Development of a cellulose-based 96-well plate vertical flow pull-down assay.

The abundance and low production cost of biomaterial cellulose paper have attracted attention for many applications. Point-of-care (PoC) diagnostic tests have been successfully developed using patterned cellulose paper. Although PoC diagnostic tests are rapid and simple to perform, their sample processing throughput is limited, allowing for only one sample to be evaluated at a time, which restricts potential applications. Thus, it was appealing to expand cellulose-based PoC tests to high-throughput versions to increase their applicability. Here, we present the development of a high-throughput cellulose-based 96-well plate vertical flow pull-down assay that can process 96 tests, is easy to prepare, and can be customized for different detection targets. The device has two key features: (i) patterned cellulose paper for 96 tests that do not require pre-immobilization of capturing reagents, and (ii) reusable sturdy housing. We believe that a variety of applications, including laboratory testing, population surveillance tests, and sizable clinical trials for diagnostic tests, can benefit from the adoption of this cellulose-based 96-well plate assay.

Finger stick blood test to assess postvaccination SARS-CoV-2 neutralizing antibody response against variants.

There is clinical need for a quantifiable point-of-care (PoC) SARS-CoV-2 neutralizing antibody (nAb) test that is adaptable with the pandemic's changing landscape. Here, we present a rapid and semi-quantitative nAb test that uses finger stick or venous blood to assess the nAb response of vaccinated population against wild-type (WT), alpha, beta, gamma, and delta variant RBDs. It captures a clinically relevant range of nAb levels, and effectively differentiates prevaccination, post first dose, and post second dose vaccination samples within 10 min. The data observed against alpha, beta, gamma, and delta variants agrees with published results evaluated in established serology tests. Finally, our test revealed a substantial reduction in nAb level for beta, gamma, and delta variants between early BNT162b2 vaccination group (within 3 months) and later vaccination group (post 3 months). This test is highly suited for PoC settings and provides an insightful nAb response in a postvaccinated population.

Generation of Thermally Stable Affinity Pairs for Sensitive, Specific Immunoassays.

Many point-of-care diagnostic tests rely on a pair of monoclonal antibodies that bind to two distinct epitopes of a molecule of interest. This protocol describes the identification and generation of such affinity pairs based on an easily produced small protein scaffold rcSso7d which can substitute monoclonal antibodies. These strong binding variants are identified from a large yeast display library. The approach described can be significantly faster than antibody generation and epitope binning, yielding affinity pairs synthesized in common bacterial protein synthesis strains, enabling the rapid generation of novel diagnostic tools.

Development and translation of a paper-based top readout vertical flow assay for SARS-CoV-2 surveillance.

Surveillance of SARS-CoV-2 infection is critical for controlling the current pandemic. Antigen rapid tests (ARTs) provide a means for surveillance. Available lateral flow assay format ARTs rely heavily on nitrocellulose paper, raising challenges in supply shortage. Vertical flow assay (VFA) with cellulose paper as test material attracts much attention as a complementary test approach. However, current reported VFAs are facing challenges in reading the test signal from the bottom face of the test cassette, complicating the test workflow and hindering translation into rapid test application. Here, we address this gap with an enhanced VFA against SARS-CoV-2 N protein that adapts a cellulose pull-down test format allowing (1) one-step sample application at the top of the test cassette and (2) readout of the test signal from the top. We also demonstrate the feasibility of translating the enhanced VFA into a point-of-care application that can help in SARS-CoV-2 surveillance.

Vertical Flow Cellulose-Based Assays for SARS-CoV-2 Antibody Detection in Human Serum.

Rapid and inexpensive serological tests for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antibodies are essential to conduct large-scale seroprevalence surveys and can potentially complement nucleic acid or antigen tests at the point of care. During the COVID-19 pandemic, extreme demand for traditional lateral flow tests has stressed manufacturing capacity and supply chains. Motivated by this limitation, we developed a SARS-CoV-2 antibody test using cellulose, an alternative membrane material, and a double-antigen sandwich format. Functionalized SARS-CoV-2 antigens were used as both capture and reporter binders, replacing the anti-human antibodies currently used in lateral flow tests. The test could provide enhanced sensitivity because it labels only antibodies against SARS-CoV-2 and the signal intensity is not diminished due to other human antibodies in serum. Three-dimensional channels in the assay were designed to have consistent flow rates and be easily manufactured by folding wax-printed paper. We demonstrated that this simple, vertical flow, cellulose-based assay could detect SARS-CoV-2 antibodies in clinical samples within 15 min, and the results were consistent with those from a laboratory, bead-based chemiluminescence immunoassay that was granted emergency use approval by the US FDA.

