Overview

Microfluidics devices are applied to deal with flow of liquid inside micrometer-sized channels, and conduct continuous observation under fluorescence microscopy. This year, we designed two microfluidic chips --a sand-clock chip and a seven-segment display chip. The sand-clock microfluidic device is a "mother machine", which consists of dozens of thin channels holding single cells, and can be used to track individual cell line for generations. And for the seven-segment display chip, cells were kept in chambers to observe fluorescence changes.

Description

Seven-segment Display Microfluidic Chip

The seven-segment display microfluidic chip consists of four distinct regions. Each region has several chambers which can be filled with different bacteria strains. Narrow fences were set between chambers to reduce flow resistance. The medium can be introduced to the chamber through three inlets. The inlets were designed to have concentric fences to avoid impurity obstructing feeding channel. Different inlets connecting the same region were used to insert medium containing different inducer.



Altogether the chip can show 1,2 and 3 pattern as a seven-segment display, when we filling the four distinct regions with our strains in the control unit.

Sand Clock Microfluidic Chip

The sand clock chip contains seven regions, and each region has its own feeding channel connecting with inlet and outlet. Thin channels housing single cell are located at the central parts of feeding channels. This sand clock microfluidic chip can be used to track individual cells of different strains at the same time.



If cells in different feeding channels are designed to give out fluorescence in order, the chip can show a sand-clock pattern.

Protocol

Fabrication of the Microfluidic Master Plate

Fabrication of the E. coli mother machine was carried out using standard UV photolithography in a clean-room environment. The device was designed using L Edit and quartz-chrome photomasks.
Note: for all spin coater steps described below, the following shorthand notation is used: speed (rpm)/acceleration (rpm/sec)/time (sec).

First Layer: Cell Channels

This set of steps lays down the channels that house the cells in the final device. The tolerances for this layer are very stringent; the exposure dose and contact between mask and wafer must be opti-mized. We recommend trying out a range of exposure parameters to ensure that a useful device is obtained. We also stress the importance of the very long post-exposure baking time in the process below.

1. Place a new 3'' Si wafer (we used 380 μm TEST-grade wafers from University Wafer) in a dish of fresh acetone. Sonicate at high power for 5 minutes.
2. Sequentially rinse the wafer with streams of methyl alcohol (MeOH), isopropyl alcohol (IPA) and H₂O (10 seconds per solvent).
3. Place the wafer on a 2'' spin chuck and spin a few seconds at 500 rpm.
4. While spinning, sequentially rinse the wafer with streams of MeOH, IPA and H₂O.
5. Spin the wafer for 1 minute at 3 000 rpm to dry.
6. Dehydrate the wafer with the smooth side upwards above clean filter paper for 5 minutes on a hot plate set to 190℃, then cool it at r.t.
7. Place the dehydrated wafer onto the spin-coater chuck and dispense a small (cover ∼2/3 of the wafer surface) amount of Su8 3005 photoresist (Microchem) using a disposable pipette. Run the spin pro-gram (Set spin program to: Step 1: 500/100/10, Step 2: 1400/300/36). This should result in a coat of ∼1.5 μm.
8. Soft-bake the wafer for 2 minutes at 95℃.
9. Expose the wafer for 40∼50 seconds (3.7 mW/cm²) through the cell-channel mask in vacuum contact mode.
10. Post exposure, bake the wafer (in order) for 2 minutes at 95℃ (according to the thickness of this layer), then cool it at r.t.

Second Layer: Feeding Channels

This layer of the device forms the culture-medium flow channels. The dimensions of these features are not critical: we have used feeding channels of widely varying dimension to similar effect. The alignment is sensitive to large errors, however. The alignment between the feeding channels and cell channels must be accurate (down to a couple of microns) in order to ensure that the cell channels are of the desired final length.

