Microscale Interfacial Polymerization on a Chip

Abstract Forming hydrogels with precise geometries is challenging and mostly done using photopolymerization, which involves toxic chemicals, rinsing steps, solvents, and bulky optical equipment. Here, we introduce a new method for in situ formation of hydrogels with a well‐defined geometry in a sealed microfluidic chip by interfacial polymerization. The geometry of the hydrogel is programmed by microfluidic design using capillary pinning structures and bringing into contact solutions containing hydrogel precursors from vicinal channels. The characteristics of the hydrogel (mesh size, molecular weight cut‐off) can be readily adjusted. This method is compatible with capillary‐driven microfluidics, fast, uses small volumes of reagents and samples, and does not require specific laboratory equipment. Our approach creates opportunities for filtration, hydrogel functionalization, and hydrogel‐based assays, as exemplified by a rapid, compact competitive immunoassay that does not require a rinsing step.


II. Fabrication of microfluidic chips
The microfluidic chips were designed using L-edit (Mentor Graphics, Oregon, USA) and patterned on a soda lime mask using a direct laser writer (DWL 2000 Heidelberg-Instruments, Heidelberg, Germany). The channel walls and capillary pinning structures of the microfluidic chips were fabricated on a 4" silicon wafer (Si-Mat, Germany) using one photolithographic step of 15 µm SU-8 3010 (MicroChem Corp., Massachusetts, USA). A thin photoresist (AZ-4562) was spin-coated on top of the microstructures to protect them from debris during the dicing process. The photoresist was removed using acetone and the chips were rinsed with isopropyl alcohol. The chips were sealed using a dry film resist (DF-1050, EMS Inc., USA). The dry film resist was exposed by a collimated 365 nm UV light (Thorlab MCWHL5-C5, 80 mW, 43 mm beam diameter, 700 mA, 4.4 V, 5.51 mW/cm 2 ) for 1 min through a photomask. The dry film resist was crosslinked only above the sample-, central-, and donor-channels in order to prevent deformation of the dry film resist due to the swelling of the hydrogel. The photomask was printed on a transparent projector sheet with a Canon Bubble Jet i9100 Printer. The contact area between the sample and the hydrogel should be maximized to avoid obstructing the diffusion of sample into the hydrogel. Hence, the gap between the capillary pinning structures should be as large as possible and the size of the capillary pinning structures should be as small as possible. With the current fabrication process, the gap between the capillary pinning structures needs to be smaller than 15 µm to ensure a reliable pinning of the 4PM solution. To include a safety margin, we used a gap of 10 µm ( Figure S1). The size of the capillary pinning structures is limited by the resolution of the photolithographic process, which for our case requires the capillary pinning structures to be at least 10 µm wide. The resulting pitch of the capillary pinning structures pattern is 20 µm.

III. Preparation of precursor solutions and fluorescent samples
4PM 2 kDa was dissolved in DMSO at a concentration of 350 mM and then diluted to 35 mM (for ξ = 6.7 nm) or 70 mM (for ξ = 5.3 nm) in MilliQ water containing 200 mM triethanolamine (final concentration). 4PM 10 kDa was dissolved in water at a concentration of 65 mM and then diluted to 10 mM (for ξ = 12.6 nm) in MilliQ water containing 200 mM triethanolamine (final concentration). PDT was dissolved in MilliQ water at a concentration of 700 mM and then diluted to 70 mM (for ξ = 6.7 nm), 140 mM (for ξ = 5.3 nm) or 20 mM (for ξ = 12.6 nm) in MilliQ water containing 200 mM triethanolamine (final concentration). Fluorescent samples were prepared in MilliQ water with the following concentrations: Rhodamine B and TRITC-dextran 10 kDa = 5 mg/mL; TRITCdextran 4.4 kDa, TRITC-dextran 20 kDa, TRITC-dextran 70 kDa, TRITC-dextran 155 kDa = 10 mg/mL.

IV. Hydrogel formation on chip
0.2 µL of 4PM were loaded in pad 1. After the central channel filled (10 s), the excess 4PM solution in pad 1 was dried with an absorbing paper in order to avoid a pressure difference between the liquids in pad 1 and pad 2 once PDT is loaded. This pressure difference would cause a capillary flow between the two pads and create inhomogeneities in the hydrogel. Similarly, 0.2 µL of PDT were loaded in pad 2. After the donor channel filled (5 s), the excess PDT solution was dried with an absorbing paper. 2 min 30 s were necessary for the PDT to diffuse through 4PM and form a hydrogel in the central channel.

V. Diffusion experiment using fluorescent molecules
The microfluidic chips were placed in a Petri dish having a 4 cm 2 wet paper to form a humidity chamber ( Figure  S2). 3 µL of sample were loaded in pad 3 and then the lid of the plastic box was closed. The humidity chamber was placed under the microscope and the measurement was started. The capillary pump provides a reliable pressure difference to pull a sample solution from the loading pad into the chip and we used a hydraulic resistance (e.g. microchannel with a narrow width) to control the flow rate of the sample through the sample channel. The hydraulic resistance was designed to provide a constant flow rate of sample in the sample channel for at least 15 minutes and at the same time to ensure a sufficiently high flow rate to make sample concentration gradients due to diffusion negligible compared to the sample channel width [1] .

VI. Competitive assay in a hydrogel
Anti-analyte antibodies were mixed with 4PM before forming a hydrogel. The final concentration of anti-analyte antibodies in the hydrogel was 1 mg/mL. Solutions containing analyte and analyte-atto488 were prepared with a constant concentration of analyte-atto488 (200 nM) and varying concentrations of analyte (16 nM, 80 nM, 400 nM, 2 µM, 10 µM, 50 µM, 250 µM and a zero analyte as control). 3 µL of sample were loaded in pad 3 following the same procedure described in the paragraph above. Fluorescence images were taken every minute for 20 minutes using a microscope.

VII. Equipment
Fluorescence images were recorded using a Nikon Eclipse 90i microscope, a 10× objective (Plan Apo Nikon, NA = 0.45) and a DS-1QM/H CCD camera (Nikon). Snapshots and videos of the chips were taken using a Leica MZ16 stereomicroscope equipped with a Nikon J1 digital camera, a custom-made microscope equipped with an 8megapixel CMOS camera and controlled by a Raspberry Pi (https://github.com/IBM/MicroscoPy) and a smartphone camera (Samsung Galaxy S7, SM-G930F).

VIII. Data processing
Images were analyzed using ImageJ and the graphs plotted using Origin 2018. Figure S1. Photographs of a capillary-driven microfluidic chip before sealing it with a dry film resist. Pads 1 to 3 are used to load 4PM, PDT and a sample, respectively. Vents allow air displaced by the filling liquids to exit the flow paths. A hydraulic resistance is used to set the desired flow rate of the sample and a capillary pump provides a constant flow rate of sample for at least 15 minutes. (i) Additional vents are placed in the sample channel to avoid formation of air bubbles while the sample fills the channel ("debubbler"). (ii) Capillary pinning structures used to pin 4PM.     The red curve shows the fluorescence intensity across the channels averaged in the direction parallel to the channels. Note the local fluorescence maximum (red arrow) that occurs due to the small hydrogel bulges, which extend toward the sample channel. In addition, the local fluorescence minimum is due to the capillary pinning structures, which contribute to a lower average fluorescence intensity at the interface between the sample channel and the hydrogel.