A colorimetric lateral flow immunoassay based on oriented antibody immobilization for sensitive detection of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). The high human-to-human transmission and rapid evolution of SARS-CoV-2 have resulted in a worldwide pandemic. To contain SARS-CoV-2, it is essential to efficiently control the transmission of the virus through the early diagnosis of infected individuals, including asymptomatic people. Therefore, a rapid and accurate assay is vital for the early diagnosis of SARS-CoV-2 in suspected individuals. In this study, we developed a colorimetric lateral flow immunoassay (LFIA) in which a CBP31-BC linker was used to immobilize antibodies on a cellulose membrane in an oriented manner. The developed LFIA enabled sensitive detection of cultured SARS-CoV-2 in 15 min with a detection limit of 5 × 104 copies/mL. The clinical performance of the LFIA for detecting SARS-CoV-2 was evaluated using 19 clinical samples validated by reverse transcription-polymerase chain reaction (RT-PCR). The LFIA detected all the positive and negative samples accurately, corresponding to 100% accuracy. Importantly, patient samples with low viral loads were accurately identified. Thus, the proposed method can provide a useful platform for rapid and accurate point-of-care testing of SARS-CoV-2 in infected individuals to efficiently control the COVID-19 pandemic.

Enzyme immobilization on porous chitosan hydrogel capsules formed by anionic surfactant gelation.

OBJECTIVES: Sodium dodecyl sulfate (SDS)-chitosan hydrogels have been employed for adsorption of anionic dyes and metallic substances. Two mutant forms of Thermoanaerobacter ethanolicus alcohol dehydrogenase (TeSADH) were used as model enzymes to develop a novel enzyme immobilization technique employing newly formulated porous chitosan hydrogels. RESULTS: The enzyme immobilized on chitosan hydrogel capsules formed by 5 g/l SDS gelation and subsequent treatment with 0.05 M NaOH was 28-35% higher in NADPH production than that formed by 20 g/l SDS gelation only under the same conditions. A 48-h asymmetric biphasic reduction of acetophenone with immobilized TeSADH enzyme at 50 °C showed 68% increase in (R)-1-phenylethanol production than the free enzyme. Compared to the free enzyme which denatured and lost its activity at 80 °C, the immobilized enzyme retained about 25% of its initial activity after 2-h incubation. CONCLUSION: In contrast to the conventional chitosan hydrogel which suffers thermal and operational stability, the newly formulated porous chitosan hydrogel capsules have excellent enzyme loading efficiency and stable at harsh temperatures. Especially, this newly developed enzyme immobilization method would be applicable for food processing.

Coexpression of CMP-sialic acid transporter reduces N-glycolylneuraminic acid levels of recombinant glycoproteins in Chinese hamster ovary cells.

Recombinant glycoproteins expressed in Chinese hamster ovary (CHO) cells contain two forms of sialic acids; N-acetylneuraminic acid (Neu5Ac) as a major type and N-glycolylneuraminic acid (Neu5Gc) as a minor type. The Neu5Gc glycan moieties in therapeutic glycoproteins can elicit immune responses because they do not exist in human. In the present work, to reduce Neu5Gc levels of recombinant glycoproteins from CHO cell cultures, we coexpressed cytidine-5'-monophosphate-sialic acid transporter (CMP-SAT) that is an antiporter and transports cytosolic CMP-sialic acids (both forms) into Golgi lumen. When human erythropoietin was used as a target human glycoprotein, coexpression of CMP-SAT resulted in a significant decrease of Neu5Gc level by 41.4% and a notable increase of Neu5Ac level by 21.2%. This result could be reasonably explained by our hypothesis that the turnover rate of Neu5Ac to Neu5Gc catalyzed by CMP-Neu5Ac hydroxylase would be reduced through facilitated transportation of Neu5Ac into Golgi apparatus by coexpression of CMP-SAT. We confirmed the effects of CMP-SAT coexpression on the decrease of Neu5Gc level and the increase of Neu5Ac level using another glycoprotein human DNase I. Therefore, CMP-SAT coexpression might be an effective strategy to reduce the levels of undesired Neu5Gc in recombinant therapeutic glycoproteins from CHO cell cultures.

