Landscape of Intercellular Crosstalk in Healthy and NASH Liver Revealed by Single-Cell Secretome Gene Analysis.

Cell-cell communication via ligand-receptor signaling is a fundamental feature of complex organs. Despite this, the global landscape of intercellular signaling in mammalian liver has not been elucidated. Here we perform single-cell RNA sequencing on non-parenchymal cells isolated from healthy and NASH mouse livers. Secretome gene analysis revealed a highly connected network of intrahepatic signaling and disruption of vascular signaling in NASH. We uncovered the emergence of NASH-associated macrophages (NAMs), which are marked by high expression of triggering receptors expressed on myeloid cells 2 (Trem2), as a feature of mouse and human NASH that is linked to disease severity and highly responsive to pharmacological and dietary interventions. Finally, hepatic stellate cells (HSCs) serve as a hub of intrahepatic signaling via HSC-derived stellakines and their responsiveness to vasoactive hormones. These results provide unprecedented insights into the landscape of intercellular crosstalk and reprogramming of liver cells in health and disease.

Bioinspired Plasmo-virus for Point-of-Care SARS-CoV-2 Detection.

Directly identifying the presence of the virus in infected hosts with an appropriate speed and sensitivity permits early epidemic management even during the presymptomatic incubation period of infection. Here, we synthesize a bioinspired plasmo-virus (BPV) particle for rapid and sensitive point-of-care (POC) detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via a self-assembled plasmonic nanoprobe array on spike proteins. The BPV enables strong near-infrared (NIR) extinction peaks caused by plasmonic nanogaps. We quantify SARS-CoV-2 in viral transport medium (VTM) at low titers within 10 min with a limit of detection (LOD) of 1.4 × 101 pfu/mL, which is 103 times more sensitive than the current gold-standard method. The high-sensitivity and high-speed POC detection may be widely used for the timely, individualized diagnosis of infectious agents in low-resource settings.

Rapid single-molecule digital detection of protein biomarkers for continuous monitoring of systemic immune disorders.

Digital protein assays have great potential to advance immunodiagnostics because of their single-molecule sensitivity, high precision, and robust measurements. However, translating digital protein assays to acute clinical care has been challenging because it requires deployment of these assays with a rapid turnaround. Herein, we present a technology platform for ultrafast digital protein biomarker detection by using single-molecule counting of immune-complex formation events at an early, pre-equilibrium state. This method, which we term "pre-equilibrium digital enzyme-linked immunosorbent assay" (PEdELISA), can quantify a multiplexed panel of protein biomarkers in 10 µL of serum within an unprecedented assay incubation time of 15 to 300 seconds over a 104 dynamic range. PEdELISA allowed us to perform rapid monitoring of protein biomarkers in patients manifesting post-chimeric antigen receptor T-cell therapy cytokine release syndrome, with ∼30-minute sample-to-answer time and a sub-picograms per mL limit of detection. The rapid, sensitive, and low-input volume biomarker quantification enabled by PEdELISA is broadly applicable to timely monitoring of acute disease, potentially enabling more personalized treatment.

An ultrahigh-throughput screening platform based on flow cytometric droplet sorting for mining novel enzymes from metagenomic libraries.

Uncultivable microbial communities provide enormous reservoirs of enzymes, but their experimental identification by functional metagenomics is challenging, mainly due to the difficulty of screening enormous metagenomic libraries. Here, we propose a reliable and convenient ultrahigh-throughput screening platform based on flow cytometric droplet sorting (FCDS). The FCDS platform employs water-in-oil-in-water double emulsion droplets serving as single-cell enzymatic micro-reactors and a commercially available flow cytometer, and it can efficiently isolate novel biocatalysts from metagenomic libraries by processing single cells as many as 108 per day. We demonstrated the power of this platform by screening a metagenomic library constructed from domestic running water samples. The FCDS assay screened 30 million micro-reactors in only 1 h, yielding a collection of esterase genes. Among these positive hits, Est WY was identified as a novel esterase with high catalytic efficiency and distinct evolutionary origin from other lipolytic enzymes. Our study manifests that the FCDS platform is a robust tool for functional metagenomics, with the potential to significantly improve the efficiency of exploring novel enzymes from nature.

