CellMag-CARWash: A High Throughput Droplet Microfluidic Device for Live Cell Isolation and Single Cell Applications.

The recent push toward understanding an individual cell's behavior and identifying cellular heterogeneity has created an unmet need for technologies that can probe live cells at the single-cell level. Cells within a population are known to exhibit heterogeneous responses to environmental cues. These differences can lead to varied cellular states, behavior, and responses to therapeutics. Techniques are needed that are not only capable of processing and analyzing cellular populations at the single cell level, but also have the ability to isolate specific cell populations from a complex sample at high throughputs. The new CellMag-Coalesce-Attract-Resegment Wash (CellMag-CARWash) system combines positive magnetic selection with droplet microfluidic devices to isolate cells of interest from a mixture with >93% purity and incorporate treatments within individual droplets to observe single cell biological responses. This workflow is shown to be capable of probing the single cell extracellular vesicle (EV) secretion of MCF7 GFP cells. This article reports the first measurement of ß-Estradiol's effect on EV secretion from MCF7 cells at the single cell level. Single cell processing revealed that MCF7 GFP cells possess a heterogeneous response to ß-Estradiol stimulation with a 1.8-fold increase relative to the control.

Accessible and Generalizable in Vitro Luminescence Assay for Detecting GPCR Activation.

G protein-coupled receptors (GPCRs) serve critical physiological roles as the most abundant family of receptors. Here, we describe the design of a generalizable and cell lysate-based method that leverages the interaction between an agonist-activated GPCR and a conformation-specific binder to reconstitute split nanoluciferase (NanoLuc) in vitro. This tool, In vitro GPCR split NanoLuc ligand Triggered Reporter (IGNiTR), has broad applications. We have demonstrated IGNiTR's use with three Gs-coupled GPCRs, two Gi-coupled GPCRs and three classes of conformation-specific binders: nanobodies, miniG proteins, and G protein peptidomimetics. As an in vitro method, IGNiTR enables the use of synthetic G protein peptidomimetics and provides easily scalable and portable reagents for characterizing GPCRs and ligands. We tested three diverse applications of IGNiTR: (1) proof-of-concept GPCR ligand screening using dopamine receptor D1 IGNiTR; (2) detection of opioids for point-of-care testing; and (3) characterizing GPCR functionality during Nanodisc-based reconstitution processes. Due to IGNiTR's unique advantages and the convenience of its cell lysate-based format, this tool will find extensive applications in GPCR ligand detection, screening, and GPCR characterization.

Multiparametric, Label-Free Analysis of Microfluidic Droplets via a Dynamic Phase Grating Approach.

Droplet-based microfluidic systems possess many fundamental advantages as a platform for the analysis of chemical and biological species. However, whereas on-chip operations have rapidly developed over the past decades, approaches for analyzing target molecules within droplets have largely remained limited to methods requiring bulky and expensive instrumentation. In this work, we describe a droplet analysis approach whereby the droplet train itself is the sensing construct. Specifically, the droplet train is interrogated as a transmission phase grating, allowing high-throughput, label-free, solution-phase, and multi-parametric analysis of droplet contents. Importantly, three distinct properties of generated droplets can be simultaneously extracted using this conceptually simple and experimentally straightforward measurement approach. Under constant droplet generation conditions, measurement of droplet viscosity is achieved by monitoring changes in zero order to first order peak separation in the far-field diffraction pattern, with a sensitivity of 2.28 × 10-4 cSt per µm change in peak separation. In parallel, measurement of droplet refractive index (RI) is achieved by measuring changes in the ratio of the zero order to first order peak intensity, with a sensitivity of 2.14 × 10-4 RI units per unit change in a diffracted peak intensity ratio. Finally, droplet generation frequency is determined from the time-varying oscillation of the peak height ratio, yielding comparable results to an expensive high-speed camera commonly used for droplet imaging. Importantly, the experimental strategy for this approach is straightforward and does not require expensive instrumentation; therefore, it may find utility in affordable and portable analysis approaches applied to diverse droplet microfluidic assays.

Chorioamnionitis-exposure alters serum cytokine trends in premature neonates.

