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. Microfluidic techniques are discussed in the context of conventional production methods, with an emphasis on the capabilities of microfluidic systems for controlling nanoparticle size and size distribution. Challenges and opportunities associated with the scaling of manufacturing throughput are discussed, together with an overview of emerging microfluidic methods for lipid nanomedicine post-processing. The impact of additive manufacturing on current and future microfluidic platforms is also considered.

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. Concentrating bacteria within the fixed volume enables the use of single-point confocal Raman detection for the rapid acquisition of spectral signatures for bacteria identification. The technology is evaluated for the analysis of E. cloacae, K. pneumoniae, and C. diphtheriae, with automated peak extraction yielding distinct spectral fingerprints for each pathogen at a concentration of 103 CFU/ml that compare favorably with spectra obtained from significantly higher concentration reference samples evaluated by conventional confocal Raman analysis. The nanogap technology offers a simple, robust, and passive approach to concentrating bacteria from dilute samples into well-defined optical detection volumes, enabling rapid and sensitive confocal Raman detection for label-free identification of focused cells.

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. 50-200 nm at production rates up to several hundred milligrams lipid per hour.

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. Sequences of individual droplet handling steps are defined through a robust Python-based scripting language. Bidirectional flow control within the microfluidic chips is provided using an H-bridge channel topology, allowing droplets to be transported to arbitrary trap locations within the array for increased operational flexibility. By enabling automated software control over all droplet operations, the system significantly expands the potential of droplet microfluidics for diverse biological and biochemical applications by combining the functionality of robotic liquid handling with the advantages of droplet-based fluid manipulation.

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. By adjusting the individual buffer and lipid flow rates within the system, the microfluidic vortex focusing technique is capable of generating liposomes with precisely controlled size and low size variance, and may be operated up to the laminar flow limit for high throughput vesicle production. The reliable formation of liposomes as small as 27 nm and mass production rates over 20 g/h is demonstrated, offering a path toward production-scale liposome synthesis using a single continuous-flow vortex focusing device.

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. By combining discretization, optical interrogation, and selective droplet release for sequential delivery to a downstream merging zone, the system enables efficient manipulation of multiple chromosomes into a defined ensemble with single macromolecule resolution. Key design and operational parameters are explored, and co-compartmentalization of three chromosome pairs is demonstrated as a first step toward formation of precisely defined chromosome ensembles for applications in genetic engineering and synthetic biology.

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. When applied to in-channel direct laser writing for microfluidic device fabrication, the process yields exceptionally strong adhesion and robust bond interfaces that can withstand pressure gradients as high as 7 MPa through proper channel design, photoinitiator selection, and processing conditions.

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. Plasma isolation is achieved without the need for external instrumentation. Device design and fabrication challenges are discussed, and effective plasma isolation within the system is demonstrated, with a peak separation efficiency above 97% using 25 × diluted blood.

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. The system employs two different multiplexer designs that reduce the number of input signals required for both sample injection and discretization. This versatile droplet-based technology offers flexible sample workflows and functionalities for the formation and manipulation of heterogeneous picoliter droplets, with particular utility for applications in biochemical synthesis and cell-based assays requiring flexible and programmable operation of parallel and multistep droplet processes. The platform is used here for the selective encapsulation of differentially labeled cells within a discrete droplet array.

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. A process for synthesizing large quantities of discrete capillary-bound monolith elements and millimeter-scale monolith bricks is described, together with the seamless integration of individual monoliths into microfluidic chips. The impact of monolith morphology, geometry, and flow conditions on cell lysis is explored, and flow regimes are identified wherein robust selective blood cell lysis and intact bacteria passage are achieved for multiple gram-negative and gram-positive bacteria. The technique is shown to enable rapid sample preparation and bacteria analysis by single-cell Raman spectrometry. The selective lysis technique presents a unique sample preparation step supporting rapid and culture-free analysis of bacteria for the point of care.

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. Performance of the microfluidic trap is investigated for variations in bypass/chamber hydrodynamic resistance ratio, micro-chamber geometry, trapped droplet volume, and overall flow rate. The integrated microfluidic platform is then utilized to demonstrate the operational steps necessary for cell-based assays requiring the isolation of defined cell populations with single cell resolution, including encapsulation of individual cells within an aqueous-phase droplet carrier, screening or incubation of the immobilized cell-encapsulated droplets, and generation of controlled combinations of individual cells through the sequential droplet merging process. Beyond its utility for cell analysis, the presented platform represents a versatile approach to robust droplet generation, storage, and merging for use in a wide range of droplet-based microfluidics applications.

