Most medical diagnostic tests are expensive, involve slow turnaround times from centralized laboratories and require highly specialized equipment with seasoned technicians to carry out the assay. To facilitate realization of precision medicine at the point of care, we have developed a mixed-scale nanosensor chip featuring high surface area pillar arrays where solid-phase reactions can be performed to detect and identify nucleic acid targets found in diseased patients. Products formed can be identified and detected using a polymer nanofluidic channel. To guide delivery of this platform, we discuss the operation of various components of the device and simulations (COMSOL) used to guide the design by investigating parameters such as pillar array loading, and hydrodynamic and electrokinetic flows. The fabrication of the nanosensor is discussed, which was performed using a silicon (Si) master patterned with a combination of focused ion beam milling and photolithography with deep reactive ion etching. The mixed-scale patterns were transferred into a thermoplastic via thermal nanoimprint lithography, which facilitated fabrication of the nanosensor chip making it appropriate for in vitro diagnostics. The results from COMSOL were experimentally verified for hydrodynamic flow using Rhodamine B as a fluorescent tracer and electrokinetic flow using single fluorescently labelled oligonucleotides (single-stranded DNAs, ssDNAs).

The presence of air bubbles boosts the shear resistance and causes pressure fluctuation within fluid-perfused microchannels, resulting in possible cell damage and even malfunction of microfluidic devices. Eliminating air bubbles is especially challenging in microscale where the adhesive surface tension force is often dominant over other forces. Here, we present an air bubble removal strategy from a novel surface engineering perspective. A microfluidic port-to-port interconnect was fabricated by modifying the peripheral of the microfluidic ports superhydrophobic, while maintaining the inner polymer microchannels hydrophilic. Such a sharp wettability contrast enabled a preferential fluidic entrance into the easy-wetting microchannels over the non-wetting boundaries of the microfluidic ports, while simultaneously filtering out any incoming air bubbles owing to the existence of port-to-port gaps. This bubble-eliminating capability was consistently demonstrated at varying flow rates and liquid analytes. Compared to equipment-intensive techniques and porous membrane-venting strategies, our wettability contrast-governed strategy provides a simple yet effective route for eliminating air bubbles and simultaneously sealing microfluidic interconnects.

An innovative method of thermal fusion bonding (TFB) using a pressure assisted boiling point (PABP) control system was characterized to determine the optimum parameters for bonding thermoplastic components containing microchannels and thin cover sheets. The PABP TFB system enables precise control of the temperature and the pressure boundary conditions by immersing a bag containing the components being bonded in boiling water and varying the vapor pressure. The original system used a human operator to manually adjust a pressure relief valve and maintain the system temperature, which introduces time delays. A closed loop control system was developed to automatically control the temperature in the pressure vessel. Test structure geometries containing microchannels of two depths, 5μm and 10μm, and four different aspect ratios were designed: 1:10, 1:50, 1:100, and 1:200. Microchannels were hot embossed in polymethyl methacrylate (PMMA) using micro-milled brass mold inserts. Results of bonding with the closed loop systems were evaluated. Mean rupture pressures decreased with increasing AR and were higher for shallower channels for the automated closed loop system. For a given aspect ratio, the rupture pressures ranged from 5%–19% higher for 5 mm channels and increased by 5%–10% in 10 mm channels with the automated closed loop system.

Liquid biopsies are becoming popular for managing a variety of diseases due to the minimally invasive nature of their acquisition, thus potentially providing better outcomes for patients. Circulating tumor cells (CTCs) are among the many different biomarkers secured from a liquid biopsy, and a number of efficient platforms for their isolation and enrichment from blood have been reported. However, many of these platforms require manual sample handling, which can generate difficulties when translating CTC assays into the clinic due to potential sample loss, contamination, and the need for highly specialized operators. We report a system modularity chip for the analysis of rare targets (SMART-Chip) composed of three task-specific modules that can fully automate processing of CTCs. The modules were used for affinity selection of the CTCs from peripheral blood with subsequent photorelease, simultaneous counting, and viability determinations of the CTCs and staining/imaging of the CTCs for immunophenotyping. The modules were interconnected to a fluidic motherboard populated with valves, interconnects, pneumatic control channels, and a fluidic network. The SMART-Chip components were made from thermoplastics via microreplication, which lowers the cost of production making it amenable to clinical implementation. The utility of the SMART-Chip was demonstrated by processing blood samples secured from colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) patients. We were able to affinity-select EpCAM expressing CTCs with high purity (0-3 white blood cells/mL of blood), enumerate the selected cells, determine their viability, and immunophenotype the cells. The assay could be completed in <4 h, while manual processing required >8 h.