OBJECTIVES: Determine if chronologic age and/or chorioamnionitis exposure alter normal serum cytokine and chemokine levels in uninfected preterm neonates during their initial NICU stay. STUDY DESIGN: A 7-plex immunoassay measured levels of serum IL-1ß, IL-6, IL-8, IL-10, TNF-α, CCL2, and CCL3 longitudinally from chorioamnionitis-exposed and unexposed preterm neonates under 33 weeks' gestation. RESULTS: Chorioamnionitis-exposed and unexposed preterm neonates demonstrated differences in the trends of IL-1ß, IL-6, IL-8, IL-10, TNF-α, and CCL2 over the first month of life. The unexposed neonates demonstrated elevated levels of these inflammatory markers in the first two weeks of life with a decrease by the third week of life, while the chorioamnionitis-exposed neonates demonstrated differences over time without a predictable pattern. Chorioamnionitis-exposed and unexposed neonates demonstrated altered IL-10 and TNF-α trajectories over the first twelve weeks of life. CONCLUSION: Chorioamnionitis induces a state of immune dysregulation in preterm neonates that persists beyond the immediate neonatal period.

Detection of biomarkers for filoviral infection with a silicon photonic resonator platform.

This protocol describes the use of silicon photonic microring resonator sensors for detection of Ebola virus (EBOV) and Sudan virus (SUDV) soluble glycoprotein (sGP). This protocol encompasses biosensor functionalization of silicon microring resonator chips, detection of protein biomarkers in sera, preparing calibration standards for analytical validation, and quantification of the results from these experiments. This protocol is readily adaptable toward other analytes, including cytokines, chemokines, nucleic acids, and viruses. For complete details on the use and execution of this protocol, please refer to Qavi et al. (2022).

Rapid detection of an Ebola biomarker with optical microring resonators.

Ebola virus (EBOV) is a highly infectious pathogen, with a case mortality rate as high as 89%. Rapid therapeutic treatments and supportive measures can drastically improve patient outcome; however, the symptoms of EBOV disease (EVD) lack specificity from other endemic diseases. Given the high mortality and significant symptom overlap, there is a critical need for sensitive, rapid diagnostics for EVD. Facile diagnosis of EVD remains a challenge. Here, we describe a rapid and sensitive diagnostic for EVD through microring resonator sensors in conjunction with a unique biomarker of EBOV infection, soluble glycoprotein (sGP). Microring resonator sensors detected sGP in under 40 min with a limit of detection (LOD) as low as 1.00 ng/mL in serum. Furthermore, we validated our assay with the detection of sGP in serum from EBOV-infected non-human primates. Our results demonstrate the utility of a high-sensitivity diagnostic platform for detection of sGP for diagnosis of EVD.

Purification of the full-length, membrane-associated form of the antiviral enzyme viperin utilizing nanodiscs.

Viperin is a radical S-adenosylmethionine enzyme that catalyzes the formation of the antiviral ribonucleotide, 3'-deoxy-3',4'-didehydroCTP. The enzyme is conserved across all kingdoms of life, and in higher animals viperin is localized to the ER-membrane and lipid droplets through an N-terminal extension that forms an amphipathic helix. Evidence suggests that the N-terminal extension plays an important role in viperin's interactions with other membrane proteins. These interactions serve to modulate the activity of various other enzymes that are important for viral replication and constitute another facet of viperin's antiviral properties, distinct from its catalytic activity. However, the full-length form of the enzyme, which has proved refractory to expression in E. coli, has not been previously purified. Here we report the purification of the full-length form of viperin from HEK293T cells transfected with viperin. The purification method utilizes nanodiscs to maintain the protein in its membrane-bound state. Unexpectedly, the enzyme exhibits significantly lower catalytic activity once purified, suggesting that interactions with other ER-membrane components may be important to maintain viperin's activity.

Risk assessment of latent tuberculosis infection through a multiplexed cytokine biosensor assay and machine learning feature selection.

Accurate detection and risk stratification of latent tuberculosis infection (LTBI) remains a major clinical and public health problem. We hypothesize that multiparameter strategies that probe immune responses to Mycobacterium tuberculosis can provide new diagnostic insights into not only the status of LTBI infection, but also the risk of reactivation. After the initial proof-of-concept study, we developed a 13-plex immunoassay panel to profile cytokine release from peripheral blood mononuclear cells stimulated separately with Mtb-relevant and non-specific antigens to identify putative biomarker signatures. We sequentially enrolled 65 subjects with various risk of TB exposure, including 32 subjects with diagnosis of LTBI. Random Forest feature selection and statistical data reduction methods were applied to determine cytokine levels across different normalized stimulation conditions. Receiver Operator Characteristic (ROC) analysis for full and reduced feature sets revealed differences in biomarkers signatures for LTBI status and reactivation risk designations. The reduced set for increased risk included IP-10, IL-2, IFN-γ, TNF-α, IL-15, IL-17, CCL3, and CCL8 under varying normalized stimulation conditions. ROC curves determined predictive accuracies of > 80% for both LTBI diagnosis and increased risk designations. Our study findings suggest that a multiparameter diagnostic approach to detect normalized cytokine biomarker signatures might improve risk stratification in LTBI.

