Facet-Dependent Passivation for Efficient Perovskite Solar Cells.

Understanding the interplay between the surface structure and the passivation materials and their effects associated with surface structure modification is of fundamental importance; however, it remains an unsolved problem in the perovskite passivation field. Here, we report a surface passivation principle for efficient perovskite solar cells via a facet-dependent passivation phenomenon. The passivation process selectively occurs on facets, which is observed with various post-treatment materials with different functionality, and the atomic arrangements of the facets determine the alignments of the passivation layers. The profound understanding of facet-dependent passivation leads to the finding of 2-amidinopyridine hydroiodide as the material for a uniform and effective passivation on both (100) and (111) facets. Consequently, we achieved perovskite solar cells with an efficiency of 25.10% and enhanced stability. The concept of facet-dependent passivation can provide an important clue on unidentified passivation principles for perovskite materials and a novel means to enhance the performance and stability of perovskite-based devices.

Highly Sensitive Immuno-CRISPR Assay for CXCL9 Detection.

CRISPR-based detection of target DNA or RNA exploits a dual function, including target sequence-specific recognition followed by trans-cleavage activity of a collateral ssDNA linker between a fluorophore (F) and a quencher (Q), which amplifies a fluorescent signal upon cleavage. In this work, we have extended such dual functionality in a modified immunoassay format to detect a target protein, CXCL9, which is markedly elevated in the urine of kidney transplant recipients undergoing acute rejection episodes. To establish the "immuno-CRISPR" assay, we used anti-CXCL9 antibody-DNA barcode conjugates to target CXCL9 and amplify fluorescent signals via Cas12a-based trans-cleavage activity of FQ reporter substrates, respectively, and in the absence of an isothermal amplification step. To enhance detection sensitivity, the DNA barcode system was engineered by introducing multiple Cas12a recognition sites. Use of biotinylated DNA barcodes enabled self-assembly onto streptavidin (SA) to generate SA-DNA barcode complexes to increase the number and density of Cas12a recognition sites attached to biotinylated anti-CXCL9 antibody. As a result, we improved the rate of CXCL9 detection approximately 8-fold when compared to the use of a monomeric DNA barcode. The limit of detection (LOD) for CXCL9 using the immuno-CRISPR assay was 14 pg/mL, which represented an ∼7-fold improvement when compared to traditional HRP-based ELISA. Selectivity was shown with a lack of crossover reactivity with the related chemokine CXCL1. Finally, we successfully evaluated the presence of CXCL9 in urine samples from 11 kidney transplant recipients using the immuno-CRISPR assay, resulting in 100% accuracy to clinical CXCL9 determination and paving the way for use as a point-of-care noninvasive biomarker for the detection of kidney transplant rejection.

Modular Assembly of Unique Chimeric Lytic Enzymes on a Protein Scaffold Possessing Anti-Staphylococcal Activity.

Lytic enzymes have been considered as potential alternatives to antibiotics. These enzymes, particularly those that target Gram-positive bacteria, consist of modular cell wall-binding and catalytic domains, which can be shuffled with those of other lytic enzymes to produce unnatural chimeric enzymes. In this work, we report the in vitro shuffling of two different modular domains using a protein self-assembly methodology. Catalytic domains (CD) and cell wall-binding domains (BD) from the bacteriocin lysostaphin (Lst) and a putative autolysin from Staphylococcus aureus (SA1), respectively, were genetically site-specifically biotinylated and assembled with streptavidin to generate 23 permuted chimeras. The specific assembly of a CD (3 equiv) and a BD (1 equiv) from Lst and SA1, respectively [CDL-BDS (3:1)], on a streptavidin scaffold yielded high lytic activity against S. aureus (at least 5.6 log reduction), which was higher than that obtained with either native Lst or SA1 alone. Moreover, at 37 °C, the initial rate of cell lysis was over 3-fold higher than that with free Lst, thereby revealing the unique catalytic properties of the chimeric proteins. In vitro self-assembly of functional domains from modular lytic enzymes on a protein scaffold likely expands the repertoire of bactericidal enzymes with improved activities.

