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. 2016 Mar 30;6:23589.
doi: 10.1038/srep23589.

Multiplexed Affinity-Based Separation of Proteins and Cells Using Inertial Microfluidics

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Free PMC article

Multiplexed Affinity-Based Separation of Proteins and Cells Using Inertial Microfluidics

Aniruddh Sarkar et al. Sci Rep. .
Free PMC article

Abstract

Isolation of low abundance proteins or rare cells from complex mixtures, such as blood, is required for many diagnostic, therapeutic and research applications. Current affinity-based protein or cell separation methods use binary 'bind-elute' separations and are inefficient when applied to the isolation of multiple low-abundance proteins or cell types. We present a method for rapid and multiplexed, yet inexpensive, affinity-based isolation of both proteins and cells, using a size-coded mixture of multiple affinity-capture microbeads and an inertial microfluidic particle sorter device. In a single binding step, different targets-cells or proteins-bind to beads of different sizes, which are then sorted by flowing them through a spiral microfluidic channel. This technique performs continuous-flow, high throughput affinity-separation of milligram-scale protein samples or millions of cells in minutes after binding. We demonstrate the simultaneous isolation of multiple antibodies from serum and multiple cell types from peripheral blood mononuclear cells or whole blood. We use the technique to isolate low abundance antibodies specific to different HIV antigens and rare HIV-specific cells from blood obtained from HIV+ patients.

Figures

Figure 1
Figure 1. Principle of multiplexed protein and cell sorting using a Dean Flow Fractionation (DFF) device.
Beads of different sizes coated with different capture agents bind in a single step to the corresponding different antibodies or cells and are then sorted based on their size using the DFF device after which they can be post-processed for downstream analysis.
Figure 2
Figure 2
(a) Fabricated PDMS spiral microchannel device showing sample (red) and sheath (green) flows. (b) Locations of streams of beads of different diameters inside the device. Solid line indicates position of centre of stream while dotted lines indicate its edges. An optimized flow rate from the highlighted range was chosen for further experiments. (c) Focused bead streams flow in separate device outlets designed to capture them. (d) Bead separation efficiency as measured using flow cytometry.
Figure 3
Figure 3. Multiplexed isolation of three different HIV antigen-specific antibodies.
(a) Fraction of antigen-specific antibodies captured from samples after binding to a mixture of beads. Simultaneous capture of antibodies against three different antigens is observed. (b) Normalized antigen binding titer of antibodies eluted from beads obtained at each outlet. Pure antibodies specific to each antigen are obtained at the respective outlet to which the beads coated with that antigen were directed. Error bars represent standard deviation of three experiments.
Figure 4
Figure 4. Optimization of bead sizes for affinity-based cell sorting.
(a) Fold-enrichment of T Cells (~6–8 μm in diameter) bound to 15 μm and 10 μm beads into the 15 μm and 10 μm bead outlets respectively. (b) Bead-cell pairs show similar focusing positions as beads with the bigger of the two sizes or the sum of two sizes depending on if they are equal or unequal in size respectively.
Figure 5
Figure 5. Demonstration of multiplexed cell sorting.
(a) Separation of T Cells and B Cells from rest of PBMC (b) Separation of CD4+ and CD8+ cells from rest of PBMC. (c) Single-step isolation of CD4+ cells from whole blood. Error bars are standard deviation of three experiments. (d) Isolation or rare gp120-specific B cells from PBMC from HIV-infected donors.

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