Diatom Biogenic Silica as a Felicitous Platform for Biochemical

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Cite This: ACS Appl. Bio Mater. 2019, 2, 2295−2316

Diatom Biogenic Silica as a Felicitous Platform for Biochemical Engineering: Expanding Frontiers Varsha Panwar and Tanmay Dutta*

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Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India ABSTRACT: Diatoms are a prolific class of single-celled photosynthetic microalgae, pervasive in aquatic habitats. More than 10 000 species of diatoms are widespread in the world and primarily sustain the food chain in marine ecosystems. An individual diatom has evolved with species-specific distinct microporous to nanoporous rigid siliceous cell walls or frustules. Exceptionally intricate pore patterns of frustules originate from the hierarchical arrangements of silica sheets. Astonishing intrinsic features of diatom frustules, such as chemical inertness, large surface area, mechanical strength, ordered 3D micropatterning to nanopatterning of pores, distinctive optical properties, biocompatibility, etc., have been tailored to utilize them in numerous applications including biomedicine, separation technology, sensing, photonics, energy conversion, and storage. The advantage of biosilica over synthetic nanoporous silica materials for their application in nanotechnology lies in its cost-effective production through in vivo biomineralization that circumvents the usage of hazardous chemicals. Various chemical and biological approaches have been implemented for surface modification, structure mimics, integration, and conversion of diatom silica shells without altering their structural attributes to manufacture nanostructured smart materials with a diverse heterogeneity. Diatom silica shells thus have gained prodigious attention as powerful tools in nanotechnology. We follow an insightful approach in this Review to deal with all different types of applications of diatom silica shells in diverse areas and associated strategies to improve their aptness to fabricate new materials or devices. KEYWORDS: biosilica, biochemical sensing, drug delivery, biophotonics, tissue engineering, supercapacitor (soils and wetlands).4 Each of these species possesses a distinctive, inordinately beautiful frustule architecture.1,2 Diatom biogenic silica (biosilica) is endowed with an interesting chemical structure with protruding free hydroxyl groups, which can be modified for tethering chemical or biological moieties.5,7 Increasing research interest in the utilization of diatom biosilica in the recent past stems from its exceptional properties like chemical inertness, biocompatibility, high mechanical and thermal stability, low thermal conductivity, uniform porous structures with a high specific surface area (SSA), tunable pore volume, etc.1,8 Unlike the production of synthetic silica materials with a micro- or nanoscale structure by an expensive process of conventional nanofabrication, diatom biosilica can be harvested in an enormous quantity and purity without substantial energy and material inputs,1,4 which makes it an unlimited, readily available, natural, low-cost, and renewable material. Moreover, the production of biosilica is immensely environment-friendly as neither toxic waste is essentially generated nor this process involves more consumption of energy compared to the production of synthetic silica-based materials.7 Aside from the advantages over

1. INTRODUCTION The evolutionary process in nature has produced enormous diversity at every level of biological organizations. One of such utmost biodiversity was perceived in diatoms, the aquatic habitats that are evolved with eccentric shells of clear silica glass around each cell. Diatoms, often called the “jewels of sea”, are microscopic (from 2 μm to 2 mm), unicellular photosynthetic algae under the class Bacillariophyceae. This phytoplankton is the originator of the food chain in marine ecosystems1,2 as nearly 25% of the primary food production on this planet is dependent on their growth.3 Nearly half of the total organic matter in the oceans of the world is contributed by diatoms. Diatom photosynthesis converts about 20% of total carbon dioxide in the biosphere into the organic form.4 Cell walls of diatoms comprise valves and are built by a biomineralization process at an ambient temperature and pressure and at a neutral pH in an aqueous environment. The distinctive protective cell wall of a diatom consists of an amorphous silica shell, termed as a frustule. A frustule comprises species-specific highly ordered complex threedimensional nanoporous to macroporous patterning, which appears to be more ostentatious than human-crafted exquisite crystal. A girdle band clutches two overlapping cell walls in a “frustule” structure, which encompasses the bulk of the single cell.1,2,5,6 Over 10 000 species of diatoms have been reported in aquatic (rivers, lakes, and oceans) and semiaquatic niches © 2019 American Chemical Society

Received: January 19, 2019 Accepted: April 29, 2019 Published: April 29, 2019 2295

DOI: 10.1021/acsabm.9b00050 ACS Appl. Bio Mater. 2019, 2, 2295−2316

Review

ACS Applied Bio Materials

Figure 1. (A) Schematic of the diatom structure. The three-dimensional structure of the frustule silica wall of a centric diatom based on SEM data has a radial symmetry and consists of hexagonal chambers in the honeycomb-like inner sheet of diatom valves, called areolae. Large holes in the areolae are called foramen. The roof of the areolae is called the cribrum, which contains a regular pattern of pores. The layer over the cribrum is a thin siliceous membrane known as a cribellum, which consists of small pores. Reproduced with permission from ref 9. Copyright 2007 Springer Nature. (B) SEM image of a Coscinodiscus sp. frustule and (C) SEM image of T. eccentrica. (D) Side view (a) and top view (b) of the SEM image of cylinder-shaped biosilica from a single T. pseudonana cell. Details of the biosilica structure (c and d) showing the highly porous surface. Reproduced with permission from ref 61. Copyright 2015 Springer Nature.

technological utilizations currently in an unprecedented fashion. Although numerous fascinating features of diatoms such as the biomineralization process, morphogenesis, etc. enliven us to analyze those aspects comprehensively, this Review will primarily focus on the exploitations of diatom biosilica in various technological applications from as simple as filtration to as complex as drug delivery, enzyme immobilizations, sensing, photonics, etc. A systematic and insightful approach has been taken here to analyze the strategies to improve the aptness of diatom silica to fabricate new materials or devices. Potentiality and advantages of biosilica over other synthetic materials will also be discussed.

synthetic nanofabricated silica materials or other polymers, diatom biogenic silica has been designated as a more promising alternate than other cellular, composite, and silk materials due to its higher mechanical strength (1700 kNm/kg) over those naturally occurring materials.4 All of these outstanding features of diatom biosilica have suitable diverse applications in various facets of study viz. wastewater treatment, enzyme immobilization, bioremediation, biomedicine, targeted drug delivery, catalysis, biophotonics, chemo- and biosensor, micro- and nanofabrication, filtration, microdevices, protein separation, microfluidics, cement additive, imaging, customized controllable drug release, cancer diagnosis and therapy, photoluminescence, detection of trace gases, etc.1,2,5,7,8 Depending on the symmetry of the frustules, diatoms are classified into two groups: (1) centric and (2) pennate diatoms. Frustules with a radial symmetry are prevailed in centric diatoms and consist of hexagonal chambers, termed as areolae (Figure 1). Each chamber comprises an outer wall with the perforation of a large round hole, which is exposed to the external environment, and an inner wall adjacent to the cell membrane, which contains a porous plate, called a sieve plate. The base of each areolae controls the flux of nutrients and exudates across the cell membrane. Pennate diatoms unlike the one with a centric symmetry are prone to be elongated in size and possess parallel striae arranged regularly to the long axis of a frustule.6,9,10 The distance between two alongside striae ranges typically from ∼0.3 to 2 μm and is strictly speciesspecific. The linear midrib of pennate diatoms or the circular midring in centric diatoms serves as a nucleation site, from which lines of silica originate. The lines of silica between the striae are called costae, which incline primarily to be arranged in combs or other similar structures. Diatom valves comprise three-dimensional and hierarchical organization of porous plates and solid walls with an invariable pore diameter. Aside from their symmetry, frustules exhibit an extensive structural diversity in their patterns and shapes.2 This remarkable characteristic of the diatom frustule is utilized in diverse

2. GENERAL PROPERTIES OF A DIATOM Eccentric physical, chemical, and optical properties of diatom silica shells have drawn remarkable research attention as a substituent to synthetic polymers and other nanomaterials.11 An easy cultivation process of diatom and minimal downstream processing essentially reduces the production cost of biosilica. Over the years, diatomaceous earth (DE), the fossilized sedimentary siliceous mineral of accumulated dead diatoms, has been utilized in various industries as the inexpensive and naturally abundant source of mesoporous silica nanomaterial with a convoluted 3D mesoporous hierarchical architecture.12,13 Unprocessed hard shell protist DE is primarily used as a stabilizing component of dynamite, filtering materials, abrasive components in metal polishes and toothpaste, biofertilizers, additives in construction materials and paints, and food integrators. DE silica has been approved by the United States Food and Drug Administration (FDA) for their usage in food and agricultural industries.11 Diatoms, an indispensable food source in the ocean’s food chain, are also the bountiful source of fatty acids, lipids, phenolic compounds, and carotenes, which are crucial to all surviving species in aquatic habitats. Natural lipids especially triglycerides are prevalent in diatoms.14 Production of lipids and chrysolaminarin is the 2296


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ACS Applied Bio Materials Table 1. Exploitation of a Diatom Silica Shell for Bio/Chemical Catalysis catalysis area organic synthesis redox reaction

modification/mimic

application/function

diatom species 19

immobilization of Pd NPs

C−C bond formation in the Heck and Suzuki coupling reaction

DE

decoration of Pt NP array

catalyze the reaction between hexacyanoferret(III) and thiosulfate

flow-through catalysis hydrolysis reaction oxidation reaction

conversion of a frustule into carbon microparticles structural mimic of a frustule

a high degree of glucose oxidase loading

Eucampia zodiacus, Coscinodiscus wailesii,20 Aulacoseira22

butyrylcholinesterase

entrapment of butyrylcholinesterase during precipitation of silica nanospheres modification by APTES and glutaraldehyde immobilization of the enzyme in biomimetically derived biosilica

tyrosinase Hab mutase B

coating of Au NPs by covalent coupling

rapid hydrolysis of organophosphorous esters, methyl parathion (insecticides and nerve agent mimics) under dark conditions. a very high Au-loading potential for the oxidation of D-glucose to Dgluconic acid Enzyme Immobilization to increase the stability of enzyme

Aulacoseira23

an increment of enzyme stability for the degradation of phenolic compounds enhancement of the mechanical stability of an immobilized enzyme and facilitates their application in flow-through reactors

diatoms from the Kızılırmak River.3 T. pseudonana29

T. pseudonana, Stephanopyxis turris, Eucampia zodiacus1 biosilica27

frustules create a highly organized periodic pattern of the refractive index, which triggers valves and girdles to act as waveguide photonic crystals. Recent explorations on the optical properties of a single valve both in visible and ultraviolet ranges revealed that spatial separation of focused light happens in different spots depending on the wavelength. This intriguing feature unfolds the feasibility of single valves to be exploited as a microbiospectrometer. Engrossment for the diatom silica shell for nanotechnology lies in its large modifiable surface with a diverge range of molecules. Tailoring the biosilica surface through chemical modifications by facile multifaceted approaches dictates its potential for the development of bioengineered materials and also its effectiveness for exploitation in numerous other areas. Hydroxyl groups on the silica surface can be easily functionalized by many reactive functional groups like COOH, NH2, SH, etc., generating robust coupling sites for tethering biological and chemical moieties like proteins, nucleic acids, antibodies, fluorescence probes, etc.12 Immobilization of these biological sensing molecules with an optical, electrochemical, or piezoelectric transducer inaugurated a new direction of research on biosensors. Diatom surface modifications with chemical groups of interest permit restraining of active biomolecules. Biosilica can also be used as a platform for biomolecular attachment though noncovalent binding like adsorption; however, the strength of that interaction largely depends on the solution condition like ionic strength, pH, etc.

