1. Introduction

Photonics and plasmonics allow manipulation of light at the nanoscale, with a plethora of applications including telecommunications, imaging, chemical and biochemical sensing, biomedicine, and quantum computing, to name a few [1]. The use of properly designed dielectric, periodic nanostructures such as photonic crystals (PhCs) makes it possible light propagation control in specific spectral ranges, the production of waveguide bends, splitters, nanocavities, and the amplification of optical fields with intrinsically low losses, which in turn can be exploited to enhance the interaction of radiation with the analyte in an optical sensing scheme [2–5]. Additionally, tight field confinement, broad bandwidth operation and small device footprint can be obtained also by exploiting the interaction of light with local oscillations of plasma electrons in a conductor (plasmons) [6]. The excitation at specific optical frequencies of surface plasmon polaritons (SPPs) along metal-dielectric interfaces or localized surface plasmons (LSPs) supported by metallic nanostructures indeed allows the control, localization or amplification of fields that can be efficiently employed in sensing platforms [7]. A typical example is given by surface enhanced Raman scattering (SERS) [8], where the near-field optical amplification induced by LSP resonances allows the boosting of Raman signal in proximity of metallized nanostructures, leading to impressive sensitivities up to single-molecule detection [9]. A precise, fine control of both amplitude and phase of optical fields can be achieved by the use of metallic metasurfaces, bi-dimensional counterpart of metamaterials, where radiation interacts with subwavelength, periodically or specially arranged metallic scatterers or apertures in a thin metallic film [10].

A first approach in the fabrication of plasmonic nanostructures is based on bottom-up chemical synthesis, which makes it feasible the production of colloidal silver and gold nanoparticles with diverse shapes (e.g. nanospheres, nanoprisms, nanocubes, nanostars, nanosheets) [11,12], silica-coated metallic nanoparticles [13], and monolayers of aligned metallic nanowires [14], among others. When used as SERS substrates, all these systems (mainly self-assembled in clusters or films) are characterized by high values of the enhancement factor but lack in tunability, robustness and reproducibility on large areas due to a randomic distribution of the hot-spots [15–17]. On the other side, top-down nanofabrication methods such as electron beam lithography (EBL) [18], focused ion beam (FIB) lithography [19], nano-imprint lithography (NIL) [20], laser-interference lithography (LIL) [21], and secondary electron lithography generated by ion milling [22] allows the production of a large variety of 2D and 3D periodic metallic nanostructures such as plasmonic nano-antenna arrays [23], silver [24] and gold [25] subwavelength gratings, optical fibers provided with metallic, nanostructured facets acting as metasurfaces [26], PhCs coupled with metallic nanoparticles [27], silver fishnet nanostructures [28], and 3D hollow plasmonic nanostructures [22]. All these solutions guarantee high performances in terms of reproducibility but generally result expensive and prohibitive for large-scale production and routine applications.

A low-cost, alternative way to obtain complex and high-reproducible plasmonic architectures on a wide scale is based on a proper metallization of nanostructured biomaterials [7,29]. Many species of animals and plants are indeed characterized by the presence of periodic or quasi-periodic structures at sub-micrometric scale with peculiar photonic properties, mainly involved in intra- and inter-species signalling [30,31]. In particular, several species of insects are provided with nanostructures acting as Bragg reflectors [32], polarization-selective reflectors [33,34], or multiple oriented PhCs [35], while floral iridescence is associated to bi-dimensional nanostructures acting as diffraction gratings [36,37]. Garrett et al. [38] obtained effective SERS substrates by thermal evaporation of gold or silver onto the conical, cuticular nanostructures found on the wings of Graphium weiskei butterfly. These structures, characterized by enhancement factors of the order of $10^6$ (for gold) and $10^7$ (for silver), have been tested in avidin-biotin assay experiments, reaching sensitivities comparable with those of a typycal enzime-linked immunosorbent assay (ELISA) system.

