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Microfluidic chain reaction of structurally programmed capillary flow events – Nature

Chip design and fabrication

The chips were designed using AutoCAD (Autodesk) and exported as .STL files for 3D printing. CCs encoding MCRs were made with a digital micromirror display (DMD) 3D printer (Miicraft 100, Creative Cadworks) using a transparent resin (Rapid Model Resin Clear, Monocure 3D) purchased from The following printing parameters were used: the layer thickness was 20 µm and the exposure time 1.5 s per layer, whereas the exposure time for the base layer was 10 s with four transition buffer layers. Following completion of the print, the chips were cleaned with isopropanol and post-cured for 1 min under ultraviolet (UV) light (Professional CureZone, Creative Cadworks).

Microchannels with cross-sections ranging from 250 × 100 to 1,500 × 1,000 µm2 were fabricated and hydrophilized by plasma activation for 10 s at approximately 30% power (PE50 plasma chamber, Plasma Etch).

CCs were sealed with a delayed tack adhesive tape (9795R microfluidic tape, 3M) forming the cover.

Paper capillary pump

Filter papers (Whatman filter paper grade 4, 1 and 50 Hardened, Cytiva) were used as paper capillary pumps for all experiments except the SARS-CoV-2 antibody assay. The pore size from 4, 1 and 50 hardened is in decreasing order, and flow resistance and capillary pressure increase with decreasing pore size.

For the SARS-CoV-2 antibody assay, absorbent pads (Electrophoresis and Blotting Paper, Grade 238, Ahlstrom-Munksjo Chromatography) were used as pumps.

Chip-to-chip connections for the 300 capillary flow events

To obtain a leakage-free connection, a thin layer of uncured photoresin, prepared by mixing poly (ethylene glycol) diacrylate (PEG-DA MW 258, Sigma-Aldrich) and Irgacure-819 (1% w/w), was applied to all of the chip-to-chip interfaces. Next, the chips were assembled and exposed to UV light in a UV chamber (320–390 nm, UVitron Intelliray 600) at 50% intensity for 30 s to cure the resin and seal the connections.

Videos and image processing

Videos and images were recorded using a Panasonic Lumix DMC-GH3K. Structural images of the chip and the embedded conduits were obtained using micro-computed tomography (Skyscan 1172, Bruker) and used to confirm the dimensions. Contact angles were measured on the basis of side view images (n = 3) and analysed using the Dropsnake extension in Image J.

Modelling and calculations

The theoretical burst pressures of capillary SVs were calculated by solving the flow field using the finite element method with COMSOL Multiphysics v.5.5. Experimentally measured contact angles (100º and 40º for the cover and the channel, respectively) were used to solve two-phase capillary flow using the level-set method. The capillary flows leading up to the SV was solved for a time period of 0–0.02 s with a time step of 1 × 10−5 s. The inlet pressure was varied with 10 Pa increment for each simulation until a burst was observed.

Experiments on pressure thresholds for capillary SV and RBV

We 3D-printed modules to evaluate SV/RBV with different cross-section areas. Each module contained three SV/RBV for replicate results. SV/RBV consisted of a two-level SV based on a geometrical channel expansion, as described elsewhere12. The chips integrated a conical inlet/outlet for tubing connection to a microfluidic flow controller system (MFCS-4C) and Fluiwell package (Fluigent) with fluidic reservoirs containing 5% red food dye in MilliQ water solution (see Extended Data Fig. 4 for setup images and Fig. 2 for contact angles). MAESFLO v.3.3.1 software (Fluigent) controlled the application of positive or negative pressure to calculate the burst pressures of the SV (liquid burst into air link) and RBV (receding meniscus), with increments of 0.1 mbar (roughly 10 Pa).