major outcome of diatom photosynthesis. The degree of lipid accumulation differs with the type of species, their growth phases, and environmental parameters.15 They primarily yield fatty acids ranging from 13 to 21 carbons and predominantly consist of saturated and monounsaturated fatty acids. Oxidation of these fatty acids produces a higher energy when compared to polyunsaturated fatty acids with an equal number of carbons. The existence of an additional fatty acid like eicosapentaenoic acid in the diatom was also recognized, which has immense importance in health and food industries.16 The high lipid content of photosynthetic diatoms is promising source for liquid biodiesel production. Diatoms exhibited a high potential in producing a huge amount of biomass, efficient biosynthesis of lipids, straightforward harvesting, and accessibility to metabolic engineering. However, the lipid storage and the degree of saturation of fatty acids can be modulated with a considerable extent by the presence/absence of silicate as a nutrient.17 An investigation on a marine diatom, Odontella aurita, for its lipid production in the presence of Si revealed that reduction of Si in the growth medium remarkably enhances its lipid production, and hence, this species was found suitable to be further explored for commercial production of biodiesel.17 High lipids and fatty acid content of diatoms have been exploited for the production of biofuel. Frustule’s distinctive physical properties like biocompatibility, unique pore arrangements for the control of nutrient uptake and restriction on viral entry, immense mechanical durability for the protection of diatoms from zooplankton predators, intricate light interaction features that promote photosynthesis and prevent the entry of harmful UV radiation,18 etc. instigated scientific communities to exploit those features to fabricate new technology or engineered materials.11 Biosilica has evolved with many astonishing features immerged within naturally periodic and tunable 2D pore arrays with micro/nanofabrication, with an unprecedented diversity in morphology and structure. The quintessence of the symmetry and the complexity of its hierarchical nanostructures, which are markedly far from those achievable through the chemical synthesis procedures in the laboratory to date, renders its photonic crystal properties that shepherd light to help photosynthesis.12 Uniform arrangements of pores on

3. EXPLOITATION OF BIOSILICA Since the past decade, diatom biosilica has enthralled scientists by its extraordinary properties and structures to deploy it in the development of new technologies and devices. Apart from chemical inertness, which renders biosilica immense scientific demands, the nanoporous diatom frustules, the naturally crafted ornate art of design, displayed its remarkable intrinsic features exploitable in technology development. Following is the meticulous analysis of how biosilica has been utilized over the years as a felicitous tool to create new devices or to fabricate new technologies. 3.1. Catalysis. Catalysis is an indispensable technique for bio/chemical industries as over 90% of all industrial syntheses directly or indirectly follow catalytic reactions. In light of the 2297


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Figure 2. (A) Illustrative diagram of diatom biosilica and nanoparticle conjugates. (a) Layer-by-layer assembly of biosilica and nanoparticle array. (b) Covalent coupling of biosilica and NPs. (B, C) Chemical modification of the biosilica surface. The amine modification (B) of the biosilica was obtained using 3-aminopropyltriethoxysilane (APTES) to react with the hydroxyl groups on the surface of the biosilica. The aminated biosilica was activated by glutaraldehyde and finally utilized to immobilize the enzyme. Mercapto coating of the biosilica surface (C) using 3-mercaptopropyltrimethoxysilane (MPTMS).

high commercial demand for catalysis, a growing urge was mounting on researchers over the years to explore and examine appropriate support material for catalytic processes. Extensive research work eventually culminated with diatom biosilica as a feasible supporting platform for catalysis (Table 1). Unlike alumina and carbon, silica-based catalytic reactions essentially produce no byproducts. On the contrary, carbon-based catalysis yields fructose and other sugar compounds (up to 50% in alkaline conditions).1 Diatomite, loaded with palladium nanoparticls, was first reported to act as a supporting platform for the efficient catalysis of the Heck and Suzuki coupling reaction, a versatile method for carbon−carbon bond formation in organic synthesis.19 Immobilized Pd nanoparticles on a diatomite (Figure 2A) appeared to be a novel air-stable catalytic system, which exhibited a high activity for the Heck and Suzuki reaction. The studies also uncover that palladium leaching into the solution during the reaction provides the catalytic site.19 A diatom-templated platinum nanoparticle array was also utilized to catalyze a notable redox reaction between hexacyanoferret(III) and thiosulfate.20,21 Platinum nanoparticles persist as catalytically active if attached to biosilica. The platinum nanoparticle, decorated on biosilica, displayed a 10 times higher reaction rate than platinum colloid while they were used as a catalyst in the aforementioned redox reaction. The specific activity of platinum when attached to biosilica has a higher specific activity than that in the colloid.20 Conversion of biosilica into its structural mimic of other materials like carbon,22 titania,23 etc. has also been investigated either to increase the degree of enzyme loading on the surface22 or to catalyze the rapid hydrolysis of the organophosphorous esters, methyl paraoxon, and methyl parathion (insecticides and nerve agent mimics), under dark conditions.23

Gold nanoparticles coated on biosilica were used as an efficient catalyst for the oxidation of D-glucose to D-gluconic acid, an important chemical intermediate in the pharmaceutical, paper, and food industries. A comparative analysis of the catalysis of that oxidative conversion was performed using biosilica of three different diatom species (Thalassiosira pseudonana, Stephanopyxis turris, and Eucampia zodiacus) as a supporting material for immobilization of gold nanoparticles by covalent coupling. Biosilica of these species exhibited a very high gold-loading potential (nearly 45 wt %) with a uniform nanoparticle disposition. This study leads to an inference that the surface of the diatom frustule renders an extremely catalytically active surface area with an improved accessibility to active sites.1 3.2. Enzyme Immobilization. Although enzymes are a natural catalyst with some fascinating characteristics like high activity, specificity, stereoselectivity, etc., certain properties of enzymes have to be improved before they can be implemented at the industrial scale.24 The stability of enzymes to withstand the hazardous industrial conditions and efficient recovery of enzymes from the catalytic reaction at the industrial reactor determine their applicability in the industrial process. Immobilization of soluble enzymes enhances their catalytic efficacy, thermal and storage stability, and also reusability. Researchers are therefore exploring the ways and means to immobilize enzymes to improve their catalytic performance (Figure 2B,C). Enzyme molecules are tethered to a solid carrier, which is insoluble in the reaction environment. An immobilized biocatalyst in consequence is developed, where the enzyme status has been changed from homogeneous (free enzyme) to heterogeneous (immobilized). Maximal rescue and recycling of the enzyme is achieved through enzyme immobilization in an uninterrupted process where the substrate is continuously supplied to the reaction for an 2298


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Figure 3. (A) Stability of butyrylcholinesterase activity at 25 °C in free and biosilica-immobilized enzyme systems. Free enzyme (■), free enzyme with antibiotic solution (⧫), and biosilica-immobilized enzyme (●). Reproduced with permission from ref 27. Copyright 2004 Springer Nature. (B) Normalized catalytic conversion of D-glucose using gold catalysts with different silicas as support materials. Reproduced with permission from ref 1. Copyright 2016 American Chemical Society.

Table 2. Bio/Chemical Sensing with a Diatom Frustule photonics and sensing area

modification/mimic

biosensing

functionalized with rabbit IgG antibody

immunoassay sensing

functionalized with goat antimouse IgG

biodetection

packing of N. soratensis frustules into C. argus frustules

SERS-based immunological assay SERS immunoassay explosive molecule sensing food sensing

APTES and glutaraldehyde modifications are followed by Ag NP self-assembly

gas sensing

Au NPs with DTNB and specific antibody tagging integration of Ag NPs functionalization of polydiallyl-dimethylammonium chloride followed by self-assembling Au NPs coating a thin (50 nm) conformal and continuous layer of a functional oxide (SnO2)


diatom species

selective and label-free photoluminescence-based detection of immunocomplex formation fabrication of an ultrasensitive immunoassay biosensor that can enhance the detection limit of fluorescence spectroscopy and fluorescence imaging fabrication of the multilayered hierarchical array to achieve an advanced sensitivity in biodetection chips enhancing the SERS sensitivity of the hybrid plasmonic biosilica nanostructured materials to detect the immune reactions between antigens and antibodies detection of an inflammatory cytokine interleukin 8 (IL-8) in human blood plasma. detection of an explosive molecule like 2,4,6-trinitro toluene from a nanoliter solution detection of illegal food adulterate like melamine, a nitrogen-rich chemical compound functions as a sensitive detector for NO gas

enhanced biocatalytic production.25 The nature of enzymes and biocatalytic processes has the determining role for selecting a bona fide support material and for the optimization of an immobilization technique. Materials should possess essential physicochemical features such as the presence of chemical moieties, large surface area or good sorption properties, biocompatibility, nontoxicity, and environmental friendly, to be able to serve as a solid carrier in the immobilization process. Many synthetic support materials have been tested for the immobilization of enzymes (Figure 2B,C);26 however, naturally occurring diatom biosilica has emerged to be the most feasible alternative for enzyme immobilization. By fulfilling of all requirements like easy availability, renewability, cost effectiveness, etc. for a solid carrier, biosilica replaces the need to synthesize artificial silica and other polymers. The suitability of diatom biosilica as a matrix for immobilization was first explored with the enzyme butyrylcholinesterase.27 A high yield of immobilization of the soluble enzyme on biosilica was obtained. Butyrylcholinesterase, entrapped on the biosilica surface, retained all of its activity and was significantly more stable than the free enzymes (Figure 3A). The enhanced stability of an immobilized enzyme

