Chiral Nanostructured Glycerohydrogel Sol-Gel Plates of Chitosan L- and D-Aspartate: Supramolecular Ordering and Optical Properties.

A comprehensive study was performed on the supramolecular ordering and optical properties of thin nanostructured glycerohydrogel sol-gel plates based on chitosan L- and D-aspartate and their individual components in the X-ray, UV, visible, and IR ranges. Our comparative analysis of chiroptical characteristics, optical collimated transmittance, the average cosine of the scattering angle, microrelief and surface asymmetry, and the level of structuring shows a significant influence of the wavelength range of electromagnetic radiation and the enantiomeric form of aspartic acid on the functional characteristics of the sol-gel materials. At the macrolevel of the supramolecular organization, a complex topography of the surface layer and a dense amorphous-crystalline ordering of polymeric substances were revealed, while at the nanolevel, there were two forms of voluminous scattering domains: nanospheres with diameters of 60-120 nm (L-) and 45-55 nm (D-), anisometric particles of lengths within ~100-160 (L-) and ~85-125 nm (D-), and widths within ~10-20 (L-) and ~20-30 nm (D-). The effect of optical clearing on glass coated with a thin layer of chitosan L-(D-)aspartate in the near-UV region was discovered (observed for the first time for chitosan-based materials). The resulting nanocomposite shape-stable glycerohydrogels seem promising for sensorics and photonics.

Surface-enhanced Raman scattering from Au nanorods, nanotriangles, and nanostars with tuned plasmon resonances.

Electromagnetic theory predicts that the optimal value of the localized plasmon resonance (LPR) wavelength for the maximal SERS enhancement factor (EF) is half the sum of the laser and Raman wavelengths. For small Raman shifts, the theoretical EF scales as the fourth power of the local field. However, experimental data often disagree with these theoretical conclusions, leaving the question of choosing the optimal plasmon resonance for the maximal SERS signal unresolved. Here, we present experimental data for gold nanorods (AuNRs), gold nanotriangles (AuNTs), and gold nanostars (AuNSTs) simulating 1D, 2D, and 3D plasmonic nanostructures, respectively. The LPR wavelengths were tuned by chemical etching within 580-1020 nm at a constant concentration of the particles. The particles were functionalized with Cy7.5 and NBT, and the dependence of the intensity at 940 cm-1 (Cy7.5) and 1343 cm-1 (NBT) on the LPR wavelength was examined for laser wavelengths of 633 nm and 785 nm. The electromagnetic SERS EFs were calculated by averaging the product of the local field intensities at the laser and Raman wavelengths over the particle surface and their random orientations. The calculated SERS plasmonic profiles were redshifted compared to the laser wavelength. For 785 nm excitation, the calculated EFs were five to seven times higher than those for 633 nm excitation. With AuNR@Cy7.5 and AuNT@ Cy7.5, the experimental SERS was 35-fold stronger than it was with NBT-functionalized particles, but with AuNST@Cy7.5 and AuNST@NBT, the SERS responses were similar. With all nanoparticles tested, the SERS plasmonic profiles after 785 nm excitation were slightly blue-shifted, as compared with the laser wavelength, possibly owing to the inner filter effect. After 633 nm excitation, the SERS profiles were red-shifted, in agreement with EM theory. In all cases, the plasmonic EF profiles were much broadened compared to the calculated ones and did not follow the four-power law.

SERS and Indicator Paper Sensing of Hydrogen Peroxide Using Au@Ag Nanorods.

The detection of hydrogen peroxide and the control of its concentration are important tasks in the biological and chemical sciences. In this paper, we developed a simple and quantitative method for the non-enzymatic detection of H2O2 based on the selective etching of Au@Ag nanorods with embedded Raman active molecules. The transfer of electrons between silver atoms and hydrogen peroxide enhances the oxidation reaction, and the Ag shell around the Au nanorod gradually dissolves. This leads to a change in the color of the nanoparticle colloid, a shift in LSPR, and a decrease in the SERS response from molecules embedded between the Au core and Ag shell. In our study, we compared the sensitivity of these readouts for nanoparticles with different Ag shell morphology. We found that triangle core-shell nanoparticles exhibited the highest sensitivity, with a detection limit of 10-4 M, and the SERS detection range of 1 × 10-4 to 2 × 10-2 M. In addition, a colorimetric strategy was applied to fabricate a simple indicator paper sensor for fast detection of hydrogen peroxide in liquids. In this case, the concentration of hydrogen peroxide was qualitatively determined by the change in the color of the nanoparticles deposited on the nitrocellulose membrane.

Sensor System Based on a Piezoelectric Resonator with a Lateral Electric Field for Virus Diagnostics.

