differences are marked by the methods used to immobilize the beads, introduce the samples and reagent solutions, and interrogate the beads at the completion of the assay.…”

Development of a low-volume, highly sensitive microimmunoassay using computational fluid dynamics-driven multiobjective optimization

“…For the validation experiments, a model system for antibody antigen binding consisting of streptavidin coated microbeads and biocytin-AlexaFluor 488 was employed. This approach has been used in prior publications as a model system of antibody-antigen binding as it provides several advantages (Yoo et al 2011; Sasso et al 2010). These include increased reaction speed, lower cost compared to antibody conjugated beads, and a one step reaction allowing for direct interrogation in real time using fluorescent microscopy.…”

Automated microfluidic processing platform for multiplexed magnetic bead immunoassays

“…The magnetic force acting on the bead can be approximated as $F=1\frac{2}{3}\pi R_{\mathrm{c}}^3\chi \nabla {\overrightarrow{B}}^{2}true/{\mu }_o{false(1+\chi false)}^2$ where B is the magnetic field from the magnet, μ o is the magnetic permeability of free space, χ is the magnetic susceptibility of the bead ( χ = 0.170) (Shevkoplyas et al 2007), and R c is the radius of the bead magnetic core (~0.5 μm). As described previously, the force on the microbead imparted by the magnetic field is approximately 7.5 pN while the shear force on the bead due to the background flow is greater than 22.3 pN so the beads are not statically trapped and can transverse through the device (Sasso et al 2010). With proper tuning the device has been shown to achieve 100 % bead stream transfer efficiency.…”