Cyclotella sp.40 Pinnularia sp.43

N. soratensis, C. argus44 Pinnularia sp.47

Pseudostaurosira trainorii48 Pinnularia sp.50 Pinnularia sp.52 Aulacoseira55

over its free form has been attributed to the fact that biosilica as a support matrix protects immobilized enzymes from extensive conformational changes due to alterations in the reaction conditions like change in temperature, pH, etc. (Figure 3A).27 Different Si materials also exhibited an assorted effect on the efficiency of enzyme catalysis (Figure 3B). Hydroxylaminobenzene mutase (HabB) from Pseudomonas pseudoalcaligenes was also reported to be immobilized on diatom silica.28,29 A comparison of the half-life measurements of free and immobilized HabB revealed that biosilicaimmobilized HabB exhibited ∼10-fold higher activity than its free form at a high/low pH and temperature.28 In the same line, the enzyme tyrosinase was immobilized on activated biosilica and modified with 3-aminopropyl triethoxysilane (APTES) and glutaraldehyde, for the removal of phenolic compounds like p-cresol, phenol, and phenyl acetate from an aqueous solution (Figure 2B). 3-Mercaptopropyl-trimethoxysilane (MPTMS) is another frustule-surface-modifying compound, which provides the SH group for further attachment with the enzymes (Figure 2C). Immobilized tyrosinase retained ∼80% of its initial activity after 10 times of repeated use in a batch culture system, which proved the higher potentiality of immobilized tyrosinase over its free form. 2299


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Figure 4. Schematic illustration of biosensing. (a) Ultrasensitive SERS-based sandwich immunoassay using hybrid plasmonic biosilica nanostructured materials. (b) SERS spectra, offset for clarity, acquired at a mouse IgG concentration from 10 μg/mL to 1 pg/mL on diatom frustules (A) and on glass slides (B). Dose−response curves of the Raman peaks at 1331 cm−1 acquired from the SERS spectra on diatom frustules (C) and glass slides (D). Average SERS intensities and error bars of human IgG at 10 μg/mL on diatom frustules and on glass slides were indicated as the solid lines and gray ribbon in parts C and D, and the detection limit on diatom frustules and on glass slides was indicated as the red line in parts C and D. Reproduced with permission from ref 47. Copyright 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. (c) On-chip chromatography-SERS detection of target molecules from heterogeneous biological samples based on plasmonic NP-coated biosilica.

3.3.1. Biosensing. Porous silicon has been extensively investigated for its capability to serve as an optical biosensor.37−39 However, exploitation of diatom silica for the same purpose has several advantages, which includes a large surface area with uniform pores as a matrix, effective monitoring of label-free binding reactions, direct investigation of biomolecules in a natural environment, and exhaustive mixing of the analytical sample and the detector for sensitive monitoring of biomolecular interactions. Immuno-complex formation between antibody and antigen, a necessary and quick process to diagnose and profile diseases reliably, is typically detected by electrical and mass measurement methods, which often bestow low signal intensities in a destructive and invasive way. Thus, the use of a label-free optical detection system as a biosensing platform has gained immense attention to circumvent those limitations. Antibody-functionalized diatom frustules have been delineated to act as a microscale biosensor platform for selective and label-free photoluminescence-based detection of immunocomplex formation.40−42 Functionalization of biosilica from the diatom Cyclotella sp. with the nucleophilic rabbit immunoglobulin G (IgG) antibody amplified the inherent blue diatom photoluminescence by a factor of 6, which is further increased by 4-fold upon binding to goat antirabbit IgG.40 Silica frustules with sub-100 nm periodic pores have also been demonstrated to act as a fluorescence immunoassay biosensor. Diatom photonic crystals possessing an increased local optical field, high spontaneous emission rate, and large surface area have been utilized to create an ultrasensitive immunoassay biosensor that can enhance the detection limit of fluorescence spectroscopy by 100-fold and that of fluorescence imaging by 10×.43 Interestingly, Nitzschia soratensis frustules were packed into Cosinodiscus argus frustules by photolitho-

A porous pattern of frustules provided a large surface area for immobilizing tyrosinase in huge quantities, which explains its high reusability.3 The living diatom silica immobilization (LiDSI) method has been reported for in vivo immobilization of enzymes like glucose oxidase, β-glucuronidase, horseradish peroxidase, etc.30 LiDSI involves genetic manipulation of specific proteins like silaffins, silacidins, cingulins, etc., which regulate the morphogenesis of diatom biosilica.31−33 An in vivo expression of a fusion protein consisting of the protein for immobilization (HabB) and one of the proteins, silaffin, involved in diatom morphogenesis, generates diatom strains that exhibit HabB activity entrapped on biosilica.28 3.3. Photonics and Bio/Chemical Sensing. Diatoms are often referred to as “living opals” as their shells are identical to the structure of opal.34−36 Diatom frustules have the properties of photonic crystals with innate optical characteristics. Biosilica as photonic crystals comprises a periodic, spatially ordered arrangement of dielectric nanostructured materials that modulate the propagation of light in a way analogous to how semiconductor crystals affect the flow of electrons. Thus, light with certain wavelengths can only pass through the crystal.2,13 Moreover, a dumbfounding fact about the optical characteristics of the diatom frustule is that the biosilica structure exhibits photoluminescence properties, since a broad blue luminescence peak at 450 nm is observed while exposed under UV light. Diatom photoluminescence is strongly species specific and depends firmly on their frustule structure and surrounding environment. These eccentric features of a diatom have been exploited to develop a wide array of biosensors and devices, in which biosilica serves as a matrix with a large surface area and an optical transducer of biomolecular interactions with a high sensitivity (Table 2). 2300


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ACS Applied Bio Materials graphic patterning to fabricate multilayered hierarchical frustule arrays to achieve advanced sensitivity in the biodetection chip.44 One of the distinctive features of diatom frustules is that they are capable of increasing the localized surface plasmon resonance of metal nanostructures.45,46 This property of frustules instigates strong enhancements of Raman signals for molecules adsorbed on nanostructures of noble metals like silver and gold, which dramatically increase Raman scattering cross sections. This phenomenon led to the development of surface-enhanced Raman scattering (SERS) in sensing applications. The SERS effect overcomes the inherent weakness of Raman spectroscopy, which is the low intensity of Raman signals. Photonic crystal properties of diatom frustules were first exploited to fabricate a SERS-based immunoassay sensor by Yang et al.47 An ultrasensitive SERSbased sandwich immunoassay was developed using hybrid plasmonic biosilica nanostructured materials. Ag-NPs were allowed to self-assemble on the surface of a diatom frustule. Adsorbed Ag-NPs were detected with enhanced surface plasmon resonances and thus increased the SERS sensitivity of the diatom frustule conjugate (Figure 4a). Subsequently, goat antimouse IgG was applied to functionalize Ag-NPs, and open adsorption sites on it were blocked with BSA to minimize the nonspecific interference in the immunoassay. The selectivity of specific antigens’ detection was examined by challenging the immunoassay sensors with complementary mouse IgG and noncomplementary human IgG antigens, respectively. The immunoassay was observed to be highly specific for the complementary mouse IgG antigen with a detection limit of 10 pg mL−1 (Figure 4b), which is 2 orders of magnitude better than a flat glass surface.47 In the same direction, another ultrasensitive SERS immunoassay based on diatom biosilica decorated with gold nanoparticles (AuNPs) for the detection of interleukin 8 (IL8) in blood plasma was developed.48 Specific antibodies and functionalized AuNPs labeled with a Raman reporter molecule 5,5′-dithiobis(2nitrobenzoic acid) were utilized to fabricate the SERS immune tag. IL8 is sandwiched between the antibody immobilized biosilica and the immune tag, which has been shown to have a higher sensitivity of detection than the sandwich ELISA method.48 Although SERS provides an outstanding limit to detect the target analyte, tremendous difficulty arises for SERS to instantaneously detect small molecules in biological fluids. The reason for this problem stems from the fact that the physiological environment contains a high concentration of salts, which has a strong influence on the stability of both the metallic colloids and the biomolecules. Moreover, macromolecules like DNA, RNA, protein, etc. in biological fluid would prevent the interaction between the target molecules and plasmonic substrate. To resolve that problem, plasmonic NP-integrated frustule film as a lab-on-chip device has been fabricated for on-chip chromatography (Figure 4c) and labelfree biosensing of small molecules from heterogeneous biological samples.49 3.3.2. Chemical Sensing. Aside from biosensing, ultrasensitive hybrid plasmonic−photonic crystal biosilica surfaceenhanced Raman scattering was employed to detect explosive molecules like 2,4,6-trinitro toluene (TNT) from a nanoliter solution.50 In situ synthesized high density Ag NPs were integrated inside the nanopores of diatom biosilica for TNT detection (Figure 5A). Biosilica functionalized with an antiTNT monoclonal antibody has also been demonstrated to

Figure 5. Schematic diagram of chemical sensing. (A) Label-free detection of explosive molecules like TNT by SERS sensing on a hydrophilic photonic diatom silica crystal. Ag NPs were integrated in frustule pores, which was utilized in inkjet printing technology for TNT detection at a nanoliter scale. (B) Synthesis of diatom@TiO2, diatom@TiO2@MnO2 3D composite supercapacitors with a frustule morphology. The pore structure is presented to show the internal and external diatom surfaces coated with TiO2 and MnO2 nanocomposites.

detect TNT by photoluminescence measurements.51 Bioenabled SERS sensing based on diatom silica was also implemented to detect illegal food adulterate like melamine. Melanin, a nitrogen-rich chemical compound, when added to food products falsely increases its protein content and eventually causes kidney disease in infants.52 A lab-on-chip photonic crystal device was also fabricated for food safety sensing to successfully monitor histamine in salmon and tuna.53 Thalassiosira rotula frustules showed an altered photoluminescence emission depending on the surrounding environment. The presence of gases and organic vapors affects both the optical intensity and peaks. Electron attracting substances, like ethanol, NO2, acetone, etc., quench the photoluminescence of a silica frustule; conversely, electron-donating compounds, like xylene, pyrimidine, etc., exhibit the opposite effect. This property of diatom frustules has been explored to detect different gases by developing gas sensors.2,4 Modification of the photoluminescence properties of different lightemitting diatoms has demonstrated to be induced by the presence of NO2.54 A microscale nitric oxide sensing system has been devised by using platinum electrodes connected to the ends of a silicon frustule replica on a silicon nitride substrate. Diatom frustules coated with a thin continuous layer of SnO2 can also act as a sensitive detector of NO gas.55,56 Inimical organic compounds like xylene present in air and water are routinely analyzed by gas chromatography, but this technique requires a complicated and costly instrument. On the contrary, diatom−Ag NP-based SERS is a rapid and easy technique for xylene detection as the porous frustules of diatom have more potential to absorb xylene.52 Interestingly, an inorganic molecular displacement reaction was conducted to convert the biosilica structure into a new composition, 2301


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ACS Applied Bio Materials Table 3. Biomedical Applications of Biosilica drug delivery area

modification/mimic

drug carrier

modification with organosilanes

antigen detection

functionalization with the model antibody rabbit IgG functionalization with IgG by genetic manipulation none

targeted drug delivery prolonged drug release sustained drug release controlled drug release water-insoluble drug delivery

modification of DE by precipitation of partially neutralized aluminum sulfate grafting of stimulus-responsive O(EG) MA copolymer solid self-emulsifying phospholipid suspension of DE and drug

siRNA delivery

APTES modification followed by siRNA attachment

bone regeneration cell growth

none


diatom species

successful loading and release of both hydrophobic (indomethacin) and hydrophilic (gentamicin) drugs quantitatively detects specific gold-nanoparticle-labeled complementary antigens

