Microfluidic multiplexors are combinatorial arrays of binary valve patterns that increase the processing power of a network by allowing complex fluid manipulations with a minimal number of controllable pressurized inputs. While simple microfluidic arrays can be designed in which each fluid channel is controlled by its own individual valve control channel, this non-integrated strategy cannot be efficiently scaled up and faces similar problems encountered in pre-LSI electronic circuits. In contrast,
multiplexors work as a binary tree (Figure 5.1) and allow control of n fluid channels with only 2log2n interconnects. Multiplexors were fabricated using previously described
Figure 5.1: Multiplexor control in a multilayer elastomeric microfluidic device. Each open/closed valve configuration within the control layer opens a single flow channel.
Valve closing pressure scales with the width of the control channel, allowing non- contiguous flow channels to be closed by the wide valves while flow channels under the narrow valves remain open.
multilayer soft lithography techniques. The two-layer monolithic silicone devices consist of a "control" layer containing the multiplexor channel network and a "flow" layer separated by a thin polymer membrane (~30 µm). The membrane at the intersection of the control and flow channels functions as a discrete valve, in which pneumatic pressure applied to the control channels causes the elastomeric membrane to deflect down, closing off the underlying flow channel. Simultaneous addressing of multiple non-contiguous flow channels is accomplished by fabricating control channels of varying width while keeping the dimension of the flow channel fixed (100 µm wide and 9 µm high). The pneumatic pressure in the control channels required to close the flow channels scales with the width of the control channel, making it simple to actuate 100 µm x 100 µm valves at relatively low pressures (~40kPa) without closing off 50 µm x 100 µm crossover regions.
By using multiplexed valve systems, the power of the binary system becomes evident, with only 20 control channels required to specifically address 1024 flow channels. This allows a large number of elastomeric valves to perform complex fluidic manipulations within these devices, while the interface between the device and the external environment is simple and robust. Introduction of fluid into these devices is accomplished through steel pins inserted into holes punched through the silicone that connect the microfluidic control and flow channels with external fluid and pneumatic inputs. Unlike
micromachined devices made out of hard materials with a high Young's modulus112, silicone is soft and forms a tight seal around the input pins, readily accepting pressures of up to 300 kPa without leakage. Actuation of the valves in the control layer is
accomplished through computer-controlled external miniature solenoid valves, making to easy to simultaneously address complex arrays of valves.
Multiplexor Mechanics - 1024 Well Serpentine Microfluidic Device
The first multiplexor-based microfluidic device was designed as an enrichment chip (Figure 5.2). The core of the chip functions as a high-density multichamber array, into which sample can be loaded, compartmentalized and analyzed. To load sample into the array region, a set of barrier valves is closed to isolate the array from the rest of the flow channel network. All valves are filled with water prior to actuation to prevent bubble formation in the flow channel that results that occurs when air passes through the elastomeric membrane between the control and flow layer channels. When the barrier valves are closed, the flow channel within the array adopts a serpentine form with a single sample input and a sample vent to purge the displaced air (Figure 5.3). After the
Figure 5.2: 1024 well serpentine chip schematic.
chip is filled with sample, the array sandwich valve is actuated at 12-15 psi, which compartmentalizes the 32 rows of sample into 1024 aliquots with volumes of ~80 pL (Figure 5.4).
Figure 5.3: A) Detailed diagram of flow channel layout in high-density array region of serpentine chip illustrating flow path for sample loading when control layer barrier valves are closed. B) Illustration of sample loading mechanics in high-density array region using bromophenol blue. Vertical valves in photo are part of the non-actuated array sandwich valve (not visible in part A for simplification purposes).
Figure 5.4: Compartmentalization of the sample into ~80 pL aliquots using the array sandwich valve in the serpentine chip.
The compartmentalized liquid can be used for applications such as in vitro protein synthesis or single-cell enzymatic assays (Chapter 6).
Sample recovery from the chip utilizes the multiplexor, which is used to isolate and recover a single row of sample within the 32 row matrix. Isolation of the contents of a single row proceeds as follows: 1) The array sandwich valve is released, mixing the contents of the compartmentalized sample within each row. 2) The purge buffer input is filled with pressurized solution (5 psi). Excess air trapped in the chip as a result of the filling process is outgassed through the elastomeric silicone. 3) All valves in the multiplexor complex are closed. The multiplexor valves are symmetrically arranged around both sides of the array to channel the purge stream to the selected row and direct the isolated sample material to the sorting junction at the output of the chip. 4) The barrier valve is opened. 4) A Labview-based program is used to operate the binary combination of multiplexor valves for a selected row, which instantly purges the
contained sample material. An illustration of the multiplexor addressing process is shown in Figure 5.5, in which every other row in the matrix is sequentially purged.
The device functions as an enrichment chip, utilizing both the multiplexor and the sorting valves near the output. For applications involving screening for rare events, in which one compartment in the entire array is likely to contain a "positive" event, the isolation of the contents of the row that it resides in translates to a 32-fold enrichment vs.
the original mixture. An example of this type of application is a directed evolution experiment using mutagenized enzyme libraries where only a small fraction of mutants are expected to be active against the selected substrate. The sorting valves are more limited it their application, as they require detection elements (such as a fluorescent tag)
to remain bound to the "positive" events within the purged sample row, which could then be detected and sorted by instruments like the fluorescence-activated droplet sorter.
Figure 5.5: Sequential row purging of the high-density chamber array using multiplexor control. The bottom micrographs correspond to the highlighted area of the design
schematic. Alternate sample rows in the array, filled with 2.4 mM bromophenol blue dye, are purged sequentially in the example.