Characterization of the impact of mixing and droplet volumes on the behavior of microfluidic ion-selective droptodes.

Droplet microfluidic optodes, or "droptodes", have emerged as a powerful technology for rapid detection of small ions in complex matrices. While using segmented aqueous phases provides the benefits of sample isolation, the influence of the liquid nature of the oil carrier phase has not yet been explored. In this paper, we examine the influence of microfluidic parameters on droptode efficiency, using potassium-sensitive droptodes as a model system. We found that while changing flow rates on device does not change droptode performance, both channel geometry and droplet size significantly impact droptode efficiency. Specifically, enhanced mixing of the droplets leads to faster equilibration on device and lowers limits of detection by about one order of magnitude. We also found that increasing the size of the sample droplet, at the expense of the size of the oil carrier/sensing phase, leads to higher sensitivity in the linear region of the droptode. These easily manipulated properties will allow one device to potentially be adapted for several different applications, based upon the type and concentration range of measurement required.

Plug-in tubes allow tunable oil removal, droplet packing, and reaction incubation for time-controlled droplet-based assays.

Here, we report a unique microfluidic technique that utilizes a membrane filter and plug-in tubes to remove oil and pack water-in-oil droplets for controlled incubation of droplet-based assays. This technique could be modularly incorporated into most droplet-generation devices without a need to alter the original designs. Our results show that removing excess oil to form tightly packed droplets allows for extended and controllable incubation for droplets traveling in microchannels. The efficiency of this technique was evaluated and confirmed using a time-dependent enzyme assay with a fluorometric readout. The system is also readily generalizable to control inter-droplet distance, crucial for studying droplet communication and pattern formation.

Coherent silicon photonic interferometric biosensor with an inexpensive laser source for sensitive label-free immunoassays.

Over the past two decades, integrated photonic sensors have been of major interest to the optical biosensor community due to their capability to detect low concentrations of molecules with label-free operation. Among these, interferometric sensors can be read-out with simple, fixed-wavelength laser sources and offer excellent detection limits but can suffer from sensitivity fading when not tuned to their quadrature point. Recently, coherently detected sensors were demonstrated as an attractive alternative to overcome this limitation. Here we show, for the first time, to the best of our knowledge, that this coherent scheme provides sub-nanogram per milliliter limits of detection in C-reactive protein immunoassays and that quasi-balanced optical arm lengths enable operation with inexpensive Fabry-Perot-type lasers sources at telecom wavelengths.

Phosphatidylethanolamine-phosphatidylserine binding synergy of seven coagulation factors revealed using Nanodisc arrays on silicon photonic sensors.

Blood coagulation is regulated through protein-protein and protein-lipid interactions that occur at the sub-endothelium following vascular damage. Soluble clotting proteins bind to membrane components in a phosphatidylserine (PS) dependent manner to assemble multi-protein complexes that regulate clot formation; however, PS is of limited abundance physiologically. In this manuscript, we investigate synergy between PS and phosphatidylethanolamine (PE)-a lipid of much higher abundance naturally. Using a label-free, silicon photonic technology, we constructed arrays of Nanodiscs having variable lipid composition and probed the binding interactions of seven different clotting factors with GLA domains that have never been studied in tandem experiments before. The factors studied were prothrombin, activated factor VII, factor IX, factor X, activated protein C, protein S, and protein Z. Equilibrium dissociation constants (Kd) for each coagulation factor binding to Nanodiscs with unique compositions of PE and PS were determined. While all factors showed greater binding affinities in the presence of PS and PE, the most dramatic improvements in binding were observed when PS quantities were lowest. This demonstrates that synergy is effective in promoting coagulation factor binding under physiological lipid compositions, as opposed to the artificially high PS content probed in most in vitro activity studies.

Real-Time Measurement of Polymer Brush Dynamics Using Silicon Photonic Microring Resonators: Analyte Partitioning and Interior Brush Kinetics.