A sensor system based on a piezoelectric resonator with a lateral electric field in the frequency range 6-7 MHz of the electric field for virus detection is described. Through use of the transmissible virus causing gastroenteritis in pigs and specific antibodies, the possibility of detecting the virus in suspension in real time was determined. It was found that the frequency dependence of the real and imaginary parts of the electrical impedance of such a resonator loaded with a virus suspension changes significantly after the addition of specific antibodies to the suspension. No changes are observed if the antibodies are not specific. Thus, the results obtained illustrate the possibility of detecting viruses in situ, directly in the liquid phase, if the change in the real or imaginary parts of the electrical impedance after the addition of antibodies is used as an analytical signal. The possibility of virus detection in the presence of foreign viral particles has been illustrated.

Improving SERS bioimaging of subcutaneous phantom in vivo with optical clearing.

Surface-enhanced Raman scattering (SERS) has proven to be a promising technique for different types of imaging including preoperative and intraoperative in vivo tumor visualization. However, the strong scattering of the turbid tissue limits its use in subcutaneous areas. In this article, we used an optical clearing technique to improve the SERS signal from a subcutaneous tumor phantom. The phantom is a 2 mm sphere of calcium alginate with incorporated petal-like gap-enhanced Raman tags. The use of optical clearing increases the SERS signal target-to-background ratio for 5 times and allow to decrease the total imaging time for at least 10 times. In addition, SERS imaging assisted with optical clearing made it possible to more precisely determine the shape and boundaries of the implanted phantom. The combination of optical clearing and SERS is a promising strategy for the clinical imaging of subcutaneous objects that are usually shielded by dermal tissue.

Immunodietica: interrogating the role of diet in autoimmune disease.

Diet is an environmental factor in autoimmune disorders, where the immune system erroneously destroys one's own tissues. Yet, interactions between diet and autoimmunity remain largely unexplored, particularly the impact of immunogenetics, one's human leukocyte antigen (HLA) allele make-up, in this interplay. Here, we interrogated animals and plants for the presence of epitopes implicated in human autoimmune diseases. We mapped autoimmune epitope distribution across organisms and determined their tissue expression pattern. Interestingly, diet-derived epitopes implicated in a disease were more likely to bind to HLA alleles associated with that disease than to protective alleles, with visible differences between organisms with similar autoimmune epitope content. We then analyzed an individual's HLA haplotype, generating a personalized heatmap of potential dietary autoimmune triggers. Our work uncovered differences in autoimmunogenic potential across food sources and revealed differential binding of diet-derived epitopes to autoimmune disease-associated HLA alleles, shedding light on the impact of diet on autoimmunity.

Advantages of Highly Spherical Gold Nanoparticles as Labels for Lateral Flow Immunoassay.

The use of lateral flow immunoassays (LFIAs) for rapid on-site testing is restricted by their relatively high limit of detection (LoD). One possible way to decrease the LoD is to optimize nanoparticle properties that are used as labels. We compare two types of Au nanoparticles: usual quasispherical gold nanoparticles (C-GNPs), obtained by the Turkevich-Frens method, and superspherical gold nanoparticles (S-GNPs), obtained by a progressive overgrowth technique. Average diameters were 18.6-47.5 nm for C-GNPs and 20.2-90.4 nm for S-GNPs. Cardiomarker troponin I was considered as the target analyte. Adsorption and covalent conjugation with antibodies were tested for both GNP types. For C-GNPs, the minimal LoD was obtained with 33.7 nm nanoparticles, reaching 12.7 ng/mL for covalent immobilization and 9.9 ng/mL for adsorption. The average diameter of S-GNPs varied from 20.2 to 64.5 nm, which resulted in a decrease in LoD for an LFIA of troponin I from 3.4 to 1.2 ng/mL for covalent immobilization and from 2.9 to 2.0 ng/mL for adsorption. Thus, we obtained an 8-fold decrease in LoD (9.9 to 1.2 ng/mL) by using S-GNPs. This effect can be related to more effective antibody immobilization and improved S-GNP optical properties. The obtained results can improve LFIAs for various practically significant analytes.

Study of the immunogenicity of the VP2 protein of canine parvovirus produced using an improved Baculovirus expression system.

BACKGROUND: Canine parvovirus (CPV) is now recognized as a serious threat to the dog breeding industry worldwide. Currently used CPV vaccines all have their specific drawbacks, prompting a search for alternative safe and effective vaccination strategies such as subunit vaccine. VP2 protein is the major antigen targeted for developing CPV subunit vaccine, however, its production in baculovirus expression system remains challenging due to the insufficient yield. Therefore, our study aims to increase the VP2 protein production by using an improved baculovirus expression system and to evaluate the immunogenicity of the purified VP2 protein in mice. RESULTS: The results showed that high-level expression of the full length VP2 protein was achieved using our modified baculovirus expression system. The recombinant virus carrying two copies of VP2 gene showed the highest expression level, with a productivity of 186 mg/L, which is about 1.4-1.6 fold that of the recombinant viruses carrying only one copy. The purified protein reacted with Mouse anti-His tag monoclonal antibody and Rabbit anti-VP2 polyclonal antibody. BALB/c mice were intramuscularly immunized with purified VP2 protein twice at 2 week intervals. After vaccination, VP2 protein could induce the mice produce high level of hemagglutination inhibition antibodies. CONCLUSIONS: Full length CPV VP2 protein was expressed at high level and purified efficiently. Moreover, it stimulated mice to produce high level of antibodies with hemmaglutination inhibition properties. The VP2 protein expressed in this study could be used as a putative economic and efficient subunit vaccine against CPV infection.

