Controlling the selection stringency of phage display using a microfluidic device Yanli Liu, ab Jonathan D. Adams, c Kelisha Turner, e Frank V. Cochran, f Sanjiv Sam Gambhir f and H. Tom Soh * bd Received 24th November 2008, Accepted 18th February 2009 First published as an Advance Article on the web 3rd March 2009 DOI: 10.1039/b820985e We report the utilization of microfluidic technology to phage selection and demonstrate that accurate control of washing stringency in our microfluidic magnetic separator (MMS) directly impacts the diversity of isolated peptide sequences. Reproducible generation of magnetic and fluidic forces allows controlled washing conditions that enable rapid convergence of selected peptide sequences. These findings may provide a foundation for the development of automated microsystems for rapid in vitro directed evolution of affinity reagents. The invention of phage display technology 1 established the paradigm of in vitro selection of affinity reagents, and this technique has become an indispensable tool for probing specific biochemical molecular interactions. 2–4 In this method, a library of peptides or proteins, typically consisting of 10 9 members, is expressed on the outer surface of a bacteriophage with the DNA encoding each variant contained within the virus particle. 5 This physical linkage between each peptide sequence and its encoding DNA allows rapid in vitro selection of molecules that bind to a target of interest with high affinity and specificity. Typically, the selection is performed through a ‘‘panning’’ process in which the target molecules are immobilized on a solid support (e.g. Petri dishes, microtiter plates or magnetic beads), incubated with the library, and washed to eliminate the weakly- or non-binding phage. 5–8 The outcome of the phage selection critically depends on the strin- gency conditions imposed during the panning process. There are many factors that govern the stringency including concentrations of the target molecule and the phage library, incubation time, temper- ature, pH, salt concentration and washing conditions. 4,5,9 Conven- tional methods of panning have proven fairly effective, but suffer from a few disadvantages. First, they require significant amounts of target molecules (e.g. mg quantities), which poses an obvious problem when the target is not abundantly available. 5 Second, the process often yields phage binders that interact with the solid support rather than the target, necessitating negative selection steps. 10 Finally, it has proven challenging to reproducibly control the washing conditions, which has a direct impact on the resulting clones that are isolated. 9,11 Microfluidic technology offers many unique advantages for molecular separations. 12–17 Here, we present the first application of microfluidics for phage selection. Using streptavidin (SA) as a model target molecule, we demonstrate micro-scale control of washing stringency, and show that this has a direct impact on the efficiency of identifying peptide binding motifs. Finally, we show that such microfluidics-based systems can enable the development of ultra- rapid, highly efficient and automated platforms for the generation of affinity reagents in a miniaturized format. We adopted the Micro-Magnetic Separation (MMS) device (Fig. 1(a)) for the panning process. The detailed fabrication procedure of the device has been previously described by our group. 18,19 Briefly, the device consists of top and bottom glass layers separated by a 30 mm thick polymer layer, with a set of patterned nickel structures on the bottom glass layer. The microfabricated ferromagnetic structures in the channel allow effective trapping and release of magnetic beads, which is essential for controlled washing (Fig. 1(b)). Due to the difference in relative magnetic permeabilities between nickel and the buffer medium (m r,nickel ¼ 200, m r,buffer 1), the placement of a neodymium–iron–boron (NeFeB) external magnet (grade N42, K&J Magnetics, Jamison, PA) on the chip results in the automatic and reproducible generation of large magnetic field gradients (VB), causing magnetic beads to be trapped at the edges of the nickel patterns (Fig. 1(b), left). 20 The magnitude of the gradient is approximately 10 4 T/m within 8 mm of the struc- tures and the resulting magnetophoretic force ( ~ F m ) is tens of nanonewtons as approximated by ~ F m ¼ mVB, where m is the magnetization of the bead. 21 During the washing step, we ensure that the fluidic drag force ( ~ F d ) is less than ~ F m so that the beads do not inadvertently elute. We calculate that ~ F d exerted on the beads is typically less than 10 pN under our experimental conditions as approximated by ~ F d ¼ 6pha ~ v f where h is the fluid viscosity, a is the diameter of the bead, and ~ v f the velocity of the fluid. After washing, removal of the external magnet de-magnetizes the nickel structures so that the beads are effectively eluted from the chip (Fig. 1(b), right). Under experimental conditions, we found that the magnetic beads were indeed tightly bound to the magnetic traps during the washing steps, and we were able to recover 99.5% of the beads that entered the device as measured via flow cytometry. We performed our phage selection with DynabeadsÒ M-270 magnetic beads coated with streptavidin (SA), purchased from Invitrogen (Carlsbad, CA). The PhD-7 phage display peptide library containing 10 9 unique sequences was purchased from New England Biolabs (Ipswich, MA). We washed the magnetic beads twice for 5 min with tris-buffered saline (TBS, 50 mM Tris- HCl, 150 mM NaCl, pH 7.5), then resuspended 10 ml of the washed a Neuroscience Research Institute, University of California, Santa Barbara, CA 93106, USA b Department of Materials, University of California, Santa Barbara, CA 93106, USA. E-mail: tsoh@engineering.ucsb.edu; Fax: +1-805-893-8651; Tel: +1-805-893-8737 c Department of Physics, University of California, Santa Barbara, CA 93106, USA d Department of Mechanical Engineering, University of California, Santa Barbara, CA 93106, USA e School of Science and Technology, Jackson State University, Jackson, MS, 39217, USA f Molecular Imaging Program at Stanford, Department of Radiology & Bio- X Program, Stanford University, Stanford, CA, 94305, USA This journal is ª The Royal Society of Chemistry 2009 Lab Chip, 2009, 9, 1033–1036 | 1033 COMMUNICATION www.rsc.org/loc | Lab on a Chip