Fc Glycan-Mediated Regulation of Placental Antibody Transfer.

Despite the worldwide success of vaccination, newborns remain vulnerable to infections. While neonatal vaccination has been hampered by maternal antibody-mediated dampening of immune responses, enhanced regulatory and tolerogenic mechanisms, and immune system immaturity, maternal pre-natal immunization aims to boost neonatal immunity via antibody transfer to the fetus. However, emerging data suggest that antibodies are not transferred equally across the placenta. To understand this, we used systems serology to define Fc features associated with antibody transfer. The Fc-profile of neonatal and maternal antibodies differed, skewed toward natural killer (NK) cell-activating antibodies. This selective transfer was linked to digalactosylated Fc-glycans that selectively bind FcRn and FCGR3A, resulting in transfer of antibodies able to efficiently leverage innate immune cells present at birth. Given emerging data that vaccination may direct antibody glycosylation, our study provides insights for the development of next-generation maternal vaccines designed to elicit antibodies that will most effectively aid neonates.

Enhanced Enzymatically Amplified Metallization on Nanostructured Surfaces for Multiplexed Point-of-Care Electrical Detection of COVID-19 Biomarkers.

Inexpensive yet sensitive and specific biomarker detection is a critical bottleneck in diagnostics, monitoring, and surveillance of infectious diseases such as COVID-19. Multiplexed detection of several biomarkers can achieve wider diagnostic applicability, accuracy, and ease-of-use, while reducing cost. Current biomarker detection methods often use enzyme-linked immunosorbent assays (ELISA) with optical detection which offers high sensitivity and specificity. However, this is complex, expensive, and limited to detecting only a single analyte at a time. Here, it is found that biomarker-bound enzyme-labeled probes act synergistically with nanostructured catalytic surfaces and can be used to selectively reduce a soluble silver substrate to generate highly dense and conductive, localized surface silver metallization on microelectrode arrays. This enables a sensitive and quantitative, simple, direct electronic readout of biomarker binding without the use of any intermediate optics. Furthermore, the localized and dry-phase stable nature of the metallization enables multiplexed electronic measurement of several biomarkers from a single drop (<10 µL) of sample on a microchip.This method is applied for the multiplexed point-of-care (POC) quantitative detection of multiple COVID-19 antigen-specific antibodies. Combining a simple microchip and an inexpensive, cellphone-interfaced, portable reader, the detection and discrimination of biomarkers of prior infection versus vaccination is demonstrated.

Detecting kinase activities from single cell lysate using concentration-enhanced mobility shift assay.

Electrokinetic preconcentration coupled with mobility shift assays can give rise to very high detection sensitivities. We describe a microfluidic device that utilizes this principle to detect cellular kinase activities by simultaneously concentrating and separating substrate peptides with different phosphorylation states. This platform is capable of reliably measuring kinase activities of single adherent cells cultured in nanoliter volume microwells. We also describe a novel method utilizing spacer peptides that significantly increase separation resolution while maintaining high concentration factors in this device. Thus, multiplexed kinase measurements can be implemented with single cell sensitivity. Multiple kinase activity profiling from single cell lysate could potentially allow us to study heterogeneous activation of signaling pathways that can lead to multiple cell fates.

Spatial configuration and composition of charge modulates transport into a mucin hydrogel barrier.

The mucus barrier is selectively permeable to a wide variety of molecules, proteins, and cells, and establishes gradients of these particulates to influence the uptake of nutrients, the defense against pathogens, and the delivery of drugs. Despite its importance for health and disease, the criteria that govern transport through the mucus barrier are largely unknown. Studies with uniformly functionalized nanoparticles have provided critical information about the relevance of particle size and net charge for mucus transport. However, these particles lack the detailed spatial arrangements of charge found in natural mucus-interacting substrates, such as certain viruses, which may have important consequences for transport through the mucus barrier. Using a novel, to our knowledge, microfluidic design that enables us to measure real-time transport gradients inside a hydrogel of mucins, the gel-forming glycoprotein component of mucus, we show that two peptides with the same net charge, but different charge arrangements, exhibit fundamentally different transport behaviors. Specifically, we show that certain configurations of positive and negative charges result in enhanced uptake into a mucin barrier, a remarkable effect that is not observed with either charge alone. Moreover, we show that the ionic strength within the mucin barrier strongly influences transport specificity, and that this effect depends on the detailed spatial arrangement of charge. These findings suggest that spatial charge distribution is a critical parameter to modulate transport through mucin-based barriers, and have concrete implications for the prediction of mucosal passage, and the design of drug delivery vehicles with tunable transport properties.

Multiplexed protease activity assay for low-volume clinical samples using droplet-based microfluidics and its application to endometriosis.

