The on-chip storage of dried reagents is an important technological challenge that must be addressed to improve the capabilities of microfluidic point-of-care (POC) chips. In this work, we investigate the use of poly (lactic-co-glycolic acid) (PLGA) as an encapsulant for the storage and controlled release of dried reagents integrated into disposable thermoplastic microfluidic chips. The PLGA layer allows multiple solid reagent deposits to remain isolated during sample introduction at room temperature and controllably released into the sample volume after heating the chip above a critical threshold temperature. Simple manual pipetting of a PLGA/ethyl acetate solution serves to form a protective PLGA shell encapsulating deposited reagents, with robust sealing between the PLGA and thermoplastic cyclic olefin polymer (COP) substrate preventing reagent leakage during sample introduction. When using a shell thickness below 20 μm to encapsulate nucleic acids as model reagents, over 90% of the deposits are retained following extended aqueous flow, while heating the chip above 40 °C leads to dramatic shrinkage of the PLGA, resulting in delamination of the encapsulating film and rapid reagent release. Using this approach, an on-chip loop-mediated isothermal amplification (LAMP) assay for the detection of methicillin-resistant Staphylococcus aureus (MRSA) is implemented using multiple encapsulated LAMP primer sets integrated directly into an array of on-chip wells. The PLGA encapsulation technique is shown to be a simple and effective method for reagent-integrated microfluidic device manufacturing, offering a new path towards true sample-in, answer-out point-of-care assays.

Nucleic acid amplification tests (NAATs) play a critical role in disease diagnostics by enabling rapid and highly sensitive detection of genomic sequences associated with specific pathogens. This paper presents a portable NAAT assay reader combining precise thermal control and spatially-multiplexed fluorescence detection that is designed to extend the applicability of nucleic acid testing across diverse environments. The compact and low cost Multiplexed Array Gene Imager (MAGI) system operates wirelessly, with assay control and readout enabled through a Web-based interface, and can be adapted to a broad range of NAAT formats and assay substrates. Thermal actuation is performed using a printed circuit board heater that provides stable closed-loop temperature control at low manufacturing cost. Performance of the MAGI system is evaluated using a spatially-multiplexed loop-mediated isothermal amplification (LAMP) assay implemented in a custom microfluidic 12-well plate. Overall, the presented system offers a manufacturable, adaptable, and functional platform for use in diverse environments where NAAT cost, portability, and assay flexibility are important considerations.

Cancer immunotherapies trigger highly variable responses in patients and in genetically identical mouse models. To assess the intrinsic stochasticity of these therapies, we performed thousands of well-controlled ex vivo immunoassays. We show that leukocyte responses and tumor cytotoxicity are highly variable at the macroscopic level and statistically distributed as a shifted Poisson process. Stochastic activation of a rare subpopulation of T cells (so-called Spark T cells), coupled with a paracrine interferon (IFN)-γ-driven positive feedback, accounts for this measured "noise" in immunotherapeutic reactions. We integrated these quantitative insights into a custom-designed machine-learning pipeline to analyze immune reactions with single-cell resolution. This led us to phenotypically and functionally identify Spark T cells in murine naive T cells and in human T cell blasts as prepared for adoptive T cell therapy. We then demonstrate their relevance in explaining variable outcomes in cancer immunotherapies.

A microfluidic platform designed to enhance the development of extracellular vesicle (EV)-based therapeutics is presented. The two-chip system combines rapid electrokinetic concentration and purification of EVs together with counterflow buffer exchange to prepare the purified vesicles for pH gradient-driven drug loading. The first chip employs electrophoretic enrichment to capture vesicles at the surface of a nanoporous membrane, with punctuated operation of the electrokinetic device supporting continuous processing of EVs from milliliter-scale volumes of cell culture supernatant. The second chip performs continuous-flow buffer exchange to protonate the vesicles and form a transmembrane pH gradient prior to passive loading of nucleic acid cargo. The modular components are shown to preserve EV bioactivity throughout the full process, using disposable thermoplastic chips that eliminate the need for device cleaning or regeneration between process runs. The technology enables a rapid and convenient workflow for EV-based therapeutic development addressing key limitations associated with established batch processes.

