Probing T-cell activation in nanoliter tumor co-cultures using membrane displacement trap arrays.

Immune responses against cancer are inherently stochastic, with small numbers of individual T cells within a larger ensemble of lymphocytes initiating the molecular cascades that lead to tumor cytotoxicity. A potential source of this intra-tumor variability is the differential ability of immune cells to respond to tumor cells. Classical microwell co-cultures of T cells and tumor cells are inadequate for reliably culturing and analyzing low cell numbers needed to probe this variability, and have failed in recapitulating the heterogeneous small domains observed in tumors.

Programmable Control of Nanoliter Droplet Arrays using Membrane Displacement Traps.

A unique droplet microfluidic technology enabling programmable deterministic control over complex droplet operations is presented. The platform provides software control over user-defined combinations of droplet generation, capture, ejection, sorting, splitting, and merging sequences to enable the design of flexible assays employing nanoliter-scale fluid volumes. The system integrates a computer vision system with an array of membrane displacement traps capable of performing selected unit operations with automated feedback control.

Microfluidic synthesis of lipid-based nanoparticles for drug delivery: recent advances and opportunities.

Microfluidic technologies are revolutionizing the synthesis of nanoscale lipid particles and enabling new opportunities for the production of lipid-based nanomedicines. By harnessing the benefits of microfluidics for controlling diffusive and advective transport within microfabricated flow cells, microfluidic platforms enable unique capabilities for lipid nanoparticle synthesis with precise and tunable control over nanoparticle properties. Here we present an assessment of the current state of microfluidic technologies for lipid-based nanoparticle and nanomedicine production.

Nanogap traps for passive bacteria concentration and single-point confocal Raman spectroscopy.

A microfluidic device enabling the isolation and concentration of bacteria for analysis by confocal Raman spectroscopy is presented. The glass-on-silicon device employs a tapered chamber surrounded by a 500 nm gap that serves to concentrate cells at the chamber apex during sample perfusion. The sub-micrometer gap retains bacteria by size exclusion while allowing smaller contaminants to pass unimpeded.

Scalable Liposome Synthesis by High Aspect Ratio Microfluidic Flow Focusing.

Microfluidic flow focusing provides an efficient approach to the generation of nanoscale lipid vesicles of tunable size and low size variance. Scalable nanoliposome synthesis over a wide range of production rates can be readily achieved using a high aspect ratio flow focusing device fabricated by widely available additive manufacturing methods. Here we detail methods for the manufacture and operation of a 3D-printed microfluidic flow focusing technology enabling the synthesis of liposomes with modal diameters ranging from ca.

Microfluidic vortex focusing for high throughput synthesis of size-tunable liposomes.

Control over vesicle size during nanoscale liposome synthesis is critical for defining the pharmaceutical properties of liposomal nanomedicines. Microfluidic technologies capable of size-tunable liposome generation have been widely explored, but scaling these microfluidic platforms for high production throughput without sacrificing size control has proven challenging. Here we describe a microfluidic-enabled process in which highly vortical flow is established around an axisymmetric stream of solvated lipids, simultaneously focusing the lipids while inducing rapid convective and diffusive mixing through application of the vortical flow field.

Deterministic assembly of chromosome ensembles in a programmable membrane trap array.

Selective spatial isolation and manipulation of single chromosomes and the controlled formation of defined chromosome ensembles in a droplet-based microfluidic system is presented. The multifunctional microfluidic technology employs elastomer valves and membrane displacement traps to support deterministic manipulation of individual droplets. Picoliter droplets are formed in the 2D array of microscale traps by self-discretization of a nanoliter sample plug, with membranes positioned over each trap allowing controllable metering or full release of selected droplets.

In situ photografting during direct laser writing in thermoplastic microchannels.

A method for in situ photografting during direct laser writing by two-photon polymerization is presented. The technique serves as a powerful approach to the formation of covalent bonds between 3D photoresist structures and thermoplastic surfaces. By leveraging the same laser for both pattern generation and localized surface reactions, crosslinking between the bulk photoresist and thermoplastic surface is achieved during polymerization.

Plasma Isolation in a Syringe by Conformal Integration of Inertial Microfluidics.

