Fig 1.
Overview of the fabrication process to produce the hydrophilic-in-hydrophobic spot array outside the cleanroom.
Created with BioRender.com.
Fig 2.
Optical setup assembled to collimate the light and photo-crosslink the resist in the UV photolithography process for fabricating hydrophilic-in-hydrophobic MDAs outside the cleanroom.
A) 3D model of the system and B) Photograph displaying the setup with the 365 nm LED powered on. Republished from www.thorslabs.com under a CC BY license, with permission from Thorlabs.
Fig 3.
A) Expanded microfluidic device composed of a clamping system printed in Formlabs black resin and a PDMS gaskets that interfaces with the patterned 2” glass wafer forming micro-channels when the device is assembled. B) Picture of the assembled microfluidic device indicating the inlets, used for loading the liquid reagents inside the channels, and the outlets, used to connect with a peristaltic pump. C) Schematic of the PDMS gasket that forms 16 identical channels when pressed on top of the glass surface, with inlets and outlets coinciding with the ones in the 3D printed holder. The channels are 1 mm wide, 15 mm long and 100 μm high. D) Illustration of the mechanism for droplet formation: the hydrophilic glass circles, which are surrounded by a hydrophobic coating (green), retain some of the liquid when a water plug is pumped over the patterned surface through the use of the PDMS micro-channels.
Fig 4.
A, B) Brightfield micrographs displaying nLOF resist spot arrays with 10 μm spot diameter and 20 μm pitch produced in soft contact mode (A) and hard contact mode (B), respectively. Scale bars are 100 μm. C) Linear regressions showing the correlation between the photomask feature sizes and the dimensions obtained in soft contact (squares, dashed line, R2 = 0.994, Y = 1.075*X + 2.25), hard contact (circles, dotted line, R2 = 0.998, Y = 1.068*X + 0.8311), hard contact with the CR-based nLOF process (triangles, full line, R2 = 0.999, Y = 1.032*X + 0.07767). The error bars represent standard deviations of 10 repetitions. D) Brightfield micrographs displaying representative nLOF resist spot arrays with varying diameters and pitches (i.e., top left 4 μm diameter and 25 μm pitch, top middle 6 μm diameter and 15 μm pitch, top right 8 μm diameter and 25 μm pitch, bottom left 10 μm diameter and 75 μm pitch, bottom middle 12 μm diameter and 25 μm pitch and bottom right 16 μm diameter and 50 μm pitch, respectively) reproduced with exposure in soft contact mode. Scale bars are 40 μm. Note that the brightfield micrographs in A-B and D are taken with different microscopes.
Fig 5.
A) Brightfield micrograph displaying a microdroplet array with 10 μm spot diameter and 25 μm pitch produced in soft contact mode. (B) Brightfield micrograph displaying a microdroplet array with 10 μm spot diameter and 5 μm pitch produced in hard contact mode with the previously published CR-based method [21]. Scale bars are 50 μm. C) Linear regressions showing the correlation between the photomask feature sizes and the droplet sizes obtained by producing the MDA substrates in soft contact with the nLOF-based CR-free method (dashed line, R2 = 0.997, Y = 1.075*X + 2.092), and in hard contact with the previously published CR-based process (full line, R2 = 0.998, Y = 0.877*X + 2.047). The error bars represent standard deviations of 10 repetitions. Droplets on 4 μm diameter spots are not available.
Fig 6.
Hydrophilic-in-hydrophobic MDA applied to enzyme linked immunosorbent assay for single molecule counting.
Representative fluorescence micrographs of the dilution series obtained with different concentrations of Anti-Tau antibody conjugated with HRP: A) 100 fM, B) 1 fM, C) 100 aM, D) 10a M and E) control. Scale bars are 100μm. F) Bar graph generated from the quantification of the number of spots in the MDA in a frame (890 μm x 1035 μm) imaged with the 10x objective for each concentration (n = 3).