As principal degrading enzymes of the extracellular matrix, metalloproteinases (MPs) contribute to various pathologies and represent a family of promising drug targets and biomarker candidates. However, multiple proteases and endogenous inhibitors interact to govern MP activity, often leading to highly context-dependent protease function that unfortunately has impeded associated clinical utility. We present a method for rapidly assessing the activity of multiple specific proteases in small volumes (<20 µL) of complex biological fluids such as clinical samples that are available only in very limited amounts. It uses a droplet-based microfluidic platform that injects the sample into thousands of picoliter-scale droplets from a barcoded droplet library (DL) containing mixtures of unique, moderately selective FRET-based protease substrates and specific inhibitors and monitors hundreds of the reactions thus initiated simultaneously by tracking these droplets. Specific protease activities in the sample are then inferred from the reaction rates using a deconvolution technique, proteolytic activity matrix analysis (PrAMA). Using a nine-member DL with three inhibitors and four FRET substrates, we applied the method to the peritoneal fluid of subjects with and without the invasive disease endometriosis. The results showed clear and physiologically relevant differences with disease, in particular, decreased MMP-2 and ADAM-9 activities.

Electronic Immunoassay Using Enzymatic Metallization on Microparticles.

We present here an inexpensive method for generating a sensitive direct electronic readout in bead-based immunoassays without the use of any intermediate optical instrumentation (e.g., lasers, photomultipliers, etc.). Analyte binding to capture antigen-coated beads or microparticles is converted to probe-directed enzymatically amplified silver metallization on microparticle surfaces. Individual microparticles are then rapidly characterized in a high-throughput manner via single-bead multifrequency electrical impedance spectra captured using a simple and inexpensive microfluidic impedance spectrometry system we develop here, where they flow through a three-dimensional (3D)-printed plastic microaperture sandwiched between plated through-hole electrodes on a printed circuit board. Metallized microparticles are found to have unique impedance signatures distinguishing them from unmetallized ones. Coupled with a machine learning algorithm, this enables a simple electronic readout of the silver metallization density on microparticle surfaces and hence the underlying analyte binding. Here, we also demonstrate the use of this scheme to measure the antibody response to the viral nucleocapsid protein in convalescent COVID-19 patient serum.

Inexpensive High-Throughput Multiplexed Biomarker Detection Using Enzymatic Metallization with Cellphone-Based Computer Vision.

Multiplexed biomarker detection can play a critical role in reliable and comprehensive disease diagnosis and prediction of outcome. Enzyme-linked immunosorbent assay (ELISA) is the gold standard method for immunobinding-based biomarker detection. However, this is currently expensive, limited to centralized laboratories, and usually limited to the detection of a single biomarker at a time. We present a low-cost, smartphone-based portable biosensing platform for high-throughput, multiplexed, sensitive, and quantitative detection of biomarkers from single, low-volume drops (<1 µL) of clinical samples. Biomarker binding to spotted capture antigens is converted, via enzymatic metallization, to the localized surface deposition of amplified, dry-stable, silver metal spots whose darkness is proportional to biomarker concentration. A custom smartphone application is developed, which uses real-time computer vision to enable easy optical detection of the deposited metal spots and sensitive and reproducible quantification of the biomarkers. We demonstrate the use of this platform for high-throughput, multiplexed detection of multiple viral antigen-specific antibodies from convalescent COVID-19 patient serum as well as vaccine-elicited antibody responses from uninfected vaccine-recipient serum and show that distinct multiplexed antibody fingerprints are observed among them.

Antibodies targeting conserved non-canonical antigens and endemic coronaviruses associate with favorable outcomes in severe COVID-19.

While there have been extensive analyses characterizing cellular and humoral responses across the severity spectrum in COVID-19, outcome predictors within severe COVID-19 remain less comprehensively elucidated. Furthermore, properties of antibodies (Abs) directed against viral antigens beyond spike and their associations with disease outcomes remain poorly defined. We perform deep molecular profiling of Abs directed against a wide range of antigenic specificities in severe COVID-19 patients. The profiles included canonical (spike [S], receptor-binding domain [RBD], and nucleocapsid [N]) and non-canonical (orf3a, orf8, nsp3, nsp13, and membrane [M]) antigenic specificities. Notably, multivariate Ab profiles directed against canonical or non-canonical antigens are equally discriminative of survival in severe COVID-19. Intriguingly, pre-pandemic healthy controls have cross-reactive Abs directed against nsp13, a protein conserved across coronaviruses. Consistent with these findings, a model built on Ab profiles for endemic coronavirus antigens also predicts COVID-19 outcome. Our results suggest the importance of studying Abs targeting non-canonical severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and endemic coronavirus antigens in COVID-19.

Enhancing protease activity assay in droplet-based microfluidics using a biomolecule concentrator.

We introduce an integrated microfluidic device consisting of a biomolecule concentrator and a microdroplet generator, which enhances the limited sensitivity of low-abundance enzyme assays by concentrating biomolecules before encapsulating them into droplet microreactors. We used this platform to detect ultralow levels of matrix metalloproteinases (MMPs) from diluted cellular supernatant and showed that it significantly (~10-fold) reduced the time required to complete the assay and the sample volume used.

Facile fabrication of microfluidic systems using electron beam lithography.