Ultrasensitive Multiparameter Phenotyping of Rare Cells Using an Integrated Digital-Molecular-Counting Microfluidic Well Plate.

Integrated microfluidic cellular phenotyping platforms provide a promising means of studying a variety of inflammatory diseases mediated by cell-secreted cytokines. However, immunosensors integrated in previous microfluidic platforms lack the sensitivity to detect small signals in the cellular secretion of proinflammatory cytokines with high precision. This limitation prohibits researchers from studying cells secreting cytokines at low abundance or existing at a small population. Herein, the authors present an integrated platform named the "digital Phenoplate (dPP)," which integrates digital immunosensors into a microfluidic chip with on-chip cell assay chambers, and demonstrates ultrasensitive cellular cytokine secretory profile measurement. The integrated sensors yield a limit of detection as small as 0.25 pg mL-1 for mouse tumor necrosis factor alpha (TNF-α). Each on-chip cell assay chamber confines cells whose population ranges from ≈20 to 600 in arrayed single-cell trapping microwells. Together, these microfluidic features of the dPP simultaneously permit precise counting and image-based cytometry of individual cells while performing parallel measurements of TNF-α released from rare cells under multiple stimulant conditions for multiple samples. The dPP platform is broadly applicable to the characterization of cellular phenotypes demanding high precision and high throughput.

An Integrated Plasmo-Photoelectronic Nanostructure Biosensor Detects an Infection Biomarker Accompanying Cell Death in Neutrophils.

Bacterial infections leading to sepsis are a major cause of deaths in the intensive care unit. Unfortunately, no effective methods are available to capture the early onset of infectious sepsis near the patient with both speed and sensitivity required for timely clinical treatment. To fill the gap, the authors develop a highly miniaturized (2.5 × 2.5 µm2 ) plasmo-photoelectronic nanostructure device that detected citrullinated histone H3 (CitH3), a biomarker released to the blood circulatory system by neutrophils. Rapidly detecting CitH3 with high sensitivity has the great potential to prevent infections from developing life-threatening septic shock. To this end, the author's device incorporates structurally engineered arrayed hemispherical gold nanoparticles that are functionalized with high-affinity antibodies. A nanoplasmonic resonance shift induces a photoconduction increase in a few-layer molybdenum disulfide (MoS2 ) channel, and it provides the sensor signal. The device achieves label-free detection of serum CitH3 with a 5-log dynamic range from 10-4 to 101 ng mL and a sample-to-answer time <20 min. Using this biosensor, the authors longitudinally measure the dynamic CitH3 profiles of individual living mice in a sepsis model at high resolution over 12 hours. The developed biosensor may be poised for future translation to personalized management of systemic bacterial infections.

Integrated on-site collection and detection of airborne microparticles for smartphone-based micro-climate quality control.

The rapid emergence of air-mediated diseases in a micro-climate demands on-site monitoring of airborne microparticles. The on-site detection of airborne microparticles becomes more challenging as the particles are highly localized and change dynamically over time. However, most existing monitoring systems rely on time-consuming sample collection and centralized off-site analysis. Here, we report a smartphone-based integrated microsystem for on-site collection and detection that enables real-time detection of indoor airborne microparticles with high sensitivity. The collection device, inspired by the Venturi effect, was designed to collect airborne microparticles without requiring an additional power supply. Our systematic analysis showed that the collection device was able to collect microparticles with consistent negative pressure, regardless of the particle concentration in the air sample. By incorporating a microfluidic-biochip based on inertial force to trap particles and an optoelectronic photodetector into a miniaturized device with a smartphone, we demonstrate real-time and sensitive detection of the collected airborne microparticles, such as Escherichia coli, Bacillus subtilis, Micrococcus luteus, and Staphylococcus with a particle-density dynamic range of 103-108 CFU mL-1. Because of its capabilities of minimal-power sample collection, high sensitivity, and rapid detection of airborne microparticles, this integrated platform can be readily adopted by the government and industrial sectors to monitor indoor air contamination and improve human healthcare.

Syntrophic co-culture amplification of production phenotype for high-throughput screening of microbial strain libraries.