A rapid simple point-of-care assay for the detection of SARS-CoV-2 neutralizing antibodies.

Background: Neutralizing antibodies (NAbs) prevent pathogens from infecting host cells. Detection of SARS-CoV-2 NAbs is critical to evaluate herd immunity and monitor vaccine efficacy against SARS-CoV-2, the virus that causes COVID-19. All currently available NAb tests are lab-based and time-intensive. Method: We develop a 10 min cellulose pull-down test to detect NAbs against SARS-CoV-2 from human plasma. The test evaluates the ability of antibodies to disrupt ACE2 receptor-RBD complex formation. The simple, portable, and rapid testing process relies on two key technologies: (i) the vertical-flow paper-based assay format and (ii) the rapid interaction of cellulose binding domain to cellulose paper. Results: Here we show the construction of a cellulose-based vertical-flow test. The developed test gives above 80% sensitivity and specificity and up to 93% accuracy as compared to two current lab-based methods using COVID-19 convalescent plasma. Conclusions: A rapid 10 min cellulose based test has been developed for detection of NAb against SARS-CoV-2. The test demonstrates comparable performance to the lab-based tests and can be used at Point-of-Care. Importantly, the approach used for this test can be easily extended to test RBD variants or to evaluate NAbs against other pathogens.

Beyond Epitope Binning: Directed in Vitro Selection of Complementary Pairs of Binding Proteins.

Many biotechnological applications require the simultaneous binding of affinity reagents to nonoverlapping target epitopes, the most prominent example being sandwich immunoassays. Typically, affinity pairs are identified via post facto functional analysis of clones that were not selected for complementarity. Here, we developed the Rapid Affinity Pair Identification via Directed Selection (RAPIDS) process, which enables the efficient identification of affinity reagents that function together as complementary pairs, from in vitro libraries of ∼109 variants. We used RAPIDS to develop highly specific affinity pairs against biomarkers of tuberculosis, Zika virus, and sepsis. Without additional trial-and-error screening, these affinity pairs exhibited utility in multiple assay formats. The RAPIDS process applies selective pressure to hundreds of thousands of potential affinity pairs to efficiently identify complementary pairs that bind to separate epitopes without binding to one another or nontargets, yielding diagnostic assays that are sensitive and specific by design.

In vitro micro-physiological model of the inflamed human adipose tissue for immune-metabolic analysis in type II diabetes.

Chronic inflammation mediated by the interaction of immune cells and adipocytes is a key underlying factor in obesity-associated type 2 diabetes mellitus (T2DM). Therefore, methods to investigate adipocyte-immune cells interaction and their immuno-metabolic status in obese/T2DM subjects not only serve as an early indicator of disease development but also provide an insight into disease mechanism. A microfluidic-based in vitro model of the human adipose that is interfaced with a co-culture of immune cell has been developed for in vitro immune-metabolic analysis. This miniaturized system integrates a biologically active in vitro cellular system within a perfusion-based microfluidic device for mimicking the major processes that characterize the interaction of adipose tissue with immune cells. A viable immune competent model of the adipocytes/PBMCs co-culture has been demonstrated and characterized. Our testing results showed that the inflammatory cytokine profile obtained from the on-chip culture agrees with those from static transwell based co-culture with more intense responses observed in the chip-based system. The microfluidic chip also allows time-resolved measurement of cytokines that provide reliable data and detailed mechanisms of inflammation. In addition, glucose uptake by the adipocytes from the chip-based cultures showed correlated insulin responsivity/resistivity to the expression of the cytokine profile in different dynamic culture conditions. Testing of the known diabetic drug, metformin, and neutraceutical compound, omega-3, on-chip show agreeable results as compared to the previously reported data. This organotypic culture system offers a physiologically relevant model that exhibits a key characteristic of type 2 diabetic adipose tissues and can be used to study the T2DM mechanisms and diabetic drug screening.

