RESEARCH PAPER Microfluidic continuous magnetophoretic protein separation using nanoparticle aggregates Su Hui Sophia Lee • T. Alan Hatton • Saif A. Khan Received: 28 February 2011 / Accepted: 8 April 2011 / Published online: 27 May 2011 Ó Springer-Verlag 2011 Abstract We demonstrate a microfluidic continuous-flow protein separation process in which silica-coated super- paramagnetic nanoparticles interact preferentially with hemoglobin in a mixture with bovine serum albumin, and the resulting hemoglobin-nanoparticle aggregates are recovered online using magnetophoresis. We present detailed modeling and analysis of this process yielding quantitative estimates of the recovery of both proteins, validated by experiments. While several previous studies utilize an average particle size in modeling magnetopho- retic particle trajectories or process design, in this study we emphasize the importance of accounting for particle size distributions in calculating particle recovery, and therefore in estimating separation efficiency. We combine experi- mentally measured size distributions of protein-nanoparti- cle aggregates with simulations of particle trajectories and provide a simple analytical method to calculate the efficiency of separation at various flow speeds, which fully accounts for heterogeneity in particle sizes. Our method can potentially be used for affinity based biomolecular separations at both analytical and preparative scales by exploiting well-established techniques to functionalize nanoparticle surfaces with selective ligands. Further, the modeling methodology presented here may be applied to provide better estimates of particle recovery in a broad range of magnetophoretic separation processes involving heterogeneity in particle sizes. Keywords Magnetic nanoclusters  Magnetic nanoparticles  Magnetophoretic separation  Protein separation  Microfluidics 1 Introduction Separation and purification of proteins, peptides, and other biomolecules are of major importance to the biosciences and biotechnology industries. Traditional separation methods are usually processes such as chromatography, electrophoresis, ultrafiltration, or precipitation (Safarik and Safarikova 2004). Microfluidic continuous-flow separation techniques offer attractive alternatives to more conven- tional batch-based methods, and several such methods based on a variety of separation principles have been developed in recent years (Lenshof and Laurell 2010; Pamme 2006; Pamme 2007; Tsutsui and Ho 2009). In a microchannel, separation can be effected simply by passive flow methods, where samples are guided to follow laminar streamlines through filters or obstacles such as pinches, weirs, or pillars (Lenshof and Laurell 2010; Pamme 2007; Tsutsui and Ho 2009). Inertial forces such as lift and centrifugal forces have been shown to enable Electronic supplementary material The online version of this article (doi:10.1007/s10404-011-0808-3) contains supplementary material, which is available to authorized users. S. H. S. Lee  T. A. Hatton  S. A. Khan Chemical and Pharmaceutical Engineering Program, Singapore- MIT Alliance, National University of Singapore, 4 Engineering Drive 3, E4-04-10, Singapore 117576, Singapore T. A. Hatton Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA S. A. Khan (&) Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, E5-02-28, Singapore 117576, Singapore e-mail: chesakk@nus.edu.sg 123 Microfluid Nanofluid (2011) 11:429–438 DOI 10.1007/s10404-011-0808-3