Enzyme-Immobilized Chitosan Nanoparticles as Environmentally Friendly and Highly Effective Antimicrobial Agents.

Highly effective and minimally toxic antimicrobial agents have been prepared by immobilizing glucose oxidase (GOx) onto biocompatible chitosan nanoparticles (CS-NPs). CS-NPs were prepared via ionotropic gelation and used for the immobilization of GOx via approaches of covalent attachment (CA), enzyme coating (EC), enzyme precipitate coating (EPC), and magnetic nanoparticle-incorporated EPC (Mag-EPC). EPC represents an approach consisting of enzyme covalent attachment, precipitation, and cross-linking, with CA and EC being control samples while Mag-EPC was prepared by mixing magnetic nanoparticles (Mag) with enzymes during the preparation of EPC. The GOx activities of CA, EC, EPC, and Mag-EPC were 8.57, 17.7, 219, and 247 units/mg CS-NPs, respectively, representing 26 and 12 times higher activity of EPC than those of CA and EC, respectively. EPC improved the activity and stability of GOx and led to good dispersion of CS-NPs, while Mag-EPC enabled facile magnetic separation. To demonstrate the expandability of the EPC approach to other enzymes, bovine carbonic anhydrase was also employed to prepare EPC and Mag-EPC samples for their characterizations. In the presence of glucose, EPC of GOx generated H2O2 in situ, which effectively inhibited the proliferation of Staphylococcus aureus in both suspended cultures and biofilms, thereby demonstrating the potential of EPC-GOx as environmentally friendly and highly effective antimicrobial materials.

3D-cultured neural stem cell microarrays on a micropillar chip for high-throughput developmental neurotoxicology.

Numerous chemicals including environmental toxicants and drugs have not been fully evaluated for developmental neurotoxicity. A key gap exists in the ability to predict accurately and robustly in vivo outcomes based on in vitro assays. This is particularly the case for predicting the toxicity of chemicals on the developing human brain. A critical need for such in vitro assays is choice of a suitable model cell type. To that end, we have performed high-throughput in vitro assessment of proliferation and differentiation of human neural stem cells (hNSCs). Conventional in vitro assays typically use immunofluorescence staining to quantify changes in cell morphology and expression of neural cell-specific biomarkers, which is often time-consuming and subject to variable specificities of available antibodies. To alleviate these limitations, we developed a miniaturized, three-dimensional (3D) hNSC culture with ReNcell VM on microarray chip platforms and established a high-throughput promoter-reporter assay system using recombinant lentiviruses on hNSC spheroids to assess cell viability, self-renewal, and differentiation. Optimum cell viability and spheroid formation of 3D ReNcell VM culture were observed on a micropillar chip over a period of 9 days in a mixture of 0.75% (w/v) alginate and 1 mg/mL growth factor reduced (GFR) Matrigel with 25 mM CaCl2 as a crosslinker for alginate. In addition, 3D ReNcell VM culture exhibited self-renewal and differentiation on the microarray chip platform, which was efficiently monitored by enhanced green fluorescent protein (EGFP) expression of four NSC-specific biomarkers including sex determining region Y-box 2 (SOX2), glial fibrillary acidic protein (GFAP), synapsin1, and myelin basic protein (MBP) with the promoter-reporter assay system.

Unprotonated Short-Chain Alkylamines Inhibit Staphylolytic Activity of Lysostaphin in a Wall Teichoic Acid-Dependent Manner.