Adipose-on-a-chip: a dynamic microphysiological in vitro model of the human adipose for immune-metabolic analysis in type II diabetes.

Infiltration of immune cells into adipose tissue is associated with chronic low-grade inflammation in obese individuals. To better understand the crosstalk between immune cells and adipocytes, in vivo-like in vitro models are required. Conventionally transwell culture plates are used for studying the adipocyte-immune cell interaction; however, the static culture nature of this approach falls short of closely recapitulating the physiological environment. Here we present a compartmentalized microfluidic co-culture system which provides a constant-rate of nutrient supply as well as waste removal, resembling the microvascular networks of the in vivo environment. Human adipocytes and U937 cells were co-cultured in close proximity in an enclosed system. The porous barrier between the adjacent compartments comprises an array of microchannels, which enables paracrine interaction between cells in adjacent compartments and improved perfusion-based long term cell feeding. Human pre-adipocytes were fully differentiated into adipocytes on the chip and remained viable for several weeks. Upon co-culturing with immune cells, adipocytes showed a tendency to develop insulin resistance. The immune-metabolic correlation has been studied by monitoring adiponectin and IL-6 expression, as well as glucose uptake upon treatment with insulin. Our microfluidic system can be potentially used to develop physiologically relevant adipose tissue models to study obesity-associated diseases such as insulin resistance and type 2 diabetes and therefore, facilitate drug development to treat these diseases.

Human adipocyte differentiation and characterization in a perfusion-based cell culture device.

Adipocytes have gained significant attention recently, because they are not only functioning as energy storage but also as endocrine cells. Adipocytes secret various signaling molecules, including adiponectin, MCP-1, and IL-6, termed collectively as "adipokines". Adipokines regulate glucose metabolism, thereby play an important role in obesity, diabetes type 2, and other metabolic disorders. Conventionally, to study the secretory function, adipocytes are cultured in vitro in static conditions. However, static culturing condition falls short of mimicking the interstitial fluid flows in living systems. Here, we developed a perfusion device which allows dynamic culture of adipocytes under constant and mild flow using a double-layered fluidic structure. Adipocytes were cultured in the bottom layer while the culture media were constantly flown in the upper layer and perfused through a porous membrane that separate the two chambers. The porous membrane between the two chambers physically separates the cells from the flow stream while maintain a fluidic connection by diffusion. This setting not only provides continuous nutrient supply to adipocytes but also maintains a steady and mild shear stress on the cell membrane. It was found the perfusion-based culture conditions promoted faster growth of primary preadipocytes and stimulated greater adipogenesis compared to static culture condition. Adipocytes cultured under perfusion systems produced more MCP-1 and IL-6, but less adiponectin. When stimulated with TNF-α, adipocytes expressed higher level of MCP-1 and IL-6, but lower level of adiponectin. No significant glucose uptake regulation was observed after treating the adipocytes with insulin in both static and perfusion-based culture. Our results demonstrate that perfusion-base culture has played a role in the adipocyte function particularly the secretion of adipokines. More future studies are required to unveil the mechanisms behind perfusion's impact on adipocytes.

On-chip electrochemical immunoassay platform for specific protein biomarker estimation in undiluted serum using off-surface membrane matrix.