SARS-CoV-2 antibody assay


SARS-CoV-2 nucleocapsid protein was purchased from Sino Biological, Inc. (40588-V08B). Human Chimeric antibody against SARS-CoV-2 nucleocapsid protein was purchased from Genscript Biotech (A02039). SIGMAFAST 3,3ʹ-diaminobenzidine tablets were purchased from Sigma-Aldrich. Biotinylated Goat-anti-Human antibody was purchased from Cedarlane (GTXHU-003-DBIO). Pierce streptavidin poly-HRP (21140) was purchased from ThermoFisher.

Nitrocellulose strips

Nitrocellulose membranes (Whatman FF80HP Plus nitrocellulose-backed membranes, Cytiva) were cut into 5.2-mm-wide strips using the Silhouette Portrait paper cutter (Silhouette). Membranes were striped with a 5-mm-wide test line of 0.25 mg ml−1 SARS-CoV-2 nucleocapsid protein delivered using a programmable inkjet spotter (sciFLEXARRAYER SX, Scienion). The test line consists of four lanes of 50 droplets of about 350 pl printed 100 µm apart from each other. Eight passes of 25 droplets were used for each lane on even and odd positions to allow solution absorption in between passes. The membranes were then dried for 1 h at 37 °C before blocking by dipping into 1% BSA in 1× PBS solution until completely wet, then retrieved and left to dry for 1 h at 37 °C and then stored with desiccant at 4 °C until use the next day.

Connection of capillary pump and nitrocellulose chip to MCR chips

Nitrocellulose strips were mounted following standard lateral flow assay assembly protocols. The nitrocellulose strip was connected to a glass fibre conjugate pad (G041 SureWick, Millipore Sigma) on one end, and to an absorbent pad (Electrophoresis and Blotting Paper, Grade 238, Ahlstrom-Munksjo Chromatography) serving as the capillary pump at the other end. All three were attached to an adhesive tape serving as the backing layer. For the saliva antibody assay, the nitrocellulose strip was sandwiched between three absorbent pads (15 × 25 mm2) and clamped with a paper clip. For the food-dye demonstrations a single absorbent pad (25 × 45 mm2) was magnetically clamped to the nitrocellulose membrane.

Saliva assay protocol

Human saliva was extracted with oral swabs (SalivaBio, Salimetrics), followed by centrifugation and 1:10 dilution with 0.22 µM filtered phosphate buffer saline containing 1% BSA, 0.1% Tween 20. Human chimeric antibody against SARS-CoV-2 nucleocapsid protein at 0 to 1,000 ng ml−1 was spiked into diluted saliva and loaded to the sample reservoirs. Three replicate measurements for concentrations of 0–10 ng ml−1, two replicate measurements for concentrations of 30–300 ng ml−1 and one measurement for 1,000 ng ml−1. Biotinylated goat anti-human antibody at 0.5 µg ml−1 and streptavidin poly-HRP at 0.5 µg ml−1 were used to detect the human antibody. Control line in the nitrocellulose strip confirms reagents delivery and colorimetric reaction completion.

Image analysis on the nitrocellulose strips

After drainage of all reservoirs, the nitrocellulose membrane strip was removed, placed on a support and left to dry for 1 h.

The dry strips were imaged using (1) a flatbed scanner (mfc-9970cdw, Brother) at a resolution of 600 dpi and (2) using a Huawei P10 smartphone with a 12 megapixel image sensor and a rear camera with a 27 mm focal length (Huawei) in a customized box. The box was cut and folded with black cardboard paper to block ambient light when imaging with the smartphone. The box had two slots fitting the size of camera and nitrocellulose strip, respectively, to ensure accurate alignment of the strip for readout. Images were taken with on-camera dual tone light-emitting diode flash at full power. Analysis of smartphone-taken and scanned images was done as follows.

Mean grey values of nitrocellulose test lines were extracted with ImageJ 1.48v (ImageJ, public domain software, W. Rasband, National Institutes of Health) within a 100 × 10 pixel rectangular area. Local background grey values were taken at 2.5 mm (0.1 inch) above each test line (following direction of the flow) for the same rectangular area, and subtracted from test line values. The normalized standard curve was then generated by subtracting negative control signal value (0 ng ml−1) from all data points.

