Rapid and quantitative detection of multiple antibodies against SARS-CoV-2 mutant proteins by photo-immobilized microarray.

A rapid automatic quantitative diagnostic system for multiple SARS-CoV-2 mutant protein-specific antibodies was developed using a microarray with photoreactive polymers. Two types of photoreactive polymers, phenylazide and polyoxyethylene, were prepared. The polymers were coated on a plastic plate. Aqueous solutions of mutant virus proteins were microspotted on the coated plate and immobilized by photoirradiation. Virus-specific IgG in the serum or blood was automatically assayed using an instrument that we developed for pipetting, reagent stirring, and washing. The results highly correlated with those of the conventional enzyme-linked immunoassay or immunochromatography. This system was successfully used to test the sera or blood from the patients recovered from the infection and the vaccinated individuals. The recovered individuals had antibodies against the nucleoprotein, in contrast to the vaccinated individuals. The amount of antibodies produced decreased with an increase in virus mutation. Blood collected from the fingertip (5 µL) and a test period of 8 min were sufficient conditions for conducting multiple antibody assays. We believe that our system would facilitate rapid and quantitative automatic assays and aid in the diagnosis of various viral infectious diseases and assessment of the immune status for clinical applications.

Synthesis of Photoreactive Poly(ethylene oxide)s for Surface Modification.

Photoreactive polymers that generate active species upon irradiation with light are very useful for modifying the surfaces of substrates. However, water solubility decreases as the number of photoreactive functional groups on the polymer increases because most photoreactive functional groups are hydrophobic. In order to improve the hydrophilicity of the photoreactive polymer, we synthesized polyethylene glycol-based photoreactive polymers bearing hydrophobic azidophenyl groups on their side chains. Because of the hydrophilicity of the ethylene glycol main chain, polymers with large numbers of azidophenyl groups were solubilized in protic solvents compared to hydrophobic alkylene chain-based polymers prepared by radical polymerization of methacrylate monomers. Polymers were immobilized on various substrates by irradiation with ultraviolet light and were shown to suppress nonspecific interactions between proteins and cells on the substrate. We conclude that such polymers are useful, highly water soluble antifouling agents.

Novel microarrays for simultaneous serodiagnosis of multiple antiviral antibodies.

We developed an automated diagnostic system for the detection of virus-specific immunoglobulin Gs (IgGs) that was based on a microarray platform. We compared efficacies of our automated system with conventional enzyme immunoassays (EIAs). Viruses were immobilized to microarrays using a radical cross-linking reaction that was induced by photo-irradiation. A new photoreactive polymer containing perfluorophenyl azide (PFPA) and poly(ethylene glycol) methacrylate was prepared and coated on plates. Inactivated measles, rubella, mumps, Varicella-Zoster and recombinant Epstein-Barr viruse antigen were added to coated plates, and irradiated with ultraviolet light to facilitate immobilization. Virus-specific IgGs in healthy human sera were assayed using these prepared microarrays and the results obtained compared with those from conventional EIAs. We observed high correlation (0.79-0.96) in the results between the automated microarray technique and EIAs. The microarray-based assay was more rapid, involved less reagents and sample, and was easier to conduct compared with conventional EIA techniques. The automated microarray system was further improved by introducing reagent storage reservoirs inside the chamber, thereby conserving the use of expensive reagents and antibodies. We considered the microarray format to be suitable for rapid and multiple serological diagnoses of viral diseases that could be developed further for clinical applications.

Recombinant human gelatin substitute with photoreactive properties for cell culture and tissue engineering.

The human recombinant collagen I α1 chain monomer (rh-gelatin) was modified by the incorporation of an azidophenyl group to prepare photoreactive human gelatin (Az-rh-gelatin), with approximately 90% of the lysine residues conjugated with azidobenzoic acid. Slight changes in conformation (circular dichroism spectra) and thermal properties (gelation and melting points) were noticed after modification. Ultraviolet (UV) irradiation could immobilize the Az-rh-gelatin on polymer surfaces, such as polystyrene and polytetrafluoroethylene. Az-rh-gelatin was stably retained on the polymer surfaces, while unmodified gelatin was mostly lost by brief washing. Human mesenchymal cells grew more efficiently on the immobilized surface than on the coated surface. The immobilized Az-rh-gelatin on the polymer surfaces was able to capture engineered growth factors with collagen affinity, and the bound growth factors stimulated the growth of cells dose-dependently. It was also possible to immobilize Az-rh-gelatin in micropatterns (stripe, grid, and so on) using photomasks, and the cells grew according to the patterns. These results suggest that the photoreactive human gelatin, in combination with collagen-binding growth factors, will be clinically useful for surface modification of synthetic materials for cell culture systems and tissue engineering.

Visible light-induced crosslinkable gelatin.

A novel visible light-crosslinkable porcine gelatin was prepared for gelation and micropatterning. The preparation employed a photo-oxidation-induced crosslinking mechanism. First, furfuryl groups were incorporated into the gelatin. Second, the modified gelatin was mixed in water with Rose Bengal, which is a visible light sensitizer. Irradiation by visible light solidified the aqueous solution. In addition, when the solution was cast on a plate, dried and photo-irradiated in the presence of a photomask a micropattern was formed that matched the micropattern on the photomask. The gelatin-immobilized regions enhanced cell adhesion. It was also confirmed that the gelatin incorporating furfuryl and Rose Bengal have no significant toxicity. The photo-crosslinkable gelatin was employed as a direct pulp capping material in the dental field. Considering these results, this system could be useful as a new type of visible light-induced crosslinkable biosealant.

A photoimmobilizable sulfobetaine-based polymer for a nonbiofouling surface.

A novel photoreactive polymer containing sulfobetaine polar groups was prepared by copolymerization of two kinds of methacrylic acids with sulfobetaine and azidoaniline. The polymer was photoimmobilized on polyester and polystyrene surfaces. Its effects on surface modification were investigated from its interactions with water, proteins and cells. Polymer immobilization altered both of the plain surfaces to becoming hydrophilic in a similar range of static contact angles (12.5±1.6° on polyester and 14.7±2.2° on polystyrene). This suggests that the surfaces were covered with sulfobetaine polar groups. Micropattern immobilization was carried out on both polymers using a photomask. The formed pattern was identical to the photomask, showing that the polymer was formed in response to ultraviolet irradiation. Measurements using atomic force microscopy showed that the polymer was formed at a thickness of 550nm, demonstrating that the polymer was cross-linked with itself and with the substrate molecules. Measurements using time-of-flight secondary ion mass spectrometry detected an abundance of sulfur-containing ions in the patterned polymer, confirming that sulfobetaine had been immobilized. Protein adsorption and mammalian cell adhesiveness were reduced markedly on the immobilized regions. The reduction of cell adhesiveness was concentration-dependent for the immobilized polymer on polyester surfaces. In conclusion, a novel sulfobetaine-containing polymer was immobilized photoreactively on conventional polymer surfaces and significantly reduced interactions with proteins and mammalian cells.