Hypothesis: The superhydrophobic lotus leaf has dual-scale surface structures, that is, nano-bumps on micro-mountains. Large hydrophilic particles, due to its high surface energy and weight, have high affility to substrates and tend to precipitate at the bottom of coating films. Small hydrophobic particles, due to its low surface energy and weight, tends to sit on the top of coating films and form porous structures. To mimic the lotus leaf surface, it may be possible to develop dual-sized particle films, in which small particles are decorated on large particles. Experiments: A one-step spin coating of a mixture of dual-sized silica particles (55/200 nm) was used. Epoxy resin was added to improve the adhesion of particle films. The single-sized and dual-sized particle films were compared. The mechanical robustness of particle films was tested by tape peeling and droplet impact. Findings: The novel combination of hydrophobic silica (55 nm) and hydrophilic silica (200 nm) is essential in creating the hierarchical structures. By combining the strong adhesion of hydrophilic silica (bottom of coating film) to polymer substrates and porous structures of hydrophobic silica (top of coating film), we first time report a one-step and versatile approach to create uniform, transparent, robust, and superhydrophobic surface. Crown Copyright (C) 2020 Published by Elsevier Inc. All rights reserved.

An improved, laser-induced fluorescence-based micro-optical biosensor was designed and fabricated, with cyclic olefin copolymer (COC) optical waveguides, a poly(methyl methacrylate) (PMMA) fluidic substrate with an array of microlenses, and a COC coupling prism integrated with the waveguide substrate or cover plate. The double-sided hot embossed fluidic substrate had sampling zone microchannels on the bottom and microlenses on the top. Dissolved COC injected into polydimethylsiloxane (PDMS) lost molds embedded the waveguides in the PMMA cover plate and formed the integrated coupling prism. The embedded COC waveguide was flycut down to 50 μm. The cover plate and shallow, 1:20 aspect ratio, microchannels were thermal fusion bonded using a pressure-assisted boiling point control system, without sagging. The large COC prism coupled better to the waveguide. The highest intensity evanescent excitation of the waveguide was obtained near the critical angle. The maximum signal-to-noise ratio (SNR) was 119 and the lowest detection limit was 7.34 × 11 -20 mol at a SNR of 2 for a 100 μm wide by 50 μm deep waveguide. The microlenses highly focused the fluorescent radiation in the sampling zone.

Existing methods for sealing chip-to-chip (or module-to-motherboard) microfluidic interconnects commonly use additional interconnect components (O-rings, gaskets, and tubing), and manual handling expertise for assembly. Novel gasketless superhydrophobic fluidic interconnects (GSFIs) sealed by transparent superhydrophobic surfaces, forming liquid bridges between the fluidic ports for fluidic passages were demonstrated. Two test platforms were designed, fabricated, and evaluated, a multi-port chip system (ten interconnects) and a modules-on-a-motherboard system (four interconnects). System assembly in less than 3 sec was done by embedded magnets and pin-in-V-groove structures. Flow tests with deionized (DI) water, ethanol/water mixture, and plasma confirmed no leakage through the gasketless interconnects up to a maximum flow rate of $100~\mu \text{L}$ /min for the multi-port chip system. The modules-on-a-motherboard system showed no leakage of water at a flow rate of $20~\mu \text{L}$ /min and a pressure drop of 3.71 psi. Characterization of the leakage pressure as a function of the surface tension of the sample liquid in the multi-port chip system revealed that lower surface tension of the liquid led to lower static water contact angles on the superhydrophobic-coated substrate and lower leakage pressures. The high-density, rapidly assembled, gasketless interconnect technology will open up new avenues for chip-to-chip fluid transport in complex microfluidic modular systems. [2020-0168]

