Figure 1. Overview of the room-temperature roll-to-roll imprinted PDMS-based centrifugal microfluidic devices. (a) Roll-to-roll additive manufacturing platform; (b) Enlarged image of R2R imprint unit; (c) Rewinder unit for collecting imprinted LoaD devices; (d) Illustration of on-chip isolation of RNA from whole blood using our R2R imprinted LoaD device; (e) and (f) R2R gravure coating unit; (g) Mass production of PDMS-based centrifugal microfluidic devices on PET substrate.

Figure 2. Design and 3D printing of the centrifugal microfluidic devices. (a) 3D model and detailed function of lab-on-a-disc (LoaD) device; (b) 3D printed LoaD device; (c) 3D design of screw valves; (d) Demonstration of device operation by food dyes, “v.1-10” stand for valve 1–10 and fluid flow sequences were indicated by dashed lines and yellow arrow, while red color circle represent closed valve and yellow one represents for opened valve.

Figure 3. Fabrication of multi-depth flexible polymer shim. (a) Fabrication steps of polymer mold; (b) The complete large-area flexible polymer mold; (c) Wrapped polymer shim on imprinting roller; (d) Image of multi-depth macro-to-micro features of the mold; (e) Demonstration of effective anti-adhesive coating layer for long lifecycle of the mold by replicating master template M10 to 10 copies from C1 to C10.

Figure 5. Roll-to-roll (R2R) replication accuracy. (a) Cross-sectional images with different magnifications from R2R imprinted samples under different operating nip pressure. Dashed areas on the left side images present the regions shown on the right; (b) Replication accuracy measuring at three positions: waste channel, S-shaped channel, and inlet hole on CAD design, 3D printed mold, PDMS mold, and R2R replicated LoaD with five samples per each.

Figure 1. (A) Patterned cell-laden hydrogel adjoining fluidic channels. (i) A silanized glass substrate treated for adhesion to the cross-linked hydrogel is (ii) reversibly bonded to a PDMS mold that is then filled with the cell-laden hydrogel and (iii) photo-cross-linked to create the patterned hydrogel on glass. (iv) The PDMS mold is removed to leave open fluidic channels that directly adjoin the patterned hydrogel. (v) The structure is surrounded with culture medium to maintain cell viability and prevent hydrogel shrinkage. (vi) An example of the patterned hydrogel with addressable open fluidic channels through which a FITC gradient was established. (B) (i) Microfluidic flow control setup. (C) (i) 3D-printed holder for fluidic integration with the patterned hydrogel and (ii) image of the patterned hydrogel with tubing to the 3D-printed holder and channel with yellow dye.

Figure 1. Schematic of device for coculture and assembloid formation. Two different types of organoids are loaded into separate channels and fed by separate media reservoirs. The geometry of the wall separating the channels shapes the organoids as they grow. When mature, the separating wall can be physically removed, allowing the organoids to interact and eventually fuse, while observing their interactions throughout culture.

Figure 3. Assembloid formation in two-piece separated devices. (A) 3D schematic of removable insert piece; shown here with a triangular wall geometry. (B) Replica molded two-piece PDMS devices, with base and insert pieces shown. (C) Assembled two-piece device, imaged from below. (D) Midbrain organoids were loaded into channels with Matrigel, and maintained viability for 7 days in culture. (E) Unguided and midbrain organoids were loaded into channels with Matrigel and cultured for 7 days before removing the separating wall. Organoids maintained the shape and spacing imposed by the separating wall before beginning to grow toward each other to form an assembloid. (F) Astrocytes identified with glial fibrillary acidic protein (GFAP; magenta) are observed on the edges of unguided organoids only. (G) Midbrain organoids uniquely express dopaminergic neuron marker tyrosine hydroxylase (TH; magenta). Both organoid types express neural marker β-tubulin III (Tuj1; green), which is observed across the separating bridge within 3 days of insert removal.

Figure 4. Axonal projection from midbrain organoids. (A) Axonal projections extending from midbrain organoid, and (B) staining positive for axonal marker tau-1 (green). (C) Axonal projections arise from all sides of the midbrain organoid. (D) Representative frequency distribution of angles of axonal projections from a midbrain organoid, showing majority of axons angled towards nearby organoid (distribution is centered around angle towards nearby organoids, 180°). Compared against a statistically random orientation, the distribution is biased toward other midbrain organoids (p < 0.05) and unguided organoids (p 0.1 by two-sample t-test comparing axon lengths that were directed toward either midbrain or unguided organoids).

Fig. 2. Circuit analysis and experiments identify operational window for MCRs. (a) The simplified equivalent electrical circuit of the MCR units shown in Fig. 1. (b) Experimental SV burst pressure (1) and RBV retention pressure (2) for valves with conduits with different, square cross-sections fitted with a numerical and an analytical model, respectively. (c) Illustration of failure for a CDV with long serpentine functional connections with very high resistance leading to liquid breach inside the air link, and premature draining of reservoir n+1. (d) Tests of 6 MCRs with increasing RFC and three different paper pumps to determine the effect of varying the flow rate (n=3 for each paper pump and RFC). All data points are shown in (b and d). Error bars are standard deviations from 3 experiments, the centre of each error bar is the mean value. As predicted, the CDVs fail when the pressure drop across the functional connection PFC(n) exceeds the CDV threshold pressure PBURS(n) + PRBV(n+1).

Fig. 3. Large-scale MCR and COVID-19 serology assay in saliva. (a) A MCR of 300 aliquots stored in 4.9 µl reservoirs across four chained and interconnected chips. See Video S2. (b) SARS-CoV-2 antibody detection in saliva. Sequential, preprogrammed release of reagents via MCR is triggered by connecting the paper pump, see Video S3. The MCR supplies 4 reagents and 4 buffers in sequence. The functionality includes delivery and removal (by flushing) of solutions, metering (40 µl – 200 µl) via reservoir size, flow speed and time control via the flow resistance of the functional connection and the capillary pressure of the paper pump. The enzymatic amplification produces a brown precipitate line visible to the naked eye. (c) Assay results and binding curve obtained by spiking antibody into saliva, and imaging by scanner and cell phone with representative images of the detection zone for each concentration, indicating the potential for quantitative point-of-care assays. (d) An assembled chip filled with colored solutions highlighting the channels for the different reagents and washing buffer.

Fig. 4. Automated thrombin generation assay (TGA) by continuous analysis of plasma subsamples (thrombochip). (a) Model thrombin generation curve (thrombograms) for plasma with normal (red) and disordered (blue) coagulation. Grey box is the time window of the thrombochip. (b) TGA operations and algorithm encoded in the thrombochip. (c) Schematic of the thrombochip with inset and (d) showing (i) timer, (ii) simultaneous release of defibrinated plasma and reagents (quencher and substrate), (iii) mixing, and (iv) flow-stop in the reaction chamber and monitoring of the fluorescence time-course signal. (e) Fluorescent thrombin substrate turnover in the ten 1 min-interval subsamples; the slope of each curve is proportional to thrombin concentration, and is one data point in the thrombogram. Abridged thrombograms of defibrinated human plasma that is (f) normal (3 replicates of pooled plasma), factor-depleted (F5, F8, F9; single measurement for each factor), and (g) mixed with anticoagulant drug (Enoxaparin) at different concentrations (single measurement at each concentration). The thrombin generation time-courses are concordant with expectations.