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. Performance of the ultrasonic micromotors is explored using devices with integrated glass rotors, using defined preload forces applied between the microfabricated stator and rotor elements. For the case of a 4.12 mm diameter PZT stator, a maximum speed of 30 rpm and stall torque of 501 mN · mm are achieved when applying a 323 mN preload force to the rotor.

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. We report the first instance of a nano-printed lens for hard x-ray microscopy, and evaluate its imaging performance. The lens consists of a spherically focusing compound refractive lens designed for 22 keV photon energy, with a tightly packed structure to provide a short total length of 1.8 mm and a focal length of 21.5 mm. The resulting lens technology was found to enable benchtop microscopy at 74x magnification and 1.1 µm de-magnified image pixel size at the object plane. It was used to image and evaluate the focal spots of tungsten-anode micro-focus x-ray sources. The overall system resolution with broadband illumination from a tungsten-anode x-ray tube at 30 kV and 10 mm focal distance was measured to be 2.30±0.22 µm.

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. The measured impedance change is independent of electrode spacing, enabling a device with low resolution electrodes to achieve a sandwich immunoassay detection limit between 1-10 ng/mL with a 4-log dynamic range, with a total assay time of 75 min.

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., its ability to be directed at a precise location, which could be particularly useful in microfluidic applications. However, existing light-responsive fluids are not suitable for these purposes since they do not convert into sufficiently strong gels that can withstand shear. Here, we address this deficiency by developing a new light-responsive system based on the well-known polysaccharide, alginate. The fluid is composed entirely of commercially available components: alginate, a photoacid generator (PAG), and a chelated complex of divalent strontium (Sr(2+)) cations. Upon exposure to ultraviolet (UV) light, the PAG dissociates to release H(+) ions, which in turn induce the release of free Sr(2+) from the chelate. The Sr(2+) ions self-assemble with the alginate chains to give a stiff gel with an elastic modulus ∼2000 Pa and a yield stress ∼400 Pa (this gel is strong enough to be picked up and held by one's fingers). The above fluid is sent through a network of microchannels and a short segment of a specific channel is exposed to UV light. At that point, the fluid is locally transformed into a strong gel in a few minutes, and the resulting gel blocks the flow through that channel while other channels remain open. When the UV light is removed, the gel is gradually diluted by the flow and the channel reopens. We have thus demonstrated a remote-controlled fluidic valve that can be closed by shining light and reopened when the light is removed. In addition, we also show that light-induced gelation of our alginate fluid can be used to deposit biocompatible payloads at specific addresses within a microchannel.

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. The droplets are then incubated in a Petri dish with the cross-linking solution, and an external magnet is placed below the Petri dish to pull the NPs together as a collective "patch" on one end of each droplet. This results in cross-linked capsules (∼150 µm in diameter) with an anisotropic (patchy) structure. When these capsules are placed in a solution of H2O2, the Pt shell of the NPs catalyzes the decomposition of H2O2 into O2 gas, which is ejected from the patchy end in the form of bubbles. As a result, the capsules (which are termed micromotors) move in a direction opposite to the bubbles. Furthermore, the micromotors can be steered along specific paths by an external magnet (the magnetic response arises due to the Fe in the core of the NPs). A given micromotor can thus be directed to meet with and adhere to an inert capsule, i.e., a model cargo. Adhesion occurs due to the soft nature of the two structures. Once the cargo is picked up, the micromotor-cargo pair can be moved along a specific path to a destination, whereupon the cargo can be released from the micromotor. We believe these soft micromotors offer significant benefits over their existing hard counterparts because of their biocompatibility, biodegradability, and ability to encapsulate a variety of payloads.

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. Here the vertical flow focusing technique is utilized to generate populations of small, unilamellar, and nearly monodisperse liposomal nanoparticles with exceptionally high production rates and remarkable sample homogeneity. By leveraging this platform, liposomes with modal diameters ranging from 80 to 200 nm are prepared at production rates as high as 1.6 mg min(-1) in a simple flow-through process.

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. On-chip storage and reconstitution of fluorescein salt sealed within defined reagent chambers are demonstrated. By taking advantage of the low gas and water permeability of cyclic olefin copolymer, the robust burst valves allow on-chip hermetic storage of reagents, making the technology well suited for the development of integrated and disposable assays for use at the point of care.

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. The droplet size and different formation modes can be tuned by varying the liquid and gas flow rates. Importantly, we identify the range of conditions that correspond to the "dripping mode", i.e., where discrete droplets are consistently generated with no satellites. We believe this is a significant development that will be beneficial for chemical and biological applications requiring clean and contaminant-free droplets, including DNA amplification, drug encapsulation, and microfluidic cell culture.