The most astonishing nanostructures found in nature are by far given by diatom frustules [39]. Diatoms are ubiquitous unicellular microalgae which colonized all seas and freshwaters throughout our planet over the last tens of millions of years [40], resulting in a massive contribution to global primary production (about 20-25% [41]). Diatom cells are encapsulated within a hydrogenated porous silica shell, the frustule, whose dimensions range between tens of micrometers to about one millimeter (depending on the species) [42]. Frustules consist in an epitheca overlapping a hypotheca in a “petri-dish-like" arrangement, every theca being formed by a valve and a series of lateral girdle bands. The symmetry of the frustule allows distinguishing between centric diatoms, characterized by a circular or polygonal valve and mainly planktonic, and pennate ones, mainly benthic and whose frustule is elongated and bilaterally symmetric (see Fig. 1) [39]. Valves and girdles are decorated by periodic, regular patterns of pores whose diameter ranges between some tens of nanometers up to about one micron (according to the species and the location within the frustule) [43]. The valve of several species is multi-layered, every layer being characterized by porous patterns with distinct lattice constant and dimensions [44]. Hypothesized frustule functions mainly include mechanical stability [45], sorting of nutrients from harmful agents [46], and light harvesting optimization [47]. Physical and chemical properties of the frustule, including high porosity [48], photonic-crystal behavior [49], optical lensing [50–54], photoluminescence [55], and biocompatibility [56], have been exploited in several technological fields such as optoelectronics [57], catalysis [58], biochemical sensing and biosensing [59,60], solar energy harvesting [61,62], biomedicine and drug delivery [63]. Furthermore, being constituted by a regularly nanostructured dielectric material, it is straightforward to imagine that a proper frustule metallization may trigger plasmonic effects. This assumption led, in recent years, to a profusion of efforts aimed at the fabrication of plasmonic, diatom-derived platforms, mainly but not exclusively directed to sensing. Being able to self-reply at high rates, diatom cultures can be envisaged as natural nanofactories able to supply on a large scale and at nearly zero cost 2D and 3D hierarchical nanostructures hardly feasible even by the most recent nano-lithographic techniques, allowing one to go beyond the limits and drawbacks of both bottom-up and top-down traditional approaches [48].

 

Fig. 1. Schematic representations of centric (circular symmetric) and pennate (bilaterally symmetric) diatom frustules, with indication of the characteristic symmetry planes. Reproduced with permission from Ref. [42].

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The aim of the present work is to offer a wide overview on the main metallization techniques which allow obtaining complex and efficient plasmonic platforms from diatom biosilica, both in the form of intact frustules and of diatomite (consisting of biosilica micro- and nanoparticles derived from fossilized diatom frustules). Finally, the most relevant applications of these bio-derived plasmonic devices in sensing, biosensing, and biomedicine have been deeply scrutinized and reported.

2. Metallic replicas of nanostructured diatom frustules

First attempts to obtain metallic periodic nanostructures starting from diatom frustules consisted in the retrieval of high-precision replicas of centric diatom valves. In particular, Losic and co-workers [64] obtained shape-preserving pure gold replicas of Coscinodiscus sp valves by gold vacuum evaporation followed by mechanical stripping of the obtained metallic film (about 1 $\mu$m thick) glued onto a glass support. The transfer of the local valve nanotopography to the negative metallic replica was verified by scanning electron microscopy (SEM) and atomic force microscopy (AFM) (see Fig. 2), while energy-dispersive X-ray analysis (EDX) revealed only gold peaks and allowed establishing that the Au surface of the obtained film was free of diatomaceous remains. The presence of a broad plasmonic resonance centered around $\lambda =580$ nm has been detected by microspectrophotometry. The resonance corresponds to the transverse plasmon absorbance band of the nano-cilynders present in the gold replica (150-200 nm in diameter, see Fig. 2(i), arrow 2) and was not present in a flat gold film with the same thickness. Applications to surface plasmon spectroscopy and surface enhanced Raman spectroscopy (SERS) have been envisaged by the authors. The same technique has been successfully extended to other diatom species (e.g. Thalassiosira eccentrica), while X-ray diffraction (XRD) measurements allowed confirming the crystal phase of the obtained gold replicas [65].

 

Fig. 2. SEM images of the inner layer (foramen) of a C. wailesii valve at different magnifcations (a-c); AFM image of an hexagonal cell of the foramen (d) and relative profile graph (e): the pores of the external layer (cribrum, arrow 2) are visible through the pores of the foramen (arrow 1); SEM (f-g) and AFM (i) images of the gold (negative) replica of the valve; profile graph (j) along the dashed line in (i) showing two kinds of gold micro- and nanostructures (arrows 1,2). Reproduced with permission from Ref. [65].

