Key factors influencing the optical detection of biomolecules by their evaporative assembly on diatom frustules

Diatoms have silica frustules with transparent and delicate micro/nano scale structures, multilevel pore arrays, and large specific surface areas. We explored the potential of diatom frustules as biomolecule support for use in optical detection, for example, in protein or DNA biochips and “lab-on-a-chip” sensors. After the solution was evaporated, most particles in the solution assembled on the frustules. Experiments indicated that this phenomenon occurs because of the large specific surface of the frustules; consequently, we studied the capacity of frustules to increase the density of antibodies. The frustules of diatoms Coscinodiscus sp., Navicula sp., and Nitzschia palea were used in this study. The colored particles for optical detection included standard protein, soybean lecithin, bovine serum albumin, and human immunoglobulin G labeled with fluorescein and carbonic black ink. The results showed that the fluorescein isothiocyanate protein was densely assembled on the frustules and exhibited a fluorescence signal that is 2.5 times stronger than that of glass. Compared with the traditional glass substrate, the frustules significantly improved the antibody density and detection signals. The evaporating assembly method was used for measuring the load capacity of frustules for different antibodies; this method can be used to quantitatively bind two or more antibodies to the frustule, which may be valuable in lab-on-a-chip sensors. The design scheme of high-throughput diatom-based biochips was discussed. Through analysis, we hypothesized that diatom frustules with a large specific surface area, high transparency and pore permeability, small sizes and heights, and flat surfaces are particularly suitable for optical detection of biomolecules.     

Enlargement of diatom frustules pores by hydrofluoric acid etching at room temperature

Based on the fact that SiO₂ can dissolve in HF solution, three kinds of diatom frustules were treated with 1% HF solution at room temperature. Given the proper reaction times (0–2 h for the diatoms Coscinodiscus and Navicula, and 0–3 h for the diatom Melosira), the size of the pores on the frustules gradually increased and the structures of the frustules remained. While HF treatment does not affect the composition, chemical bonds, or photoluminescence signature of the diatom frustules, the treatment reduces their surface areas. This method may be beneficial to diatom studies, diatom nanotechnology, and diatom device applications that make use of diatom pores.     

Controlled nanoporous structures of a marine diatom

We demonstrated chemical etching of a marine diatom shell with 1 N NaOH for controlling the pore size of nanoporous structures of the shell under various conditions. Scanning electron microscopy (SEM) images clearly revealed that the pore size of the diatom shells was regulated in the case of etching at 25 degrees C. In contrast, fluctuations in the etched structures was relatively high even during short periods degradation at 40, 60, and 90 degrees C; therefore, controlled nanoporous structures could not be fabricated. This is the first example of artificial modification of natural diatom shells at the nanoscale although diatom shells have been widely used in industry. In addition, a backbone-like structure was observed during the etching process. The structure was similar to the intermediate structure observed during the primitive stage of the diatom cell growth. Probably, this information is valuable for studying the mechanism of nanoporous structures of diatoms.     

Morphology and physical-chemical properties of baked nanoporous frustules

We investigated the morphology and physical-chemical properties of baked and unbaked nanoporous frustules. Scanning electron microscopy (SEM) observations showed that the nanoporous structures of frustules unchanged at 400 degrees C even after baking for 6 h. During baking at 800 degrees C, the frustule structures changed dramatically. On the other hand, Fourier transform infrared spectroscopy (FTIR) of bulk frustule samples indicated that physical-chemical properties of the frustules had clearly changed after baking at not only 800 degrees C but also 400 degrees C. These results showed that the reconstruction of the structures had occurred inside the frustules, even though the morphology of the frustules had not apparently changed at 400 degrees C. In order to characterize the exact shape of the frustules, living diatom cells were grown on a functionalized mica surface, and then baked without any chemical treatment for SEM study. This 'direct baking' technique is effective for comparing minute structures of the frustules, because completed combination of every part of the frustules can be observed.     