The manuscript presents a new biosensor platform using bioreceptors modified porous 2-dimensional (2D) membrane based off-surface matrix for on-chip electrochemical immunoassay. Antibody based bioreceptors modified 2D matrix of porous polycarbonate (PC) membrane with densely packed 20µm holes as off-surface matrix was incorporated in very close proximity of the sensor surface and integrated with fluidic system for reagent flow and incubation chamber. Covalent attachment of antibodies on 2D PC membrane based off-matrix was achieved using 4-fluoro-3-nitro-azidobenzene (FNAB) cross-linker. Anti-TNF-α/FNAB/PC membrane was integrated over array of micro fingers of gold based sensor chip using double side tape spacer and StartingBlock phosphate buffer saline- Tween-20; (PBS-T20) blocking buffer was utilized to minimize nonspecific binding. Differential pulse voltammetric studies of Anti-Tnf-α/FNAB/PC-Au for protein biomarker (TNF-α) detection and estimation in undiluted serum indicated that the immunosensor system can detect TNF-α linearly in 100pg/ml to 100ng/ml range with insignificant interference from other cytokines and serum proteins. Further, immunosensor exhibited high sensitivity of 194nA/(ng/ml) and 240nA/(ng/ml), respectively for single and double membrane based system. Thus, use of 2D membrane based off surface matrix may present the new platform to sensitively measure biomarkers electrochemically to pg/ml range with insignificant nonspecific binding and false signal in undiluted serum.

Off surface matrix based on-chip electrochemical biosensor platform for protein biomarker detection in undiluted serum.

The manuscript describes a concept of using off surface matrix modified with capturing biomolecule for on-chip electrochemical biosensing. 3D matrix made by laser engraving of polymethyl methacrylate (PMMA) sheet as off surface matrix was integrated in very close vicinity of the electrode surface. Laser engraving and holes in PMMA along with spacing from surface provide fluidic channel and incubation chamber. Covalent binding of capturing biomolecule (anti-TNF-α antibody) on off-surface matrix was achieved via azide group activity of 4-fluoro-3-nitro-azidobenzene (FNAB), which act as cross-linker and further covalently binds to anti-TNF-α antibody via thermal reaction. Anti-TNF-α/FNAB/PMMA matrix was then integrated over comb structured gold electrode array based sensor chip. Separate surface modification followed by integration of sensor helped to prevent the sensor chip surface from fouling during functionalization. Nonspecific binding was prevented using starting block T20 (PBS). Results for estimating protein biomarker (TNF-α) in undiluted serum using Anti-TNF-α/FNAB/PMMA/Au reveal that system can detect TNF-α in 100pg/ml to 100ng/ml range with high sensitivity of 119nA/(ng/ml), with negligible interference from serum proteins and other cytokines. Thus, use of off surface matrix may provide the opportunity to electrochemically sense biomarkers sensitively to ng/ml range with negligible nonspecific binding and false signal in undiluted serum.

On-chip immune cell activation and subsequent time-resolved magnetic bead-based cytokine detection.

Cytokine profiling and immunophenotyping offer great potential for understanding many disease mechanisms, personalized diagnosis, and immunotherapy. Here, we demonstrate a time-resolved detection of cytokine from a single cell cluster using an in situ magnetic immune assay. An array of triple-layered microfluidic chambers was fabricated to enable simultaneous cell culture under perfusion flow and detection of the induced cytokines at multiple time-points. Each culture chamber comprises three fluidic compartments which are dedicated to, cell culture, perfusion and immunoassay. The three compartments are separated by porous membranes, which allow the diffusion of fresh nutrient from the perfusion compartment into the cell culture compartment and cytokines secretion from the cell culture compartment into the immune assay compartment. This structure hence enables capturing the released cytokines without disturbing the cell culture and without minimizing benefit gain from perfusion. Functionalized magnetic beads were used as a solid phase carrier for cytokine capturing and quantification. The cytokines released from differential stimuli were quantified in situ in non-differentiated U937 monocytes and differentiated macrophages.

EIS-based biosensor for ultra-sensitive detection of TNF-α from non-diluted human serum.

Serum background is a critical issue for biosensor development as it interferes with the detection of target molecules and may give rise to false positive signal. We present here highly sensitive and selective TNF-α biosensor which is able to detect TNF-α from non-diluted human serum using magnetic bead coupled antibody and electrochemical impedance spectroscopy (EIS) techniques. The process is designed to detect TNF-α from human serum in three stages; (1) abundant protein backgrounds are depleted from the serum using magnetic bead coupled albumin and IgG antibodies, (2) after background depletion TNF-α is captured using magnetic bead coupled TNF-α antibody, and (3) the captured TNF-α is eluted from the magnetic beads and measured using EIS technique in which comb structured gold microelectrodes array (CSGM) is utilized to enhance the detection sensitivity. The system is able to achieve the limit of detection (LOD) at 1 pg/ml (57 fM) and a linear relationship between increasing TNF-α concentrations and charge-transfer resistance in a dynamic range of 1-1000 pg/ml.