The limit of detection was calculated using the three-sigma criterion using a non-linear four-parameter logistic curve fit of the log-transformed data with OriginPro 8.5 SPR (OriginLab Corporation).

Automated microfluidic TGA (Thrombochip)

Citrated human plasma (P9523, lot number SLBX8880), fluorogenic thrombin substrate Z-GGR-AMC and Enoxaparin were purchased from Sigma-Aldrich; Batroxobin was from Prospec; Technothrombin TGA RC High reagent was from Diapharma; Human thrombin, non-patient plasma that were immuno-depleted of Factor V and Factor IX, and Factor VIII inactivated were from Haematologic Technologies; (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), and ethylenediaminetetraacetic acid (EDTA) and CaCl2 were from Sigma-Aldrich.

The purchased pooled human plasma (collected in the United States in a Food and Drug Administration licensed centre site no. 268, as specified in the Certificate of Origin supplied by the manufacturer) was prepared by the manufacturer from whole blood collected by standard industry method using 4% trisodium citrate as an anticoagulant, pooled and then centrifuged. The resulting plasma was 0.45 µm filtered and lyophilized. Factor V- and Factor IX-depleted plasma were immune depleted; Factor VIII-depleted plasma was prepared by chemical depletion. The plasma preparations were assayed to ensure the activity of the remaining factors by the manufacturer.

Human plasma (pooled normal or factor depleted) were defibrinated by the addition of batroxobin (final concentration 0.6 BU ml−1). The mixtures were incubated at room temperature for 20 min, followed by an extra incubation at 4 °C for 1 h. The mixtures were then centrifuged at 10,000g for 10 min to remove the fibrin clot and other debris. Defibrinated plasma were collected from the supernatant.

A solution containing 21% defibrinated plasma (plasma defibrination is needed to prevent clogging of the microfluidic channels by the fibrin clot), 48% Technothrombin TGA RC High reagent (high phospholipid and relipidated tissue factor content) and 20 mM CaCl2 in 25 mM HEPES at pH 7.4 was loaded into the sample reservoirs of the thrombochip. A substrate solution containing 420 µM Z-GGR-AMC, 30 mM EDTA in 25 mM HEPES at pH 7.4 was loaded into the reagent reservoirs. The concentration of plasma, activation agent and substrate were optimized to yield a peak thrombin concentration and time of 150 nM and 200 s. All solutions were equilibrated to room temperature for 20 min before loading. Coagulation-inhibited plasma contained Enoxaparin at final concentrations of 0 to 1.0 anti-Xa units ml−1 or IU ml−1. The samples and reagents were loaded on the chip after initiating the coagulation cascade. The paper pump was connected to the chip to start the flow after 5 min from initiating the coagulation cascade. Fluorescence signals generated in the reaction chambers were monitored by illuminating the thrombochip with UV light at 365 nm with 20 W (realUV LED Flood Light, Waveform Lighting) and the visible 440 nm fluorescence emission signals measured by imaging at 5 s intervals using a Panasonic Lumix DMC-GH3K digital camera (f/3.5, Exposure time: 2 s, ISO-200). The rate of fluorescence signal generation in each reaction chamber (that is, the slope of the recorded fluorescence generation curve) is a measure of the rate of substrate turnover by thrombin and was used to deduce the amount of thrombin generated using a standard curve. Image J was used to analyse the images for fluorescence intensity.

Standard curve for thrombin quantification

Ten human thrombin solutions at concentrations ranging from 0 to 300 nM in 25 mM HEPES at pH 7.4 were loaded into the ten sample reservoirs in the thrombochip. A substrate solution containing 420 µM Z-GGR-AMC, 30 mM EDTA in 25 mM HEPES at pH 7.4 was loaded into the reagent reservoirs. The standard curve was constructed by plotting the slope of the recorded fluorescence generation curve in each reaction chamber against the known thrombin concentration of the solution that was loaded to the corresponding sample reservoir.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.