µFBI: a microfluidic bead-based immunoassay for multiplexed detection of proteins from a µL sample volume

Abstract

Background: Over the last ten years, miniaturized multiplexed immunoassays have become robust, reliable research tools that enable researchers to simultaneously determine a multitude of parameters. Among the numerous analytical protein arrays available, bead-based assay systems have evolved into a key technology that enables the quantitative protein profiling of biological samples whilst requiring only a minimal amount of sample material.

Methodology/principal findings: A microfluidic bead-based immunoassay, µFBI, was developed to perform bead-based multiplexed sandwich immunoassays in a capillary. This setup allows the simultaneous detection of several parameters and only requires 200 ng of tissue lysate in a 1 µL assay volume. In addition, only 1 µL of detection antibodies and 1 µL of the reporter molecule Streptavidin-Phycoerythrin were required. The µFBI was used to compare the expression of seven receptor tyrosine kinases and their degree of tyrosine phosphorylation in breast cancer tissue and in normal tissue lysates. The total amount of HER-2, as well the degree of tyrosine phosphorylation was much higher in breast cancer tissue than in normal tissue. µFBI and a standard bead-based assay led to identical protein expression data. Moreover, it was possible to reduce the quantity of sample material required by a factor of 100 and the quantity of reagents by a factor of 30.

Conclusions/significance: The µFBI, microfluidic bead-based immunoassay, allows the analysis of multiple parameters from a very small amount of sample material, such as tumor biopsies or tissue sections.

Figure 1. Schematic illustration of microfluidic bead-based immunoassay (µFBI).

(A) The assay is performed in a capillary by controlling the solution flow into or out of the capillary with a syringe pump. A filter (pore diameter = 0.5 µm) is used to keep the antibody-coated beads at the incubation site to capture target analytes and to react with detection reagents. After incubation, the beads are pumped out and submitted to a Luminex 100 IS instrument for fluorescent signal readout. (B) The structure of the incubation zone consists of a filter and an adaptor to connect two fused silica-capillaries at both sides. The left capillary is connected to a syringe pump and the right capillary is connected to a sample solution reservoir.

Figure 2. Optimization of µFBI-relevant parameters.

(A – D) denote the optimization of bead number, capillary coating surface, analyte volume, and incubation time, respectively. In the experiment, anti-biotin antibody-coated beads were incubated in the capillary with biotin-PE (100 ng/ml). The resulting beads were then pumped out and diluted in 100 µL with assay diluent and analyzed with the Luminex 100 IS system using fluorescent detection. The numbers in gray given in C and D represent the flow rate of the sample solution. The MFI value (column for A and B, circle for C and D) was read out and the recovered beads (square) were counted with the Luminex 100 IS system.

Figure 3. Quantification of Biotin-Phycoerythrin using the µFBI assay.

1,000 anti-biotin antibody-coated beads were incubated with different concentrations of biotin-phycoerythrin (1 µl) for 5 min, respectively. A BSA-coated bead served as a control. The MFI generated with the anti-biotin antibody (triangle) or the BSA-coated beads (circle) are shown.

Figure 4. Protein expression analysis using the µFBI.

Total protein expression and phosphotyrosine-specific expression of seven RTKs were determined by µFBI (A and B) and a normal bead-based assay (C and D). For µFBI, seven mixed capture antibody-coated beads (1,000 beads per analyte) were incubated with tissue lysates (1 µl) for 60 min, mixed biotinylated detection antibodies (1 µl) for 60 min and Strep-PE (1 µl) for 45 min. The MFI value was obtained with a Luminex 100 IS system.

Figure 5. Simulation of a 100-plex assay.

800 beads of each bead type were incubated in the capillary for 1 h. Beads were pumped out and counted with a Luminex-100 instrument. (A) Counted beads of the 100 different color-coded bead types which can be spectrally distinguished by their colour code. (B) number of counted beads for each bead type.