Rapid, low-cost fabrication of electronic microfluidics via inkjet-printing and xurography (MINX).

Microfluidics has shown great promise for point-of-care assays due to unique chemical and physical advantages that occur at the micron scale. Furthermore, integration of electrodes into microfluidic systems provides additional capabilities for assay operation and electronic readout. However, while these systems are abundant in biological and biomedical research settings, translation of microfluidic devices with embedded electrodes are limited. In part, this is due to the reliance on expensive, inaccessible, and laborious microfabrication techniques. Although innovative prior work has simplified microfluidic fabrication or inexpensively patterned electrodes, low-cost, accessible, and robust methods to incorporate all these elements are lacking. Here, we present MINX, a low-cost <1 USD and rapid (∼minutes) fabrication technique to manufacture microfluidic device with embedded electrodes. We characterize the structures created using MINX, and then demonstrate the utility of the approach by using MINX to implement an electrochemical bead-based biomarker detection assay. We show that the MINX technique enables the scalable, inexpensive fabrication of microfluidic devices with electronic sensors using widely accessible desktop machines and low-cost materials.

A Multidatabase ExTRaction PipEline (METRE) for facile cross validation in critical care research.

Transforming raw EHR data into machine learning model-ready inputs requires considerable effort. One widely used EHR database is Medical Information Mart for Intensive Care (MIMIC). Prior work on MIMIC-III cannot query the updated and improved MIMIC-IV version. Besides, the need to use multicenter datasets further highlights the challenge of EHR data extraction. Therefore, we developed an extraction pipeline that works on both MIMIC-IV and eICU Collaborative Research Database and allows for model cross validation using these 2 databases. Under the default choices, the pipeline extracted 38,766 and 126,448 ICU records for MIMIC-IV and eICU, respectively. Using the extracted time-dependent variables, we compared the Area Under the Curve (AUC) performance with ​​prior works on clinically relevant tasks such as in-hospital mortality prediction. METRE achieved comparable performance with AUC 0.723-0.888 across all tasks with MIMIC-IV. Additionally, when we evaluated the model directly on MIMIC-IV data using a model trained on eICU, we observed that the AUC change can be as small as +0.019 or -0.015. Our open-source pipeline transforms MIMIC-IV and eICU into structured data frames and allows researchers to perform model training and testing using data collected from different institutions, which is of critical importance for model deployment under clinical contexts. The code used to extract the data and perform training is available here: https://github.com/weiliao97/METRE.

Accelerating the optimization of vertical flow assay performance guided by a rational systematic model-based approach.

Rapid diagnostic tests (RDTs) have shown to be instrumental in healthcare and disease control. However, they have been plagued by many inefficiencies in the laborious empirical development and optimization process for the attainment of clinically relevant sensitivity. While various studies have sought to model paper-based RDTs, most have relied on continuum-based models that are not necessarily applicable to all operation regimes, and have solely focused on predicting the specific interactions between the antigen and binders. It is also unclear how the model predictions may be utilized for optimizing assay performance. Here, we propose a streamlined and simplified model-based framework, only relying on calibration with a minimal experimental dataset, for the acceleration of assay optimization. We show that our models are capable of recapitulating experimental data across different formats and antigen-binder-matrix combinations. By predicting signals due to both specific and background interactions, our facile approach enables the estimation of several pertinent assay performance metrics such as limit-of-detection, sensitivity, signal-to-noise ratio and difference. We believe that our proposed workflow would be a valuable addition to the toolset of any assay developer, regardless of the amount of resources they have in their arsenal, and aid assay optimization at any stage in their assay development process.

A sample-to-answer electrochemical biosensor system for biomarker detection.

Biomarker detection is critical for the diagnosis and treatment of numerous diseases. Typically, target biomarkers in blood samples are measured through tests conducted at centralized laboratories. Testing at central laboratories increases wait times for results, in turn increasing healthcare costs and negatively impacting patient outcomes. Alternatively, point-of-care platforms enable the rapid measurement of biomarkers, expand testing location capabilities and mitigate manual processing steps through integration and automation. However, many of these systems focus on sample detection rather than the equally important sample preparation. Here we present a fully integrated and automated sample-to-answer electrochemical biosensing platform which incorporates each aspect of the biomarker testing workflow from blood collection to sample preparation to assay operation and readout. The system combines a commercial microneedle blood sampling device with membrane-based plasma filtration upstream of a bead-based electrochemical immunoassay. We characterize the high separation efficiency (>99%) and low non-specific binding of the whole blood-to-plasma filtration membrane under a range of operating conditions. We demonstrate a full sample-to-answer workflow through the analysis of interlukin-6-spiked blood samples.