Coiled-coil peptide based sensor for ultra-sensitive thrombin detection.

Comb structured gold microelectrode array (CSGMA) functionalized with self-assembled monolayer of thiol terminated coiled-coil peptide (CCP) linked together by the thrombin specific cleavage site (Leu-Val-Pro-Arg-Gly-Ser) has been used to fabricate an ultrasensitive, disposable, electrochemical thrombin biosensor. CCP with thiol at one end provides the ease of CSGMA functionalization and the presence of thrombin specific peptide in the middle of coiled-coil peptide provides site for thrombin capture and detection. CCP/CSGMA electrodes were characterized using label-free electrochemical impedance (EIS) technique and exposed to solutions with different thrombin concentrations for its estimation. Results reveal that CCP/CSGMA electrodes have a limit of detection (LOD) of 10 fg/ml (28 fM) and are able to detect catalytic activity of thrombin within 30 min time frame. CCP/CSGMA electrodes were found to be selective against other IgG anti-bodies such as DO1 and HA. Thus, CCP/CSGMA electrodes provide high specificity toward thrombin detection and mechanistic details of binding and cleavage process.

Aptamer-based array electrodes for quantitative interferon-γ detection.

Present work describes the methylene blue tagged thiolated aptamer-modified gold micro-array based biosensor for specific detection of IFN-γ. The microchips with the microelectrode array were fabricated using standard silicon microfabrication technologies, and modified with methylene blue tagged aptamer using standard gold thiol chemistry. Electrodes were characterized and tested using Cyclic Voltammetric (CV) and Square Wave Voltammetry (SQW) measurements in a standard three-electrode format at room temperature. On an aptamer modified electrode, aptamer density was estimated to be about 4.4 × 10(12)molecules/cm(2). In IFN-γ studies, oxidation peak currents were found to decrease and more than 50% signal suppression was achieved at 500 ng/ml. Further, the magnitude of signal suppression was found to be logarithmically proportional to the IFN-γ in the concentration range of 1-500 ng/ml, with a detection limit of 1.3 ng/ml (i.e. 0.8 fmol in used sample volume of 10 µl). Biosensor showed negligible signal changes (5%) in a very high non-specific protein background, while still able to differentiate target protein IFN-γ at 5 ng/ml. The results indicated that our sensor binds selectively to target molecules, and the non-specific binding where adsorption of BSA protein molecules may be effectively omitted from consideration.

Control of TMEM16A by INO-4995 and other inositolphosphates.

BACKGROUND AND PURPOSE: Ca(2+)-dependent Cl(-) secretion (CaCC) in airways and other tissues is due to activation of the Cl(-) channel TMEM16A (anoctamin 1). Earlier studies suggested that Ca(2+) -activated Cl(-) channels are regulated by membrane lipid inositol phosphates, and that 1-O-octyl-2-O-butyryl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(propionoxymethyl) ester (INO-4995) augments CaCC. Here we examined whether TMEM16A is the target for INO-4995 and if the channel is regulated by inositol phosphates. EXPERIMENTAL APPROACH: The effects of INO-4995 on CaCC were examined in overexpressing HEK293, colonic and primary airway epithelial cells as well as Xenopus oocytes. We used patch clamping, double electrode voltage clamp and Ussing chamber techniques. KEY RESULTS: We found that INO-4995 directly activates a TMEM16A whole cell conductance of 6.1 ± 0.9 nS pF(-1) in overexpressing cells. The tetrakisphosphates Ins(3,4,5,6)P(4) or Ins(1,3,4,5)P(4) and enzymes controlling levels of InsP(4) or PIP(2) and PIP(3) had no effects on the magnitude or kinetics of TMEM16A currents. In contrast in Xenopus oocytes, human airways and colonic cells, which all express TMEM16A endogenously, Cl(-) currents were not acutely activated by INO-4995. However incubation with INO-4995 augmented 1.6- to 4-fold TMEM16A-dependent Cl(-) currents activated by ionomycin or ATP, while intracellular Ca(2+) signals were not affected. The potentiating effect of INO-4995 on transient ATP-activated TMEM16A-currents in cystic fibrosis (CF) airways was twice of that observed in non-CF airways. CONCLUSIONS AND IMPLICATIONS: These data indicate that TMEM16A is the target for INO-4995, although the mode of action appears different for overexpressed and endogenous channels. INO-4995 may be useful for the treatment of CF lung disease.