Petal-like Gap-Enhanced Raman Tags with Controllable Structures for High-Speed Raman Imaging.

Surface-enhanced Raman scattering (SERS) is widely used for in vitro and in vivo bioimaging applications. However, reproducible and controllable fabrication of SERS tags with high density of electromagnetic hot-spots is still challenging. We report an improved strategy for the synthesis of core/shell Raman tags with high density of hot-spots and high immobilization of reporter molecules. The strategy is based on simultaneous growth and functionalization of an Au shell around Au nanospheres coated with 4-nitrobenzenethiol (NBT). The amount of added 4-NBT is key factor to control the structure SERS response of the resulting particles. Specifically, we demonstrate the formation of gap-enhanced Raman tags (GERTs) with a smooth solid shell (sGERTs), petal-like GERTs (pGERTs), and mesoporous Au particles (mGERTs) filled with Raman molecules. In contrast to NBT molecules, similar thiols such as 1,4-benzenedithiol (BDT) and 2-naphtalenethiol (NT) do not support the formation of pGERTs and mGERTs. To explain this finding, we proposed a growth mechanism based on the unique chemical structure of NBT. The SERS response of optimized pGERTs is 50 times higher than that from usual sGERTs, which makes pGERTs suitable for single-particle spectroscopy. We demonstrate successful application of pGERTs for high-speed cell imaging using 10 ms accumulation time per pixel and a total imaging time of about 1 min. Because of the high SERS response and unique porous structure, these nanoparticles have great potential for bioimaging and other applications.

Two forms of substrate for the bioluminescent reaction in three species of basidiomycetes.

The luminescent response of the enzymatic system of Armillaria borealis on the cold and hot extracts from cell-free culture liquids of Inonotus obliquus, Pholiota sp. and A. borealis was examined. The greatest influence on the light emission produced by the luminescent system of A. borealis was provided by the temperature at which the probes were prepared for assay. Boiling a culture liquid on water bath for a few minutes promoted a multifold increase in the luminescence. The results of luminescence assay suggest that the substance involved in the bioluminescent reaction in higher fungi is presented in culture liquids and mycelia in two forms. In one form, it is ready to interact with the enzymatic system and in the second form, it becomes accessible for the reaction after heat treatment. The pool of thermoactivated substance was found to be much large than the amount of the ready accessible one. We suggest that predecessors of hispidin, which is fungal luciferin precursor, are responsible for this phenomenon. They are not involved in bioluminescence at their original state and are converted into the substrate under the influence of high temperature.

Polydopamine-coated Au nanorods for targeted fluorescent cell imaging and photothermal therapy.

Au nanorods (AuNRs) have attracted a great interest as a platform for constructing various composite core/shell nanoparticles for theranostics applications. However, the development of robust methods for coating AuNRs with a biocompatible shell of high loading capacity and with functional groups still remains challenging. Here, we coated AuNRs with a polydopamine (PDA) shell and functionalized AuNR-PDA particles with folic acid and rhodamine 123 (R123) to fabricate AuNR-PDA-R123-folate nanocomposites. To the best of our knowledge, such AuNR-PDA-based composites combining fluorescent imaging and plasmonic phothothermal abilities have not been reported previously. The multifunctional nanoparticles were stable in cell buffer, nontoxic and suitable for targeted fluorescent imaging and photothermal therapy of cancer cells. We demonstrate the enhanced accumulation of folate-functionalized nanoparticles in folate-positive HeLa cells in contrast to the folate-negative HEK 293 cells using fluorescent microscopy. The replacement of folic acid with polyethylene glycol (PEG) leads to a decrease in nanoparticle uptake by both folate-positive and folate-negative cells. We performed NIR light-mediated targeted phototherapy using AuNR-PDA-R123-folate and obtained a remarkable cancer cell killing efficiency in vitro in comparison with only weak-efficient nontargeted PEGylated nanoparticles. Our work illustrates that AuNR-PDA could be a promising nanoplatform for multifunctional tumor theranostics in the future.

Green synthesis of nanoparticles with extracellular and intracellular extracts of basidiomycetes.