Inflammation resolution circuits are uncoupled in acute sepsis and correlate with clinical severity.

Sepsis is a critical illness characterized by dysregulated inflammatory responses lacking counter-regulation. Specialized proresolving mediators are agonists for antiinflammation and for promoting resolution, and they are protective in preclinical sepsis models. Here, in human sepsis, we mapped resolution circuits for the specialized proresolving mediators resolvin D1 and resolvin D2 in peripheral blood neutrophils and monocytes, their regulation of leukocyte activation and function ex vivo, and their relationships to measures of clinical severity. Neutrophils and monocytes were isolated from healthy subjects and patients with sepsis by inertial microfluidics and resolvin D1 and resolvin D2 receptor expression determined by flow cytometry. The impact of these resolvins on leukocyte activation was determined by isodielectric separation and leukocyte function by stimulated phagolysosome formation. Leukocyte proresolving receptor expression was significantly higher in sepsis. In nanomolar concentrations, resolvin D1 and resolvin D2 partially reversed sepsis-induced changes in leukocyte activation and function. Principal component analyses of leukocyte resolvin receptor expression and responses differentiated sepsis from health and were associated with measures of sepsis severity. These findings indicate that resolvin D1 and resolvin D2 signaling for antiinflammation and resolution are uncoupled from leukocyte activation in early sepsis and suggest that indicators of diminished resolution signaling correlate with clinical disease severity.

Fully Automated, Sample-to-Answer Leukocyte Functional Assessment Platform for Continuous Sepsis Monitoring via Microliters of Blood.

We report a fully automated, sample-to-answer, and label-free leukocyte activation analysis platform for monitoring immune responses in sepsis, by integrating the multidimensional double spiral (MDDS) and isodielectric separation (IDS) subplatforms. The integrated platform can provide rapid and fully automated identification of clinically diagnosed sepsis patients from only 50 µL of peripheral blood volume within 25 min. Many critical innovations were implemented in direct interconnection between the two subplatforms, such as intermediate sample storage and sample transfer, addressing flow rate mismatch (from mL/min to µL/min), and integration of a ridge array for upstream cell focusing in the IDS subplatform. The ridge array in the IDS subplatform can prevent the distortion of electrical profiling due to the residual red blood cells even after the MDDS process. We showed that the integrated platform can separate leukocytes (up to >99.9% red blood cell removal) in the MDDS subplatform and automatically transfer them to the downstream ridge-integrated IDS subplatform for their activation analysis without any apparent ex vivo cell activation and any human intervention. We also demonstrated that the integrated platform can identify differences between leukocytes from human sepsis and healthy subjects significantly (p = 0.0024, 95% confidence interval) by looking into differences in the intrinsic electrical properties of leukocytes. The integrated platform could enable monitoring of host leukocyte function daily or even hourly as a bedside assessment tool, which is currently a critical yet unmet need for managing many critical care patients.

An integrated model for bead-based immunoassays.

Bead-based immunoassays have shown great promise for rapid and sensitive protein quantification. However, there still lacks holistic understanding of assay performance that can inform assay design and optimization. In this paper, we present an integrated mathematical model for surface coverage bead-based assays. This model examines the building blocks of surface coverage assays, including heterogeneous binding of analyte molecules on bead or sensor surfaces, attachment of bead labels to sensor surfaces, and generation of electrochemical current by bead labels. To demonstrate and validate this model, we analyze a semi-homogeneous bead-based electronic enzyme-linked immunosorbent assay and find that experimental results agree with various model predictions. We show that the model can provide design guidance for choice of various assay parameters including bead size, bead number, antibody affinity and assay time, and provide a perspective to reconcile the performance of various implementations of surface coverage assays.