Diatom auxospore scales and early stages in diatom frustule morphogenesis: their potential for use in nanotechnology

Incomplete, forming diatom cell wall components have potential uses in nanotechnology that differ from those of mature frustules. Diminutive, discoid auxospore scales, produced after sexual reproduction, could also be evaluated for their value. The structure of developing diatom valves and girdle bands is generally simple, since features such as cribra and other ornamentation have yet to be added. They are also more lightly silicified. Bullulae and honeycomb structures give strength to the final framework of some species using a minimal amount of silica. The morphogenesis of girdle bands is shown to be unidirectional in a marine centric species, forming open-ended cylinders, another potential shape for the arsenal of structures useful to nanotechnologists.     

Preparation of photocatalyst using diatom frustules by liquid phase deposition method

We prepared a photocatalyst using diatom frustules by the liquid phase deposition (LPD) method. Purified native frustules were reacted with boric acid (H3BO3) and ammonium hexafluorotitanate ((NH4)2TiF6) in order to cover the frustule surface with a TiO2 film. Scanning electron microscopy (SEM) observation revealed that the nanoporous structures of the frustules and grown TiO2 layers were co-existent after the LPD treatment when the incubation period was 6 and 12 h. Furthermore, photocatalytic activity of the functionalized frustules was clearly proved by the decomposition of methylene blue (MB) molecules. When the incubation periods were 12 and 24 h, reasonable photocatalytic activity was obtained. The results suggested that higher photocatalytic activity was achieved without losing nanoporous structures when the frustules were treated for 12 h by the LPD method.     

Analysis of nanoindentation response of diatom frustules

Diatom frustules have been suggested for numerous nanotechnological applications. Experimental studies using nanoindenter have shown that the hardness and the stiffness of the frustules vary with location of indentation. To gain further insight, a computational framework has been developed where the Berkovich nanoindentation experiments were simulated by a rigid-deformable contact process. Three different approaches that provide progressively increasing level of understanding of the deformation behavior of frustules were adopted. The differences in the mechanical responses of the frustule due to variation of indentation location, size of pores, and distribution of pores were analyzed. It has been found that the effective stiffness of the frustule is linearly related to the porosity level and does not depend on the frustule size or its pore architecture. It has been shown that a 3D porous shell computational model is more appropriate to simulate the experimentally obtained mechanical response of diatom frustules.     

Investigation of mechanical properties of diatom frustules using nanoindentation

Diatom frustules have been identified as potential candidate materials for nanotechnology applications. However, for successful engineering applications, their mechanical properties must be fully determined. Toward this end, indentation hardness and elastic properties frustules of the centric diatom Coscinodiscus concinnus were evaluated using nanoindentation. A series of nanoindentation tests were performed on the outer surfaces of frustules at various locations. Analysis of the indentations revealed that the Young's modulus and hardness values appear to be strongly dependent on the location of the indentation. The modulus varied from 0.591 to 2.768 GPa in the center and 0.347 to 2.446 GPa at locations away from the center. Similarly, frustule hardness varied between 0.033 and 0.116 GPa in the center and between 0.076 and 0.12 GPa away from the center. Another series of nanoindentation tests were performed on the frustules (positioned in both concave and convex orientations) at various locations to analyze the failure mode. It was found that the failure modes in each of the orientations were also drastically different. In convex orientation, cracks initiated along the sharp edges of the indentation were followed by circular ring cracks, whereas in concave orientation only cracks along the sharp edges (corresponding to the three edges of the indenter) were revealed. The porosity and the nonplanar nature of the frustules make it difficult to extract the mechanical properties accurately at each location.     

The evolution of advanced mechanical defenses and potential technological applications of diatom shells

Diatoms are unicellular algae with silicified cell walls, which exhibit a high degree of symmetry and complexity. Their diversity is extraordinarily high; estimates suggest that about 10(5) marine and limnic species may exist. Recently, it was shown that diatom frustules are mechanically resilient, statically sophisticated structures made of a tough glass-like composite. Consequently, to break the frustules, predators have to generate large forces and invest large amounts of energy. In addition, they need feeding tools (e.g., mandibles or gastric mills) which are hard, tough, and resilient enough to resist high stress and wear, which are bound to occur when they feed on biomineralized objects such as diatoms or other biomineralized protists. Indeed, many copepods feeding on diatoms possess, in analogy to the enamelcoated teeth of mammals, amazingly complex, silica-laced mandibles. The highly developed adaptations both to protect and to break diatoms indicate that selection pressure is high to optimize material properties and the geometry of the shells to achieve mechanical strength of the overall structure. This paper discusses the mechanical challenges which force the development of mechanical defenses, and the structural components of the diatom frustules which indicate that evolutionary optimization has led to mechanically sophisticated structures. Understanding the diatom frustule from the nanometer scale up to the whole shell will provide new insights to advanced combinations of nanostructured composite ceramic materials and lightweight architecture for technological applications.     

