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

Aniruddh Sarkar,Galit Alter,Alison E Mahan,Han Wei Hou,Jongyoon Han

doi:10.1038/srep23589

Aniruddh Sarkar, Galit Alter + Show 3 more

Open Access

https://doi.org/10.1038/srep23589

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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.

Highlights

Current affinity purification methods for proteins and cells use binary separation of binding and non-binding fractions of the sample mixture
Inertial microfluidics offers the advantage of high sample throughput in relatively inexpensive yet robust and easy-to-use devices, and can be adapted for use with a wide range of downstream assays
The mixture is flowed through a spiral microchannel device, which sorts the mixture into different outlets based on size. This device works on the principle of Dean Flow Fractionation (DFF)[11]

Summary

Introduction

Current affinity purification methods for proteins and cells use binary separation of binding and non-binding fractions of the sample mixture. Isolation of multiple targets is performed serially using multiple binding, washing and elution steps using resins or magnetic beads coated with bait molecules[1,2] This approach, while traditionally effective, is time consuming, low-throughput, and difficult to standardize and use for limited volume clinical samples due to the unavoidable loss and degradation of sample with repeated purifications. Earlier work using inertial microfluidic devices, which has been reviewed recently[9], has demonstrated cell and particle focusing, isolation and analysis and has been widely applied to isolation of circulating tumor cells (CTC) in cancer These methods have used size, shape or deformability of particles and cells, which can directly affect their inertial focusing[10]. We develop and demonstrate this technique and use it in the context of HIV-specific antibody and cell isolation from blood obtained from HIV-positive patients

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