Effects of Chemical Fixatives on Kinetic Measurements of Biomolecular Interaction on Cell Membrane.

Understanding the interaction between ligands and membrane proteins is important for drug design and optimization. Although investigation using live cells is desirable, it is not feasible in some circumstances and cell fixation is performed to reduce cell motion and degradation. This study compared the effects of five fixatives, i.e., formaldehyde vapor (FV), paraformaldehyde (PFA), acetone, methanol, and ethanol, on kinetic measurements via the LigandTracer method. We found that all five fixatives exerted insignificant effects on lectin-glycan interaction. However, antibody-receptor interaction is markedly perturbed by coagulant fixatives. The acetone fixation changed the binding of the anti-human epidermal growth factor receptor 2 (HER2) antibody to HER2 on the cell membrane from a 1:2 to a 1:1 binding model, while methanol and ethanol abolished the antibody binding possibly by removal of the HER2 receptors on the cell membrane. The capability of binding was retained when methanol fixation was performed at lower temperatures, albeit with a binding model of 1:1 instead. Moreover, whereas cell morphology does not exert a substantial impact on lectin-glycan interaction, it can indeed modify the binding model of antibody-receptor interaction. Our results provided insights into the selection of fixatives for cell-based kinetic studies.

Live Cells versus Fixated Cells: Kinetic Measurements of Biomolecular Interactions with the LigandTracer Method and Surface Plasmon Resonance Microscopy.

Cell-based kinetic studies of ligand or candidate drug binding to membrane proteins have produced affinity and kinetic values that are different from measurements using purified proteins. However, ligand binding to fixated cells whose membrane constituents (e.g., proteins and their glycosylated forms) are partially connected by a cross-linking reagent has not been compared to that to live cells. Under the same experimental conditions for the LigandTracer method, we measured the interactions of fluorophore-labeled lectins and antibody molecules with glycans at HFF cells and the human epithelial growth receptor 2 at SKBR3 cells, respectively. In conjunction with surface plasmon resonance microscopy, the effects of labels and cell/sub-cell heterogeneity on binding kinetics were investigated. Our results revealed that, for cell constituents whose structures and functions are not closely dependent on cell viability, the ligand binding kinetics at fixated cells is only slightly different from that at live cells. The altered kinetics is explained on the basis of a less mobile receptor confined in a local environment created by partially interconnected protein molecules. We show that cell/sub-cell heterogeneity and labels on the ligands can alter the binding reaction more significantly. Thus, fixating cells not only simplifies experimental procedures for drug screening and renders assays more robust but also provides reliable kinetic information about drug binding to cell constituents whose structures are not changed by chemical fixation.

Microfluidically Partitioned Dual Channels for Accurate Background Subtraction in Cellular Binding Studies by Surface Plasmon Resonance Microscopy.

Unlike conventional surface plasmon resonance (SPR) using an antifouling film to anchor biomolecules and a reference channel for background subtraction, SPR microscopy for single-cell analysis uses a protein- or polypeptide-modified gold substrate to immobilize cells and a cell-free area as the reference. In this work, we show that such a substrate is prone to nonspecific adsorption (NSA) of species from the cell culture media, resulting in false background signals that cannot be correctly subtracted. To obtain accurate kinetic results, we patterned a dual-channel substrate using a microfluidic device, with one channel having poly-l-lysine deposited in situ onto a preformed polyethylene glycol (PEG) self-assembled monolayer for cell immobilization and the other channel remaining as PEG-covered for reference. The two 2.0 mm-wide channels are separated by a 75 µm barrier, and parts of the channels can be readily positioned into the field of view of an SPR microscope. The use of this dual-channel substrate for background subtraction is contrasted with the conventional approach through the following binding studies: (1) wheat germ agglutinin (WGA) attachment to the N-acetyl glucosamine and N-acetyl-neuraminic acid sites of glycans on HFF cells, and (2) the S1 protein of the COVID-19 virus conjugation with angiotensin-converting enzyme 2 (ACE2) on the HEK293 cells. Both studies revealed that interferences by NSA and the surface plasmon polariton wave diffracted by cells can be excluded with the dual-channel substrate, and the much smaller refractive index changes caused by the injected solutions can be correctly subtracted. Consequently, sensorgrams with higher signal-to-noise ratios and shapes predicted by the correct binding model can be obtained with accurate kinetic and affinity parameters that are more biologically relevant. The affinity between S1 protein and ACE2 is comparable to that measured with recombinant ACE2, yet the binding kinetics is different, suggesting that the cell membrane does impose a kinetic barrier to their interaction.