A multi-scale fluidic motherboard, which can be used in a universal molecular processing system (uMPS) integrated with task-specific processing modules, was designed and fabricated in thermoplastics. The motherboard consists of a coverplate and a substrate. The coverplate included fluidic interconnects and thermal grooves on the top side, and the corresponding interconnects on the bottom side. The substrate was comprised of cell lysis microchannels, micromixers, and flow-connecting microchannels on the top side, and reservoirs for sample inputs and waste output, thermal grooves, and valve seats for flow control on the bottom side. The coverplates and substrates were fabricated with double-sided hot embossing of polycarbonate (PC) using four micromilled brass molds, two for the coverplate and another two for the substrate. Evaluation of the relative front-to-backside alignment for the double-sided hot embossing yielded an accuracy of 25 μm ± 14 μm (average ± standard deviation) for the coverplates and 30 μm ± 20 μm for the substrates. Thermal fusion bonding (TFB) of the coverplate and substrate was done using a spring plunger bonding setup with a range of temperatures and pressures. The motherboard bonded at 154 °C and 12.0 psi for 2 hours in a convection oven produced complete bonding with a little deformation of the valve seats. The complete motherboard will be integrated with the task-specific processing modules in the uMPS for investigating circulating markers from whole blood for precision molecular diagnosis of disease at low cost and with high fidelity.

HYPOTHESIS:Compared to vertical micro-pillars, re-entrant micro-structures exhibited superior omniphobicity for suspending liquids to Cassie-Baxter state. However, the existing re-entrant structures rely on complex multi-step deposition and etching procedures. The conventional, rigid-templated imprinting would instead damage the re-entrant structures. This leads to the question: is it possible to preserve the re-entrant curvatures by a flexible-templated imprinting? EXPERIMENTS:We facilely imprinted the re-entrant structures on a plastic substrate using a flexible nylon-mesh template. The effect of imprinting time (15-35 min), temperature (110-120 °C) and pressure (15-50 Bar) was investigated. To further improve the liquid-repellency and abrasion resistance, the silica nanoparticles (30-650 nm) along with epoxy resin binder (10 mg/mL) were pre-coated. FINDINGS:A one-step imprinting is sufficient to fabricate the re-entrant structures by utilizing flexible nylon-mesh template, without damaging the imprinted structures after the demolding process. The pre-coated silica nanoparticles and epoxy resin (1) improved liquid repellency by introducing hierarchical surface structures (e.g. contact angle hysteresis of olive oil reduced > 10°), and (2) acted as a protective layer against mechanical abrasion (omniphobicity maintained after 25 cycles, ~1.6 kPa sand paper abrasion). Additionally, the fluorine-free post-treatment was sufficient for the omniphobicity on the obtained plastic structures.

A microfluidic chip is described that facilitates research and quality control analysis of zebrafish sperm which, due to its miniscule (i.e., 2–5 μl) sample volume and short duration of motility (i.e., <1 min), present a challenge for traditional manual assessment methods. A micromixer molded in polydimethylsiloxane (PDMS) bonded to a glass substrate was used to activate sperm samples by mixing with water, initiated by the user depressing a transfer pipette connected to the chip. Sample flow in the microfluidic viewing chamber was able to be halted within 1 s, allowing for rapid analysis of the sample using established computer-assisted sperm analysis (CASA) methods. Zebrafish sperm cell activation was consistent with manual hand mixing and yielded higher values of motility at earlier time points, as well as more subtle time-dependent trends in motility, than those processed by hand. Sperm activation curves, which indicate sample quality by evaluating percentage and duration of motility at various solution osmolalities, were generated with on-chip microfabricated gold floor electrodes interrogated by impedance spectroscopy. The magnitude of admittance was linearly proportional to osmolality and was not affected by the presence of sperm cells in the vicinity of the electrodes. This device represents a pivotal step in streamlining methods for consistent, rapid assessment of sperm quality for aquatic species. The capability to rapidly activate sperm and consistently measure motility with CASA using the microfluidic device described herein will help improve the reproducibility of studies on sperm and assist development of germplasm repositories.