Polymer brushes are found in biomedical and industrial technologies, where they exhibit functionalities considerably dependent on polymer brush-solvent-analyte interactions. It remains a difficult challenge to quickly analyze solvent-swollen polymer brushes, both at the solvent-polymer brush interface and in the brush interior, as well as to monitor the kinetics of interaction of solvent-swollen brushes with key analytes. Here, we demonstrate the novel use of silicon photonic microring resonators to characterize in situ swollen polymer brush-analyte interactions. By monitoring resonant wavelength shifts, we find that brush-solvent-analyte interaction parameters can be extracted from a single set of data or from successive analyte introductions using a single brush-coated sensor. The partition coefficient of three industrially relevant plasticizers into hydrophobic and hydrophilic brushes was determined and found to be in agreement with known solubility trends. We found that the diffusion coefficient of the plasticizer into the brush decreases as brush thickness increases, supporting a model of a dense inner brush layer and diffuse outer layer. pKa's of pH-sensitive brushes were determined on the microring resonator platform; upon increasing the dry brush thickness, the pKa for poly(2-dimethylamino ethyl methacrylate) decreased from 8.5 to approach the bulk material pKa of 7.3 and showed dependence on the presence and concentration of salt. These proof-of-concept experiments show how the surface-sensitive nature of the microring resonator detection platform provides valuable information about the interaction of the polymer brushes with the solvents and analytes, not easily accessed by other techniques.

Biotoxoid Photonic Sensors with Temperature Insensitivity Using a Cascade of Ring Resonator and Mach-Zehnder Interferometer.

The great advances in silicon photonic-sensing technology have made it an attractive platform for wide sensing applications. However, most silicon photonic-sensing platforms suffer from high susceptibility to the temperature fluctuation of an operating environment. Additional complex and costly chemical signal-enhancement strategies are usually required to improve the signal-to-noise ratio (SNR). Here, a biotoxoid photonic sensor that is resistant to temperature fluctuation has been demonstrated. This novel sensor consists of a ring resonator coupled to a Mach-Zehnder interferometer (MZI) readout unit. Instead of using costly wavelength interrogation, our photonic sensor directly measures the light intensity ratio between the two output ports of MZI. The temperature dependence (TD)-controlling section of the MZI is used to eliminate the adverse effects of ambient temperature fluctuation. The simulation and experimental results show a linear relationship between the interrogation function and the concentration of an analyte under operation conditions. The thermal drift of the proposed sensor is just 0.18%, which is a reduction of 567-fold for chemical sensing and 28-fold for immuno-biosensing compared to the conventional single-ring resonator. The SNR increases from 6.85 to 19.88 dB within a 2 °C temperature variation. The high SNR optical sensor promises great potential for amplification-free detection of nucleic acids and other biomarkers.

Impact of Silanization Parameters and Antibody Immobilization Strategy on Binding Capacity of Photonic Ring Resonators.

Ring resonator-based biosensors have found widespread application as the transducing principle in "lab-on-a-chip" platforms due to their sensitivity, small size and support for multiplexed sensing. Their sensitivity is, however, not inherently selective towards biomarkers, and surface functionalization of the sensors is key in transforming the sensitivity to be specific for a particular biomarker. There is currently no consensus on process parameters for optimized functionalization of these sensors. Moreover, the procedures are typically optimized on flat silicon oxide substrates as test systems prior to applying the procedure to the actual sensor. Here we present what is, to our knowledge, the first comparison of optimization of silanization on flat silicon oxide substrates to results of protein capture on sensors where all parameters of two conjugation protocols are tested on both platforms. The conjugation protocols differed in the chosen silanization solvents and protein immobilization strategy. The data show that selection of acetic acid as the solvent in the silanization step generally yields a higher protein binding capacity for C-reactive protein (CRP) onto anti-CRP functionalized ring resonator sensors than using ethanol as the solvent. Furthermore, using the BS3 linker resulted in more consistent protein binding capacity across the silanization parameters tested. Overall, the data indicate that selection of parameters in the silanization and immobilization protocols harbor potential for improved biosensor binding capacity and should therefore be included as an essential part of the biosensor development process.

Translational Opportunities for Microfluidic Technologies to Enable Precision Epigenomics.

Personalizing health care by taking genetic, environmental, and lifestyle factors into account is central to modern medicine. The crucial and pervasive roles epigenetic factors play in shaping gene-environment interactions are now well recognized. However, identifying robust epigenetic biomarkers and translating them to clinical tests has been difficult due in part to limitations of available platforms to detect epigenetic features genome-wide (epigenomic assays). This Feature introduces several important prospects for precision epigenomics, highlights capabilities and limitations of current laboratory technologies, and emphasizes opportunities for microfluidic tools to facilitate translation of epigenetic analyses to the clinic, with a particular focus on methods to profile gene-associated histone modifications and their impacts on chromatin structure and gene expression.

A linear mass concentration detector for solvent gradient polymer separations.

