Ultra‐sensitive immunoassay biosensors using hybrid plasmonic‐biosilica nanostructured materials

We experimentally demonstrate an ultra‐sensitive immunoassay biosensor using diatom biosilica with self‐assembled plasmonic nanoparticles. As the nature‐created photonic crystal structures, diatoms have been adopted to enhance surface plasmon resonances of metal nanoparticles on the surfaces of diatom frustules and to increase the sensitivity of surface‐enhanced Raman scattering (SERS). In this study, a sandwich SERS immunoassay is developed based on the hybrid plasmonic‐biosilica nanostructured materials that are functionalized with goat anti‐mouse IgG. Our experimental results show that diatom frustules improve the detection limit of mouse IgG to 10 pg/mL, which is ?100× better than conventional colloidal SERS sensors on flat glass.

Ultra‐sensitive immunoassay biosensor using diatom biosilica with self‐assembled plasmonic nanoparticles.     

来源于海绵的纳米生物硅材料及其相关酶研究进展

水生生物海绵的纳米结构的硅质骨针及其相关酶,在微电子、光纤及生物医学等方面具有诱人的前景,硅生物技术研究的发展有望成为纳米生物技术的一个新亮点。简要综述了海绵骨针的结构、组成及其形态发生过程,骨针合成代谢相关酶——硅聚合酶、骨针分解代谢相关酶——硅分解酶基因的克隆及表达影响因子,海绵骨针硅材料及其相关酶的获得、体外催化活性及潜在应用。     

Enzyme immobilization via silaffin‐mediated autoencapsulation in a biosilica support

Enzymes and other biomolecules are often immobilized in a matrix to improve their stability or to improve their ability to be reused. Performing a polycondensation reaction in the presence of a biomolecule of interest relies on random entrapment events during polymerization and may not ensure efficient, homogeneous, or complete biomolecule encapsulation. To overcome these limitations, we have developed a method of incorporating autosilification activity into proteins without affecting enzymatic functionality. The unmodified R5 silaffin peptide from Cylindrotheca fusiformis is capable of initiating silica polycondensation in vitro at ambient temperatures and pressures in aqueous solution. In this study, translational fusion proteins between R5 and various functional proteins (phosphodiesterase, organophosphate hydrolase, and green fluorescent protein) were produced in Escherichia coli. Each of the fusion proteins initiated silica polycondensation, and enzymatic activity (or fluorescence) was retained in the resulting silica spheres. Under certain circumstances, the enzymatically‐active biosilica displayed improved stability relative to free enzyme at elevated temperatures. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Micro‐photoluminescence of single living diatom cells

Diatoms are single‐celled microalgae that possess a nanostructured, porous biosilica shell called a frustule. This study characterized the micro‐photoluminescence (μ‐PL) emission of single living cells of the photosynthetic marine diatom Thalassiosira pseudonana in response to UV laser irradiation at 325 nm using a confocal Raman microscope. The photoluminescence (PL) spectrum had two primary peaks, one centered at 500–510 nm, which was attributed to the frustule biosilica, and a second peak at 680 nm, which was attributed to auto‐fluorescence of photosynthetic pigments. The portion of the μ‐PL emission spectrum associated with biosilica frustule in the single living diatom cell was similar to that from single biosilica frustules isolated from these diatom cells. The PL emission by the biosilica frustule in the living cell emerged only after cells were cultivated to silicon depletion. The discovery of the discovery of PL emission by the frustule biosilica within a single living diatom itself, not just its isolated frustule, opens up future possibilities for living biosensor applications, where the interaction of diatom cells with other molecules can be probed by μ‐PL spectroscopy. Copyright © 2016 John Wiley & Sons, Ltd.     

Cd2+ affects the growth,hierarchical structure and peptide composition of the biosilica of the freshwater diatom Nitzschia palea (Kützing) W. Smith

Biosilica from diatoms is formed at ambient conditions under the control of biological and physicochemical processes. The changes in growth and biosilica formation through uptake of different concentrations of Cd²⁺ by the diatom Nitzschia palea (Kützing) W. Smith was investigated, correlating Cd²⁺ effects to changes in the biosilica nanostructure and the relative content of the encapsulated biomolecules. Diatom growth rates at different Cd²⁺ concentrations (as 1, 2, 3, 4, and 5 × 10^?1 mg L^?1 CdCl₂) were studied in order to determine the concentrations at which sublethal effects were visible, allowing the harvest of sufficient diatom cells for further experiments. We found a clear correlation between the Cd²⁺ concentrations and both the nanostructure of the biosilica and content of encapsulated peptides. Cd²⁺ induced biosilica deformation was assessed by scanning electron microscopy and attenuated total reflectance‐Fourier Transformed Infrared Spectroscopy (FTIR), revealing that micromorphological changes in frustule features (striae, costae, pores) and nanostructural modifications (structure of the silica and conformation of the encapsulated peptides) occurred at applied Cd²⁺ concentrations of 2 and 3 × 10^?1 mg L^?1. In particular the FTIR contribution of peptides decreased at elevated Cd²⁺ concentrations, whereas shifts in wave number of several relevant organic bonds as C = O stretching (1765 cm^?1) and possibly hydrated sulfate (1160, 1110 and 980 cm^?1) were assigned. Additional analysis of the amide I band showed a relative increase in β‐sheet structure (1680–1620 cm^?1) when Cd²⁺ concentration increased. Cadmium uptake clearly affected the molecular ordering of the biosilica in Nitzschia palea, most probably by interfering in biological or physicochemical processes involved in diatom biosilicification.     