Leukocyte function assessed via serial microlitre sampling of peripheral blood from sepsis patients correlates with disease severity.

Dysregulated leukocyte responses underlie the pathobiology of sepsis, which is a leading cause of death. However, measures of leukocyte function are not routinely available in clinical care. Here we report the development and testing of an inertial microfluidic system for the label-free isolation and downstream functional assessment of leukocytes from 50 µl of peripheral blood. We used the system to assess leukocyte phenotype and function in serial samples from 18 hospitalized patients with sepsis and 10 healthy subjects. The sepsis samples had significantly higher levels of CD16dim and CD16- neutrophils and CD16+ 'intermediate' monocytes, as well as significantly lower levels of neutrophil-elastase release, O2- production and phagolysosome formation. Repeated sampling of sepsis patients over 7 days showed that leukocyte activation (measured by isodielectric separation) and leukocyte phenotype and function were significantly more predictive of the clinical course than complete-blood-count parameters. We conclude that the serial assessment of leukocyte function in microlitre blood volumes is feasible and that it provides significantly more prognostic information than leukocyte counting.

Caring for cells in microsystems: principles and practices of cell-safe device design and operation.

Microfluidic device designers and users continually question whether cells are 'happy' in a given microsystem or whether they are perturbed by micro-scale technologies. This issue is normally brought up by engineers building platforms, or by external reviewers (academic or commercial) comparing multiple technological approaches to a problem. Microsystems can apply combinations of biophysical and biochemical stimuli that, although essential to device operation, may damage cells in complex ways. However, assays to assess the impact of microsystems upon cells have been challenging to conduct and have led to subjective interpretation and evaluation of cell stressors, hampering development and adoption of microsystems. To this end, we introduce a framework that defines cell health, describes how device stimuli may stress cells, and contrasts approaches to measure cell stress. Importantly, we provide practical guidelines regarding device design and operation to minimize cell stress, and recommend a minimal set of quantitative assays that will enable standardization in the assessment of cell health in diverse devices. We anticipate that as microsystem designers, reviewers, and end-users enforce such guidelines, we as a community can create a set of essential principles that will further the adoption of such technologies in clinical, translational and commercial applications.

Designable 3D Microshapes Fabricated at the Intersection of Structured Flow and Optical Fields.

3D structures with complex geometric features at the microscale, such as microparticles and microfibers, have promising applications in biomedical engineering, self-assembly, and photonics. Fabrication of complex 3D microshapes at scale poses a unique challenge; high-resolution methods such as two-photon-polymerization have print speeds too low for high-throughput production, while top-down approaches for bulk processing using microfabricated template molds have limited control of microstructure geometries over multiple axes. Here, a method for microshape fabrication is presented that combines a thermally drawn transparent fiber template with a masked UV-photopolymerization approach to enable biaxial control of microshape fabrication. Using this approach, high-resolution production of complex microshapes not producible using alternative methods is demonstrated, such as octahedrons, dreidels, and axially asymmetric fibers, at throughputs as high as 825 structures/minute. Finally, the fiber template is functionalized with conductive electrodes to enable hierarchical subparticle localization using dielectrophoretic forces.

Microfluidics in structured multimaterial fibers.

Traditional fabrication techniques for microfluidic devices utilize a planar chip format that possesses limited control over the geometry of and materials placement around microchannel cross-sections. This imposes restrictions on the design of flow fields and external forces (electric, magnetic, piezoelectric, etc.) that can be imposed onto fluids and particles. Here we report a method of fabricating microfluidic channels with complex cross-sections. A scaled-up version of a microchannel is dimensionally reduced through a thermal drawing process, enabling the fabrication of meters-long microfluidic fibers with nonrectangular cross-sectional shapes, such as crosses, five-pointed stars, and crescents. In addition, by codrawing compatible materials, conductive domains can be integrated at arbitrary locations along channel walls. We validate this technology by studying unexplored regimes in hydrodynamic flow and by designing a high-throughput cell separation device. By enabling these degrees of freedom in microfluidic device design, fiber microfluidics provides a method to create microchannel designs that are inaccessible using planar techniques.

Sequentially multiplexed amperometry for electrochemical biosensors.