DE66,67

successful delivery of drugs to the targeted cells like neuroblastoma and tumor cells drug permeation across colon cancer cells Caco-2/HT-29 cocultured monolayers

T. pseudonana61

extended release of adsorbed diclofenac sodium

DE8

thermoresponsive delivery of levofloxacin

Aulacoseira sp.75

self-emulsifying phospholipid suspension approach combined with diatom particles as solid drug carriers as new oral formulations for water-insoluble drugs gene silencing in the cancerous cells

DE77

Cyclotella sp.72

DE7

DE86

Tissue Engineering source of silicon for bone tissue engineering

DE105

covalent functionalization of biosilica shells with TEMPO silicatein-mediated biosilicification of collagen-coated surfaces

support material for bone cell growth

T. weissflogii106

to stimulate osteoblast growth and function by promoting hydroxyapatite formation

bone morphogenesis

enzyme-mediated biosilicification

modulate the expression of genes involved in bone morphogenetic proteins like amelogenin, ameloblastin, and enamelin

bone resorption

none

to treat and prevent osteoporotic disorders

biosilica formation by silicatein107 biosilica formation by silicatein108 biosilica formation by silicatein109

hemostatic agent

chitosan coating

Hemorrhage Control fast fluid absorption leading to efficient hemorrhage control

hydroxyapetite formation

chitosan/dopamine/diatombiosilica composite beads

rapid blood coagulation

Coscinodiscus sp.111 Coscinodiscus sp.114

distinctive structural, chemical, and mechanical features.64,65 Feasibility of biosilica as a drug delivery carrier was first assessed by Losic and his colleagues.66,67 The effectiveness of the pill box structure of diatom microcapsules in terms of biocompatibility, successful loading, and release of both hydrophobic and hydrophilic drugs has proved the potentiality of diatom silica as a preferable drug carrier66 versus the existing synthetic nanoporous silica materials. Multifaceted properties of biosilica allow both the tailoring of drug loading and release properties by chemical functionalization68−71 and the covalent attachment of a specific antibody on its surface (Figure 6A).72,73 Although tremendous effort has been devoted to synthesize mesoporous silica nanoparticles and to utilize it as a carrier for drug delivery, a majority of the attempts were ineffective until Cai and his co-workers reported a successful image-guided drug delivery and in vivo tumor targeting with antibody-conjugated mesoporous silica nanoparticles.74 Interestingly, diatom silica as a drug carrier has been utilized in the long run by manipulating the T. pseudonana genome to modify its frustule surface. A generic method was developed for the attachment of both antibodies and hydrophobic drug molecules on the biosilica surface.61 The LiDSI method was used to achieve the incorporation of an IgG-binding domain of protein G on the surface of biosilica, which in turn enabled the attachment of cancer cell-targeting antibodies. Drug loading was achieved through the encapsulation of a hydrophobic drug in cationic micelles and eventually attached to the diatom shell

preserving the size, shape, and morphology of diatom frustules. Magnesium converted SiO2 diatoms by a vapor phase reaction at 900 °C to MgO with a similar shape and size.57 Likewise, the diatom structure has been shown to be assembled of TiO2, a material used in commercial solar cells, by exposing frustules to TiO2 gas. In this process, titanium replaces the silicon in the biosilica structure (Figure 5B).57 3.4. Biomedical Application. 3.4.1. Drug Delivery. Physicochemical properties of porous silicon, e.g., pore size and shape, layer dimensions, surface functional groups, etc., can be customized to a high extent during the synthesis− functionalization process, and thus, silica-based nanoporous materials have attracted considerable attention over the years as a vehicle for targeted drug delivery (Table 3).58,59 A high efficiency and specificity to deliver hydrophobic drugs and extended drug release profiles have established nanoporous silica materials as a linchpin in the proliferating field of nanomedicine.60 However, the downside of the usage of these nanomaterials lies in their synthesis process, which is tedious, expensive, and requires toxic reagents such as hydrofluoric acid and silanes. Therefore, the natural nanoporous biosilica derived from frustules of a diatom, in lieu of synthetic mesoporous silica, has gained additional engrossment as an effective tool for delivering chemotherapeutic drugs to cancerous cells.61−63 Diatom silica shells can overcome the constraints of conventional delivery of therapeutic agents through its 2302


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Figure 6. Illustration of drug delivery. (A) Drug delivery system using diatom biosilica through its functionalization with sensing biomolecules or immunotargeting bioreceptors, optically active dyes (for imaging) and/or magnetic nanoparticles (for controlled movement to target diseased tissue or cancer cells). (B) Genetically engineered biosilica (green) consisting of surface immobilized cell-specific IgG (red) and liposomeencapsulated drug molecules (yellow) can be successfully applied to adherent neuroblastoma cells (red) or lymphocyte cells (purple). The release of drug molecules happens in the immediate surrounding of the target cells. Reproduced with permission from ref 61. Copyright 2015 Springer Nature. (C) Antibody-labelled diatom biosilica loaded with SN38 reduces neuroblastoma SH-SY5Y tumor growth and histochemical analysis of SH-SY5Y xenografts. (a) Average tumor volumes measured over 17 days in immunodeficient BALB/c nude mice that were implanted with SHSY5Y neuroblastoma cells and treated with a single dose of different diatom biosilica-based materials: GB1-biosilica (n = 4, black squares), antip75NTR-GB1-biosilica (n = 4, blue triangles), anti-p75NTR-GB1-biosilica-SN38 (n = 4, green diamonds), and SN38-CTAB micelles alone (n = 4, red triangles). (b) Comparison of average tumor volumes after 17 days of treatment with anti-p75NTR-GB1-SN38 (green), GB1-biosilica (black), SN38-CTAB micelles and anti-p75NTR-GB1-biosilica (blue). Arrow indicates day on which the intraperitoneal injection was administered. All data are presented as mean ± S.E.M. statistics by an analysis of variance (a) and Student’s t-test (b). Letters that are not shared are significantly different (a, P = 0.002; b, P = 0.004). Frozen sections from individual tumors (28 days after tumor cell inoculation) from the following treatment groups (n = 4) were stained with H&E. (c) Untreated tumor control. Tumor treated with (d) GB1-biosilica, (e) anti-p75NTR-GB1-labeled biosilica, (f) antip75NTR-GB1-biosilica-SN38-CTAB micelle, and (g) SN38-CTAB micelles. Treatments via intraperitoneal injection occurred 12 days after tumor cell inoculation. Images are representative of tissue sections selected from four independent experiments (three images for each sample). Insets: dark spots show the diatom biosilica. Scale bar, 50 μm. Reproduced with permission from ref 61. Copyright 2015 Springer Nature.

drug, diclofenac sodium, on its modified surface. An in vitro study reveals an extended release of diclofenac sodium from the diatomite surface.8 Likewise, a broad-spectrum antibiotic, levofloxacin, was loaded on the surface of diatom silica microcapsules, which was previously grafted with thermoresponsive copolymers of oligo (ethylene glycol) methacrylates.75,76 A temperature-dependent drug release resulted in a strong antimicrobial activity against two common wound pathogens, Staphylococcus aureus and Pseudomonas aeruginosa.75 Utilization of a diatom frustule has been successful as a solid carrier of water-insoluble drug-like carbamazepine (CBZ).77 A poor water solubility of CBZ to primarily treat epilepsy and neuropathic pain demands a relatively high therapeutic dose (100−200 mg increment; daily 0.8−1.2 g) and hence, the time to reach the optimum drug concentration after oral administration varies from 4 to 8 h or longer.78 An oral application of a self-emulsifying phospholipid suspension combined with diatom silica particles as a dispersion medium of CBZ was capable of rapidly dissolving high amounts of water-insoluble CBZ upon dilution with a biological medium (Figure 7A).77 In the same direction, an unconventional nanohybrid consisting of graphene oxide-decorated diatom

surface (Figure 6B). The compelling aspect of this study was that a strategy was devised to simultaneously attach both antibodies and hydrophobic drug molecules to the biosilica of T. pseudonana without using covalent cross-linking and organic solvents. The antibodies, presented on the biosilica surface, specifically targeted and killed cancerous cells like B-lymphoma and neuroblastoma (Figure 6C).61 Diatom silica microparticles (DSMs) have also been assessed for their potential in oral delivery of gastrointestinal drugs like mesalamine and prednisone. DSMs proved to be essentially nontoxic even at high concentrations of 1000 μg/mL in colon cancer cells (Caco-2, HT-29, HCT-116 cells) and Caco-2/HT-29 cocultured cells.7 In stimulated gastrointestinal conditions, diatoms showed a prolonged release of both prednisone and mesalamine. In addition, diatoms exhibited the competency to augment drug permeability across Caco-2/HT-29 monolayers.7 Aside from the codelivery of the antibody and drug to the target cell, the modified diatomite has also been reported to be capable of carrying analgesic drugs through adsorption. The remarkably modifiable surface chemistry of diatomite allowed for successful adsorption of a nonsteroidal anti-inflammatory 2303


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Figure 7. (A) Solid self-emulsifying drug delivery system (SEDDS). SEPS, self-emulsifying phospholipid suspension; SSEPS, solid self-emulsifying phospholipid suspension. (B) Fabrication process of GO−DE nanohybrids through electrostatic and covalent attachment methods. (a) Plain diatomaceous earth (DE) silica particles, (b) organosilane (APTES)-functionalized DE particles, (c) GO−DE nanohybrids fabricated via covalent attachment, and (d) electrostatic attachment of graphene oxide (GO) sheets. Reproduced with permission from ref 69. Copyright 2013 Royal Society of Chemistry. (C) In vitro drug release plots of the (a) burst release for the first 2 h and (b) the complete release process until the entire loaded drug is eluted. The inset shows the position of drug (indomethacin) molecules on the GO surface and hydrogen bond interactions affected by pH. Reproduced with permission from ref 69. Copyright 2013 Royal Society of Chemistry.