Internalization kinetics and cytoplasmic localization of functionalized diatomite nanoparticles in cancer cells by Raman imaging

Porous biosilica nanoparticles obtained from diatomites (DNPs) have been recently demonstrated to be non‐toxic nanovectors of therapeutic agents in cancer cells. In this work, the internalization kinetics and intracellular spatial distribution of functionalized DNPs incubated with human lung epidermoid carcinoma cell line (H1355) up to 72 hours are investigated by Raman imaging. The label‐free Raman results are compared with confocal fluorescence microscopy and photoluminescence (PL) data. Raman bands specifically assigned to DNPs and cellular components provide evidence that the nanovectors are internalized and co‐localize with lipid environments. A considerable DNPs uptake in cells is observed within 6 hours, with equilibrium being achieved after 18 hours. The obtained data show the presence of DNPs up to 72 hours, without damage to cell viability or morphology. The PL measurements performed on DNPs not penetrating the cells at different incubation times are strongly correlated with the results obtained by Raman imaging and confocal microscopy analyses.      

Inside Cover: Internalization kinetics and cytoplasmic localization of functionalized diatomite nanoparticles in cancer cells by Raman imaging (J. Biophotonics 4/2018)

The internalization kinetics and intracellular spatial distribution of functionalized diatomite nanoparticles in human lung epidermoid carcinoma cell line have been investigated by confocal fluorescence and Raman microscopy. In this context, Raman imaging due to its non‐destructive, chemically selective and label‐free working principle provides evidence that the nanovectors are internalized and co‐localize with lipid environments, suggesting an endocytic internalisation route. Nanoparticle uptakes and intracellular persistence are observed up to 72 hours, without damage to cell viability or morphology. Further details can be found in the article by Stefano Managò et al. ( e201700207 )

     

NANOSTRUCTURE OF THE DIATOM FRUSTULE AS REVEALED BY ATOMIC FORCE AND SCANNING ELECTRON MICROSCOPY

The cell wall (frustule) of the freshwater diatom Pinnularia viridis (Nitzsch) Ehrenberg is composed of an assembly of highly silicified components and associated organic layers. We used atomic force microscopy (AFM) to investigate the nanostructure and relationship between the outermost surface organics and the siliceous frustule components of live diatoms under natural hydrated conditions. Contact mode AFM imaging revealed that the walls were coated in a thick mucilaginous material that was interrupted only in the vicinity of the raphe fissure. Analysis of this mucilage by force mode AFM demonstrated it to be a nonadhesive, soft, and compressible material. Application of greater force to the sample during repeated scanning enabled the mucilage to be swept from the hard underlying siliceous components and piled into columns on either side of the scan area by the scanning action of the tip. The mucilage columns remained intact for several hours without dissolving or settling back onto the cleaned valve surface, thereby revealing a cohesiveness that suggested a degree of cross-linking. The hard silicified surfaces of the diatom frustule appeared to be relatively smooth when living cells were imaged by AFM or when field-emission SEM was used to image chemically cleaned walls. AFM analysis of P. viridis frustules cleaved in cross-section revealed the nanostructure of the valve silica to be composed of a conglomerate of packed silica spheres that were 44.8 ± 0.7 nm in diameter. The silica spheres that comprised the girdle band biosilica were 40.3 ± 0.8 nm in diameter. Analysis of another heavily silicified diatom, Hantzschia amphioxys (Ehrenberg) Grunow, showed that the valve biosilica was composed of packed silica spheres that were 37.1 ± 1.4 nm and that silica particles from the girdle bands were 38.1 ± 0.5 nm. These results showed little variation in the size range of the silica particles within a particular frustule component (valve or girdle band), but there may be differences in particle size between these components within a diatom frustule and significant differences are found between species.     

Mineralogical characterization of biosilicas versus geological analogs

Non-crystalline silica mineraloids are essential to life on Earth as they provide architectural structure to dominant primary producers, such as plants and phytoplankton, as well as to protists and sponges. Due to the difficulty in characterizing and quantifying the structure of highly disordered X-ray amorphous silica, relatively little has been done to understand the mineralogy of biogenic silica and how this may impact the material properties of biogenic silica, such as hardness and strength, or how biosilica might be identified and differentiated from its inorganic geological counterparts. Typically, geologically formed opal-A and hyalite opal-A_N are regarded as analogs to biogenic silica, however, some spectroscopic and imaging studies suggest that this might not be a reasonable assumption. In this study, we use a variety of techniques (X-ray diffraction, Raman spectroscopy, and scanning electron microscopy) to compare differences in structural disorder and bonding environments of geologically formed hydrous silicas (Opal-A, hyalite, geyserite) and silica glass versus biogenic silicas from an array of organisms. Our results indicate differences in the levels of structural disorder and the Raman-observed bonding environments of the SiO₂ network modes (D₁ mode) and the Q-species modes (~1015 cm⁻¹) between varieties of biogenic silicas and geologically formed silicas, which aligns with previous studies that suggest fundamental differences between biogenic and geologically formed silica. Biosilicas also differ structurally from one another by species of organism. Our mineralogical approach to characterizing biosilicas and differentiating them from other silicas may be expanded to future diagenesis studies, and potentially applied to astrobiology studies of Earth and other planets.