Multiplexed electrochemical biosensors are intriguing due to their capability to permit high-throughput and low-cost assays. While commercial single-chip potentiostats are one promising approach for rapidly prototyping portable and low-cost electrochemical biosensors, it is still challenging to utilize them to achieve parallel multiplexing due to the limited resources integrated onto the chips. In this paper, we provide a methodology for incorporating multiplexing into commercial single-chip potentiostats by using a sequential architecture. In the sequential architecture, the multiplexed biosensors are interfaced to the single-chip potentiostat via single-pole single-throw switches, and the measurements alternate across the sensors. We build analytical and finite element models to investigate the behavior of the sensors, particularly when they are disconnected from the potentiostat, and find that we can take advantage of the dynamics of the sensors to achieve improved sensitivity over conventional chronoamperometry. We also investigate and compare different strategies to interface the multiplexed sensors to the single-chip potentiostat. Using the proposed multiplexing architecture, we demonstrate the implementation of 16-fold multiplexed amperometry, which is validated using ferricyanide measurement. Finally, the sequential multiplexing methodology is applied to a multiplexed bead-based electronic enzyme-linked immunosorbent assays of human interleukin-6.

Multi-frequency dielectrophoretic characterization of single cells.

We explore the use of dielectrophoresis to discern the electrical properties of single cells by observing them at multiple frequencies. We first simulate experimental conditions to show that as we increase the number of measured frequencies, we are able to better discriminate among different cells. Furthermore, we use the simulation to find the optimal number and value of frequencies to use to best discriminate among different cells in general. We then fabricate a microfluidic device, calibrate it with polystyrene beads, and characterize it with BA/F3 cells. With this device, we test three different activation levels of HL60 cells treated with cytochalasin D using the optimal frequency sequence obtained in simulation to determine the differences in discrimination abilities depending on the number of frequencies used. We quantify the discrimination abilities of the optimal one, two, and three frequencies by minimizing 0-1 loss.

Miniaturized Impedance Flow Cytometer: Design Rules and Integrated Readout.

A dual-channel credit-card-sized impedance cell counter featuring a throughput of 2000 cell/s and detection of single yeast cells (5 µm) with a signal-to-noise ratio of 20 dB is presented. Its compactness is achieved by a CMOS ASIC combining a lock-in impedance demodulator with an oversampling 20-bit ΣΔ ADC and real-time peak detection embedded in field-programmable gate array. The module is coupled to a dielectrophoretic cell-sorting microfluidic device, offering compact and label-free electrical readout that replaces the need for a fluorescence microscope and, thus, is suitable for point-of-care diagnostics. The independent role of each dimension of the planar sensing microelectrodes is demonstrated, with simulations and experiments, along with its relevant effect on the spectrum of thin channels, deriving useful design guidelines.

Multiplexed Cell-Based Sensors for Assessing the Impact of Engineered Systems and Methods on Cell Health.

Bioinstrumentation engineers have long been creating platforms to study cell health and disease. It becomes necessary to ensure that such cell-probing tools do not themselves harm cells through complex stressors resulting from their design or operational conditions. Here, we present multiplexed cell-based sensors to simultaneously quantify stress induced by diverse mechanisms such as shear stress, DNA damage, and heat shock. Our sensors do not require additional reagents and can be conveniently quantified by flow cytometry and real-time imaging. Successful adaptation of our sensors by external users enabled systematic assessment of multiple flow sorters, alongside their operational parameters using the same cells and preparation. Our results provide insight into "gentle" and stressful sorting parameters that had not been quantified previously. Overall, this work presents a facile and quantitative approach to investigate multifactorial cell-stress emergent from diverse bioinstrumentation, which can be utilized to discover design and operation conditions ideal for cell health.

Monitoring sepsis using electrical cell profiling.