Engineering and medical applications of diatoms

Biologists, and diatomists in particular, have long studied the properties of single-cell algae, and engineers are just discovering how to exploit features unique to these organisms. Their uniform nanopore structure, microchannels, chemical inertness, and silica microcrystal structure suggest many nanoscale applications. This paper proposes three potential research initiatives taking advantage of diatom morphology and mechanical and chemical properties: (1) embedding diatom frustules in a metal-film membrane; (2) magnetizing frustules for pinpoint drug delivery; and (3) producing silica nanopowders from frustules. The potential benefits of each initiative and its technical challenges are outlined.     

Growth of copper on diatom silica by electroless deposition technique

In this study, the growth of copper on porous diatom silica by electroless deposition method has been demonstrated for the first time. Raman peaks of copper (145, 213, and 640 cm^?1) appeared in the copper-coated, Amphora sp. and Skeletonema sp. diatom samples, confirming the successful deposition of copper. Scanning electron microscopy (SEM) indicated the presence of copper on the diatom silica surface. The 3D intricate structure of diatom was still evident by optical and scanning electron microscopy analyses when the diatom samples were immersed in the copper bath for only 5 hours. Incubating the diatom samples in the copper bath for 24 h produced a dense coating on the diatom surface and covered the intricate 3D structure of the diatom silica. These results present possibilities of the fabrication of hierarchically organized copper with 3D diatom replica structures.     

Prospects of manipulating diatom silica nanostructure

A key to the development of nanotechnology will be the ability to make complex nanoscaled three-dimensional structures at low cost and in large numbers. The wide variety of structures in the silicified cell walls of diatoms offers a promising natural source of such materials. Diatom silica can be converted into other materials, with maintenance of detailed morphology. To facilitate the use of diatoms in nanotechnology, specific manipulation of the structure in vivo will be desirable. This article explores the possibilities of manipulating diatom silica structure, by nongenetic and genetic means. Nongenetic influences that affect silica structure include changes in environmental conditions and life cycle stages and the presence or absence of particular compounds. The genetically based natural variation in structure in different diatom species indicates that genetic manipulation is possible. To achieve this, however, several goals must be met. The first is to identify cell wall synthesis (CWS) genes involved in structure formation. The recently completed genome sequence of Thalassiosira pseudonana opens the door for genomic and proteomic approaches to accomplish this. An important method to determine the function of CWS genes will be to modify gene sequences or expression and monitor the effect on structure. Performing gene modifications is straightforward, and modified genes can be introduced into diatoms, but the current inability to replace native diatom genes with modified copies could be a problem. However, there are feasible approaches yet to be applied to achieve this goal. It is very likely that continued development and application of molecular genetic techniques will enable us to specifically modify diatom silicified structures and provide a detailed understanding of the underlying mechanism of their formation.     

Frustules to fragments, diatoms to dust: how degradation of microfossil shape and microstructures can teach us how ice sheets work

In a laboratory experiment we investigated micro- and nanoscale changes in fossil diatom valves and in the texture of diatomaceous sediments that result from ice sheet overburden and subglacial shearing. Our experiment included compression and shearing of Antarctic diatom-rich sediments in a ring shear device and comparison of experimental samples with natural glacial sediments from the Antarctic continental shelf. The purpose of the experiment is to establish objective criteria for analyzing subglacial processes and interpreting the origin of glacial-geologic features on the Antarctic continental shelf. We find distinct changes resulting from different glacial settings, with respect to whole diatom frustules, diatom micromorphology, and microtextural properties of sedimentary units. By providing constraints on subglacial shearing, these observations of genetically controlled micro- and nanoscale diatom structures and architecture are contributing to the understanding of large-scale glacial processes, aiding the development of models of modern ice sheet processes, and guiding interpretation of past ice sheet configurations.     