Lysostaphin (Lst) is a potent bacteriolytic enzyme that kills Staphylococcus aureus, a common bacterial pathogen of humans and animals. With high activity against both planktonic cells and biofilms, Lst has the potential to be used in industrial products, such as commercial cleansers, for decontamination. However, Lst is inhibited in the presence of monoethanolamine (MEA), a chemical widely used in cleaning solutions and pharmaceuticals, and the underlying mechanism of inhibition remains unknown. In this study, we examined the cell binding and killing capabilities of Lst against S. aureus ATCC 6538 in buffered salt solution with MEA at different pH values (7.5 to 10.5) and discovered that only the unprotonated form of MEA inhibited Lst binding to the cell surface, leading to low Lst activity, despite retention of its secondary structure. This reduced enzyme activity could be largely recovered via a reduction in wall teichoic acid (WTA) biosynthesis through tunicamycin treatment, indicating that the suppression of Lst activity was dependent on the presence and amount of WTA. We propose that the decreased cell binding and killing capabilities of Lst are associated with the influence of uncharged MEA on the conformation of WTA. A similar effect was confirmed with other short-chain alkylamines. This study offers new insight into the impact of short-chain alkylamines on both Lst and WTA structure and function and provides guidance for the application of Lst in harsh environments.IMPORTANCE Lysostaphin (Lst) effectively and selectively kills Staphylococcus aureus, the bacterial culprit of many hospital- and community-acquired skin and respiratory infections and food poisoning. Lst has been investigated in animal models and clinical trials, industrial formulations, and environmental settings. Here, we studied the mechanistic basis of the inhibitory effect of alkylamines, such as monoethanolamine (MEA), a widely used chemical in commercial detergents, on Lst activity, for the potential incorporation of Lst in disinfectant solutions. We have found that protonated MEA has little influence on Lst activity, while unprotonated MEA prevents Lst from binding to S. aureus cells and hence dramatically decreases the enzyme's bacteriolytic efficacy. Following partial removal of the wall teichoic acid, an important component of the bacterial cell envelope, the inhibitory effect of unprotonated MEA on Lst is reduced. This phenomenon can be extended to other short-chain alkylamines. This mechanistic report of the impact of alkylamines on Lst functionality will help guide future applications of Lst in disinfection and decontamination of health-related commercial products.

In vitro gene expression-coupled bacterial cell chip for screening species-specific antimicrobial enzymes.

Targeting infectious bacterial pathogens is important for reducing the evolution of antibiotic-resistant bacteria and preserving the endogenous human microbiome. Cell lytic enzymes including bacteriophage endolysins, bacterial autolysins, and other bacteriolysins are useful antibiotic alternatives due to their exceptional target selectivity, which may be used to lysins rapidly kill target bacteria and their high specificity permit the normal commensal microflora to be left undisturbed. Genetic information of numerous lysins is currently available, but the identification of their antimicrobial function and specificity has been limited because most lysins are often poorly expressed and exhibit low solubilities. Here, we report the development of bacterial cell chip for rapidly accessing the function of diverse genes that are suggestive of encoding lysins. This approach can be used to evaluate rapidly the species-specific antimicrobial activity of diverse lysins synthesized from in vitro transcription and translation (TNT) of plasmid DNA. In addition, new potent lysins can be assessed that are not expressed in hosts and display low solubility. As a result of evaluating the species-specific antimicrobial function of 11 (un)known lysins with an in vitro TNT-coupled bacterial cell chip, a potent recombinant lysin against Staphylococcus strains, SA1, was identified. The SA1 was highly potent against not only S. aureus, but also both lysostaphin-resistant S. simulans and S. epidermidis cells. To this end, the SA1 may be applicable to treat both methicillin-resistant S. aureus (MRSA) and lysostaphin-resistant MRSA mutants. Biotechnol. Bioeng. 2017;114: 1648-1657. © 2017 Wiley Periodicals, Inc.

Detection of cerebrospinal fluid leakage by specific measurement of transferrin glycoforms.