Airway epithelial cells--functional links between CFTR and anoctamin dependent Cl- secretion.

Airways consist of a heterogeneous population of cells, comprising ciliated cells, Clara cells and goblet cells. Electrolyte secretion by the airways is necessary to produce the airway surface liquid that allows for mucociliary clearance of the lungs. Secretion is driven by opening of Cl(-) selective ion channels in the apical membrane of airway epithelial cells, through either receptor mediated increase in intracellular cAMP or cytosolic Ca(2+). Traditionally cAMP-dependent and Ca(2+)-dependent secretory pathways are regarded as independent. However, this concept has been challenged recently. With identification of the Ca(2+) activated Cl(-) channel TMEM16A (anoctamin 1) and with detailed knowledge of the cAMP-regulated cystic fibrosis transmembrane conductance regulator (CFTR), it has become possible to look more closely into this relationship.

Expression and function of epithelial anoctamins.

Endogenous Ca(2+)-activated Cl(-) currents (CaCCs) are abundant and present in very different cell types. Very good evidence has been provided that endogenous CaCC is produced by anoctamin 1 (Ano1) and Ano2. Insight into the physiological role of anoctamins has been provided for Ano1, Ano2 and Ano6; however, the physiological role of the other seven members of the anoctamin family remains obscure. Anoctamins 1 and 2 may operate as individual Ca(2+)-sensitive channel proteins or may require accessory subunits for complete function. We find that overexpressed Ano1 has properties resembling all those of endogenous CaCCs, although with some noticeable biophysical and regulatory differences when compared with endogenous channels. Apart from Ano1 and Ano2, expression of Ano6 also produces a Cl(-) conductance. Depending on the cellular background, Ano6 currents may have variable properties. Anoctamins 1 and 6 are frequent in epithelial cells, often coexpressed together with Ano8, Ano9 and Ano10. Most available data on anoctamins were obtained from mouse tissues and from cultured cells, which may not be representative of native human tissues.

CFTR and TMEM16A are separate but functionally related Cl- channels.

Previous reports point out to a functional relationship of the cystic fibrosis transmembrane conductance regulator (CFTR) and Ca(2+) activated Cl(-) channels (CaCC). Recent findings showing that TMEM16A forms the essential part of CaCC, prompted us to examine whether CFTR controls TMEM16A. Inhibition of endogenous CaCC by activation of endogenous CFTR was found in 16HBE human airway epithelial cells, which also express TMEM16A. In contrast, CFBE airway epithelial cells lack of CFTR expression, but express TMEM16A along with other TMEM16-proteins. These cells produce CaCC that is inhibited by overexpression and activation of CFTR. In HEK293 cells coexpressing TMEM16A and CFTR, whole cell currents activated by IMBX and forskolin were significantly reduced when compared with cells expressing CFTR only, while the halide permeability sequence of CFTR was not changed. Expression of TMEM16A, but not of TMEM16F, H or J, produced robust CaCC, which that were inhibited by CaCCinh-A01 and niflumic acid, but not by CFTRinh-172. TMEM16A-currents were attenuated by additional expression of CFTR, and were completely abrogated when additionally expressed CFTR was activated by IBMX and forskolin. On the other hand, CFTR-currents were attenuated by additional expression of TMEM16A. CFTR and TMEM16A were both membrane localized and could be coimmunoprecipitated. Intracellular Ca(2+) signals elicited by receptor-stimulation was not changed during activation of CFTR, while ionophore-induced rise in [Ca(2+)](i) was attenuated after stimulation of CFTR. The data indicate that both CFTR and TMEM16 proteins are separate molecular entities that show functional and molecular interaction.