Calcined and natural frustules filled epoxy matrices: The effect of volume fraction on the tensile and compression behavior

The effects of calcined diatom (CD) and natural diatom (ND) frustules filling (0–12 vol.%) on the quasi-static tensile and quasi-static and high strain rate compression behavior of an epoxy matrix were investigated experimentally. The high strain rate testing of frustules-filled and neat epoxy samples was performed in a compression Split Hopkinson Pressure Bar set-up. The frustules filling increased the stress values at a constant strain and decreased the tensile failure strains of the epoxy matrix. Compression tests results showed that frustules filling of epoxy increased both elastic modulus and yield strength values at quasi-static and high strain rates. While, a higher strengthening effect and strain rate sensitivity were found with ND frustules filling. Microscopic observations revealed two main compression deformation modes at quasi-static strain rates: the debonding of the frustules from the epoxy and/or crushing of the frustules. However, the failure of the filled composites at high strain rates was dominated by the fracture of epoxy matrix.     

Superductile,Wavy Silica Nanostructures Inspired by Diatom Algae

Biology implements intriguing structural design principles that allow for attractive mechanical properties—such as high strength, toughness, and extensibility despite being made of weak and brittle constituents, as observed in biomineralized structures. For example, diatom algae contain nanoporous hierarchical silicified shells, called frustules, which provide mechanical protection from predators and virus penetration. These frustules generally have a morphology resembling honeycombs within honeycombs, meshes, or wavy shapes, and are surprisingly tough when compared to bulk silica, which is one of the most brittle materials known. However, the reason for its extreme extensibility has not been explained from a molecular level upwards. By carrying out a series of molecular dynamics simulations with the first principles‐based reactive force field ReaxFF, the mechanical response of the structures is elucidated and correlated with underlying deformation mechanisms. Specifically, it is shown that for wavy silica, unfolding mechanisms are achieved for increasing amplitude and allow for greater ductility of up to 270% strain. This mechanism is reminiscent to the uncoiling of hidden length from proteins that allows for enhanced energy dissipation capacity and, as a result, toughness. We report the development of an analytical continuum model that captures the results from atomistic simulations and can be used in multiscale models to bridge to larger scales. Our results demonstrate that tuning the geometric parameters of amplitude and width in wavy silica nanostructures are beneficial in improving the mechanical properties, including enhanced deformability, effectively overcoming the intrinsic shortcomings of the base material that features extreme brittleness.     

Diatomaceous Lessons in Nanotechnology and Advanced Materials

Silicon, in its various forms, finds widespread use in electronic, optical, and structural materials. Research on uses of silicon and silica has been intense for decades, raising the question of how much diversity is left for innovation with this element. Shape variation is particularly well examined. Here, we review the principles revealed by diatom frustules, the porous silica shells of diatoms, microscopic, unicellular algae. The frustules have nanometer‐scale detail, and the almost 100 000 species with unique frustule morphologies suggest nuanced structural and optical functions well beyond the current ranges used in advanced materials. The unique frustule morphologies have arisen through tens of millions of years of evolutionary selection, and so are likely to reflect optimized design and function. Performing the structural and optical equivalent of data mining, and understanding and adopting these designs, affords a new paradigm in materials science, an alternative to combinatorial materials synthesis approaches in spurring the development of new material and more nuanced materials.     

Diatom Frustules Decorated with Co Nanoparticles for the Advanced Anode of Li-Ion Batteries

Silica is regarded as a promising anode material for lithium-ion batteries (LIBs) because of its high theoretical capacity. However, large volume variation and poor electrical conductivity are limiting factors for the development of SiO₂ anode materials. To solve this problem, combining SiO₂ with a conductive phase and designing hollow porous structures are effective ways. In this work, The Co(II)-EDTA chelate on the surface of diatom biosilica (DBS) frustules and obtained DBS@C-Co composites decorated with Co nanoparticles by calcination without a reducing atmosphere is first precipitated. The unique three-dimensional structure of diatom frustules provides enough space for the volume change of silica during lithiation/delithiation. Co nanoparticles effectively improve the electrical conductivity and electrochemical activity of silica. Through the synergistic effect of the hollow porous structure, carbon layer and Co nanoparticles, the DBS@C-Co-60 composite delivers a high reversible capacity of >620 mAh g⁻¹ at 100 mA g⁻¹ after 270 cycles. This study provides a new method for the synthesis of metal/silica composites and an opportunity for the development of natural resources as advanced active materials for LIBs.     