Most flow visualizations and flow measurements to understand particle mobility in porous media are typically performed in transparent microfluidic devices (micro-models) with 2D pore-throat networks. Nano-particle mobility studies to date have been limited to micro-models made of transparent thermoplastic or silicone-based materials. In an effort to fabricate materials close to reservoir rock, ceramic micro-model has been designed and micro fabricated by our group to study nano-particle transport in rock-based ceramic micro-model. A Confocal Micro-Particle Image Velocimetry (C-μPIV) technique augmented with associated post processing algorithms [1] is used in obtaining 3D distributions of nano-particle velocity and concentration at selected locations of the ceramic micro-model. Furthermore, a novel in-situ, nondestructive method of measuring 3D geometry of non-transparent ceramic micro-model is described and validated. The particle experiment uses 860 nm fluorescence labeled polystyrene neutrally buoyant, and electrically neutral nano-particles. The data was acquired using confocal laser-scanning microscope to quantify 3D particle transport at selected observation locations. In addition, fluorescence microscope was used to measure in-situ geometry of porous media. Results of detailed 3D measurements of nano-particle velocity and particle concentration from experiment conducted at a constant flow rate of 30 nL/min in the rock-based micro-model are presented and discussed. Particle velocities range from 0 to 20.93 μm/sec in magnitude, and average concentration range from 6.02 × 103 to 6.79 × 103 particles at inlet channel while velocities range from 0 to 73.63 μm/sec and concentration range from 4.9 × 101 to 1.45 × 103 particles at selected observation locations of the ceramic micro-model. 3D velocity fields at selected locations also indicate that mean velocity closer to the top wall is comparatively higher than bottom wall, because of higher planar porosity and smooth pathway for the nano-particles closer to the top wall. The three dimensional micro-model geometry reconstructed from the fluorescence data can be used to conduct numerical simulations of the flow in the as-tested micro-model for future comparisons to experimental results after incorporating particle transport and particle-wall interaction models.

A ceramic-based micromodel was fabricated with batching of green alumina ceramics mixed with polymer binders, extrusion of the green alumina tapes, and hot embossing of the green tapes with a metal mold. The metal mold fabricated using optical lithography of SU8 and electroforming of nickel contained 2.5D pore network geometry in 13 layers of a rock, Boise sandstone. The hot embossing process enabled the generation of the pore network geometries with a minimum feature size of 25 μm and for distinct formation of the 13 layers of the 2.5D pore geometry of the rock. The green ceramic micromodels were processed with solvent extraction, thermal debinding, and sintering. The sintered micromodels showed significant shrinkages at all directions of the micromodels, which were 17.6% in x, 17.5% in y, and 14.6% in z. The sintered, 2.5D rock-based ceramic micromodel was capped with a thin glass cover slide and used for flow visualization with a fluorescent dye and fluorescent nano-particles. The dye-filled micromodel showed good flow connectivity and fluorescence signal intensity dependence on depth. It was observed that the peak particle concentration close to the observation window and gradual decrease in particle concentration along the depth. The higher velocities were measured in the low flow resistance region with velocity variations along the depth. The microfabricated 2.5D ceramic micromodels will allow resistance to harsh experimental conditions such as high temperature and pressure, and opportunity for investigation of the complex flow patterns in 3D.

Plastic microfluidic device for the efficient isolation (>90% recovery) of cell free DNA from plasma for molecular profiling.

Microfluidic devices where the microchannel walls were decorated with micro and nanostructures were fabricated using 3D nanomolding. Using 3D molded microfluidic devices with microchannel walls decorated with microscale gratings, the fluid mixing behavior was investigated through experiments and numerical simulation. The use of microscale gratings in the micromixer was predicated by the fact that large obstacles in a microchannel enhances the mixing performance. Slanted ratchet gratings on the channel walls resulted in a helical flow along the microchannel, thus increasing the interfacial area between fluids and cutting down the diffusion length. Increasing the number of walls decorated with continuous ratchet gratings intensified the strength of the helical flow, enhancing mixing further. When ratchet gratings on the surface of the top cover plate were aligned in a direction to break the continuity of gratings from the other three walls, a stack of two helical flows was formed one above each other. This work concludes that the 3D nanomolding process can be a cost-effective tool for scaling-up the fabrication of microfluidic mixers with improved mixing efficiencies. Graphical abstract In this paper we show that a micromixer with patterned walls can be fabricated using 3D nanomolding and solvent-assisted bonding to manipulate the flow patterns to improve mixing.

Fabrication of 2.5D rock-based micromodels with high resolution features is presented using SU-8 multi-layer lithography and nickel electroforming for nickel molds. Processes associated with SU-8 were carefully optimized by the use of the vacuum contact, the use of UV filter, and controls of UV exposure doses and baking times. The use of SU-8 Micro Spray enabled the easy fabrication of multi-layers of SU-8, while exhibiting some total thickness variations. The thirteen layered SU-8 samples showed reliable patterning results for features at 10 and 25 gm resolutions, and minor pattern distortions of features at the 5 gm resolution. Flycutting method employed in multi-layer lithography of SU-8 yielded accurate total thickness control within 1.5 gm and excellent pattern formation for all of 5, 10, and 25 gm features. Electroforming of nickel was optimized with electroplating bath composition and electroplating parameters such as current density to realize the high resolution nickel mold. The fabricated nickel molds from flycutting based SU-8 samples revealed the feasibility of manufacturing the minimum features down to 5 gm for thirteen layers without any pattern distortions. The replication-based micromolding method will allow for fabrication of micromodels in a variety of materials such as polymers and ceramics. The high resolution, 2.5D micromodels will be used for investigation of pore-scale fluid transport, which will aid in understanding the complicated fluidic phenomena occurring in the 3D reservoir rock.