A Four-Channel Surface Plasmon Resonance Sensor Functionalized Online for Simultaneous Detections of Anti-SARS-CoV-2 Antibody, Free Viral Particles, and Neutralized Viral Particles.

Current tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detect either the constituent nucleic acids/proteins of the viral particles or antibodies specific to the virus, but cannot provide information about viral neutralization by an antibody and the efficacy of an antibody. Such information is important about individuals' vulnerability to severe symptoms or their likelihood of showing no symptoms. We immobilized online SARS-CoV-2 spike (S1) protein and angiotensin-converting enzyme 2 (ACE2) into separate surface plasmon resonance (SPR) channels of a tris-nitrilotriacetic acid (tris-NTA) chip to simultaneously detect the anti-S1 antibody and viral particles in serum samples. In addition, with a high-molecular-weight-cutoff filter, we separated the neutralized viral particles from the free antibody molecules and used a sensing channel immobilized with Protein G to determine antibody-neutralized viral particles. The optimal density of probe molecules in each fluidic channel can be precisely controlled through the closure and opening of the specific ports. By utilizing the high surface density of ACE2, multiple assays can be carried out without regenerations. These three species can be determined with a short analysis time (<12 min per assay) and excellent sensor-to-sensor/cycle-to-cycle reproducibility (RSD < 5%). When coupled with an autosampler, continuous assays can be performed in an unattended manner at a single chip for up to 6 days. Such a sensor capable of assaying serum samples containing the three species at different levels provides additional insights into the disease status and immunity of persons being tested, which should be helpful for containing the SARS-CoV-2 spread during the era of incessant viral mutations.

Regenerable and high-throughput surface plasmon resonance assay for rapid screening of anti-SARS-CoV-2 antibody in serum samples.

Current serological antibody tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) require enzyme or fluorescent labels, and the titer well plates cannot be reused. By immobilizing histidine (His)-tagged SARS-CoV-2 spike (S1) protein onto tris‒nitrilotriacetic acid (tris-NTA) sensor and using the early association phase for mass-transfer-controlled concentration determination, we developed a rapid and regenerable surface plasmon resonance (SPR) method for quantifying anti-SARS-CoV-2 antibody. On a five-channel SPR instrument and with optimized S1 protein immobilization density, each of the four analytical channels is sequentially used for multiple measurements, and all four channels can be simultaneously regenerated once they have reached a threshold value. Coupled with a programmable autosampler, each sensor can be regenerated at least 20 times, enabling uninterrupted assays of more than 800 serum samples. The accuracy and speed of our method compare well with those of the enzyme-linked immunosorbent assay (ELISA), and the detection limit (0.057 µg mL-1) can easily meet the requirement for screening low antibody levels such as those in convalescent patients. In addition, our method exhibits excellent channel-to-channel (RSD = 1.9%) and sensor-to-sensor (RSD = 2.1%) reproducibility. Obviation of an enzyme label drastically reduced the assay cost, rending our method (<60 cents) much more cost effective than those of commercial ELISA kits ($4.4-11.4). Therefore, our method offers a cost-effective and high-throughput alternative to the existing methods for serological measurements of anti-SARS-CoV-2 antibody levels, holding great promise for rapid screening of clinical samples without elaborate sample pretreatments and special reagents.

Design and Synthesis of a New Mannitol Stearate Ester-Based Aluminum Alkoxide as a Novel Tri-Functional Additive for Poly(Vinyl Chloride) and Its Synergistic Effect with Zinc Stearate.

Thermal stabilizers, lubricant, and plasticizers are three crucial additives for processing poly(vinyl chloride) (PVC). In this study, a new mannitol stearate ester-based aluminum alkoxide (MSE-Al) was designed and synthesized as a novel additive for PVC. The thermal stability and processing performance of PVC stabilized by MSE-Al were evaluated by the Congo red test, conductivity measurement, thermal aging test, ultravioletevisible (UV-Vis) spectroscopy test, and torque rheometer test. Results showed that the addition of MSE-Al could not only markedly improve the long-term thermal stability of PVC, but also greatly accelerate the plasticizing and decrease the balance torque, which demonstrated that MSE-Al possessed a lubricating property. Thus, MSE-Al was demonstrated to be able to provide tri-functional additive roles, e.g., thermal stabilizer, plasticizer, and lubricant. The test results for the thermal stability of PVC indicated that the initial whiteness of PVC stabilized by MSE-Al was not good enough, thus the synergistic effect of MSE-Al with zinc stearates (ZnSt2) on the thermal stability of PVC was also investigated. The results showed that there is an appreciable synergistic effect between MSE-Al and ZnSt2. The thermal stabilization mechanism and synergism effect of MSE-Al with ZnSt2 are then discussed.