I86A/C295A mutant secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus has broadened substrate specificity for aryl ketones.

Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase (SADH) reduces aliphatic ketones according to Prelog's Rule, with binding pockets for small and large substituents. It was shown previously that the I86A mutant SADH reduces acetophenone, which is not a substrate of wild-type SADH, to give the anti-Prelog R-product (Musa, M. M.; Lott, N.; Laivenieks, M.; Watanabe, L.; Vieille, C.; Phillips, R. S. ChemCatChem2009, 1, 89-93.). However, I86A SADH did not reduce aryl ketones with substituents larger than fluorine. We have now expanded the small pocket of the active site of I86A SADH by mutation of Cys-295 to alanine to allow reaction of substituted acetophenones. As predicted, the double mutant I86A/C295A SADH has broadened substrate specificity for meta-substituted, but not para-substituted, acetophenones. However, the increase of the substrate specificity of I86A/C295A SADH is accompanied by a decrease in the kcat/Km values of acetophenones, possibly due to the substrates fitting loosely inside the more open active site. Nevertheless, I86A/C295A SADH gives high conversions and very high enantiomeric excess of the anti-Prelog R-alcohols from the tested substrates.

Mechanically Durable and Biologically Favorable Protein Hydrogel Based on Elastic Silklike Protein Derived from Sea Anemone.

As biodegradable scaffolds, protein hydrogels have considerable potential, particularly for bioartificial organs and three-dimensional space-filling materials. However, their low strength and stiffness have been considered to be limitations for enduring physiological stimuli. Therefore, protein hydrogels have been commonly utilized as delivery vehicles rather than as supporting materials. In this work, sea anemone tentacle-derived recombinant silk-like protein (aneroin) was evaluated as a potential material for a mechanically durable protein hydrogel. Inspired by the natural hardening mechanism, photoinitiated dityrosine cross-linking was employed to fabricate an aneroin hydrogel. It was determined that the fabricated aneroin hydrogel was approximately 10-fold stiffer than mammalian cardiac or skeletal muscle. The aneroin hydrogel provided not only structural support but also an adequate environment for cells. It exhibited an adequate swelling ability and microstructure, which are beneficial for facilitating mass transport and cell proliferation. Based on its mechanical and biological properties, this aneroin hydrogel could be used in various biomedical applications, such as cell-containing patches, biomolecule carriers, and artificial extracellular matrices.

Optical detection of paraoxon using single-walled carbon nanotube films with attached organophosphorus hydrolase-expressed Escherichia coli.

In whole-cell based biosensors, spectrophotometry is one of the most commonly used methods for detecting organophosphates due to its simplicity and reliability. The sensor performance is directly affected by the cell immobilization method because it determines the amount of cells, the mass transfer rate, and the stability. In this study, we demonstrated that our previously-reported microbe immobilization method, a microbe-attached single-walled carbon nanotube film, can be applied to whole-cell-based organophosphate sensors. This method has many advantages over other whole-cell organophosphate sensors, including high specific activity, quick cell immobilization, and excellent stability. A device with circular electrodes was fabricated for an enlarged cell-immobilization area. Escherichia coli expressing organophosphorus hydrolase in the periplasmic space and single-walled carbon nanotubes were attached to the device by our method. Paraoxon was hydrolyzed using this device, and detected by measuring the concentration of the enzymatic reaction product, p-nitrophenol. The specific activity of our device was calculated, and was shown to be over 2.5 times that reported previously for other whole-cell organophosphate sensors. Thus, this method for generation of whole-cell-based OP biosensors might be optimal, as it overcomes many of the caveats that prevent the widespread use of other such devices.

Genetically engineered bacteriophages as novel nanomaterials: applications beyond antimicrobial agents.

Bacteriophages, also known as phages, are viruses that replicate in bacteria and archaea. Phages were initially discovered as antimicrobial agents, and they have been used as therapeutic agents for bacterial infection in a process known as "phage therapy." Recently, phages have been investigated as functional nanomaterials in a variety of areas, as they can function not only as therapeutic agents but also as biosensors and tissue regenerative materials. Phages are nontoxic to humans, and they possess self-assembled nanostructures and functional properties. Additionally, phages can be easily genetically modified to display specific peptides or to screen for functional peptides via phage display. Here, we demonstrated the application of phage nanomaterials in the context of tissue engineering, sensing, and probing.