A simple and rapid detection of cerebrospinal fluid (CSF) leakage would benefit spine surgeons making critical postoperative decisions on patient care. We have assessed novel approaches to selectively determine CSF ß2-transferrin (ß2TF), an asialo-transferrin (aTF) biomarker, without interference from serum sialo-transferrin (sTF) in test samples. First, we performed mild periodate oxidation to selectively generate aldehyde groups in sTF for capture with magnetic hydrazide microparticles, and selective removal with a magnetic separator. Using this protocol sTF was selectively removed from mixtures of CSF and serum containing CSF aTF (ß2TF) and serum sTF, respectively. Second, a two-step enzymatic method was developed with neuraminidase and galactose oxidase for generating aldehyde groups in sTF present in CSF and serum mixtures for magnetic hydrazide microparticle capture. After selectively removing sTF from mixtures of CSF and serum, ELISA could detect significant TF signal only in CSF, while the TF signal in serum was negligible. The new approach for selective removal of only sTF in test samples will be promising for the required intervention by a spine surgeon.

High sensitivity detection of active botulinum neurotoxin by glyco-quantitative polymerase chain-reaction.

The sensitive detection of highly toxic botulinum neurotoxin (BoNT) from Clostridium botulinum is of critical importance because it causes human illnesses if foodborne or introduced in wounds and as an iatrogenic substance. Moreover, it has been recently considered a possible biological warfare agent. Over the past decade, significant progress has been made in BoNT detection technologies, including mouse lethality assays, enzyme-linked immunosorbent assays, and endopeptidase assays and by mass spectrometry. Critical assay requirements, including rapid assay, active toxin detection, sensitive and accurate detection, still remain challenging. Here, we present a novel method to detect active BoNTs using a Glyco-quantitative polymerase chain-reaction (qPCR) approach. Sialyllactose, which interacts with the binding-domain of BoNTs, is incorporated into a sialyllactose-DNA conjugate as a binding-probe for active BoNT and recovered through BoNT-immunoprecipitation. Glyco-qPCR analysis of the bound sialyllactose-DNA is then used to detect low attomolar concentrations of BoNT and attomolar to femtomolar concentrations of BoNT in honey, the most common foodborne source of infant botulism.

Ultrasensitive ImmunoMag-CRISPR Lateral Flow Assay for Point-of-Care Testing of Urinary Biomarkers.

Rapid, accurate, and noninvasive detection of biomarkers in saliva, urine, or nasal fluid is essential for the identification, early diagnosis, and monitoring of cancer, organ failure, transplant rejection, vascular diseases, autoimmune disorders, and infectious diseases. We report the development of an Immuno-CRISPR-based lateral flow assay (LFA) using antibody-DNA barcode complexes with magnetic enrichment of the target urinary biomarkers CXCL9 and CXCL10 for naked eye detection (ImmunoMag-CRISPR LFA). An intermediate approach involving a magnetic bead-based Immuno-CRISPR assay (ImmunoMag-CRISPR) resulted in a limit of detection (LOD) of 0.6 pg/mL for CXCL9. This value surpasses the detection limits achieved by previously reported assays. The highly sensitive detection method was then re-engineered into an LFA format with an LOD of 18 pg/mL for CXCL9, thereby enabling noninvasive early detection of acute kidney transplant rejection. The ImmunoMag-CRISPR LFA was tested on 42 clinical urine samples from kidney transplant recipients, and the assay could determine 11 positive and 31 negative urinary samples through a simple visual comparison of the test line and the control line of the LFA strip. The LFA system was then expanded to quantify the CXCL9 and CXCL10 levels in clinical urine samples from images. This approach has the potential to be extended to a wide range of point-of-care tests for highly sensitive biomarker detection.

Split fluorescent protein-mediated multimerization of cell wall binding domain for highly sensitive and selective bacterial detection.