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Even though, as remarked above, this methodology guarantees an effective transfer of the multi-layered valve configuration to a 2D metallic film, it does not allow obtaining a free-standing replica retaining the global 3D morphology of the frustule. This task has been accomplished by Bao et al. [66] by means of a two-step process comprising i) the conversion of Aulacoseira sp frustules into nanocrystralline silicon replicas via a low-temperature (650 $^{\circ }$C) magnesiothermic reaction; ii) electroless deposition of a noble metal onto the silicon replica of the frustule followed by selective dissolution of silicon by immersing the specimens in an aqueous NaOH solution. In this way, gold, silver, and palladium self-supporting replicas of the frustules retaining the morphology of the starting structure have been obtained. Yu et al. [67] applied, for the same species, a simpler, single-step methodology making use of direct electroless gold deposition [68,69] followed by silica removal by dissolving the gold-coated frustules in a hydrofluoric (HF) solution. After the process, self-supporting gold replicas of the frustules inheriting their 3D configuration, size and pore pattern distribution have been obtained. Pores diameter is generally reduced after the process and can be tuned by acting on the deposition time. The surface of the gold replicas results clustered, with cluster dimensions of about 15-20 nm. The presence of clusters is strictly related to the characteristic gold nucleation taking place during the deposition process. The combination of the 3D porous structure of the replicas and the nanoscale features of their surface are favorable when considering their plasmonic response. These structures worked as excellent gold catalysts for the reduction of 4-nitrophenol (4-NP) into 4-aminophenol (4-AP) in the presence of sodium borohydride (NaBH$_4$) as reductant.

Metallized frustule replicas employed as SERS substrates have been first introduced by Payne at al. [70]. Titanium (5 nm) and silver (30 nm) films have been evaporated onto Synedra sp and Thalassiosira sp silica shells. The deposition has been followed by HF etching in order to remove silica and titanium layer. The obtained 3D silver replicas have been tested as SERS substrates for the detection of rhodamine 6G (R6G) at different concentrations (100 nM, 1 $\mu$M, 1 mM), obtaining an enhancement factor of about $1\times 10^6$ for Synedra and $7\times 10^5$ for Thalassiosira, assuming a 100% coverage of the dye on the substrate and being the enhancement factor EF defined as:

Coscinodiscus sp valves have been recently used as bio-template in the fabrication of polydimethylsiloxane (PDMS) replicas via nanoimprint technique [71]. The obtained structures are characterized by the presence of micro- and nano-pillars (minimum diameter and minimum height $\sim$ 40 nm and $\sim$ 25 nm, respectively) being the negative counterpart of the hierarchical pore-structure of the foramen (inner layer of the valve) and cribrum (external layer of the valve). The polymeric replicas have then been sputtered with Au nanoparticles (AuNPs), leading to effective SERS substrates tested in the detection of R6G and Carbendazim in aqueous solution. The adjustement of the sputtering duration allowed controlling the diameter and gap of the deposited nanoparticles. The intensity of the SERS signal reached a maximum for a sputtering duration of 12 minutes, corresponding to an almost complete AuNPs covering of the PDMS micro-nano pillars with the generation of a large quantity of sub-10 nm gaps. Raman spectra have been acquired at different R6G concentrations ($10^{-7}-10^{-12}$ M) and in different random spots over the substrate, leading to an average relative standard deviation (RSD) evaluated over the intensities of the peaks at 612 cm$^{-1}$ less than 20% and an EF of about $2.24\times 10^7$. In the case of Carbendazim the tested concentrations were comprised between $10^{-4}$ and $10^{-9}$ M, with a RSD relative to the intensities of the peaks at 1268 cm$^{-1}$ of about 21% and an EF appoximately equal to $1.01\times 10^7$.

Besides Raman enhanced scattering, other plasmonic phenomena sustained by metallic replicas of frustules have been investigated. It is well known [72] that a metallic film perforated by periodic arrays of holes can be characterized by sharp peaks in transmission at wavelengths larger than the array period. This behavior, referred to as extraordinary optical transmission (EOT), is essentially due to the coupling of incoming light with the surface plasmons supported by the periodically patterned metal film and and can find applications mainly in sensing and biosensing due to the high sensitivity of the EOT peak position to the changes in the dielectric function at the metal surface [73–75]. In 2012 Fang and co-workers [76] reported on the presence of transmission maxima and reflectance minima through free-standing gold replicas of Coscinodiscus asteromphalus centric valves, characterized by the presence of an approximately hexagonal local arrangement of holes with a diameter of $1.2\pm 0.2$ $\mu$m and an equivalent lattice constant of $2.3\pm 0.3$ $\mu$m. The metallization procedure consisted in a wet aminosilanization treatment of the frustules followed by a dendritic layer-by-layer process leading to an amine-enriched silica matrix subsequently exposed to chloroauric acid solution in order to bind Au(III) complexes to the functionalized surface. A final reaction with sodium borohydride allows reducing the gold complexes in elemental gold while treatment in a HF solution induces selective dissolution of silica and, ultimately, the retrieval of a free-standing gold replica of the valve. The transmission maximum detected around $\lambda \simeq 4.3$ $\mu$m has to be ascribed to interference processes arising by the interaction of the surface plasmons generated by the sub-wavelength, periodically patterned metallic surface and the incoming light.