Sepsis is a potentially lethal condition that may be ameliorated through early monitoring of circulating activated leukocytes for faster stratification of severity of illness and improved administration of targeted treatment. Characterization of the intrinsic electrical properties of leukocytes is label-free and can provide a quick way to quantify the number of activated cells as sepsis progresses. Iso-dielectric separation (IDS) uses dielectrophoresis (DEP) to characterize the electrical signatures of cells. Here, we use IDS to show that activated and non-activated leukocytes have different electrical properties. We then present a double-sided version of the IDS platform to increase throughput to characterize thousands of cells. This new platform is less prone to cell fouling and allows faster characterization. Using peripheral blood samples from a cecal ligation and puncture (CLP) model of polymicrobial sepsis in mice, we estimate the number of activated leukocytes by looking into differences in the electrical properties of cells. We show for the first time using animal models that electrical cell profiling correlates with flow cytometry (FC) results and that IDS is therefore a good candidate for providing rapid monitoring of sepsis by quantifying the number of circulating activated leukocytes.

Longitudinal multiparameter assay of lymphocyte interactions from onset by microfluidic cell pairing and culture.

Resolving how the early signaling events initiated by cell-cell interactions are transduced into diverse functional outcomes necessitates correlated measurements at various stages. Typical approaches that rely on bulk cocultures and population-wide correlations, however, only reveal these relationships broadly at the population level, not within each individual cell. Here, we present a microfluidics-based cell-cell interaction assay that enables longitudinal investigation of lymphocyte interactions at the single-cell level through microfluidic cell pairing, on-chip culture, and multiparameter assays, and allows recovery of desired cell pairs by micromanipulation for off-chip culture and analyses. Well-defined initiation of interactions enables probing cellular responses from the very onset, permitting single-cell correlation analyses between early signaling dynamics and later-stage functional outcomes within same cells. We demonstrate the utility of this microfluidic assay with natural killer cells interacting with tumor cells, and our findings suggest a possible role for the strength of early calcium signaling in selective coordination of subsequent cytotoxicity and IFN-gamma production. Collectively, our experiments demonstrate that this new approach is well-suited for resolving the relationships between complex immune responses within each individual cell.

Microfluidic neurite guidance to study structure-function relationships in topologically-complex population-based neural networks.

The central nervous system is a dense, layered, 3D interconnected network of populations of neurons, and thus recapitulating that complexity for in vitro CNS models requires methods that can create defined topologically-complex neuronal networks. Several three-dimensional patterning approaches have been developed but none have demonstrated the ability to control the connections between populations of neurons. Here we report a method using AC electrokinetic forces that can guide, accelerate, slow down and push up neurites in un-modified collagen scaffolds. We present a means to create in vitro neural networks of arbitrary complexity by using such forces to create 3D intersections of primary neuronal populations that are plated in a 2D plane. We report for the first time in vitro basic brain motifs that have been previously observed in vivo and show that their functional network is highly decorrelated to their structure. This platform can provide building blocks to reproduce in vitro the complexity of neural circuits and provide a minimalistic environment to study the structure-function relationship of the brain circuitry.

Iso-acoustic focusing of cells for size-insensitive acousto-mechanical phenotyping.

Mechanical phenotyping of single cells is an emerging tool for cell classification, enabling assessment of effective parameters relating to cells' interior molecular content and structure. Here, we present iso-acoustic focusing, an equilibrium method to analyze the effective acoustic impedance of single cells in continuous flow. While flowing through a microchannel, cells migrate sideways, influenced by an acoustic field, into streams of increasing acoustic impedance, until reaching their cell-type specific point of zero acoustic contrast. We establish an experimental procedure and provide theoretical justifications and models for iso-acoustic focusing. We describe a method for providing a suitable acoustic contrast gradient in a cell-friendly medium, and use acoustic forces to maintain that gradient in the presence of destabilizing forces. Applying this method we demonstrate iso-acoustic focusing of cell lines and leukocytes, showing that acoustic properties provide phenotypic information independent of size.

Spatially and temporally controlled immune cell interactions using microscale tools.

Many critical immunological responses are mediated by cell-cell interactions. Despite their capabilities, traditional techniques that rely on snapshot analysis or ensemble measurements can only provide a fragmentary picture of the complexity of these interactions. Emerging classes of new and versatile microscale tools hold great potential for enabling detailed investigation of these interactions with precise control in space and time, multiplexed measurement capability and high-throughput single-cell analysis. These features allow new ways of examining immune cell interactions that are not possible with traditional methods, improve the extent of information extracted from experiments, and reveal new findings. Here, we review recent developments in microscale tools that are paving the way for comprehensive analyses of cell-cell interactions in the immune system.