Estimation of scattering error in spectrophotometric measurements of light absorption by aquatic particles from three-dimensional radiative transfer simulations

We present an approach based on three-dimensional Monte Carlo radiative transfer simulations for estimating scattering error in measurements of light absorption by aquatic particles with a typical laboratory double-beam spectrophotometer. The scattering error is calculated by combining the weighting function describing the angular distribution of photon losses that are due to scattering on suspended particles with the volume scattering function of particles. We applied this method to absorption measurements made on marine phytoplankton, a diatom Thalassiosira pseudonana and a cyanobacterium Synechococcus. Assuming that the scattering phase function is described by the Henyey-Greenstein formula, we determined the backscatter probability of phytoplankton, which yields the best correction for scattering error at a light wavelength of 750 nm, where true absorption is null. The backscattering ratio estimated for both phytoplankton species is significantly higher than previously reported data based on Mie-scattering calculations for homogeneous spheres. Depending on the type of particles, the corrected absorption spectra obtained with our method may be similar or significantly different from spectra obtained with the null-point correction based on wavelength-independent scattering error.     

Two-stage photobioreactor process for the metabolic insertion of nanostructured germanium into the silica microstructure of the diatom Pinnularia sp.

There is significant interest in imbedding nanoscale germanium (Ge) into dielectric silica for optoelectronic applications. In this study, a bioreactor process was developed to metabolically insert nanostructured Ge into a patterned silica matrix of the diatom Pinnularia sp. at levels ranging from 0.24 to 0.97 wt.% Ge. In Stage I, the diatom cell culture was grown up to silicon starvation. In Stage II, soluble silicon and germanium were co-fed to the silicon-starved culture to promote one cell division during Ge uptake. In Stage II, soluble Si and Ge were transported into the silicon-starved diatom cell by a surge uptake process, and Ge uptake preceded its incorporation into the frustule. STEM–EDS line scans of the frustule in the newly-divided cells revealed that the Ge was uniformly incorporated into the biosilica. The overall shape of the new frustule was intact, but Si–Ge oxides filled the frustule areolae and altered their nanoscale pore size and geometry. Ge-rich pockets imbedded within the silica frustule and Ge-rich nanoparticles littering the frustule surface were also found. These results suggest that a two-stage diatom cultivation process can biologically fabricate and self-assemble new types of Ge–Si nanocomposite hierarchical materials that possess intricate submicron features.     

Photoluminescence of silica nanostructures from bioreactor culture of marine diatom Nitzschia frustulum

The marine diatom Nitzschia frustulum is a single-celled photosynthetic organism that uses soluble silicon as the substrate to fabricate intricately patterned silica shells called frustules consisting of 200 nm diameter pores in a rectangular array. Controlled photobioreactor cultivation of the N. frustulum cell suspension to silicon starvation induced changes in the nanostructure of the diatom frustule, which in turn imparted blue photoluminescence (PL) to the frustule biosilica. The photoluminescent properties were imbedded within a patterned substrate precisely ordered at the nano, submicron and microscales. The peak PL intensity increased by a factor of 18 from the mid-exponential to late stationary phase of the cultivation cycle, and the peak PL wavelength increased from 440 to 500 nm. TEM analysis revealed that the emergence of blue photoluminescence was associated with the appearance of fine structures on the frustule surface, including 5 nm nanopore arrays lining the base of the frustule pores, which were only observed at the late stationary phase when both silicon consumption and cell division were complete for two photoperiods. Photoluminescence was quenched by thermal annealing of diatom biosilica in air at 800 degrees C for 1.0 hr, commensurate with the loss of silanol (triple bond Si-OH) groups on the diatom biosilica, as confirmed by FT-IR. Consequently, the likely origin of blue photoluminescence in the diatom biosilica was from surface silanol groups and their distribution on the frustule fine structures.