plays a central role in precipitating calcium phosphate in early stages of biocalcification of bone.90 Silica−collagen composites has been shown to exhibit in vitro osteoinductive properties when exposed to a simulated body fluid solution.91 The bioactivity of that composite is further increased by the integration of calcium phosphate as a third component.92 It is worth mentioning that silicon binds to glycosaminoglycans and regulates the cross-links between two of the extracellular matrix components, collagen and proteoglycans, and improves the matrix quality to facilitate bone mineralization.93,94 Moreover, silicon directly inhibits osteoclast formation and bone resorption.92 Dietary supplements of silicon in growing animals are reported to improve bone quality in terms of bone strength and density.95,96 The prevalence of silicon in an osteoid is nearly 25 times higher than its concentration in surrounding areas, and enhanced calcification diminishes silicon content gradually.88 Silicon has been shown to be essential for the formation of connective tissue and the stimulation of osteoblast proliferation.97 Thus, biomaterials consisting of silicon integrated into hydroxyapatite have largely been used in orthopedic and dental areas. Their high osteoconduction capacities help facilitate fast bone remodeling.98,99 Synthetic silicate nanoplatelets, which are cytocompatible and strongly interact with the cells, have also been shown to induce stem cell differentiation in osteoblasts and osteocytes in the absence of any external osteoinductive factors (e.g., dexamethasone).100,101 A diverse array of amorphous silica materials from synthetic sources have been utilized for bone tissue regeneration. However, these silica particles are frequently associated with

silica (Figure 7B) as microcarriers of indomethacin, a natural anti-inflammatory drug, displayed a pH-dependent prolonged drug release profile (Figure 7C).69,79 A major shortcoming in chemotherapy and targeted therapies for effective cancer treatment is that tumors often develop multidrug resistance.80,81 Thus, great attention is being paid to restrain the multidrug resistance by combination therapy through gene-silencing RNA, such as small interfering RNA (siRNA) and microRNA (miRNA).82−84 Although siRNA-mediated genetic interference is a powerful molecular tool to treat cancer,85 siRNA is inefficient to penetrate the cell membrane, and in consequence, inadequate siRNA delivery resulted in a limited silencing of its target gene. Thus, an effective transportation of siRNA across the cell membrane requires a nanocarrier. A nanoconjugate of siRNA and biosilica has successfully been demonstrated to deliver abundant siRNA in the cancer cells (H1355).86 3.4.2. Tissue Engineering. Bone development relies on the interaction of multiple factors within a functionally defined spatiotemporal context. The interplay between protein (e.g., bone morphogenetic protein and alkaline phosphatase), organic (organophosphates) and inorganic (silica) chemicals, and physical factors (stress) is essential for bone reconstitution.87 Polymeric porous matrices loaded with calcium phosphate-based ceramics have a decisive role in formulating strategies for tissue engineering for bone regeneration (Table 3). Silicon is believed to possess a critical role in the bone mineralization process in mammals. Some physiological systems control different stages of calcification in the presence of trace elements like silicon, magnesium, and zinc.88,89 Silicon 2304


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Figure 8. (a) The biomimetic approach. A reactive ester polymer (2) was allowed to functionalize the template (1) and the linker (3). (4) Recombinant silicatein is bound via His-tag and Ni2+ to the linker−polymer and subsequently mediates the formation and assembly of polysilica (5). (b) Synthesis and immobilization of the TEMPO-APTES adduct on diatom biosilica. Reproduced with permission from ref 106. Copyright 2015 Wiley-VCH Verlag GmbH & Co. KGaA. (c) SEM images of MG63 osteoblast-like cells seeded onto F- (A, B) and FT-coated (C, D) substrates for 48 h (A, C) and 7 days (B, D). Few cells adhered to the F-coated substrates at 48 h, while an almost uniform sheet was observed after 7 days of culture. Diatom frustules before and after TEMPO grafting are designated as F and FT, respectively. Diatom shells (*). Scale bars: 50 μm. Inset scale bars: 10 μm. Reproduced with permission from ref 106. Copyright 2015 Wiley-VCH Verlag GmbH & Co. KGaA.

reported to promote hydroxyapatite formation107 and provoke alteration of amelogenin, ameloblastin, and enamelin expression in human osteoblast-like SaOS-2 cells.108 SaOS-2 cells while growing on biosiliceous matrices also induce osteoprotegerin (OPG) expression but does not affect the steady-state expression of the receptor activator of the NF-kB ligand (RANKL). Consequently, an enhanced cytokine synthesis and release happens in then extracellular space. At the end, OPG-cytokine binds to RANKL and abolishes its function to activate osteoclast differentiation. Thus, biosilicainduced OPG synthesis in osteoblast-like cells counteracts those pathways that control RANKL expression and function (e.g., maturation of preosteoclasts and activation of osteoclasts).109 3.4.3. Hemorrhage Control. Unmanageable hemorrhaging caused by civilian and military war or by complicated orthopedic, cardiovascular, and hepatic surgeries, where the incidence of severe bleeding is frequent, can lead to hypovolemic shock, which eventually can culminate in death without fluid replacement and bleeding stoppage.11,110,111 A colossal amount of effort has been made in the past few years to erect hemostatic agents with a high efficacy. Current hemostatic agents are associated with a major drawback of a lower efficiency of hemorrhage control. QuickClot zeolite, a widely used blood-coagulating agent, generates heat with the temperature increasing from 44 to 95 °C. This leads to burning of tissues in the surrounding area.112 The HemCon bandage consisting of a lyophilized chitosan derivative has overcome the aforementioned problem, but its specific shape renders it incapable of treating a large or deep wound.113 The specifications of an ideal hemostatic agent include the following: the aptness for rapid blood clotting at all scales, the capacity to minimize damage at the secondary site, nontoxic, nonimmunogenic, and inexpensive. Porous silica materials have acquired remarkable attention to fabricate hemostatic materials as they have a rapid plasma absorbability without the increment of temperature and efficient contact activated homeostasis.111 However, the

hazardous chemicals and surfactants, which are used for its synthesis.102 Hence, biogenic silica from a diatom as an alternative to synthetic silica has gained immense attention for bone remodeling. Naturally synthesized biosilica lacks any toxic materials associated with it and has a high bioactivity, which essentially depends on its size, shape, and surface properties.103,104 Diatom silica microparticles and nanoparticles have successfully been shown to release silicon ions, which has absolute potential for bone tissue engineering,105 and can be used as a biological blueprint for biomedical and biotechnological applications. In a biomimetic approach, polymer-functionalized inert matrices were utilized for the attachment of His-tagged silicatein, which has the capacity to synthesize nanoparticulate biosilica (Figure 8a).87 Inductively coupled plasma optical emission spectrometry revealed that the dissolution of diatom skeleton-derived particles in deionized water liberates silicon ions, the kinetics of which is influenced by the diatomite purification method and particle size. Diatom-derived microparticles and nanoparticles showed essentially no cytotoxic effect in vitro depending on the administration conditions.105 Diatom silica has shown enormous prospective to act as a single platform for multipurpose effects like bone regeneration, drug delivery, and antioxidant function.11,106 Thalassiosira weissflogii frustules were implemented to increase the adhesion and proliferation of L murine fibroblasts and human osteosarcoma MG63 cell lines (Figure 8c). Nanostructured biosilica from T. weissflogii was functionalized covalently with cyclic nitroxide 2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), an efficient scavenger of reactive oxygen species in biological systems to avoid any inflammation. The attachment of TEMPO on biosilica was achieved by APTES (Figure 8b). The resulting TEMPO− biosilica was used for physical adsorption of ciprofloxacin, a common drug to treat bacterial infections associated with teeth and bones. The TEMPO−biosilica platform combining ciprofloxacin drug delivery with antioxidant properties is demonstrated to be an appropriate material for fibroblasts and osteoblast-like cell growth.106 Biosilica has also been 2305


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Figure 9. (a) Data from the rat-tail amputation model among the diatomite, diatom, chitosan-coated diatom (1-CS-diatom), gauze, and commercial Quikclot zeolite. (A) Photograph of the hemostatic effect by contacting with the wound. (B) Clotting time. (C) Blood loss. Data represents the mean ± SD (n = 6). Reproduced with permission from ref 111. Copyright 2016 American Chemical Society. (b) Cytotoxicity of diatomite, diatom, and CS-diatom exposure on mouse embryonic fibroblasts (MEFs) at different concentrations after (A) 24, (B) 48, and (C) 72 h incubation. Data represent the mean ± SD (n = 6). (D) Fluorescence microscopy images of MEFs treated with diatomite, diatom, and 1-CSdiatom at 5 and 10 mg/mL for 24 h incubation. Reproduced with permission from ref 111. Copyright 2016 American Chemical Society.

(CdS) was deposited on the Pinnularia sp. frustule to accord optoelectric properties to biosilica. CdS is a direct band gap (2.42 eV) semiconductor at room temperature, and its conductivity increases when it is irradiated, leading to its usage as a photoresistor. It forms a core component of a photovoltaic cell in combination with a p-type semiconductor. Hence, the CdS/biosilica nanocomposite has a high potential to develop photodetectors, solar cells, and various other optoelectronic devices.117 The evolved frustules had a compact, homogeneous, and nanostructured coverage, and their photoluminescence spectra showed a sharp yellow emission typical of CdS nanoparticles; thus, a new pathway to synthesize nanostructured materials with tunable photoluminescent properties was unravelled. Likewise, frustules were coated with a TiO2 nanosphere and MnO2 mesosphere nanosheet to resolve the nonconductive nature of a diatomite. This deposition is based on the hydrolysis and methathetic reaction of the TiF4 precursor followed by the reaction with KMnO4, which allows the coating of internal and external hollow structures with TiO2 and MnO2 (Figure 5B). The template-based synthesis provides a strong vertical and horizontal interconnection of building structures in both layers, which maximize their conductivity.118 The pseudocapacitive behavior of MnO2119 and the charge storage nature of its electrode120 have fascinated researchers to use it as an electrode material for supercapacitors. The presence of TiO2 on the diatom silica structure improves specific capacitance and rate performance and lowers resistance when compared with the explored MnO2 electrode devoid of diatom and TiO2.118 The 3D composite diatomite@TiO2@MnO2 electrode, which is prepared with a low-cost natural material and a simple scalable process, has been described to possess an enhanced supercapacitor behavior (Figure 5B) and, thus, has extraordinary potential for an energy storage/conservation application.118 A frustule from the Pinnularis sp. containing metabolically inserted germanium (Ge) by a two-stage

shortcomings of these synthetic silica materials in terms of high production cost, prolonged time requirement for their synthesis involving toxic chemicals, etc. urged us to use lowcost mesoporous silica materials like biosilica. Diatomite was coated with a chitosan derivative, a natural, highly biocompatible, biodegradable polymer with an efficient hemostatic quality. Chitosan-decorated biosilica has been reported to possess a higher plasma absorption ability, blood compatibility, and essentially no cytotoxicity, which ultimately are reflected in a higher efficiency in hemorrhage control (Figure 9).111 Wang et al. have recently reported the utilization of composite beads consisting of chitosan, dopamine, and diatom silica for rapid blood coagulation.114,115 Dopamine in that composite was used as a bioglue. The composite beads have been represented as a good biocompatible material with less than a 5% hemolytic rate and above an 80% cell viability. 3.5. Diatoms for Energy Applications. Limitation of amorphous biosilica to develop an optoelectric device lies in its dielectric nature. Numerous attempts have been made to modify silica without altering frustule shapes and morphologies to develop functional materials, which will be technologically more fitted. This type of synergistic combination of natural nanostructures from biological sources and functionalization of synthetic chemical moieties with the desired optical, electrical, and magnetic properties established the foundation for the synthesis of bioengineered 3D micro/nanostructured materials for energy applications. Silver nanoparticles, for instance, were deposited on a frustule surface by a chemical reaction-based route, in which process plasma-pretreated frustules were added to a silver nitrate solution to facilitate electrostatic adhesion of AgNO3 to the biosilica surface. The addition of sodium hydroxide followed by a glucose redox reaction produces silver nanoparticles attached to biosilica. This material acts as an efficient photocatalyst in the sodium borohydrate fast reduction of rose bengal dye.116 In a similar line, a thin film of cadmium sulfide 2306