A confocal Micro-Particle Image Velocimetry (C-μPIV) technique along with associated post image processing algorithms is established to quantify three dimensional distributions of nano-particle velocity and concentration at the micro-scale (pore-scale) in 2.5D porous media designed from a Boise rock sample. In addition, an in-situ, non-destructive method for measuring the geometry of the micro-model, including its depth, is described and demonstrated. The particle experiments use 900 nm fluorescence labeled polystyrene particles at a flow rate of 10 nLmin−1 and confocal laser scanning microscopy (CLSM), while in-situ geometry measurements use regular microscope along with Rhodamine dye and a depth-to-fluorescence-intensity calibration. Image post-processing techniques include elimination of background noise and signal from adsorbed nano-particle on the inner surfaces of the micro-model. In addition, a minimization of depth of focus technique demonstrates a capability of optically thin slice allowing us to measure depth wise velocity in 2.5D micro-model. The mean planar components of the particle velocity of the steady-state flow and particle concentration distributions were measured in three dimensions. Particle velocities range from 0.01 to 122 μm s−1 and concentrations from 2.18 × 103 to 1.79 × 104 particles mm−2. Depth-wise results show that mean velocity closer to the top wall is comparatively higher than bottom walls, because of higher planar porosity and smooth pathway for the nano-particles closer to the top wall. The three dimensional micro-model geometry reconstructed from the fluorescence data can be used to conduct numerical simulations of the flow in the as-tested micro-model for future comparisons to experimental results after incorporating particle transport and particle-wall interaction models.

Sperm cell activation plays a critical role in a range of biological and engineering processes, from fertilization to cryopreservation protocol evaluation. Across a range of species, ionic and osmotic effects have been discovered that lead to activation. Sperm cells of zebrafish (Danio rerio) initiate motility in a hypoosmotic environment. In this study, we employ a microfluidic mixer for the purpose of rapidly diluting the extracellular medium to initiate the onset of cell motility. The use of a microchannel offers a rapid and reproducible mixing profile throughout the device. This greatly reduces variability from trial to trial relative to the current methods of analysis. Coupling these experiments with numerical simulations, we were able to investigate the dynamics of intracellular osmolality as each cell moves along its path through the micromixer. Our results suggest that intracellular osmolality, and hence intracellular ion concentration, only slightly decreases, contrary to the common thought that larger changes in these parameters are required for activation. Utilizing this framework, microfluidics for controlled extracellular environments and associated numerical modeling, has practical applicability in standardizing high-throughput aquatic sperm activation, and more fundamentally, investigations of the intracellular environment leading to motility.

Low-cost modular polymer microfluidic platforms integrating several different functional units may potentially reduce the cost of molecular and environmental analyses, and enable broader applications. Proper function of such systems depends on well-characterized assembly of the instruments. Passive alignment is one approach to obtaining such assemblies. Model modular devices containing passive alignment features, hemispherical pins in v-grooves, and integrated alignment standards for characterizing the accuracy of the assemblies were replicated in polycarbonate using doubled-sided injection molding. The dimensions and locations of the assembly features and alignment standards were measured. The assemblies had mismatches from 16 ± 4 to 20 ± 6 μm along the x-axis and from 103 ± 7 to 118 ± 11 μm along the y-axis. The vertical variation from the nominal value of 287 μm ranged from -10 ± 4 to 34 ± 7 μm. An assembly tolerance model was used to estimate the accuracy of the assemblies based on the manufacturing variations of the alignment structures. Variation of the alignment structure features were propagated through the assembly using Monte Carlo methods. The estimated distributions matched the measured experimental results well, with differences of 2%-13% due to unmodeled aspects of the variations Accurate assembly of advanced polymer microsystems is feasible and predictable in the design phase. [2014-0125].