3. Diatom frustules conjugated to metallic nanoparticles as platforms for biochemical sensing

Since the seminal work by Fuhrmann et al. [49], several authors focused their studies on the optical properties of diatom frustule girdles and valves seen as photonic crystal slabs. In particular, a lot of effort has been devoted to the analysis of the guided-mode resonances (GMRs) supported by these structures, both numerically and experimentally, mainly by means of plane wave expansion (PWE) and finite difference time domain (FDTD) calculations [49,77,78], microscatterometry characterization [79] and near-field scanning optical microscopy (SNOM) imaging [80,81]. In the case of artificial PhCs, it has been demonstrated how a properly designed coupling of metallic nanoparticles to the resonant modes of the PhC surface may provide an additional electromagnetic gain to SERS [27]. Furthermore, the porous morphology of a diatom frustule coupled to metallic NPs and used as SERS substrate may maximize both the number of field hot-spots and of adsoprtion sites for a given analyte if compared to planar SERS substrates [82]. In 2013 Ren et al. [83] theoretically and experimentally studied the coupling of Pinnularia sp frustule GMRs with localized surface plasmons (LSPs) supported by silver nanoparticles (AgNPs) integrated onto the frustules (see Fig. 3). Optical transmission and electric field enhancement factor (E-field EF, defined as the maximum electric field inside the frustule normalized to the peak electric field amplitude of an incident gaussian beam) have been simulated by three-dimensional finite element method (FEM) considering several possible locations and coupling configurations of the metallic nanoparticles within a schematized CAD model of the frustule. The total E-field EF approximately equals the product of E-field EF from the nanoparticle and the photonic crystal structure, respectively, and ranges between $16.9 \times$ (50 nm Ag spherical NP between two corner holes of the frustule unit cell, with hole diameter $\simeq 100$ nm) and $235 \times$ (dimer formed by two NPs with 24 nm diameter and a gap size of 2 nm located inside the central pore). Experimentally, diatom-coated glass substrates have been functionalized by aminopropyltriethoxil-sylane (APTES) in order to promote adhesion to colloidal AgNPs. Various nanoparticle morphologies are obtained, including isolated NPs, dimers, trimers, short chains and nanorods, giving rise to multiple plasmonic resonances in the visible spectral range. In this first, pioneering study, the NPs resulted mainly located onto the frustule without entering the silica nanopores since their average size resulted larger than pores diameter, thus not standing in an optimal configuration in terms of coupling with the GMRs. Nevertheless, when the frustule-NPs hybrids are used as SERS substrates in the detection of R6G (1 $\mu$M in ethanol), the Raman signal intensity results enhanced by a factor $4.79 \pm 0.8$ if compared to the SERS signal coming from the glass substrate conjugated with the same AgNPs. The described methodology has been refined and optimized by the same authors both in resonant and non-resonant conditions and further verified also by Raman mapping [85]. In resonant conditions (affected by quenching), when using diatom frustules hybridized with AgNPs as SERS substrate, an additional EF of $4-6\times$ has been observed (compared to AgNPs assembled on a glass slide) for R6G concentrations ranging from $10^{-9}$ to $10^{-5}$ M. Under non resonant-conditions, an EF $9-12\times$ compared to AgNPs assembled on a quartz slide has been obtained over R6G concentrations from $10^{-7}$ to $10^{-4}$ M. The additional EF is again attributed to the coupling of the nanoparticle LSPs with the frustule GMRs. Amplified Raman scattering signals ($3.9\times$ additional EF) of R6G on frustules in absence of NPs have also been detected, confirming the contribution of frustule GMRs to the overall amplification process. An accurate study on reliability and reproducibility of an hybridized diatom frustule used as SERS substrate has been performed by Chamuah et al. by using Cosmioneis hawaiiensis valves conjugated with self-assembled gold nanoparticles (AuNPs) for the detection of malachite green (MG) and fluoride in drinking water [86]. SERS signals were optimized when AuNPs were synthesized with a diameter of 40 nm, the pores of the frustule being about 100 nm wide. After interrogation in 10 different locations of the SERS substrate, RSD values for the Raman peaks at 808, 1186 and 1618 cm$^{-1}$ equal to 6.81%, 7.57% and 4.74% have been obtained for MG, with a detection limit of 1 nM. In case of fluoride solutions in water, the RSD values corresponding to the Raman shifts at 1084 and 1288 cm$^{-1}$ were found to be 17.26% and 18.49%, respectively, with a detection limit of 100 nM, far below the established danger limit of about 79 $\mu$M. The stability of the substrate has been tested recording MG Raman signals everyday for one week, resulting in a maximum fluctuation of about 4% of the average value of the peak intensity at 1186 cm$^{-1}$.