A thermoplastic microfluidic substrate is conformally integrated onto the cylindrical barrel of a conventional venipuncture syringe, forming a spiral inertial separation element supporting the isolation of plasma from diluted whole blood. The cylindrical shape of the syringe itself serves to define the flow path required for inertial separation by transforming a linear microchannel to a spiral topology. The hybrid system enables inertial plasma separation by Dean flow focusing within the same syringe used for a patient blood draw, with the seamlessly interconnected microfluidic element operated by automated or manual actuation of the syringe plunger.

A programmable microfluidic platform for multisample injection, discretization, and droplet manipulation.

A programmable microfluidic platform enabling on-demand sampling, compartmentalization, and manipulation of multiple aqueous volumes is presented. The system provides random-access actuation of a microtrap array supporting selective discretization of picoliter volumes from multiple sample inputs. The platform comprises two interconnected chips, with parallel T-junctions and multiplexed microvalves within one chip enabling programmable injection of aqueous sample plugs, and nanoliter volumes transferred to a second microtrap array chip in which the plugs are actively discretized into picoliter droplets within a static array of membrane displacement actuators.

Isolation of intact bacteria from blood by selective cell lysis in a microfluidic porous silica monolith.

Rapid and efficient isolation of bacteria from complex biological matrices is necessary for effective pathogen identification in emerging single-cell diagnostics. Here, we demonstrate the isolation of intact and viable bacteria from whole blood through the selective lysis of blood cells during flow through a porous silica monolith. Efficient mechanical hemolysis is achieved while providing passage of intact and viable bacteria through the monoliths, allowing size-based isolation of bacteria to be performed following selective lysis.

Microfluidic on-demand droplet generation, storage, retrieval, and merging for single-cell pairing.

A multifunctional microfluidic platform combining on-demand aqueous-phase droplet generation, multi-droplet storage, and controlled merging of droplets selected from a storage library in a single integrated microfluidic device is described. A unique aspect of the technology is a microfluidic trap design comprising a droplet trap chamber and lateral bypass channels integrated with a microvalve that supports the capture and merger of multiple droplets over a wide range of individual droplet sizes. A storage unit comprising an array of microfluidic traps operates in a first-in first-out manner, allowing droplets stored within the library to be analyzed before sequentially delivering selected droplets to a downstream merging zone, while shunting other droplets to waste.

Miniature bulk PZT traveling wave ultrasonic motors for low-speed high-torque rotary actuation.

Traveling wave ultrasonic micromotors fabricated from a single layer of homogeneous bulk piezoelectric lead zirconate titanate (PZT) are described. The miniature motors are capable of bi-directional rotary motion with controllable speeds. By taking advantage of transverse interdigitated electrodes to excite traveling waves in a patterned bulk PZT substrate, the monolithic micromotor stators are patterned using a simple and low cost fabrication technique based on micropowder blasting.

Active or Passive On-Demand Droplet Merging in a Microfluidic Valve-Based Trap.

A microfluidic valve-based trap enabling controlled capture, release, and temporary immobilization of droplets together with on-demand merging of selected droplets is presented in this paper. The microfluidic trap technology can merge droplets passively or in active manner via a pneumatically actuated membrane. A microchip is developed with two functional units of droplet generator and merging mechanism to implement the passive or active merging performance of the microfluidic valve-based trap using a low and high surfactant concentrated continuous oil-phase.

Nano-printed miniature compound refractive lens for desktop hard x-ray microscopy.

Hard x-ray lenses are useful elements in x-ray microscopy and in creating focused illumination for analytical applications such as x-ray fluorescence imaging. Recently, polymer compound refractive lenses for focused illumination in the soft x-ray regime (< 10 keV) have been created with nano-printing. However, there are no such lenses yet for hard x-rays, particularly of short focal lengths for benchtop microscopy.

Impedimetric Immunosensing in a Porous Volumetric Microfluidic Detector.

A sensitive and rapid impedemetric immunosensor is demonstrated utilizing porous volumetric microfluidic detection elements and silver enhanced gold nanoparticle probes. The porous detection elements significantly increase capture probe density and decrease diffusion length scales compared to conventional planar sensors to improve target capture efficiency and enhance impedance signal. In this work, a packed bed of silica beads functionalized with antibody probes serves as a porous sensor element within a thermoplastic microchannel, with an interdigitated gold electrode microarray used to measure impedance changes caused by the concentration dependent formation of silver aggregates.

Light-Directed Self-Assembly of Robust Alginate Gels at Precise Locations in Microfluidic Channels.