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Figure 10. (A) Schematic diagram of the shape-preserving conversion of diatom frustules to nanostructured silicon by magnesiothermic reduction. Thiol-modified nanostructured silicon frustules are attached to the surface of a gold-plated electrode, which is utilized in an assembly of the working electrode into a three-electrode electrochemical cell for light conversion and photocurrent measurements. Reproduced with permission from ref 127. Copyright 2014 Royal Society of Chemistry. (B) Photocurrent measurements after CdS deposition on hydrosilylated diatoms and dependence on bias potential, which was increased in steps of 100 mV. The inset shows the SEM image of the arrangement of hydrosilylated silicon diatom frustules coated with CdS on the fabricated electrode surface. Further, the high-resolution SEM image and a corresponding EDXS confirm the presence of a CdS coating on the hydrosilylated silicon diatom frustules. Reproduced with permission from ref 127. Copyright 2014 Royal Society of Chemistry.

times to increase the connectivity of the titania layer, and the resulting modified biosilica in association with ethylcellulose and terpineol was used as a substrate coating for the photoanode of DSSC. The effectiveness of this substrate for the adsorption of N719 ruthenium dye was high, which led to enhanced light harvesting and energy conversion efficiency (∼30%) compared to a standard titania cell developed without frustules.125 A pioneering work by Sandhage et al. on the conversion of a cylindrical biosilica frustule from Aulacoseira sp. into the MgO/ Si composite,57 or Si,56 or TiO2126 nanostructures that retain the frustule shape was based on the magnesiothermal reduction or the methatetic reaction. The resulting silica mimic can be further modified chemically to fine-tune their features for diverse applications including solar energy conversion. A hydrosilylation reaction of the Si replica of Aulacoseira frustules with allyl mercaptan functionalized its surface with thiol groups to assist their stable binding to a goldplated glass slide, which was used as the working electrode in a photoelectrochemical cell for solar energy conversion (Figure 10A).127 To further enhance the photocurrent generation, a secondary chemical bath deposition of the CdS photocatalyst on the surface of Si replicas on the gold electrode was performed (Figure 10B). Interestingly, a silica frustule from Aulacoseira sp. was also doped with boron, and then magnesiothermal conversion generated B-doped silica mimics, which turned out to be efficient p-type semiconducting materials. Additionally, B-doped silica replicas were utilized to fabricate efficient photocathodes of electrochemical cells for hydrogen production by water splitting.128 Magnesiothermic reduction was also employed to convert biosilica to nanosilicon (nanoSi) for use as anodes in lithium-ion batteries.129 The nanoSi powder was carbon coated through chemical vapor deposition of ethylene. Carbon-coated silica nanostructures were deposited with polyacetylene black and poly(acrylic acid) binder on a copper foil electrode, which was eventually used as the anode in a Li-ion battery.129 Carbon-coated silicon with a high porosity presented a higher specific surface area (162.6

photobioreactor cultivation process has been shown to possess both photoluminescence and electroluminescence in the visible spectral range.121 Since Ge is a semiconductor, the Si−Ge oxide nanostructure composite has potential to be utilized in optoelectronics, thin-film displays, solar cells, and electroluminescent devices.122 A distinctive process involved the assembly of mesoporous TiO 2 inside the macropores of diatom frustules by sonochemical condensation of a titanium chloride precursor and a subsequent thermal treatment at 550 °C to accumulate TiO2 inside the biosilica pore. When the sonication time was altered, the amount of TiO2 inside the periodic macropores of diatom was regulated. The sonochemical method of titania deposition inside the frustule pores does not alter the original 3D shape and geometry of the pores. The resulting composite with 30% TiO2 loading delivered a high photocatalytic performance.123 A similar ultrasound-based method was also applied to modify the Coscinodiscus lineatus frustule surface with ZnS nanoparticles, which have a high refractive index and are primarily used in optic and photonic applications.124 Surface-modified diatom frustules with titania nanoparticles have been reported to improve the light harvesting efficiency of dye sensitized solar cells (DSSC),125 which has developed into promising technology for high solar energy conversion efficiencies.123 Titania has an excellent electron conductivity and, thus, is used as a semiconducting material. Titania NPs were attached to the plasma-treated surface of diatom frustules. Plasma treatment is a clean and reagentless process, in which an air plasma source of 40 kHz at 110 W was used to treat the frustule surface for 5 min with agitation between each ion bombardment. This treatment effectively removed methyl groups bound to the silica surface and helped attach the hydrophilic hydroxyl group to titania. NPs were synthesized by the controlled hydrolysis of titanium(IV) isopropoxide at room temperature in purged hexane, carried out in the presence of plasma-treated frustules. A subsequent thermal process at 500 °C resulted in a TiO2 NP deposition at an anatase phage on the frustule surface. The same procedures were repeated three 2307


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ACS Applied Bio Materials cm2 g−1) than the original diatomite. The DE-based nanoSi anodes display good cyclability with a specific discharge capacity of 1102.1 mAh g−1 after 50 cycles and high areal loading (2 mg cm−2).129,130 The metathetic displacement reaction implemented to yield an anatase TiO2 frustule mimics a reaction between Aulacosera biosilica with TiF4 gas at 350 °C.126 These mimics were utilized in a hydrothermal treatment with molten Ba(OH)2 or Sr(OH)2 to acquire BaTiO3 or SrTiO3 titanate frustule replicas, which exhibit a nearly phase pure, nanocrystalline perovskite structure. Piezoelectric and ferroelectric properties of these structures accomplish the yardsticks for an appropriate active material for capacitors, transducers, piezoelectric actuators, passive memory storage devices, and electro-optical devices.131 A combination of Si with other elements like Zn, Ge, and Ti132 when incorporated in a diatom frustule is considered as an advanced material with felicitous properties for utilization in solar cell technology. Based on the current knowledge on the element uptake and biosilicification processes in a diatom, an innovative approach for doping a silica frustule with titanium oxide, of which is a semiconductor with a higher band gap, was developed through biological incorporation of Ti into the frustules of the pennate diatom Pinnularia sp.132 Chauton et al. reported that the distribution of the incorporated Ti is inhomogeneous both between and within valves. Ti was shown to be predominant around the pores on the valves, which could be utilized in the enhancement of photon transport through the nanoscaled pores. Bioincorporation of group IV elements in the silica shells was also examined in Synedra acus, and germanium was shown to be substantially incorporated. The consequences of this metal incorporation in the silica shell are altered (valve shape and thickness, change in areolae rows, etc.).133 The advantage of bioincorporation of elements like Ti, Ge, etc. lies in the employment of biological machinery, which eliminates the usage of hazardous chemicals. 3.6. Sorting. 3.6.1. Microfluidics. In an aquatic environment, diatoms are exposed to a continuous flow of living and nonliving Brownian particles, which includes small nutrient molecules, ions, bacteria, etc. Lattice-like silica frustules of the diatom act as a protecting barrier, which shield intracellular DNA from UV radiation,134 and concurrently, their microchannel wall promotes the particle control process that excludes the uptake of large particles.135 Brownian particles including nutrients and bacteria are only permitted to reach to the cell surface of a nonmotile diatom by a diffusive and advective transport mechanism.136 Thus, frustules follow a particle sorting mechanism for diatoms and have a decisive role in allowing the materials to reach the cell membrane and its receptors. Diatoms reside in the environment where the diameter of particles varies from a few nanometers to 1 μm; hence, the sorting of particles across distances of 10 nm to 10 μm is a crucial function. Diatom frustules with a unique complex 3D disposition of silica pores provide angularity, hardness, and inertness. The combination of all of these properties is not present in any other biologically derived structures and render diatoms the microfluidic sorting capabilities of particles.137 In contrast to the flexible exterior of other microbes where the sorting functions limit at hundreds of micrometers, the diatom biosilica exterior follows the sorting process on submicrometer mechanisms. The exterior diatom microstructure under flow conditions controls the behavior of particles at the surface, including the particles in the colloidal size range. In addition to that, frustule

microtopographies may also control the flows above the surface. Diatom silica shells have two sets of staked pores: one with a nearly 40 nm diameter arranged in the interior region of frustules and another set with a 1 μm diameter positioned at the exterior part. The hexagonal silica cell walls in conjunction with these pores establish the 3D chambers, which only can enable constrained and asymmetric diffusion.137 The synthetic mesoporous silicon membrane emulating the frustule geometry, fabricated to possess the periodic asymmetric variation in the pore diameter, exhibited the potential of such structures for submicrometer particle separation.138 This likelihood ascertains the asymmetric diffusion of macromolecules across the frustule valves. Nature ameliorated frustule features through evolution for the selective transportation of macromolecules through frustule microchannels in a highly regulated asymmetric fashion even when exposed to the oceans’ periodic pressure oscillations. Hence, incorporation of functionalized silica frustules of diatoms into microfluidic or lab-on-a-chip systems might be achieved to develop well-controlled, efficient, high-resolution separation devices for the analysis of macromolecules like protein, DNA, and RNA and also for perusal of cell sorting. Research interest in the development of microfluidic diatomite analytical devices (μDAD) has escalated over the years to promote a new spectrum of biomedical devices for point-of-care diagnosis and biosensing. μDADs comprise nanoporous photonic crystal biogenic silica channels for label-free biosensing of felonious drugs like cocaine from biofluidic samples using on-chip chromatography in association with the SERS-sensing process.139 Nanoscale dimensions of diatomite microfluidic channels in conjugation with its photonic crystal property enabled μDADs to possess an unprecedented sensitivity down to the parts-per-billion (ppb) level, while detecting pyrene (1 ppb) from the mixed sample with Raman dye and cocaine (10 ppb) from human plasma.139 3.6.2. Chromatography. Chromatography is an important tool to purify cellular proteins. Gel filtration is distinctive among all chromatographic techniques in that fractionation depends on the relative size of protein molecules. Unlike traditional filtration, none of the proteins are retained by a gel filtration column. Generally, porous beads are utilized to construct the chromatographic support of the gel filtration column. A column built with such permeable beads will have two measurable liquid volumes. Liquid between the beads constitutes the external volume; however, the internal volume is determined by the volume of liquid within the pores of the beads. Larger molecules will settle an equilibrium in the external volume, while a small molecule will equilibrate with both internal and external volumes. Larger molecules in a mixture of proteins applied on the top of the column will emerge first. In contrast, smaller proteins will traverse through the pores of the beads and, thus, will travel a longer path than the larger proteins.140 Since diatom frustules contain a uniform patterning of pores with a specific size, proteins in a mixture can be separated by selecting frustules from a particular species of diatom and the pore size of which is permeable to one protein but not to the other. Frustules have also been reported to be designed by the assistance of Compustat to provide a constant and highly specific size, which could then be utilized as porous beads in the gel filtration column. The key advantages of the usage of biosilica over the synthetic cross-linked dextran gel material like Sephadex include a low production cost and a wide 2308