 

Fig. 3. Schematic of a diatom frustule hybridized with silver nanoparticles and used as SERS substrate (left). Detail of the valve showing the coupling between incident radiation (in this case coherent radiation at $\lambda =532$ nm), frustule GMRs and nanoparticle LSPs inducing enhanced SERS signal (right). $I_0$ and $I$ stand for the signal intensity coming from a planar and a frustule-based SERS substrate, respectively. Modified with permission from Ref. [84].

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Onesto et al. [87] reported on the hybridization of intact frustules derived from diatomaceous earth (including cylindrical Alaucoseira sp frustules) with AuNPs using a photo-deposition process based on biosilica shells suspension in chloroauric acid (HAuCl$_4$) solutions followed by UVB/UVA illumination. The obtained structures were used as SERS substrates in the detection of BSA and mineral oil emulsions at concentrations down to 10$^{-16}$ M and 50 ppm, respectively. Metallized frustules are exploited in solution with the target molecules and their use as tracers in microcirculation circuits or in vivo systems is envisaged by the authors. Numerical simulations based on FEM have been performed on CAD models directly derived from SEM images of actual diatom shells, showing how the relative orientation of the incoming EM field with respect to the frustule influences field enhancement, with the amplified field oscillating between $\sim 1.5\times 10^8$ V/m and $\sim 4 \times 10^8$ V/m in the range $\theta =0-70^{\circ }$, with $\theta$ incident angle.

In recent years, Pinnularia sp has been the object of an increasing number of experimentations in the field of plasmonics, representing a sort of model organism for the achievement of biosilica/metallic NPs hybrids optimized for sensing applications. Different frustule hybridization methods have been implemented, including AuNPs self-assembly via electrostatic interaction and in-situ growth of AgNPs after SnCl$_2$ treatment [82,88], which ensures a higher density of AgNPs penetrating frustule pores, thus providing even higher EFs [82]. Improved computational techniques based on FDTD method allowed simulating the coupling of a large number of randomly distributed metallic NPs with the GMRs supported by a multi-layered Pinnularia sp valve, showing a 50% increase of the total volume of hot-spots respect to a flat substrate, the hot-spots being defined as the points where the local field enhancement $|E/E_0|^4$ is bigger than 100 [88]. Pinnularia sp frustules conjugated to metallic NPs have been applied also in the detection of melamine (C$_3$H$_6$N$_6$) for food safety sensing, xylene for air-water quality monitoring, and in the detection of trace level of organic matter in soil, in all cases surpassing traditional colloidal SERS substrates in terms of sensitivity [88]. Exploiting the high hydrophilicity of the porous, nanostructured diatom biosilica (which allows a fast flow of a liquid analyte towards the frustule due to capillary forces) in combination with an inkjet printing device able to dispense miniature amounts of analyte (100 pL per droplet) on a single frustule, Kong et al. [82] succeeded in the label-free detection of 2,4,6-trinitrotoluene (TNT) down to $10^{-10}$ M in concentration, making use of Pinnularia sp frustules decorated with in-situ synthesized AgNPs as SERS substrates. The same structures have been tested in 2019 by Sivashanmugan et al. [89] as multiscale, hierarchical mesocapsules for ultrasensitive optofluidic-SERS sensing (see Fig. 4). When used within a microfluidic channel, the mesocapsules allowed reaching a detection limit of $10^{-13}$ M for R6G (essentialy down to single molecule detection) with average EF values as high as $2.2\times 10^{10}$, $100\times$ greater than the ones achieved by using regular colloidal AgNPs. In case of benzene and chlorobenzene detection in tap water, diatom-derived mesocapsules were able to detect analyte concentrations down to $10^{-9}$ M corresponding to 0.05 $\mu$g L$^{-1}$, which is $10\times$ lower than the established safety level for the considered pollutants. The use of core-shell nanoparticles such as gold-silica core-shell (Au@SiO$_2$) NPs conjugated to the frustule allows for stronger binding affinities, higher adsorption capabilities and improved molecular trapping of the analyte due to the shell porosity and can represent a great advantage in vapor sensing. Following this assumption, Squire et al. [90] made use of Pinnularia sp frustules hybridized with Au@SiO$_2$ NPs as multiscale SERS sensors for the rapid and sensitive detection of vapor DNT (below 100 ppb within 3 min at room temperature and making use of a flow chamber). AgNPs-Pinnularia sp frustule monolayers have been recently used in conjunction with ultrathin layer chromatography (UTLC) for the detection of nile red and malachite green [84]. The combination of UTCL separation, SERS detection of the separate analytes and photonic enhancement of SERS signals induced by diatom frustules allowed to obtain detection limits down to 0.6 pg in the line scan.