Recently there has been much interest in using light to activate self-assembly of molecules in a fluid, leading to gelation. The advantage of light over other stimuli lies in its spatial selectivity, i.e.

Catalytic Propulsion and Magnetic Steering of Soft, Patchy Microcapsules: Ability to Pick-Up and Drop-Off Microscale Cargo.

We describe the creation of polymeric microcapsules that can exhibit autonomous motion along defined trajectories. The capsules are made by cross-linking aqueous microdroplets of the biopolymer chitosan using glutaraldehyde. A coflow microfluidic tubing device is used to generate chitosan droplets containing nanoparticles (NPs) with an iron (Fe) core and a platinum (Pt) shell.

High-Throughput Continuous Flow Production of Nanoscale Liposomes by Microfluidic Vertical Flow Focusing.

Liposomes represent a leading class of nanoparticles for drug delivery. While a variety of techniques for liposome synthesis have been reported that take advantage of microfluidic flow elements to achieve precise control over the size and polydispersity of nanoscale liposomes, with important implications for nanomedicine applications, these methods suffer from extremely limited throughput, making them impractical for large-scale nanoparticle synthesis. High aspect ratio microfluidic vertical flow focusing is investigated here as a new approach to overcoming the throughput limits of established microfluidic nanoparticle synthesis techniques.

Single-use thermoplastic microfluidic burst valves enabling on-chip reagent storage.

A simple and reliable method for fabricating single-use normally closed burst valves in thermoplastic microfluidic devices is presented, using a process flow that is readily integrated into established workflows for the fabrication of thermoplastic microfluidics. An experimental study of valve performance reveals the relationships between valve geometry and burst pressure. The technology is demonstrated in a device employing multiple valves engineered to actuate at different inlet pressures that can be generated using integrated screw pumps.

Microfluidic generation of uniform water droplets using gas as the continuous phase.

Microfluidic schemes for forming uniform aqueous microdroplets usually rely on contacting the aqueous liquid (dispersed phase) with an immiscible oil (continuous phase). Here, we demonstrate that the oil can be substituted with gas (nitrogen or air) while still retaining the ability to generate discrete and uniform aqueous droplets. Our device is a capillary co-flow system, with the inner flow of water getting periodically dispersed into droplets by the external flow of gas.

A chitosan coated monolith for nucleic acid capture in a thermoplastic microfluidic chip.

A technique for microfluidic, pH modulated DNA capture and purification using chitosan functionalized glycidyl methacrylate monoliths is presented. Highly porous polymer monoliths are formed and subsequently functionalized off-chip in a batch process before insertion into thermoplastic microchannels prior to solvent bonding, simplifying the overall fabrication process by eliminating the need for on-chip surface modifications. The monolith anchoring method allows for the use of large cross-section monoliths enabling high flowrates and high DNA capture capacity with a minimum of added design complexity.

Ex Situ Integration of Multifunctional Porous Polymer Monoliths into Thermoplastic Microfluidic Chips.

A unique method for incorporating functional porous polymer monolith elements into thermoplastic microfluidic chips is described. Monolith elements are formed in a microfabricated mold, rather than within the microchannels, and chemically functionalized off chip before insertion into solvent-softened thermoplastic microchannels during chip assembly. Because monoliths may be trimmed prior to final placement, control of their size, shape, and uniformity is greatly improved over photopolymerization methods.

Development of a microchip Europium nanoparticle immunoassay for sensitive point-of-care HIV detection.

Rapid, sensitive and specific diagnostic assays play an indispensable role in determination of HIV infection stages and evaluation of efficacy of antiretroviral therapy. Recently, our laboratory developed a sensitive Europium nanoparticle-based microtiter-plate immunoassay capable of detecting target analytes at subpicogram per milliliter levels without the use of catalytic enzymes and signal amplification processes. Encouraged by its sensitivity and simplicity, we continued to miniaturize this assay to a microchip platform for the purpose of converting the benchtop assay technique to a point-of-care test.

A facile route to the synthesis of monodisperse nanoscale liposomes using 3D microfluidic hydrodynamic focusing in a concentric capillary array.

A novel microscale device has been developed to enable the one-step continuous flow assembly of monodisperse nanoscale liposomes using three-dimensional microfluidic hydrodynamic focusing (3D-MHF) in a concentric capillary array. The 3D-MHF flow technique displays patent advantages over conventional methods for nanoscale liposome manufacture (i.e.