Microbes can be engineered to synthesize a wide array of bioproducts, yet production phenotype evaluation remains a frequent bottleneck in the design-build-test cycle where strain development requires iterative rounds of library construction and testing. Here, we present Syntrophic Co-culture Amplification of Production phenotype (SnoCAP). Through a metabolic cross-feeding circuit, the production level of a target molecule is translated into highly distinguishable co-culture growth characteristics, which amplifies differences in production into highly distinguishable growth phenotypes. We demonstrate SnoCAP with the screening of Escherichia coli strains for production of two target molecules: 2-ketoisovalerate, a precursor of the drop-in biofuel isobutanol, and L-tryptophan. The dynamic range of the screening can be tuned by employing an inhibitory analog of the target molecule. Screening based on this framework requires compartmentalization of individual producers with the sensor strain. We explore three formats of implementation with increasing throughput capability: confinement in microtiter plates (102-104 assays/experiment), spatial separation on agar plates (104-105 assays/experiment), and encapsulation in microdroplets (105-107 assays/experiment). Using SnoCAP, we identified an efficient isobutanol production strain from a random mutagenesis library, reaching a final titer that is 5-fold higher than that of the parent strain. The framework can also be extended to screening for secondary metabolite production using a push-pull strategy. We expect that SnoCAP can be readily adapted to the screening of various microbial species, to improve production of a wide range of target molecules.

AC Electroosmosis-Enhanced Nanoplasmofluidic Detection of Ultralow-Concentration Cytokine.

Label-free, nanoparticle-based plasmonic optical biosensing, combined with device miniaturization and microarray integration, has emerged as a promising approach for rapid, multiplexed biomolecular analysis. However, limited sensitivity prevents the wide use of such integrated label-free nanoplasmonic biosensors in clinical and life science applications where low-abundance biomolecule detection is needed. Here, we present a nanoplasmofluidic device integrated with microelectrodes for rapid, label-free analysis of a low-abundance cell signaling protein, detected by AC electroosmosis-enhanced localized surface plasmon resonance (ACE-LSPR) biofunctional nanoparticle imaging. The ACE-LSPR device is constructed using both bottom-up and top-down sensor fabrication methods, allowing the seamless integration of antibody-conjugated gold nanorod (AuNR) biosensor arrays with microelectrodes on the same microfluidic platform. Applying an AC voltage to microelectrodes while scanning the scattering light intensity variation of the AuNR biosensors results in significantly enhanced biosensing performance. The AC electroosmosis (ACEO) based enhancement of the biosensor performance enables rapid (5-15 min) quantification of IL-1ß, a pro-inflammatory cytokine biomarker, with a sensitivity down to 158.5 fg/mL (9.1 fM) for spiked samples in PBS and 1 pg/mL (58 fM) for diluted human serum. Together with the optimized detection sensitivity and speed, our study presents the first critical step toward the application of nanoplasmonic biosensing technology to immune status monitoring guided by low-abundance cytokine measurement.

Centrifugal microfluidics for sorting immune cells from whole blood.

Sorting and enumeration of immune cells from blood are critical operations involved in many clinical applications. Conventional methods for sorting and counting immune cells from blood, such as flow cytometry and hemocytometers, are tedious, inaccurate, and difficult for implementation for point-of-care (POC) testing. Herein we developed a microscale centrifugal technology termed Centrifugal Microfluidic Chip (CMC) capable of sorting immune cells from blood and in situ cellular analysis in a laboratory setting. Operation of the CMC entailed a blood specimen layered on a density gradient medium and centrifuged in microfluidic channels where immune cell subpopulations could rapidly be sorted into distinct layers according to their density differentials. We systematically studied effects of different blocking molecules for surface passivation of the CMC. We further demonstrated the applicability of CMCs for rapid separation of minimally processed human whole blood without affecting immune cell viability. Multi-color imaging and analysis of immune cell distributions and enrichment such as recovery and purity rates of peripheral blood mononuclear cells (PBMCs) were demonstrated using CMCs. Given its design and operation simplicity, portability, blood cell sorting efficiency, and in situ cellular analysis capability, the CMC holds promise for blood-based diagnosis and disease monitoring in POC applications.

Fully Automated Portable Comprehensive 2-Dimensional Gas Chromatography Device.