The collection and separation of aerosols is necessary to support the capture and analysis of particulate matter that can impact human health, enabling effective monitoring and prediction of exposure risk, and supporting fundamental studies of aerosol generation, transport, and distribution. Of particular concern are sub-micrometer particles which can deeply infiltrate the lungs, but efficient separation and capture of these smaller particles is challenging. In the case of bioaerosols, effective recovery of biological particles following capture often involves significant dilution of the collected sample, hindering downstream analysis. Here we report a miniature 3D printed cyclone-based platform capable of highly effective separation and recovery of submicron bioaerosols with well-defined cut-off sizes. Using a set of geometrically similar microcyclones fabricated at different size scales, with minimum channel dimensions of 0.75 mm, cut diameters as low as 0.05 μm are achieved at aerosol flow rates up to 5 L/min, with sharp cut-offs yielding nearly 100 % capture efficiency for larger particulates. Furthermore, captured influenza virus is recovered from the devices with up to 85 % efficiency and minimal dilution via a rapid elution process that takes advantage of the low internal microcyclone volume. The high capture efficiency for submicron particles enabled by the microcyclone platform, together with its compact form factor, effective sample recovery, tunable cut size, and cost-effective manufacture, makes the technology a promising tool for broad applications in aerosol and bioaerosol monitoring.

Quantifying infectious virus is essential for vaccine development, clinical diagnostics, and infectious disease research, but current assays are constrained by long turnaround times, high costs, and laborious procedures. To address these limitations, we present a digital focus assay employing an array of independent nanoliter cell cultures. The microfluidic platform allows cells in each nanowell to be inoculated with virus, followed by oil discretization to prevent cross-contamination. After incubation, infected cells are visualized through immunofluorescence staining, and a binary map of wells positive for viral antigen is generated by automated image analysis, allowing infectious viral titer to be calculated by statistical analysis. The platform requires significantly smaller sample and reagent volumes than conventional focus assays while enhancing assay automation and endpoint time flexibility. The technology is applied to the quantification of infectious influenza A using both model virus and clinical specimens, demonstrating the digital platform as an accurate, rapid, cost-effective, and convenient tool for viral load quantification with broad utility in clinical, pharmaceutical, and research applications.

In this study, we present a scalable multilayer platform enabling rapid buffer exchange and sample purification by counterflow microdialysis. The microfluidic system features a compact design employing polycarbonate substrates that contain integrated microchannels, with mixed cellulose ester membrane filters bonded between the mating polycarbonate layers. The unique design allows microdialysis layers to be stacked within a single device while employing standard membrane filters to increase the transport of ions and small molecules between the buffer compartments to enhance microdialysis performance. Devices with up to 4 microdialysis layers are demonstrated using a manufacturing process that supports an arbitrary level of scaling. Performance of the fabricated multilayer microdialysis chips is evaluated for application to continuous-flow buffer exchange for pH shifting and lipid nanoparticle purification.

A quadruple mass Coriolis vibratory gyroscope operating in the mode-matched condition has been redesigned with the singular focus of minimizing nonlinear transduction mechanisms, thereby allowing for angle random walk (ARW) noise reduction when operating at amplitudes higher than 2 mu m. This is achieved through the following steps: (i) redesigning the Coriolis mass folded flexures and shuttle springs, (ii) linearizing the antiphase coupler spring rate while maintaining parasitic modal separation, (iii) replacing parallel plate transducers with linear combs, (iv) implementing dedicated force-balanced electrostatic frequency tuners, and (v) microTorr vacuum packaging enabling operation at the thermoelastic dissipation limit of silicon. Additionally, cross-axis stiffness is reduced through folded-flexure moment balancing to further reduce ARW. By the balancing of positive and negative Duffing frequency contributions, net frequency nonlinearity was further reduced to -20 ppm. The gyroscope presented in this study has achieved an ARW of 0.0005 deg/root hr, with an uncompensated bias instability of 0.08 deg/hr. These advancements hold promise for enhancing the performance of precision vibratory gyroscopes for navigation and North-finding applications. [2023-0144]