We present two fast and generic methods for the fabrication of polymeric microfluidic systems using electron beam lithography: one that employs spatially varying electron-beam energy to expose to different depths a negative electron-beam resist, and another that employs a spatially varying electron-beam dose to differentially expose a bi-layer resist structure. Using these methods, we demonstrate the fabrication of various microfluidic unit structures such as microchannels of a range of geometries and also other more complex structures such as a synthetic gel and a chaotic mixer. These are made without using any separate bonding or sacrificial layer patterning and etching steps. The schemes are inherently simple and scalable, afford high resolution without compromising on speed and allow post CMOS fabrication of microfluidics. We expect them to prove very useful for the rapid prototyping of complete integrated micro/nanofluidic systems with sense and control electronics fabricated by upstream processes.

Multiplexed Affinity-Based Separation of Proteins and Cells Using Inertial Microfluidics.

Isolation of low abundance proteins or rare cells from complex mixtures, such as blood, is required for many diagnostic, therapeutic and research applications. Current affinity-based protein or cell separation methods use binary 'bind-elute' separations and are inefficient when applied to the isolation of multiple low-abundance proteins or cell types. We present a method for rapid and multiplexed, yet inexpensive, affinity-based isolation of both proteins and cells, using a size-coded mixture of multiple affinity-capture microbeads and an inertial microfluidic particle sorter device. In a single binding step, different targets-cells or proteins-bind to beads of different sizes, which are then sorted by flowing them through a spiral microfluidic channel. This technique performs continuous-flow, high throughput affinity-separation of milligram-scale protein samples or millions of cells in minutes after binding. We demonstrate the simultaneous isolation of multiple antibodies from serum and multiple cell types from peripheral blood mononuclear cells or whole blood. We use the technique to isolate low abundance antibodies specific to different HIV antigens and rare HIV-specific cells from blood obtained from HIV+ patients.

High-throughput protease activity cytometry reveals dose-dependent heterogeneity in PMA-mediated ADAM17 activation.

As key components of autocrine signaling, pericellular proteases, a disintegrin and metalloproteinases (ADAMs) in particular, are known to impact the microenvironment of individual cells and have significant implications in various pathological situations including cancer, inflammatory and vascular diseases. There is great incentive to develop a high-throughput platform for single-cell measurement of pericellular protease activity, as it is essential for studying the heterogeneity of protease response and the corresponding cell behavioral consequences. In this work, we developed a microfluidic platform to simultaneously monitor protease activity of many single cells in a time-dependent manner. This platform isolates individual microwells rapidly on demand and thus allows single-cell activity measurement of both cell-surface and secreted proteases by confining individual cells with diffusive FRET-based substrates. With this platform, we observed dose-dependent heterogeneous protease activation of HepG2 cells treated with phorbol 12-myristate 13-acetate (PMA). To study the temporal behavior of PMA-induced protease response, we monitored the pericellular protease activity of the same single cells during three different time periods and revealed the diversity in the dynamic patterns of single-cell protease activity profile upon PMA stimulation. The unique temporal information of single-cell protease response can help unveil the complicated functional role of pericellular proteases.

Microfluidic probe for single-cell analysis in adherent tissue culture.

Single-cell analysis provides information critical to understanding key disease processes that are characterized by significant cellular heterogeneity. Few current methods allow single-cell measurement without removing cells from the context of interest, which not only destroys contextual information but also may perturb the process under study. Here we present a microfluidic probe that lyses single adherent cells from standard tissue culture and captures the contents to perform single-cell biochemical assays. We use this probe to measure kinase and housekeeping protein activities, separately or simultaneously, from single human hepatocellular carcinoma cells in adherent culture. This tool has the valuable ability to perform measurements that clarify connections between extracellular context, signals and responses, especially in cases where only a few cells exhibit a characteristic of interest.

Non-linear and linear enhancement of enzymatic reaction kinetics using a biomolecule concentrator.

In this work we investigate concentration-enhanced enzyme activity assays in nanofluidic biomolecule concentrator chips which can be used to detect and study very low abundance enzymes from cell lysates and other low volume, low concentration samples. A mathematical model is developed for a mode of operation of the assay (J. H. Lee, B. D. Cosgrove, D. A. Lauffenburger and J. Han, J. Am. Chem. Soc., 2009, 131, 10340-10341) in which enzyme and substrate are concentrated together into a plug on chip which results in a non-linear enhancement of the reaction rate. Two reaction phases, an initial quadratic enzyme-limited phase and a later, linear substrate-limited phase, are predicted and then verified with experiments. It is determined that, in most practical situations, the reaction eventually enters a substrate-limited phase, therefore mitigating the concern for non-specific reactions of biosensor substrates with off-target enzymes in such assays. We also use this mode to demonstrate a multiplexed concentration-enhanced enzyme activity assay. We then propose and demonstrate a new device and mode of operation, in which only the enzyme is concentrated and then mixed with a fixed amount of substrate in an adjacent picolitre-scale reaction chamber. This mode results in a linear enhancement of the reaction rate and can be used to perform mechanistic studies on low abundance enzymes after concentrating them into a plug on chip.