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carbon,152 polymeric resin,153 chitosan, starch, clay, perlite,151 mesoporous silica,154 zeolite,155 etc. have been utilized as adsorbents; however, the synthesis of these materials is expensive and entails toxic materials. Hence, the focus on diatom silica with an intricate mesoporosity was intensified to use it as an inexpensive natural sorbent of heavy metals from water. The surface of diatom microparticles was modified with a self-assembled monolayer of organosilanes (MPTMS, APTES, and n-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (AEAPTMS)). Surface-functionalized diatom frustules exhibited high-efficiency Hg(II) ion adsorption.144 Diatom particles modified with mercapto groups (MPTMS) showed a higher adsorption efficiency at a pH range of 5−9 than that modified with amino groups. This fact confirms that sulfurdonating functional groups (SH) have a higher capability of capturing the Hg(II) ion (Figure 11A).144 Diatom frustules

number of different kinds of frustules with species-specific uniform pore sizes. DE or diatomite, a heterogeneous mixture of the fossilized remains of diatoms, has long been known as an excellent filter as they have a high porosity with very fine pores. DE has been reported to possess a wide range of applications such as filters in swimming pools and water purification, removal of dissolved uranium from water, etc.13,141 The use of a diatom also has a benefit over DE, as DE furnishes a medium of heterogeneous filtration, while diatom biosilica provides a homogeneous porous environment,142 which is essential for separating proteins with a higher precision in size exclusion chromatography.6 Siliceous valves from diatom Synedra acus were also reported to be utilized as high-performance liquid chromatography (HPLC) sorbents with well-developed flow macropores (>100 nm in size). Somewhat concave rectangular or needle-shaped silica shells 20−100 μm in length, 2−5 μm in width, and nearly 200 nm in thickness have been successfully used in liquid chromatography as an economical and efficacious substituent of synthetic chromatographic silica particles. Uniform micro- and mesopores with a specific surface area and high mechanical strength of biosilica appeared to be efficient in filtration and to withstand high pressure (up to 40 MPa), which are the eminently desired aspects of the HPLC matrix.143 3.7. Removal of Heavy Metals. Heavy metals like mercury, arsenic, cadmium, lead, etc. are commonly identified as the most deleterious pollutants in the environment because of their high toxicity as well as bioaccumulation and bioconcentration through the food chain. Natural processes like volcanoes, river floods, etc. and many man-made industrial bustle and processes such as gaseous emissions of fossil fuel combustion, paper pulp, pharmaceutical, rubber, fertilizer, oil refineries, waste treatment, etc. are the sources of metal pollution.144,145 Many among the elemental heavy metals like mercury, cadmium, lead, etc. and their compounds have no known usual metabolic function, and their association with living organisms is elucidated as the contamination from anthropogenic sources. Cd as an environmental toxicant is found to be frequently contaminated in foods like spinach, potato, lettuce, etc. in the form of cadmium chloride, cadmium oxide, and cadmium sulfide.146 Exposure to Cd causes alteration in the expression of signaling cascade proteins with a deleterious effect on human sertoli cell viability.147,148 The toxicity of metalloid arsenic depends on the chemical form in which it is found and on its metabolism; arsenic in its trivalent form is more toxic than its pentavalent state.149 Damages are predominantly found in the liver, skin, and cardiovascular and reproductive systems. Lead causes occupational health problems that damage the renal, skeletal, hematopoietic, nervous, and reproductive systems.150 The toxicity of mercury largely depends on its redox state. A divalent form of mercury is more toxic as it reduces disulfide bonds and binds to amino acid cysteine in a polypeptide chain. Elemental mercury (Hg0) and organo-mercury compounds like monomethyl and dimethyl mercury have the ability to penetrate membranes and also to cross the blood−brain barrier.151 Several techniques like electrodeposition, ion exchange, coagulation, bioadsorption and photoreduction, membrane filtration, reverse osmosis, etc. have been implemented to eliminate heavy metals from the air, water, and soil. Adsorption using solid porous materials has been recognized as a trustworthy method among them in terms of efficiency and cost-effectiveness. Many synthetic materials, e.g., activated

Figure 11. (A) Removal of Hg(II) ions through adsorption. A biosilica surface was chemically modified by MPTMS. Free thiol groups covalently attached to the modified biosilica surface adsorb Hg2+ ions from the medium. (B) Illustrative diagram of biosilica replica modeling. The external concave surface (1) of the diatom frustule was immobilized (a). Generation of a negative replica of the frustule into PDMS (b and c). The PDMS replica was replicated into a UV-curable, mercaptol ester polymer (NOA60) (d and e). (f) and (g) Convex orientations (2) of frustules and their positive replica.

also showed a good efficiency for the cationic exchange of metals like As3+, Pb2+, etc. Diatom silica also showed a good retention capacity for heavy metals.145 The potential of functionalized diatom frustules was examined for the frustules’ ability to act as a bioadsorbent for trivalent arsenic from water. Silica shells of a diatom Melosira sp. were chemically modified with MPTMS and 3-aminopropyl-trimethoxysilane (APTMS), which allowed the frustule surface to possess bifunctional (amino and thiol) groups. The presence of both amino and thiol groups effectively removed arsenite, a toxic form of arsenic abundant in an aqueous environment, by adsorption. Within 26 h, a maximum adsorption capacity of 10.9 mg g−1 was attained for a solution containing 12 mg L−1 As(III) at pH 4 and a sorbent dosage of 2 g L−1.156

4. ADDITIONAL DIVERSE AREAS OF BIOSILICA APPLICATIONS Aside from its extensive utilization in diverse areas, researchers are progressively exploring diatom frustules in additional fields, where its potentiality was never been examined (Table 4). A wide array of smart nanostructured materials were synthesized by chemical and biochemical methods that integrate, decorate, convert, or mimic biosilica shells of diatoms without amending their structural attributes. One important area of investigation 2309


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ACS Applied Bio Materials Table 4. Representative Examples of Biosilica Applications in Additional Diverse Areas energy applications area

modification/mimic


diatom species 116

photocatalyst

decoration with Ag NPs deposition of titania

reduction of rose bengal dye degradation of methylene blue dye upon UV irradiation

photoresistor

deposition of a thin CdS film

fabrication of the 3D composite electrode semiconductor

diatom@TiO2@MnO2 composite

optics and photonics solar cells photoelectrode

deposition of ZnS NPs surface modification with titania NPs surface functionalization by allyl mercaptan deposition of CdS on the surface of Si replicas deposition of C-coated nano-Si powder on a copper foil electrode replicas of frustules

development of photodetectors, solar cells, and various other optoelectronic devices enhancement of the supercapacitor behavior and has potential for energy storage/conservation applications utilized for optoelectronics, thin-film displays, solar cells. and electroluminescent devices nano-optics and nanophotonics device development improvement of harvesting efficiency of dye sensitized solar cells utilized as a working electrode in photoelectrochemical cells for solar energy conversion photocathodes of electrochemical cells for hydrogen production by water splitting used as an anode in a Li-ion battery

lithium-ion battery capacitor, transducer, etc.

metabolic insertion of Ge

particle separation

none

size exclusion separation

none modified by hexadecyltrimethylammonium

adsorption of Hg(II) ions cation exchange bioadsorbent

modification by organosilanes modification by organosilanes modification by MPTMS and APTMS

mammalian cell growth

modification by organosilanes

used in capacitors, transducers, piezoelectric actuators, passive memory storage devices, and electro-optical devices Microfluidics submicrometer particle separation

DE diatom species from Yunan Province, China123 Pinnularia sp.117 Coscinodiscus sp.118 Pinnularis sp.121 Coscinodiscus lineatus124 DE125 Aulacoseira sp.127 Aulacoseira sp.128 DE129 Aulacosera sp.131

Coscinodiscus sp., T. eccentrica139

Chromatography separation of biomolecules removal of uranium ions from water

DE6 DE141

Removal of Heavy Metals removal of Hg(II) ions from water

DE144

elimination of toxic cations like As3+ and Pb2+ from solution removal of arsenite in an aqueous environment 3D Cell Growth effective platform for 3D mammalian cell growth

diatoms from Mexico145 Melosira sp.156 T. weissflogii5

bacterial fermentation and very slow growth of plant-based setups. To overcome the above shortcomings, diatom-based PHB manufacturing is gaining considerable attention for the following reasons: easy to handle, no requirement of adding an external organic carbon source in media, and demands less light and energy for cultivation. PHB synthesis in a diatom was first reported by Hempel et al., by the introduction of the in vivo PHB synthesis pathway of bacteria Ralstonia eutropha H16 into the diatom Phaeodactylum tricornutum.158 Interestingly, the expression level of PHB in P. tricornutum was around 100fold higher than that in plant cytosols.159 Hence, the insertion or alteration of metabolic pathways in a diatom for the synthesis of biologically active molecules or raw materials may have numerous applications in the nanobiotechnological industry and the production of renewable biofuel.17 The addition of fillers to polymer materials significantly reduces polymer consumption and also modifies polymer properties like the enhancement of mechanical rigidity, noise insulation, and fire resistance, alteration in dielectric nature, and reduction in the toxicity of the combustion products. Currently, poly(vinyl chloride) (PVC) is predominantly expended as fillers among all other known polymers. The introduction of 2−3% diatomite into PVC-based plastisol significantly reduces its combustibility, inflammability, and smoke-forming ability. Diatomite addition to plastisol considerably increases the viscosity and also improves its foaming and carbonization properties.160 DE as fillers also efficiently promotes the formation of coke with a high porosity, which

is the utilization of diatom frustules as multifunctional support for mammalian cell growth in three dimensions.106 Bioactive mesoporous materials have been shown experimentally and clinically a lot of promise to act as scaffolds for cell attachment, proliferation, and differentiation because of their regular distribution of pores, large specific surface area, and tunable pore size.157 The intriguing ability of biosilica as an alternative of synthetic mesoporous materials for regenerative medicine applications was first demonstrated by Cicco et al.106 They assessed the effect of TEMPO-functionalized biosilica from T. weissflogii on the bone environment using L murine fibroblasts and the human osteosarcoma cell line MG63 and established that TEMPO functionalization positively affects cell viability (Figure 8c).106 Apart from TEMPO functionalization, T. weissflogii frustule surfaces were chemically modified with organosilanes like MPTMS and APTES, which decorate diatom microcapsule surfaces with mercapto amino groups. An analysis of adhesion and growth of normal human dermal fibroblasts and the human osteosarcoma Saos-2 cell line (Saos2) on bare and silane-coated diatom frustules revealed that both cell types were not affected either by mesostructure or by the SH group but were negatively affected when exposed to NH2 groups.5 Hence, the urge to identify a proficient approach, which will fabricate diatom silica as an effective platform for 3D mammalian cell growth, is mounting high. Low-cost commercial production of poly(R)-3-hydroxybutyrate (PHB), a biodegradable thermoplastic, is still a challenge, owing to the high cost of their production by 2310