Structural evaluation of GM1-related carbohydrate-cholera toxin interactions through surface plasmon resonance kinetic analysis.

Surface plasmon resonance (SPR) can provide kinetic information about an interaction, and it can also be used to rapidly monitor dynamic processes, such as adsorption and degradation, without the need for sample labeling. Here, we employed SPR to analyze carbohydrate-protein interactions, particularly GM1-related carbohydrate-Vibrio cholera toxin interactions. The interaction between cholera toxin subunits A (ctxA) and B (ctxB) was similar to general ligand-receptor interactions. After the direct immobilization of thiol-containing GM1 pentasaccharide on a gold surface, the GM1-ctxB interaction kinetics were evaluated, and they showed a similar degree of kinetics as reported in previous reports. We found that ctxA had a high affinity for the GM1-ctxAB complex, although its equilibrium dissociation constant was 10 times lower than that of GM1-ctxB binding. Comparative analyses of GM1-related carbohydrate-ctxAB interactions were also conducted to determine the kinetic values of several GM1 analogues with different structures, although their kinetic values were one order of magnitude lower than those of the GM1-ctxAB interaction. The kinetic analysis results for the interactions of GM1 analogues and ctxAB indicated that the sialic acid thumb is important for recognition, and the terminal galactose and N-acetylgalactosamine fingers are required to stabilize the GM1-ctxAB interaction. Taken together, our results indicate that the direct immobilization of carbohydrate in an SPR-based analytical system can be used to evaluate the structural contribution of carbohydrate moieties in carbohydrate-protein interactions, as well as provide valuable information that can be used to understand the interactions.

The final conformation of the complete ectodomain of the HA2 subunit of influenza hemagglutinin can by itself drive low pH-dependent fusion.

One of the best characterized fusion proteins, the influenza virus hemagglutinin (HA), mediates fusion between the viral envelope and the endosomal membrane during viral entry into the cell. In the initial conformation of HA, its fusogenic subunit, the transmembrane protein HA2, is locked in a metastable conformation by the receptor-binding HA1 subunit of HA. Acidification in the endosome triggers HA2 refolding toward the final lowest energy conformation. Is the fusion process driven by this final conformation or, as often suggested, by the energy released by protein restructuring? Here we explored structural properties as well as the fusogenic activity of the full sized trimeric HA2(1-185) (here called HA2*) that presents the final conformation of the HA2 ectodomain. We found HA2* to mediate fusion between lipid bilayers and between biological membranes in a low pH-dependent manner. Two mutations known to inhibit HA-mediated fusion strongly inhibited the fusogenic activity of HA2*. At surface densities similar to those of HA in the influenza virus particle, HA2* formed small fusion pores but did not expand them. Our results confirm that the HA1 subunit responsible for receptor binding as well as the transmembrane and cytosolic domains of HA2 is not required for fusion pore opening and substantiate the hypothesis that the final form of HA2 is more important for fusion than the conformational change that generates this form.

Functional interaction analysis of GM1-related carbohydrates and Vibrio cholerae toxins using carbohydrate microarray.

The development of analytical tools is important for understanding the infection mechanisms of pathogenic bacteria or viruses. In the present work, a functional carbohydrate microarray combined with a fluorescence immunoassay was developed to analyze the interactions of Vibrio cholerae toxin (ctx) proteins and GM1-related carbohydrates. Ctx proteins were loaded onto the surface-immobilized GM1 pentasaccharide and six related carbohydrates, and their binding affinities were detected immunologically. The analysis of the ctx-carbohydrate interactions revealed that the intrinsic selectivity of ctx was GM1 pentasaccharide ≫ GM2 tetrasaccharide > asialo GM1 tetrasaccharide ≥ GM3trisaccharide, indicating that a two-finger grip formation and the terminal monosaccharides play important roles in the ctx-GM1 interaction. In addition, whole cholera toxin (ctxAB(5)) had a stricter substrate specificity and a stronger binding affinity than only the cholera toxin B subunit (ctxB). On the basis of the quantitative analysis, the carbohydrate microarray showed the sensitivity of detection of the ctxAB(5)-GM1 interaction with a limit-of-detection (LOD) of 2 ng mL(-1) (23 pM), which is comparable to other reported high sensitivity assay tools. In addition, the carbohydrate microarray successfully detected the actual toxin directly secreted from V. cholerae, without showing cross-reactivity to other bacteria. Collectively, these results demonstrate that the functional carbohydrate microarray is suitable for analyzing toxin protein-carbohydrate interactions and can be applied as a biosensor for toxin detection.