Cell wall peptidoglycan binding domains (CBDs) of cell lytic enzymes, including bacteriocins, autolysins and bacteriophage endolysins, enable highly selective bacterial binding, and thus, have potential as biorecognition molecules for nondestructive bacterial detection. Here, a novel design for a self-complementing split fluorescent protein (FP) complex is proposed, where a multimeric FP chain fused with specific CBDs ((FP-CBD)n) is assembled inside the cell, to improve sensitivity by enhancing the signal generated upon Staphylococcus aureus or Bacillus anthracis binding. Flow cytometry shows enhanced fluorescence on the cell surface with increasing FP stoichiometry and surface plasmon resonance reveals nanomolar binding affinity to isolated peptidoglycan. The breadth of function of these complexes is demonstrated through the use of CBD modularity and the ability to attach enzymatic detection modalities. Horseradish peroxidase-coupled (FP-CBD)n complexes generate a catalytic amplification, with the degree of amplification increasing as a function of FP length, reaching a limit of detection (LOD) of 103 cells/droplet (approximately 0.1 ng S. aureus or B. anthracis) within 15 min on a polystyrene surface. These fusion proteins can be multiplexed for simultaneous detection. Multimeric split FP-CBD fusions enable use as a biorecognition molecule with enhanced signal for use in bacterial biosensing platforms.

Proximal High-Index Metamaterials based on a Superlattice of Gold Nanohexagons Targeting the Near-Infrared Band.

Plasmonic nanoparticles can be assembled into a superlattice, to form optical metamaterials, particularly targeting precise control of optical properties such as refractive index (RI). The superlattices exhibit enhanced near-field, given the sufficiently narrow gap between nanoparticles supporting multiple plasmonic resonance modes only realized in proximal environments. Herein, the planar superlattice of plasmonic Au nanohexagons (AuNHs) with precisely controlled geometries such as size, shape, and edge-gaps is reported. The proximal AuNHs superlattice realized over a large area with selective edge-to-edge assembly exhibited the highest-ever-recorded RI values in the near-infrared (NIR) band, surpassing the upper limit of the RI of the natural intrinsic materials (up to 10.04 at λ = 1.5 µm). The exceptionally enhanced RI is derived from intensified in-plane surface plasmon coupling across the superlattices. Precise control of the edge-gap of neighboring AuNHs systematically tuned the RI as confirmed by numerical analysis based on the plasmonic percolation model. Furthermore, a 1D photonic crystal, composed of alternating layers of AuNHs superlattices and low-index polymers, is constructed to enhance the selectivity of the reflectivity operating in the NIR band. It is expected that the proximal AuNHs superlattices can be used as new optical metamaterials that can be extended to the NIR range.

p-Phenylenediamine-Bridged Binder-Electrolyte-Unified Supramolecules for Versatile Lithium Secondary Batteries.

The binder is an essential component in determining the structural integrity and ionic conductivity of Li-ion battery electrodes. However, conventional binders are not sufficiently conductive and durable to be used with solid-state electrolytes. In this study, a novel system is proposed for a Li secondary battery that combines the electrolyte and binder into a unified structure, which is achieved by employing para-phenylenediamine (pPD) moiety to create supramolecular bridges between the parent binders. Due to a partial crosslinking effect and charge-transferring structure of pPD, the proposed strategy improves both the ionic conductivity and mechanical properties by a factor of 6.4 (achieving a conductivity of 3.73 × 10-4 S cm-1 for poly(ethylene oxide)-pPD) and 4.4 (reaching a mechanical strength of 151.4 kPa for poly(acrylic acid)-pPD) compared to those of conventional parent binders. As a result, when the supramolecules of pPD are used as a binder in a pouch cell with a lean electrolyte loading of 2 µL mAh-1 , a capacity retention of 80.2% is achieved even after 300 cycles. Furthermore, when it is utilized as a solid-state electrolyte, an average Coulombic efficiency of 99.7% and capacity retention of 98.7% are attained under operations at 50 °C without external pressure or a pre-aging process.

FGF-FGFR signaling mediated through glycosaminoglycans in microtiter plate and cell-based microarray platforms.