Au, Ag, Se, and Si nanoparticles were synthesized from aqueous solutions of HAuCl4, AgNO3, Na2SeO3, and Na2SiO3 with extra- and intracellular extracts from the xylotrophic basidiomycetes Pleurotus ostreatus, Lentinus edodes, Ganoderma lucidum, and Grifola frondosa. The shape, size, and aggregation properties of the nanoparticles depended both on the fungal species and on the extract type. The bioreduction of the metal-containing compounds and the formation rate of Au and Ag nanoparticles depended directly on the phenol oxidase activity of the fungal extracts used. The biofabrication of Se and Si nanoparticles did not depend on phenol oxidase activity. When we used mycelial extracts from different fungal morphological structures, we succeeded in obtaining nanoparticles of differing shapes and sizes. The cytotoxicity of the noble metal nanoparticles, which are widely used in biomedicine, was evaluated on the HeLa and Vero cell lines. The cytotoxicity of the Au nanoparticles was negligible in a broad concentration range (1-100 µg/mL), whereas the Ag nanoparticles were nontoxic only when used between 1 and 10 µg/mL.

Antitumor Activity of Extracts from Medicinal Basidiomycetes Mushrooms.

Aqueous extracts from the vegetative submerged mycelia of cultivated Basidiomycetes Ganoderma lucidum, Lentinus edodes, and Grifola frondosa, as well as from the fruiting bodies of G. lucidum, were found to have antitumor activity. The antitumor effect of the mycelial extracts from all 3 fungal species was ascertained in vivo in rats with implanted kidney cancer. Dystrophic changes in tumor cells and tumor necrosis (up to 90%) were noted. In vitro cytotoxicity studies of the A549 human lung adenocarcinoma cell line and HEp-2 human laryngeal epidermoid carcinoma cells showed that the extracts from the G. lucidum fruiting bodies and from the L. edodes vegetative mycelium were the most effective. The animals' immune systems were activated, and the fungal extracts displayed no toxicity when administered orally.

Biodistribution of Different Sized Nanodiamonds in Mice.

The particle size is one of critical parameters influencing the biodistribution of detonation nanodiamonds (DND) after their administration into the body. As DNDs are prone to aggregation, the difference between their sizes in aqueous and physiological solutions has to be taken into account. Radioactive I125-BSA molecules were covalently immobilized on DNDs divided in three fractions of different average size. The DND-BSAI125 conjugates were intravenously administrated into adult mice and the particle allocation in the animal's organs and blood was evaluated based on the radioactivity distribution. We conclude that most of the conjugates were taken from the bloodstream and trapped in the liver and spleen. The short-term distribution pattern for all DNDs was similar regardless of size and practically unchanged with time. No significant clearance of the particles was observed for 4 h, but the presence of DNDs was detected in the blood. It was found that the largest particles tend to accumulate more into the liver as compared to the smaller ones. However, the size effect was not well pronounced for the studied size range.

Enzymatic formation of gold nanoparticles by submerged culture of the basidiomycete Lentinus edodes.

We report for the first time that the medicinal basidiomycete Lentinus edodes can reduce Au(III) from chloroauric acid (HAuCl4) to elemental Au [Au(0)], forming nanoparticles. Several methods, including transmission electron microscopy, electron energy loss spectroscopy, X-ray fluorescence, and dynamic light scattering, were used to show that when the fungus was grown submerged, colloidal gold accumulated on the surface of and inside the mycelial hyphae as electron-dense particles mostly spherical in shape, with sizes ranging from 5 to 50nm. Homogeneous proteins (the fungal enzymes laccase, tyrosinase, and Mn-peroxidase) were found for the first time to be involved in the reduction of Au(III) to Au(0) from HAuCl4. A possible mechanism forming Au nanoparticles is discussed.

Stimulation of luminescence of mycelium of luminous fungus Neonothopanus nambi by ionizing radiation.

The luminescent system of higher luminous fungi is not fully understood and the enzyme/substrate pair of the light emission reaction has not been isolated. It was suggested that luminescence of fungi involves oxidase-type enzymes, and reactive oxygen species are important for fungal light production. Generation of reactive oxygen species can be stimulated by ionizing irradiation, which has not been studied for luminous fungi. We report the effect of X-irradiation on the luminescence of fungus Neonothopanus nambi. Experiments were performed with mycelium on a home-built setup based on an X-ray tube and monochromator/photomultiplier tube. Application of X-rays does not change the emission spectrum, but after approximately 20 min of continuous irradiation, light production from unsupported mycelium starts growing and increases up to approximately five times. After peaking, its level decreases irrespective of the presence of X-irradiation. After staying at a certain level, light production collapses to zero, which is not related to the drying of the mycelium or thermal impact of radiation. The observed shape of kinetics is characteristic of a multistage and/or chain reaction. The time profile of light production must reflect the current levels of radicals present in the system and/or the activity of enzyme complexes involved in light production.