1. Set the spin program to: Step 1: 500/100/10, Step 2:1200/300/40.
2. Place the wafer onto the spin-coater chuck and dispense a small amount of Su8 3005 photoresist (cover ∼2/3 of the wafer surface) with a pipette being careful not to introduce bubbles. Run the spin program. This should result in a coat of - 9 μm.
3. Soft-bake the wafer (in order) for 8 min at 95℃.
4. With an Su8-developer-soaked swab, clean the newly-deposited photoresist off the alignment marks to make them visible for the alignment process.
5. Align the feeding channel mask to the alignment marks on the wafer. Apply vacuum contact and check alignment again. If the vacuum application skewed the alignment, repeat the alignment process.
6. Expose the wafer for 30 seconds (3.7 mW/cm²) through the aligned feeding channel mask.
7. Bake the wafer for 10 min at 95℃.
8. Develop the wafer for 2 minutes in Su8 Developer with mild agitation.
9. Rinse the wafer for 10 seconds in IPA. Check to ensure that the development is finished. If undesired photoresist remains, develop again for 20 seconds.
10. Verify channel height using a profilometer. The expected height is 10.5 μm. If the channel dimensions lie outside of your expected tolerance bounds, the process must be repeated with modified spin coat-ing parameters.

Chip Preparation

Dimethyl siloxane monomer (Sylgard 184) was mixed at a 10:1 ratio with curing agent, poured onto the silicon wafer, defoamed, degassed for 1 h, and cured at 75℃for 1h. Individual chips were then cut, and the inlets and outlets were punched using a puncher (in-ner diameter: 0.9 mm, outer diameter: 1.3 mm), and cleaned with Scotch tape. Bonding to water-cleaned and nitrogen-dried coverslips was ensured using oxygen plasma treatment (60 s at LOW and O₂ pressure at 170 mTorr) on the day the experiments were started. Attach the treated surfaces of the chip and coverslip together gently to prevent cavity collapse. The chips were then incubated at 75℃ overnight to reinforce the bonding.

Cell Preparation

Escherichia coli strains were grown overnight in LB with appropriate antibiotics and diluted 1:100 ap-proximately 2-3 h before the beginning of the experiments in the imaging media, which consisted of M9 salts,10% (v/v) LB, 0.2% (w/v) glucose, 2mM MgSO₄, 0.1mM CaCl₂, 1.5 μM thiamine hydrochloride (Sigma Aldrich, included as a passivating agent). The cells were centrifuged on a holder that could fit into a standard table-top centrifuge at 4000 × g for 10 min to insert them into the single straight channels. The feeding channels were con-nected to syringes filled with imaging media using Tygon tubing (VWR), and media was pumped us-ing syringe pumps (New Era Pump System) initially at a high rate of 100 μl/min for 5 min, to clear the inlets and outlets.The media was then pumped at 2.5-5 μl/min for the duration of the experiment and the cells were allowed to adapt to the device for several hours before imaging was started.

IPTG Synchronization

To synchronize the phase of the oscillators in the population, we diluted the strains in imaging medium supplemented with appropriate antibiotics and 1 mM IPTG to obtain a density of the early expo-nential phase (OD₆₀₀=0.2) 8 h later (∼1×10^-6) at 37℃.

Microscopy and Image Acquisition

Images were acquired using a Nikon ECLIPSE Ti inverted microscope equipped with a temperature-controlled incubator, an Orca R2 CCD camera (Hamamatsu), a 60×Plan Apo oil objective (numerical aperture (NA) 1.4, Nikon), an automated xy-stage (Ludl) and Nikon HG Pre-centered Fiber Illuminator (INTENSILIGHT C-HGFIE). All experiments were performed at 37℃. Typical exposure was low (50-100 ms) to reduce photobleaching, and the reporter channels were acquired using 2 × 2 binning (CCD chip dimension of 1,344 × 1,024 pixels, effective pixel size of 129 × 129 nm). Then, 16-bit TIFF images were taken every 5-8 min, and focal drift was controlled via the Nikon PerfectFocus system, as well as a custom routine based on z-stack images of a sacrificial position (a position that was not used for further analysis). The following filter sets were used for acquisition: GFP (Nikon GFPHQ), RFP (Nikon TxRed), YFP (Nikon YFPHQ) and CFP (Nikon CFPHQ).




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