We developed a fully automated portable 2-dimensional (2-D) gas chromatography (GC x GC) device, which had a dimension of 60 cm × 50 cm × 10 cm and weight less than 5 kg. The device incorporated a micropreconcentrator/injector, commercial columns, micro-Deans switches, microthermal injectors, microphotoionization detectors, data acquisition cards, and power supplies, as well as computer control and user interface. It employed multiple channels (4 channels) in the second dimension (2D) to increase the 2D separation time (up to 32 s) and hence 2D peak capacity. In addition, a nondestructive flow-through vapor detector was installed at the end of the 1D column to monitor the eluent from 1D and assist in reconstructing 1D elution peaks. With the information obtained jointly from the 1D and 2D detectors, 1D elution peaks could be reconstructed with significantly improved 1D resolution. In this Article, we first discuss the details of the system operating principle and the algorithm to reconstruct 1D elution peaks, followed by the description and characterization of each component. Finally, 2-D separation of 50 analytes, including alkane (C6-C12), alkene, alcohol, aldehyde, ketone, cycloalkane, and aromatic hydrocarbon, in 14 min is demonstrated, showing the peak capacity of 430-530 and the peak capacity production of 40-80/min.

Low-Power Miniaturized Helium Dielectric Barrier Discharge Photoionization Detectors for Highly Sensitive Vapor Detection.

This paper presents the design, fabrication, and characterization of a microhelium dielectric barrier discharge photoionization detector (µHDBD-PID) on chip with dimensions of only ∼15 mm × âˆ¼10 mm × âˆ¼0.7 mm and weight of only ∼0.25 g. It offers low power consumption (<400 mW), low helium consumption (5.8 mL/min), rapid response (up to ∼60 ms at a flow rate of 1.5 mL/min), quick warm-up time (∼5 min), an excellent detection limit (a few picograms), a large linear dynamic range (>4 orders of magnitude), and maintenance-free operation. Furthermore, the µHDBD-PID can be driven with a miniaturized (∼5 cm × âˆ¼2.5 cm × âˆ¼2.5 cm), light (22 g), and low cost (∼$2) power supply with only 1.5 VDC input. The dependence of the µHDBD-PID performance on bias voltage, auxiliary helium flow rate, carrier gas flow rate, and temperature was also systematically investigated. Finally, the µHDBD-PID was employed to detect permanent gases and a sublist of the EPA 8260 standard reagents that include 51 analytes. The µHDBD-PID developed here can have a broad range of applications in portable and microgas chromatography systems for in situ, real-time, and sensitive gas analysis.

Integrated monolithic 3D MEMS scanner for switchable real time vertical/horizontal cross-sectional imaging.

We present an integrated monolithic, electrostatic 3D MEMS scanner with a compact chip size of 3.2 × 2.9 mm(2). Use of parametric excitation near resonance frequencies produced large optical deflection angles up to ± 27° and ± 28.5° in the X- and Y-axes and displacements up to 510 µm in the Z-axis with low drive voltages at atmospheric pressure. When packaged in a dual axes confocal endomicroscope, horizontal and vertical cross-sectional images can be collected seamlessly in tissue with a large field-of-view of >1 × 1 mm(2) and 1 × 0.41 mm(2), respectively, at 5 frames/sec.

In situ calibration of micro-photoionization detectors in a multi-dimensional micro-gas chromatography system.

A photoionization detector (PID) is widely used as a gas chromatography (GC) detector. By virtue of its non-destructive nature, multiple PIDs can be used in multi-dimensional GC. However, different PIDs have different responsivities towards the same chemical compound with the same concentration or mass due to different aging conditions of the PID lamps and windows. Here, we carried out a systematic study regarding the response of 5 Krypton µPIDs in a 1 × 4-channel 2-dimensional µGC system to 7 different volatile organic compounds (VOCs) with the ionization potential ranging from 8.45 eV to 10.08 eV and the concentration ranging from ∼1 ng to ∼2000 ng. We used one of the PIDs as the reference detector and calculated the calibration factor for each of the remaining 4 PIDs against the first PID, which we found is quite uniform regardless of the analyte, its concentration, or chromatographic peak width. Based on the above observation, we were able to quantitatively reconstruct the coeluted peaks in the first dimension using the signal obtained with a PID array in the second dimension. Our work will enable rapid and in situ calibration of PIDs in a GC system using a single analyte at a single concentration. It will also lead to the development of multi-channel multi-dimensional GC where multiple PIDs are employed.