Virus-like nanoparticles as a theranostic platform for cancer.

Virus-like nanoparticles (VLPs) are natural polymer-based nanomaterials that mimic viral structures through the hierarchical assembly of viral coat proteins, while lacking viral genomes. VLPs have received enormous attention in a wide range of nanotechnology-based medical diagnostics and therapies, including cancer therapy, imaging, and theranostics. VLPs are biocompatible and biodegradable and have a uniform structure and controllable assembly. They can encapsulate a wide range of therapeutic and diagnostic agents, and can be genetically or chemically modified. These properties have led to sophisticated multifunctional theranostic platforms. This article reviews the current progress in developing and applying engineered VLPs for molecular imaging, drug delivery, and multifunctional theranostics in cancer research.

Interactive configuration through force analysis of GM1 pentasaccharide-Vibrio cholera toxin interaction.

Understanding of the molecular relationships in carbohydrate-protein interactions provides useful information on biological processes in living organisms and is also helpful for development of potent biomedical agents. Herein, the interaction unbinding force between GM1 pentasaccharide and Vibrio cholera toxin (ctx) proteins was measured using atomic force microscopy (AFM), which enabled us to determine the interaction of ctx holotoxin (ctxAB) with GM1 and the interactive formation. First, the interaction force measured between A and B subunits (ctxA-ctxB) was 184.2 ± 4.5 pN, and the unbinding forces were evaluated to confirm the role of ctxA in ctxAB complex formation and were determined to be 443.7 ± 7.5 and 535.7 ± 25.9 pN for GM1-ctxB and GM1-ctxAB complexes, respectively. The force difference of ∼90 pN between GM1-ctxB and GM1-ctxAB might be due to the formation of the cholera toxin complex. Importantly, from the analogue analyses, we understand how structural and binding positional differences in complex carbohydrates affect the interaction with protein and surmise that the GM1-ctxAB complex makes a "two-finger grip" formation through the conformational change of a flexible carbohydrate. In conclusion, using AFM force analysis, we successfully quantified and characterized the interactive configuration of carbohydrate-protein molecules.

Recombinant mussel adhesive protein fp-5 (MAP fp-5) as a bulk bioadhesive and surface coating material.

Mussel adhesive proteins (MAPs) attach to all types of inorganic and organic surfaces, even in wet environments. MAP of type 5 (fp-5), in particular, has been considered as a key adhesive material. However, the low availability of fp-5 has hampered its biochemical characterization and practical applications. Here, soluble recombinant fp-5 is mass-produced in Escherichia coli. Tyrosinase-modified recombinant fp-5 showed ∼1.11 MPa adhesive shear strength, which is the first report of a bulk-scale adhesive force measurement for purified recombinant of natural MAP type. Surface coatings were also performed through simple dip-coating of various objects. In addition, complex coacervate using recombinant fp-5 and hyaluronic acid was prepared as an efficient adhesive formulation, which greatly improved the bulk adhesive strength. Collectively, it is expected that this work will enhance basic understanding of mussel adhesion and that recombinant fp-5 can be successfully used as a realistic bulk-scale bioadhesive and an efficient surface coating material.

Glycan chip based on structure-switchable DNA linker for on-chip biosynthesis of cancer-associated complex glycans.