 

Fig. 4. Schematic of an optofluidic-SERS sensing system based on mesocapsules derived from Pinnularia sp frustules hybridized with in-situ grown AgNPs and inserted in a microfluidic channel. Mixture fluids containing target molecules and mesocapsules are injected in the microfluidic channel, allowing for high-sensitivity, real time analyte detection at different concentrations. Reproduced with permission from Ref. [89].

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Besides providing substantial enhancement of the Raman signal, AgNPs assembled onto a diatom frustule may be used also to attach bioprobes (e.g. antibodies) to the substrate. In this way, Yang et al. obtained a SERS-based sandwhich immunoassay starting from the above mentioned Pinnularia sp frustules hybridized with AgNPs aimed at the detection of immune reactions between goat-anti mouse IgG antibody and mouse IgG antigen [91]. Frustules have been deposited onto a glass slide and the overall substrate (frustules plus underlying flat glass) underwent the following treatments: functionalization by APTES in order to allow AgNPs binding to the surface; AgNPs self-assembling onto the surface; functionalization of the AgNPs with goat-anti mouse IgG; bovine serum albumin (BSA) treatment in order to block the non-specific adsorption sites on the surface of the AgNPs. Furthermore, AuNP aggregates functionalized with antibodies and DTNB as Raman-active reporter have been introduced in order to provide an even stronger SERS enhancement when antibody/antigen recognition took place. Non complimentary antigen (human IgG) has been used as nonspecific control. $4\times$ stronger Raman signals from DTNB have been detected from the hybridized frustules used as SERS substrates repsect to NPs-flat glass substrates. A detection limit of mouse IgG of 10 pg/mL has been obtained on diatom frustules, to be compared to the one retrieved on flat glass substrates (1 ng/mL), thus achieving an improvement of two orders of magnitude. A similar sandwhich structure has been later designed by Kamińska et al. [92] in the development of a SERS immunoassay based on the functionalization of amino-modified Pseudostaurosira trainorii frustules with anti-interleukin-8 (anti-IL-8) antibodies for ultrasensitive detection of interleukins in blood plasma. The SERS tags consisted in 70-nm Au nanoparticles functionalized with anti-IL-8 as marker and 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) as Raman reporter. The combined effect of SERS signal amplification mediated by frustule GMRs, rapid mass transport inside the biosilica porous matrix, large frustule surface area and consequent magnification of the number of hot-spots and adsorption sites, high hydrophilicity of biosilica and resulting elevated concentration of target molecules, turned out in a detection limit for interleukin IL-8 of 6.2 pg mL$^{-1}$, which is two orders of magnitude better than the one achieved by a conventional glass-based SERS immunoassay.

One of the intrinsic limitations of ultra-sensitive nanosensors lies in the long time required for low-concetration analyte detection, which can reach several hours to detect molecules at 1 pM-1 fM [93]. In order to overcome this restriction, Guo et al. [94] designed and fabricated an opto-plasmonic micromotor-sensor based on an Alaucoseira sp frustule hybridized with AgNPs for SERS detection and covered on one side by a thin Ni/Au bilayered film (deposited by electron-beam evaporation) for magnetic actuation (see Fig. 5). The microsensor can be transported in individual microfluidic wells or channels and brought at high-speed rotation by applying a tunable rotating magnetic field. First tests allowed rapid and sensitive detection of salmon sperm DNA. The rotation notably accelerates the capture efficiency of DNA molecules by at least 4 times at 1200 rpm, boosting the detection speed of the analyte at low concentrations (down to 80 nM).