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have drawn significant research attention as a highly promising replacement of synthetic materials, especially those with nanopatterning. Diatom silica shells, in particular, have evolved with their own attributes in their composition and structures, which have been exploited in diverse applications. Each individual feature of biosilica was maneuvered to contrive technological devices. Enormous growth in the discoveries of biosilica utilization for technology development intensified our engrossment in understanding the aspects of their uniqueness. A methodical approach has been taken in this Review to comprehensively describe all different kinds of applications of diatom biosilica to date in the literature. This Review also includes outstanding advancements in understanding diatoms’ genomic, structural, optical, photonic, and mechanical properties, leading to the nanofabrication and engineering of new materials and devices using biosilica. Biogenic silica shells with an unparalleled diversity in its structure and morphology have been utilized in a wide range of applications. The principal advantage of biosilica over other synthetic mesoporous materials lies in the process of in vivo production of silica structures by a diatom without the involvement of complex chemical procedures, thus, presenting an extraordinarily cheap material for analytical sciences.13 Numerous biological and chemical approaches to modify frustule composition of a diatom preserving their genuine morphological characteristics provide a powerful resource for the low-cost and scalable production of nanostructured materials, the attributes of which can be tailored for a specific application. Biofunctionalization or a chemical modification of the diatom silica surface has uplifted its application into an advanced stage for the production of a new set of bioactive Si nanostructures that can be conjugated with biomolecules. Nanostructured materials with the covalently or noncovalently attached molecules like proteins, drugs, or metal-based nanoparticles have been efficiently produced with the retention properties of both nanostructures and surface modifiers.57 Surface modification and its intricate photoluminescence properties have enabled biosilica to be applicable in bio/ chemical sensor development. Likewise, the biosilica platform with an immobilized antibody and attached drugs has been successfully used in targeted drug delivery. Genetic engineering-based in vivo modification of the diatom silica surface has been established the more effective surface modification method for changing both the bulk and surface composition of diatom frustules. Bioclastic and morphology-preserving processes produced a diverse heterogenic nanostructured mimic with altered chemical composition and characteristics from those of diatom silica. These materials are extensively used in microfluidics, optics, nanoimprint lithography electronics, etc. Diatoms also exhibited its stupendous potential to serve as a solar-fueled expression system for recombinant proteins. Completely assembled and functional IgG antibodies can be expressed in a sufficient concentration, and more interestingly, it can be secreted into the culture medium where the antibody maintains its stability for several days. This process substantially alleviates the difficulties of the extended complex downstream purification steps. Phaeodactylum tricornutum was engineered to effectively secrete fully functional and assembled human IgG antibodies in the culture medium to destroy the hepatitis B virus surface protein.17 Thus, diatom microalgae are emerging as novel machineries for the expression of high-valueadded pharmaceutically important proteins. A fascinating

demonstrates the polymer’s heat shielding nature. The effectiveness of diatomite appeared to be substantially higher than that of other natural Si-containing fillers like muscovite, vermiculite, palygorskite, phlogopite, etc.160 A soft-lithographic method was employed to synthesize the organic polymer mimic of diatom silica shells, preserving its 3D morphology.161 An emerging application of these mimics lies in the synthesis of dendritic materials by grafting the appropriate polymers on its surface, which also can alleviate the post-tailoring process. Dendritic materials have gained tremendous attention for their applications in multidisciplinary areas ranging from drug delivery to nanobuilding blocks. A hyperbranched dendritic copolymer of poly(divinyl-benzene) and poly(ethylene glycol dimethacrylate) was successfully prepared from homopolymerizations of commercially available multifunctional vinyl monomers.162 Implementation of this reaction favorably grafted a unique three-dimensional poly 2(dimethylamino) ethyl methacrylate-co-ethylene glycol dimethacrylate onto T. weissflogii frustule surfaces.163 The presence of a large number of vinyl functional groups on the modified hyperbranched surface of frustules allows the ease of posttailoring. Furthermore, free vinyl groups generate a rigid polymer coating on diatom surfaces as a consequence of crosslinking with each other.163 Frustules, apart from serving as templates for the synthesis of positive replicas with an unaltered 3D nanostructure, have also been shown to generate negative replicas. Frustule valves from centric diatom species, Coscinodiscus sp. and T. eccentrica, were immobilized in a controlled fashion to expose their external concave or internal convex surface to the substrate. The elastomeric soft polymer mold poly(dimethylsiloxane) (PDMS), a widely adopted material for transferring patterns onto various surfaces, was utilized as a negative replica to transfer the diatom’s porous pattern on it by replica modeling. The PDMS replica was then converted into a positive replica using a desired material such as the UV-curable, mercaptol ester polymer NOA60 (Figure 11B).164 Optically transparent and cost-effective PDMS and NOA60 have regular uses in microfluidics, photonics, etc. The polymeric facsimiles have the potential to be utilized in the development of different biomimetic shapes, microlense development, nanofabrication, biosensing, etc. Although the single diatom replication is inciting, the random assembly of natural diatoms creates problems in the controlled large area fabrication of diatom templates for replication. Hence, most technical implications require highly ordered and controlled diatom frustules regularly distributed over a large area. Threedimensional multiphoton laser lithography is a divergent approach to fabricate a diatom-like structure by exquisitely mimicking the frustule structure. This technique also restores the tailoring and other structural properties of frustules in the mimic for an optimized utilization in the technical application. A negative tone acrylic photoresist was utilized to fabricate the microstructures by 3D lased lithography for the demands of two-photon-polymerization-based 3D direct-laser writing or nanoimprint lithography. The resists manifested a high mechanical stability and a high spatial fabrication resolution.165

5. CONCLUSIONS Nature has selected living organisms to possess the optimum capacity to create specific functional elements indispensable for their survival, growth, and reproduction. These functional components may comprise macroscale or nanoscale structures with a hallmark of highly replicative characteristics and, thus, 2311


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(5) Cicco, S. R.; Danilo, V.; Roberto, G.; Eloisa, S.; Roberta, R.; Presti, M. L.; Farinola, G. M. Biosilica from Living Diatoms: Investigations on Biocompatibility of Bare and Chemically Modified Thalassiosira weissf logii Silica Shells. Bioengineering 2016, 3, 35. (6) Parkinson, J.; Gordon, R. Beyond micromachining: the potential of diatoms. Trends Biotechnol. 1999, 17 (5), 190−196. (7) Zhang, H.; Shahbazi, M. A.; Makila, E. M.; da Silva, T. H.; Reis, R. L.; Salonen, J. J.; Hirvonen, J. T.; Santos, H. A. Diatom silica microparticles for sustained release and permeation enhancement following oral delivery of prednisone and mesalamine. Biomaterials 2013, 34 (36), 9210−9219. (8) Janićijević, J.; Krajisnik, D.; Calija, B.; Vasiljevic, B. N.; Dobricic, V.; Antonijevic, M. D.; Milic, J. Modified local diatomite as potential functional drug carrierA model study for diclofenac sodium. Int. J. Pharm. 2015, 496, 466−474. (9) Losic, D.; Pillar, R. J.; Dilger; Mitchell, J. G.; Voelcker, N. H. Atomic force microscopy (AFM) characterisation of the porous silica nanostructure of two centric diatoms. J. Porous Mater. 2007, 14, 61− 69. (10) Schroder, H. C.; Wang, X.; Tremel, W.; Ushijima, H.; Muller, W. E. G. Biofabrication of biosilica glass by living organisms. Nat. Prod. Rep. 2008, 25, 455−474. (11) Maher, S.; Kumeria, T.; Aw, M. S.; Losic, D. Diatom silica for biomedical application: recent progress and advances. Adv. Healthcare Mater. 2018, 7, 1800552. (12) Ragni, R.; Cicco, S. R.; Vona, D.; Farinola, G. M. Multiple routes to smart nanostructured materials from diatom microalgae: a chemical perspective. Adv. Mater. 2018, 30, 1704289. (13) Yang, W.; Lopez, P. J.; Rosengarten, G. Diatoms: Selfassembled silica nanostructures and templates for bio/chemical sensors and biomimetic membranes. Analyst 2011, 136, 42. (14) Chen, Y. C. The biomass and total lipid content and composition of twelve species of marine diatoms cultured under various environments. Food Chem. 2012, 131, 211−219. (15) Levitan, O.; Dinamarca, J.; Hochman, G.; Falkowski, P. G. Diatoms: a fossil fuel of the future. Trends Biotechnol. 2014, 32 (3), 117−124. (16) Hu, Q.; Sommerfeld, M.; Jarvis, E.; Ghirardi, M.; Posewitz, M.; Seibert, M.; Darzins, A. Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J. 2008, 54 (4), 621−639. (17) Hempel, F.; Bozarth, A. S.; Lindenkamp, N.; Klingl, A.; Zauner, S.; Linne, U.; Maier, U. G. Microalgae as bioreactors for Bioplastic production. Microb. Cell Fact. 2011, 10 (1), 81. (18) Aguirre, L. E.; Ouyang, L.; Elfwing, A.; Hedblom, M.; Wulff, A.; Inganas, O. Diatom frustules protect DNA from ultraviolet light. Sci. Rep. 2018, 8, 5138. (19) Zhang, Z.; Wang, Z. Diatomite-supported palladium nanoparticle: an efficient catalyst for the Heck and Suzuki reaction. J. Org. Chem. 2006, 71, 7485−7487. (20) Jantschke, A.; Herrmann, A. K.; Lesnyak, V.; Eychmuller, A.; Brunner, E. Decoration of diatom biosilica with noble metal and semiconductor nanoparticles (

Diatom Biogenic Silica as a Felicitous Platform for Biochemical

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