Magnetic force micropiston: an integrated force/microfluidic device for the application of compressive forces in a confined environment

Abstract

Cellular biology takes place inside confining spaces. For example, bacteria grow in crevices, red blood cells squeeze through capillaries, and chromosomes replicate inside the nucleus. Frequently, the extent of this confinement varies. Bacteria grow longer and divide, red blood cells move through smaller and smaller passages as they travel to capillary beds, and replication doubles the amount of DNA inside the nucleus. This increase in confinement, either due to a decrease in the available space or an increase in the amount of material contained in a constant volume, has the potential to squeeze and stress objects in ways that may lead to changes in morphology, dynamics, and ultimately biological function. Here, we describe a device developed to probe the interplay between confinement and the mechanical properties of cells and cellular structures, and forces that arise due to changes in a structure's state. In this system, the manipulation of a magnetic bead exerts a compressive force upon a target contained in the confining space of a microfluidic channel. This magnetic force microfluidic piston is constructed in such a way that we can measure (a) target compliance and changes in compliance as induced by changes in buffer, extract, or biochemical composition, (b) target expansion force generated by changes in the same parameters, and (c) the effects of compression stress on a target's structure and function. Beyond these issues, our system has general applicability to a variety of questions requiring the combination of mechanical forces, confinement, and optical imaging.

Figure 1

Compression with and without confinement. In part (a) compressive forces are applied to a molecule in a system without complete confinement, allowing the molecule to reposition itself in response to the force. (b) Compressive force applied in a system that does not permit large scale repositioning of the target.

Figure 2

Cartoon representation of the operation of the micropiston system showing the entire device (a) and zoomed–in view of the microchannels [(b)–(f)]. In part (a), valves at the upper right and lower left of the device are open, allowing fluid to flow from the upper main channel, across the small channels (compression chambers) with constrictions and into the second large channel. The direction of fluid flow is indicated with green arrows, closed valves preventing fluid flow are indicated with a red X. Parts (b)–(f) depict loading the device. Empty compression chambers (b) are first loaded with polystyrene blocking beads that prevent passage of the compression target (c). (d) Using fluid flow, compression targets (tan line) are loaded into the chambers. (e) Magnetic beads are then loaded into the chambers using weak fluid flow. After magnetic bead loading, all valves are closed and the system is allowed to equilibrate prior to the application of force. (f) Compressive forces are applied by actuating the magnetic beads in the direction of the polystyrene blocking beads (down the channel).

Figure 3

Device construction. (a) Small channels (“compression chambers”) are created first by etching away the surrounding silicon wafer. (b) Two large “main” channels made from SU–8 are added to the wafer, completing the silicon master. (c) Etched thin magnetic foils are placed atop the Si master, covered in PDMS, and cured for 2 h at 65 ºC. The foils are cut along the dashed white lines to separate the micropiston chip from the alignment frame. (d) Completed micropiston chip peeled away from the Si wafer and cut from the alignment frame. Holes punched in the backside of the PDMS (indicated with blue ‘‘X’’) are used to deliver fluid to the microfluidic channels. (e) Zoomed–in view of the microfluidic channels and the etched magnetic materials.

Figure 4

Fluidic circuit. The micropiston “chip” containing the compression chambers is connected to an external fluid delivery system using four valves (numbered 1–4) that either deliver fluid to the input/output side of a main channel or isolate the chip from the fluid delivery system.

Figure 5

(a) The complete micropiston system, with the electromagnet drive unit in the center flanked by acrylic fluid delivery manifolds. These three components sit atop a heated plate (surface covered with black insulation) inserted into the 96–well plate adapter of a motorized stage (Proscan II, Prior Scientific, Rockland, MA). (b) Illustrated side view of the micropiston chip (dashed black rectangle), fluid manifold, and electromagnets. (c) Multiple compression chambers (small channels) of the micropiston system loaded with 4.5 μm blocking beads. (d) Non–specific adsorption to the inner channel surfaces is reduced significantly using a lipid treatment. The top two images are unprocessed fluorescence, bottom two have fluorescent intensity mapped to surface height. Blue lines indicate channel walls. The adsorption of 200 nm polystyrene beads to channel surfaces after a 2 h incubation is shown for a bare channel (left) and treated channel (right).

Figure 6

Blocking beads in microfluidic channels capturing lambda DNA. The passage of fluorescently labeled lambda DNA through the microfluidic channels is significantly hindered by the introduction of 4.5 μm blocking beads.

Figure 7

Bead movement driven by chromatin expansion. Exposure of the sperm chromatin (bright material between dark beads) to egg extract causes the chromatin to decompact, pushing the bead towards the top of the channel. Here, chromatin expansion pushes the magnetic bead over 10 μm in the compression chamber.

Figure 8

Magnetic force as a function of drive current. (Upper left) Magnetic force as a function of distance to the pole tip for a 2.5 A current. (Lower right) Example trajectories for 4.5 μm beads using in these experiments.

Figure 9

Piston in action. (a) Montage showing individual frames of a compression cycle in the micropiston. Force towards the constricted end of the channel is generated by the magnetic force sub–system, return force generated by fluid flow. (b) Two compression chambers, each loaded with four magnetic beads, are actuated producing approximately 1.2 nN in each chamber.