Polymer-coated micro-optofluidic ring resonator detector for a comprehensive two-dimensional gas chromatographic microsystem: µGC ×µGC-µOFRR.

We describe first results from a micro-analytical subsystem that integrates a detector comprising a polymer-coated micro-optofluidic ring resonator (µOFRR) chip with a microfabricated separation module capable of performing thermally modulated comprehensive two-dimensional gas chromatographic separations (µGC ×µGC) of volatile organic compound (VOC) mixtures. The 2 × 2 cm µOFRR chip consists of a hollow, contoured SiO(x) cylinder (250 µm i.d.; 1.2 µm wall thickness) grown from a Si substrate, and integrated optical and fluidic interconnection features. By coupling to a 1550 nm tunable laser and photodetector via an optical fiber taper, whispering gallery mode (WGM) resonances were generated within the µOFRR wall, and shifts in the WGM wavelength caused by transient sorption of eluting vapors into the PDMS film lining the µOFRR cylinder were monitored. Isothermal separations of a simple alkane mixture using a PDMS coated 1st-dimension ((1)D) µcolumn and an OV-215-coated 2nd-dimension ((2)D) µcolumn confirmed that efficient µGC ×µGC-µOFRR analyses could be performed and that responses were dominated by film-swelling. Subsequent tests with more diverse VOC mixtures demonstrated that the modulated peak width and the VOC sensitivity were inversely proportional to the vapor pressure of the analyte. Modulated peaks as narrow as 120 ms and limits of detection in the low-ng range were achieved. Structured contour plots generated with the µOFRR and a reference FID were comparable.

µGC × µGC: comprehensive two-dimensional gas chromatographic separations with microfabricated components.

The development and characterization of a microanalytical subsystem comprising silicon-micromachined first- and second-dimension separation columns and a silicon-micromachined thermal modulator (µTM) for comprehensive two-dimensional (i.e., µGC × µGC) separations are described. The first dimension consists of two series-coupled 3.1 cm × 3.1 cm µcolumn chips with etched channels 3 m long and 250 µm × 140 µm in cross section, wall-coated with a poly(dimethylsiloxane) (PDMS) stationary phase. The second dimension consists of a 1.2 cm × 1.2 cm µcolumn chip with an etched channel 0.5 m long and 46 µm × 150 µm in cross section wall-coated with either a trigonal tricationic room-temperature ionic liquid (RTIL) or a commercial poly(trifluoropropylmethyl siloxane) (OV-215) stationary phase. The two-stage, cryogen-free µTM consists of a Si chip containing two series-coupled, square spiral channels 4.2 cm and 2.8 cm long and 250 µm × 140 µm in cross section wall-coated with PDMS. Conventional injection methods and flame ionization detection were used. Temperature-ramped separations of a simple alkane mixture using the RTIL-coated second-dimension ((2)D) µcolumn produced reasonably good peak shapes and modulation numbers; however, strong retention of polar compounds on the RTIL-coated (2)D µcolumn led to excessively broad peaks with low (2)D resolution. Substituting OV-215 as the (2)D µcolumn stationary phase markedly improved the performance, and a structured 22 min chromatogram of a 36-component mixture spanning a vapor pressure range of 0.027 to 13 kPa was generated with modulated peak fwhm (full width at half-maximum) values ranging from 90 to 643 ms and modulation numbers of 1-6.

INFLAMMATORY RESPONSES TO POLYMICROBIAL INTRA-ABDOMINAL SEPSIS ARE HIGHLY VARIABLE BUT STRONGLY CORRELATED TO ENTEROBACTERIACEAE OUTGROWTH.