On-chip glycan biosynthesis is an effective strategy for preparing useful complex glycan sources and for preparing glycan-involved applications simultaneously. However, current methods have some limitations when analyzing biosynthesized glycans and optimizing enzymatic reactions, which could result in undefined glycan structures on a surface, leading to unequal and unreliable results. In this work, a glycan chip is developed by introducing a pH-responsive i-motif DNA linker to control the immobilization and isolation of glycans on chip surfaces in a pH-dependent manner. On-chip enzymatic glycosylations are optimized for uniform biosynthesis of cancer-associated Globo H hexasaccharide and its related complex glycans through stepwise quantitative analyses of isolated products from the surface. Successful interaction analyses of the anti-Globo H antibody and MCF-7 breast cancer cells with on-chip biosynthesized Globo H-related glycans demonstrate the feasibility of the structure-switchable DNA linker-based glycan chip platform for on-chip complex glycan biosynthesis and glycan-involved applications.

Bone Graft Biomineral Complex Coderived from Marine Biocalcification and Biosilicification.

Bone graft materials have been mainly developed based on inorganic materials, including calcium phosphate. However, these graft materials usually act as osteoconductive rather than osteoinductive scaffolds. To improve bone reconstruction, a combination of several materials has been proposed. However, there are still no alternatives that can completely replace the existing animal-derived bone graft materials. In this work, a marine-inspired biomineral complex was suggested as a potential bone graft material. The proposed biosilicified coccolithophore-derived coccoliths using bioengineered mussel adhesive proteins show osteopromotive ability through the synergistic effects of osteoconductivity from calcium carbonate and osteoinductivity from silica. Its possibility of use as a bone substitute was determined by evaluating the in vitro osteogenic behaviors of multipotent mesenchymal stem cells and in vivo bone regeneration in a rat calvarial defect model. Therefore, the marine-inspired biomineral complex developed in this study could be successfully used for bone tissue engineering.

A functional carbohydrate chip platform for analysis of carbohydrate-protein interaction.

A carbohydrate chip based on glass or other transparent surfaces has been suggested as a potential tool for high-throughput analysis of carbohydrate-protein interactions. Here we proposed a facile, efficient, and cost-effective method whereby diverse carbohydrate types are modified in a single step and directly immobilized onto a glass surface, with retention of functional orientation. We modified various types of carbohydrates by reductive amination, in which reducing sugar groups were coupled with 4-(2-aminoethyl)aniline, which has di-amine groups at both ends. The modified carbohydrates were covalently attached to an amino-reactive NHS-activated glass surface by formation of stable amide bonds. This proposed method was applied for efficient construction of a carbohydrate microarray to analyze carbohydrate-protein interactions. The carbohydrate chip prepared using our method can be successfully used in diverse biomimetic studies of carbohydrates, including carbohydrate-biomolecule interactions, and carbohydrate sensor chip or microarray development for diagnosis and screening.

Expression and purification of the full-length N-acetylgalactosaminyltransferase and galactosyltransferase from Campylobacter jejuni in Escherichia coli.

The successful enzymatic synthesis of various ganglioside-related oligosaccharides requires many available glycan-processing enzymes. However, the number of available glycan-processing enzymes remains limited. In this study, the full-length CgtA43456 (ß-(1→4)-N-acetylgalactosaminyltransferase) and CgtB11168 (ß-(1→3)-galactosyltransferase) were successfully produced from Escherichia coli through the optimization of E. coli-preferable codon usage, selection of E. coli strain, and use of the molecular chaperone GroEL-GroES (GroEL/ES). The CgtA43456 enzyme was produced as a soluble form in E. coli C41(DE3) co-expressed with codon-optimized CgtA43456 and GroEL/ES. However, soluble CgtB11168 was well expressed in E. coli C41(DE3) with only the codon-optimized CgtB11168. Rather, when co-expressed with GroEL/ES, total production of CgtB11168 was reduced. Using immobilized-metal affinity chromatography, the CgtA43456 and CgtB11168 proteins were obtained with approximately 75-78 % purity. The purified CgtA43456 showed a specific activity of 21 mU/mg using UDP-N-acetylgalactosamine and GM3 trisaccharide as donor and acceptor, respectively. The purified CgtB11168 catalyzed the transfer of galactose from UDP-Gal to GM2 tetrasaccharide with a specific activity of 16 mU/mg. We propose that they could be used as catalysts for enzymatic synthesis of GM1 ganglioside-related oligosaccharides.