Characterization of copolymers requires the measurement of two distributions-molecular weight (MW) and chemical composition (CC). Molecular weight distributions (MWD) are traditionally determined using size exclusion chromatography (SEC) run under isocratic solvent conditions. Chemical composition distributions (CCD) are often determined using liquid adsorption chromatography (LC) with solvent gradients. The use of solvent gradients, however, often limits options of compatible detectors. A gradient compatible, universal linear mass concentration detector is a longstanding unmet need. Many industrially-relevant polymers lack chromophores or other discriminating moieties requiring detectors with a universal response. Differential refractive index (dRI) is incompatible with gradient elution due to its small dynamic range. Charged aerosol detectors (CAD) and evaporative light scattering detectors (ELSD) are probably the most promising options for gradient elution detection, but both suffer from a nonlinear mass concentration response. Silicon photonic microring resonators are optical sensors that are responsive to changes in the local refractive index (RI). The substantial dynamic range of this technology makes it attractive for refractive index-based detection during solvent gradient elution. Previously, the microring resonator platform was used as a SEC detector to characterize the MWD of broadly dispersed polystyrene (PS) standards. In this study, we demonstrate the gradient compatibility of the microring resonator platform for polymer detection by quantifying the CCD of polymer blend components. Control experiments were run with UV and ELSD detection, highlighting the uniqueness of the platform as a linear mass concentration detector with a universal detector response.

Silicon Photonic Microring Resonator Arrays as a Universal Detector for Capillary Electrophoresis.

Electrophoretic separations conventionally rely on chromogenic, fluorogenic, or redox properties for analyte detection that, in many instances, involve chemical modification of samples prior to analysis. For analytes natively lacking chemical signatures, refractive index-based measurements are appealing as a method to detect these molecules without pretreatment. Microring resonators are a type of whispering gallery mode sensor capable of detecting bulk changes in refractive index. Here, we demonstrate the use of silicon photonic microring resonator arrays as a postcolumn detector for capillary electrophoresis. In this approach, we establish the universal detection capabilities of microrings through calibration with analytes lacking unique spectral signatures. Separations of small molecule mixtures are demonstrated using capillary zone electrophoresis. For these separations, the microring resonators maintain a linear response over several orders of magnitude in concentration for three candidate small molecules. Successful separation of three sugars with direct detection is also demonstrated. We further present the successful separation and detection of three model proteins, exemplifying the promise of microring resonators arrays as a biocompatible detector for capillary electrophoresis. Additionally, the spatially offset, array-based nature of the sensing platform enables real-time analysis of analyte mobility and performance characterization-a combination that is not typically provided using single-point detectors.

Droplet CAR-Wash: continuous picoliter-scale immunocapture and washing.

To address current limitations in adapting solid phase sample capture and washing techniques to continuously flowing droplet microfluidics, we have developed the "Coalesce-Attract-Resegment Wash" (CAR-Wash) approach. This module provides efficient, high-throughput magnetic washing by electrocoalescing magnetic bead-laden input droplets with a washing buffer flow and magnetophoretically transporting beads through the buffer into a secondary droplet formation streamline. In this work, we first characterized the technology in terms of throughput, sample retention, and flow-based exclusion of waste volume, demonstrating >500 Hz droplet processing with >98% bead retention and >100-fold dilution in final droplets. Next, we showed that the technique can be adapted to alternative commercially available magnetic beads with lower magnetite content per particle. Then, we demonstrated the CAR-Wash module's effectiveness in washing away a small molecule competitive inhibitor to restore the activity of magnetic bead-immobilized ß-galactosidase. Finally, we applied the system to immunomagnetically enrich a green fluorescent protein-histone H2B fusion protein from cell lysate while washing away mCherry and other lysate components. We believe this approach will bridge the gap between powerful biochemical and bioanalytical techniques and current droplet microfluidic capabilities, and we envision future application in droplet-based immunoassays, solid phase extraction, and other complex, multi-step operations.

Ionophore-Based Biphasic Chemical Sensing in Droplet Microfluidics.

Droplet microfluidics is an enabling platform for high-throughput screens, single-cell studies, low-volume chemical diagnostics, and microscale material syntheses. Analytical methods for real-time and in situ detection of chemicals in the droplets will benefit these applications, but they remain limited. Reported herein is a novel heterogeneous chemical sensing strategy based on functionalization of the oil phase with rationally combined sensing reagents. Sub-nanoliter oil segments containing pH-sensitive fluorophores, ionophores, and ion-exchangers enable highly selective and rapid fluorescence detection of physiologically important electrolytes (K+ , Na+ , and Cl- ) and polyions (protamine) in sub-nanoliter aqueous droplets. Electrolyte analysis in whole blood is demonstrated without suffering from optical interference from the sample matrix. Moreover, an oil phase doped with an aza-BODIPY dye allows indication of H2 O2 in the aqueous droplets, exemplifying sensing of targets beyond ionic species.