ABSTRACT: Sepsis is a common, heterogeneous, and frequently lethal condition of organ dysfunction and immune dysregulation due to infection. The causes of its heterogeneity, including the contribution of the pathogen, remain unknown. Using cecal slurry, a widely used murine model of intraperitoneal polymicrobial sepsis, as well as 16S ribosomal RNA sequencing and measurement of immune markers, we performed a series of translational analyses to determine whether microbial variation in cecal slurry composition (representing intra-abdominal pathogens) mediated variation in septic response. We found wide variation in cecal slurry community composition that changed markedly over the 24-h course of infection. This variation in cecal slurry bacteria led to large variation in physiologic and inflammatory responses. Severity of inflammatory response was positively correlated with intraperitoneal enrichment with Enterobacteriaceae. Likewise, in a human cohort of patients with intra-abdominal abscesses, Enterobacteriaceae was also associated with increased inflammatory markers. Taken together, these data demonstrate that intra-abdominal Enterobacteriaceae drives inflammation in sepsis both in animal models and human subjects. More broadly, our results demonstrate that pathogen identity is a major driver of the host response in polymicrobial sepsis and should not be overlooked as a major source of phenotypic heterogeneity.

Preprogrammed, parallel on-chip immunoassay using system-level capillarity control.

Fully manual use of conventional multiwell plates makes enzyme-linked immunosorbent assay (ELISA)-based immunoassays highly time-consuming and labor-intensive. Here, we present a capillarity-driven on-chip immunoassay that greatly saves time and labor with an inexpensive setup. Our immunoassay process starts with pipetting multiple solutions into multiwells constructed on a microfluidic device chip. Subsequently, capillarity spontaneously transports multiple sample solutions and common reagent solutions into assigned detection channels on the chip in a purely passive and preprogrammed manner. Our device implements capillarity-driven immunoassays involving four sample and six reagent solutions within 30 min by orchestrating the functions of on-chip passive components. Notably, our immunoassay technique reduces the total number of pipetting processes by ~5 times, as compared to assays on multiwell plates (48 vs 10). This assay technique allows us to quantify the concentrations of C-reactive protein and suppressor of tumorigenicity 2 with a detection limit of 8 and 90 pM, respectively. This device should be useful for sophisticated, parallel biochemical microfluidic processing in point-of-care settings under limited resources.

Smart three-dimensional gas chromatography.

We developed a complete computer-controlled smart 3-dimensional gas chromatography (3-D GC) system with an automation algorithm. This smart 3-D GC architecture enabled independent optimization of and control over each dimension of separation and allowed for much longer separation time for the second- and third-dimensional columns than the conventional comprehensive 3-D GC could normally achieve. Therefore, it can potentially be employed to construct a novel GC system that exploits the multidimensional separation capability to a greater extent. In this Article, we introduced the smart 3-D GC concept, described its operation, and demonstrated its feasibility by separating 22 vapor analytes.

A tissue chip with integrated digital immunosensors: In situ brain endothelial barrier cytokine secretion monitoring.

Organ-on-a-chip platforms have potential to offer more cost-effective, ethical, and human-resembling models than animal models for disease study and drug discovery. Particularly, the Blood-Brain-Barrier-on-a-chip (BBB-oC) has emerged as a promising tool to investigate several neurological disorders since it promises to provide a model of the multifunctional tissue working as an important node to control pathogen entry, drug delivery and neuroinflammation. A comprehensive understanding of the multiple physiological functions of the tissue model requires biosensors detecting several tissue-secreted substances in a BBB-oC system. However, current sensor-integrated BBB-oC platforms are only available for tissue membrane integrity characterization based on permeability measurement. Protein secretory pathways are closely associated with the tissue's various diseased conditions. At present, no biosensor-integrated BBB-oC platform exists that permits in situ tissue protein secretion analysis over time, which prohibits researchers from fully understanding the time-evolving pathology of a tissue barrier. Herein, the authors present a platform named "Digital Tissue-BArrier-CytoKine-counting-on-a-chip (DigiTACK)," which integrates digital immunosensors into a tissue chip system and demonstrates on-chip multiplexed, ultrasensitive, longitudinal cytokine secretion profiling of cultured brain endothelial barrier tissues. The integrated digital sensors utilize a novel beadless microwell format to perform an ultrafast "digital fingerprinting" of the analytes while achieving a low limit of detection (LoD) around 100-500 fg/mL for mouse MCP1 (CCL2), IL-6 and KC (CXCL1). The DigiTACK platform is extensively applicable to profile temporal cytokine secretion of other barrier-related organ-on-a-chip systems and can provide new insight into the secretory dynamics of the BBB by sequentially controlled experiments.