Fibroblast growth factor (FGF) signals cell growth through its interaction with a fibroblast growth factor receptor (FGFR) and a glycosaminoglycn (GAG) coreceptor. Here, we examine the signaling of five different FGFs (1, 2, 6, 8, and 8b) through FGFR3c. A small library of GAG and GAG-derivative coreceptors are screened to understand better the structure-activity relationship of these coreceptors on signaling. Initially, data were collected in a microtiter plate well-based cell proliferation assay. In an effort to reduce reagent requirements and improve assay throughput, a cell-based microarray platform was developed. In this cell-based microarray, FGFR3c-expressing cells were printed in alginate hydrogel droplets of ∼30 nL and incubated with FGF and GAG. Heparin was the most effective GAG coreceptor for all FGFs studied. Other GAGs, such as 2-O-desulfated heparin and chondroitin sulfate B, were also effective coreceptors. Signaling by FGF 8 and FGF 8b showed the widest tolerance for coreceptor structure. Finally, this on-chip cell-based microarray provides comparable data to a microtiter plate well-based assay, demonstrating that the coreceptor assay can be converted into a high-throughput assay.

Highly Sensitive Detection of Bacteria by Binder-Coupled Multifunctional Polymeric Dyes.

Infectious diseases caused by pathogens are a health burden, but traditional pathogen identification methods are complex and time-consuming. In this work, we have developed well-defined, multifunctional copolymers with rhodamine B dye synthesized by atom transfer radical polymerization (ATRP) using fully oxygen-tolerant photoredox/copper dual catalysis. ATRP enabled the efficient synthesis of copolymers with multiple fluorescent dyes from a biotin-functionalized initiator. Biotinylated dye copolymers were conjugated to antibody (Ab) or cell-wall binding domain (CBD), resulting in a highly fluorescent polymeric dye-binder complex. We showed that the unique combination of multifunctional polymeric dyes and strain-specific Ab or CBD exhibited both enhanced fluorescence and target selectivity for bioimaging of Staphylococcus aureus by flow cytometry and confocal microscopy. The ATRP-derived polymeric dyes have the potential as biosensors for the detection of target DNA, protein, or bacteria, as well as bioimaging.

Unveiling facet-dependent degradation and facet engineering for stable perovskite solar cells.

A myriad of studies and strategies have already been devoted to improving the stability of perovskite films; however, the role of the different perovskite crystal facets in stability is still unknown. Here, we reveal the underlying mechanisms of facet-dependent degradation of formamidinium lead iodide (FAPbI3) films. We show that the (100) facet is substantially more vulnerable to moisture-induced degradation than the (111) facet. With combined experimental and theoretical studies, the degradation mechanisms are revealed; a strong water adhesion following an elongated lead-iodine (Pb-I) bond distance is observed, which leads to a δ-phase transition on the (100) facet. Through engineering, a higher surface fraction of the (111) facet can be achieved, and the (111)-dominated crystalline FAPbI3 films show exceptional stability against moisture. Our findings elucidate unknown facet-dependent degradation mechanisms and kinetics.

Suramin binds and inhibits infection of SARS-CoV-2 through both spike protein-heparan sulfate and ACE2 receptor interactions.

SARS-CoV-2 receptor binding domains (RBDs) interact with both the ACE2 receptor and heparan sulfate on the surface of host cells to enhance SARS-CoV-2 infection. We show that suramin, a polysulfated synthetic drug, binds to the ACE2 receptor and heparan sulfate binding sites on the RBDs of wild-type, Delta, and Omicron variants. Specifically, heparan sulfate and suramin had enhanced preferential binding for Omicron RBD, and suramin is most potent against the live SARS-CoV-2 Omicron variant (B.1.1.529) when compared to wild type and Delta (B.1.617.2) variants in vitro. These results suggest that inhibition of live virus infection occurs through dual SARS-CoV-2 targets of S-protein binding and previously reported RNA-dependent RNA polymerase inhibition and offers the possibility for this and other polysulfated molecules to be used as potential therapeutic and prophylactic options against COVID-19.

Percolated Plasmonic Superlattices of Nanospheres with 1 nm-Level Gap as High-Index Metamaterials.