Impact of Expanded Small Alkyl-Binding Pocket by Triple Point Mutations on Substrate Specificity of Thermoanaerobacter ethanolicus Secondary Alcohol Dehydrogenase.

Site-directed mutagenesis was employed to generate five different triple point mutations in the double mutant (C295A/I86A) of Thermoanaerobacter ethanolicus alcohol dehydrogenase (TeSADH) by computer-aided modeling with the aim of widening the small alkyl-binding pocket. TeSADH engineering enables the enzyme to accept sterically hindered substrates that could not be accepted by the wild-type enzyme. The underline in the mutations highlights the additional point mutation on the double mutant TeSADH introduced in this work. The catalytic efficiency (kcat/KM) of the M151A/C295A/I86A triple TeSADH mutant for acetophenone increased about 4.8-fold higher than that of the double mutant. A 2.4-fold increase in conversion of 3'-methylacetophenone to (R)-1-(3-methylphenyl)-ethanol with a yield of 87% was obtained by using V115A/C295A/I86A mutant in asymmetric reduction. The A85G/C295A/I86A mutant also produced (R)-1-(3-methylphenyl)-ethanol (1.7-fold) from 3'-methylacetophenone and (R)-1-(3-methoxyphenyl)-ethanol (1.2-fold) from 3'- methoxyacetophenone, with improved yield. In terms of thermal stability, the M151A/ C295A/I86A and V115A/C295A/I86A mutants significantly increased ΔT1/2 by +6.8°C and +2.4°C, respectively, with thermal deactivation constant (kd) close to the wild-type enzyme. The M151A/C295A/I86A mutant reacts optimally at 70 °C with almost 4 times more residual activity than the wild type. Considering broad substrate tolerance and thermal stability together, it would be promising to produce (R)-1-(3-methylphenyl)-ethanol from 3'- methylacetophenone by V115A/C295A/I86A, and (R)-1-phenylethanol from acetophenone by M151A/C295A/I86A mutant, in large-scale bioreduction processes.

On-chip biosynthesis of GM1 pentasaccharide-related complex glycans.

A functional glycan chip combined with on-chip enzymatic glycosylation was developed to prepare complex glycan sources and to apply glycan-involved applications simultaneously. GM3 trisaccharide, GM2 tetrasaccharide, and GM1 pentasaccharide were successfully directly biosynthesized on lactose-immobilized surfaces through three consecutive glycosyltransferase reactions along with small amounts of enzymes and donors, without any additional processes. Biosynthesized GM1 pentasaccharide-related complex glycans were demonstrated to provide information on the substrate specificity of whole cholera toxin. Thus, the proposed on-chip glycan biosynthesis system can provide a new direction toward obtaining complex glycan sources and complex glycan-involved applications such as glycan-protein interaction analysis and glycan biomarker-based diagnosis.

Mutagenesis of Met-151 and Thr-153 to alanine in Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase changes substrate specificity for acetophenones.

Secondary alcohol dehydrogenase (SADH) from Thermoanaerobacter ethanolicus reduces ketones to chiral alcohols, and generally obeys Prelog's Rule, with binding pockets for large and small alkyl substituents, giving (S)-alcohols. We have previously shown that mutations in both the large and small pockets can alter both substrate specificity and stereoselectivity. In the present work, Met-151 and Thr-153, residues located in the small pocket, were mutated to alanine. The M151A mutant SADH shows significantly lower activity and lower stereoselectivity for reduction of aliphatic ketones than wild-type SADH. Furthermore, M151A showed non-linear kinetics for reduction of acetone. T153A SADH shows lower activity but similar stereoselectivity for ketone reduction compared to wild-type SADH. The I86A/M151A/C295A and I86A/T153A/C295A triple mutant SADH show altered specificity for reduction of substituted acetophenones. These results confirm that these mutations are useful to combine with I86A/C295A SADH to expand the small pocket of SADH and broaden the substrate specificity.