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. Here we leverage a membrane displacement trap array technology that overcomes limitations of conventional microwell plates for immunodynamic studies. The microfluidic platform supports on-demand formation of dense nanowell cultures under continuous perfusion reflecting the tumor microenvironment, with real-time monitoring of T cell proliferation and activation within each nanowell. The system enables selective ejection of cells for profiling by fluorescence activated cell sorting, allowing observed on-chip variability in immune response to be correlated with off-chip quantification of T cell activation. The technology offers new potential for probing the molecular origins of T cell heterogeneity and identifying specific cell phenotypes responsible for initiating and propagating immune cascades within tumors. Insight Box Variability in T cell activation plays a critical role in the immune response against cancer. New tools are needed to unravel the mechanisms that drive successful anti-tumor immune response, and to support the development of novel immunotherapies utilizing rare T cell phenotypes that promote effective immune surveillance. To this end, we present a microfluidic cell culture platform capable of probing differential T cell activation in an array of nanoliter-scale wells coupled with off-chip cell analysis, enabling a high resolution view of variable immune response within tumor / T cell co-cultures containing cell ensembles orders of magnitude smaller than conventional well plate studies.

The current landscape of microfluidic technologies for lipid nanoparticle production, size control, scaling, and nanomedicine post-processing is described in a review of this evolving field.

Coriolis vibratory gyroscope drive amplitude of 16 $\upmu\mathrm{m}$ is demonstrated with $+30$ ppm frequency shift nonlinearity. Here, MEMS quadruple mass gyroscope (QMG) mechanical flexures are specifically designed to separate spurious modes without affecting linear operation of gyroscope drive over large amplitudes. A simulated frequency change of $+480$ ppm was initially predicted, however, when electrostatically driven at large amplitudes comb drive and sense tips begin to introduce spring softening reducing nonlinearity to $+30$ ppm. Careful consideration was also taken to electrostatically tune as-fabricated frequency mismatch without further exacerbating nonlinearity. Angle random walk (ARW) versus drive amplitude is plotted with a demonstrated $8\times$ improvement compared to similar quadruple mass gyroscopes.

Thousands of patients have benefited from the growing use of cancer immunotherapies. However, the success of these therapies can be highly variable. Strikingly, some murine tumor models show large variability in the outcome of cancer immunotherapies, even when the mice, tumor cells and anti-tumor immune cells injected into mice are all genetically identical. Here, we sought to analyze this variability in adoptive cell therapies, in order to identify the immune population driving this variability. To search for the occurrence of large variability ex vivo, we extracted mouse TCR-transgenic CD8+ T cells, and co-cultured them with antigen-expressing tumor cells. By using multiplexed in vitro assays and single-cell analysis of thousands of samples, we identified conditions (e.g. cell numbers, antigen quality, tumor cell types) where large variations in the immune activation against cancer cells is observed even between technical replicates. We then developed a quantitative framework that uses statistical modeling and machine learning to extract useful information from this immunotherapeutic variability. Our framework allowed the prediction and identification of a rare population of naïve CD8+ T cells (“Spark T cells”) that is necessary and sufficient to spark massive anti-tumor immune reactions. We are currently performing experiments to test the efficacy and functional significance of the identified immune population in vivo. We are also applying this framework in human TCR-engineered T cell blasts to identify the equivalent of the Spark T cells in humans. We envision this framework being applied to identify other relevant immune cell types that act as catalysts for successful cancer immunotherapies. Supported by the Intramural Research Program of the National Institutes of Health, the National Cancer Institute

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.

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.

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.

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.

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.

The seamless integration of reagents into microfluidic devices can serve to significantly reduce assay complexity and cost for disposable diagnostics. In this work, the integration of multiplexed reagents into thermoplastic 2D microwell arrays is demonstrated using a scalable pin spotting technique. Using a simple and low-cost narrow-bore capillary spotting pin, high resolution deposition of concentrated reagents within the arrays of enclosed nanoliter-scale wells is achieved. The pin spotting method is further employed to encapsulate the deposited reagents with a chemically modified wax layer that serves to prevent disruption of the dried assay components during sample introduction through a shared microchannel, while also enabling temperature-controlled release after sample filling is complete. This approach supports the arbitrary patterning and release of different reagents within individual wells without crosstalk for multiplexed analyses. The performance of the in-well spotting technique is characterized using on-chip rolling circle amplification to evaluate its potential for nucleic acid-based diagnostics.

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.

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.