Nanophotonics relies on precise control of refractive index (RI) which can be designed with metamaterials. Plasmonic superstructures of nanoparticles (NPs) can suggest a versatile way of tuning RI. However, the plasmonic effects in the superstructures demand 1 nm-level exquisite control over the interparticle gap, which is challenging in a sub-wavelength NPs. Thus far, a large-area demonstration has been mostly discouraged. Here, heteroligand AuNPs are prepared, which are stable in oil but become Janus particles at the oil-water interface, called "adaptive Janus particles." NPs are bound at the interface and assembled into 2D arrays over square centimeters as toluene evaporates, which distinctively exhibits the RI tunability. In visible and NIR light, the 2D superstructures exhibit the highest-ever RI (≈7.8) with varying the size and interparticle gap of NPs, which is successfully explained by a plasmonic percolation model. Furthermore, fully solution-processable 2D plasmonic superstructures are proved to be advantageous in flexible photonic devices such as distributed Bragg reflectors.

Controlled synthesis of solid-shelled non-spherical and faceted microbubbles.

The ability to control the shape of hollow particles (e.g., capsules or bubbles) holds great promise for enhancing the encapsulation efficiency and mechanical/optical properties. However, conventional preparation methods suffer from a low yield, difficulty in controlling the shape, and a tedious production process, limiting their widespread application. Here, we present a method for fabricating polyhedral graphene oxide (GO)-shelled microbubbles with sharp edges and vertices, which is based on the microfluidic generation of spherical compound bubbles followed by shell deformation. Sphere-to-polytope deformation is a result of the shell instability due to gradual outward gas transport, which is dictated by Laplace pressure across the shell. The shape-variant behaviours of the bubbles can also be attributed to the compositional heterogeneity of the shells. In particular, the high degree of control of microfluidic systems enables the formation of non-spherical bubbles with various shapes; the structural motifs of the bubbles are easily controlled by varying the size and thickness of the mid-shell in compound bubbles, ranging from tetrahedra to octahedra. The strategy presented in this study provides a new route for fabricating 3D structured solid bubbles, which is particularly advantageous for the development of high-performance mechanical or thermal material applications.

Interactions between heparin and SARS-CoV-2 spike glycoprotein RBD from omicron and other variants.

Heparan sulfate (HS) acts as a co-receptor of angiotensin-converting enzyme 2 (ACE2) by interacting with severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) spike glycoprotein (SGP) facilitating host cell entry of SARS-CoV-2 virus. Heparin, a highly sulfated version of heparan sulfate (HS), interacts with a variety of proteins playing key roles in many physiological and pathological processes. In this study, SARS-CoV-2 SGP receptor binding domain (RBD) wild type (WT), Delta and Omicron variants were expressed in Expi293F cells and used in the kinetic and structural analysis on their interactions with heparin. Surface plasmon resonance (SPR) analysis showed the binding kinetics of SGP RBD from WT and Delta variants were very similar while Omicron variant SGP showed a much higher association rate. The SGP from Delta and Omicron showed higher affinity (K D ) to heparin than the WT SGP. Competition SPR studies using heparin oligosaccharides indicated that binding of SGP RBDs to heparin requires chain length greater than 18. Chemically modified heparin derivatives all showed reduced interactions in competition assays suggesting that all the sulfo groups in the heparin polysaccharide were critical for binding SGP RBDs with heparin. These interactions with heparin are pH sensitive. Acidic pH (pH 6.5, 5.5, 4.5) greatly increased the binding of WT and Delta SGP RBDs to heparin, while acidic pH slightly reduced the binding of Omicron SGP RBD to heparin compared to binding at pH 7.3. In contrast, basic pH (pH 8.5) greatly reduced the binding of Omicron SGP RBDs to heparin, with much less effects on WT or Delta. The pH dependence indicates different charged residues were present at the Omicron SGP-heparin interface. Detailed kinetic and structural analysis of the interactions of SARS-CoV-2 SGP RBDs with heparin provides important information for designing anti-SARS-CoV-2 molecules.