 

Fig. 5. Schematic of an opto-plasmonic micromotor sensor based on a metallized diatom frustule coupled to a rotating magnetic field (a). DNA capture by the rotating sensor (b). Detail of the porous structure of the frustule (c). SERS spectra of DNA molecules at different concentrations (80 nM-4 $\mu$M) (d). Reproduced with permission from Ref. [94].

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4. Uniformly metallized diatom frustules as efficient SERS subsrates

Even though they can provide high levels of sensitivity and detection limits competing or even surpassing those of traditional SERS substrates, metallic NPs/frustules hybrids may be affected by an uneven spatial distribution of EM hot-spots throughout the volume of interest, the field enhancement being preferentially localized around the metallic nanoparticles [87]. An improvement in the reproducibility of the acquired SERS spectra can be obtained by an uniform metallization of the starting frustule, with no need of biosilica removal usually performed in the 3D-replicas fabrication techniques reviewed in Section 2. An example is given in Ref. [95], where electroless deposition of gold onto Aulacoseira sp frustules is reported. This metallization process included three main steps (a sensitization step in a SnCl$_2$ and C$_2$HF$_3$O$_2$ solution, an activation step in an ammoniacal solution of AgNO$_3$, and a final plating/galvanic Ag/Au displacement step) and led to the deposition of a high density of AuNPs onto the biosilica, acting as preferred sites for further Au nucleation and growth during exposure to the plating solution. The final result is a complete coverage of the frustule with a nanometric, uniform Au film preserving the original morphology of the diatom shell. The obtained substrate was tested in SERS sensing of p-mercaptoaniline (pMA), which self-assembles into monolayers over the substrate surface. Looking at the spectra acquired in different points of single Au-frustule substrates and assuming a total coverage of the sensing surface by pMA, a maximum enhancement factor EF$_{max}\simeq 1.0\times 10^{5}$ was estimated. The collected spectra showed a reproducible overall scattering pattern (same series of peaks and constant ratio between the main peaks), even though the intensity of the single peaks was still affected by a significant degree of variability, suggesting the need of more efficient metallization strategies in terms of homogeneity of the deposition.

A simple but effective approach based on Au thermal evaporation has been applied to Pseudotizchia multistriata single valves [96]. The peculiar 3D, hierarchical morphology of the frustule, comprising several interrelated periodicities (see Fig. 6(a) and (b)), allows for an efficient coupling with external optical radiation. Single valves were deposited onto a quartz slide which was then covered by nanometric Au layers (20-50 nm) by thermal evaporation. The metalized valves, characterized by a plasmonic resonance centered around 695 nm for a gold thickness of 40 nm, have been tested as SERS substrates for the detection of biphenyl-4-thiol (BPT, see Fig. 6(c)), which self-assembles in a monolayer onto the substrate with a packing density of 4 molecules per nm$^2$, allowing for an accurate estimation of the average number of molecules adsorbed to the frustule in the scattering area. The obtained EF resulted optimized for a gold thickness of 40 nm and was equal to ($4.6\pm 0.9)\times 10^6$, in accordance with numerical simulations where the relative electromagnetic near-field amplification $G=|\frac {E_{loc}+E_0}{E_0}|^4$ (with $E_0$ and $E_{loc}$ incident and local field amplitudes, respectively) has been retrieved starting from an accurate CAD model of the metallized valve derived from AFM characterization (see Fig. 6(d)). The homogeneity of the 40 nm thick Au layer guaranteed high performances in terms of reproducibility, with 80% of SERS spectra measured in different regions of the valve edge being well overlapped. The presence of a lateral, extruded band on one side of the valve characterized by extremely sharp edges (with a radius of curvature of about 13 nm) not only ensures strong near-field localization (see Fig. 6(d)), but also allows for an easy access to complex biological environments avoiding steric hindrance, as is the case of cellular membranes. Chemical composition of cell membranes is of fundamental importance in the assessment of several pathologies, and the SERS substrates derived from P. multistriata valves have been succesfully tested in probing red blood cells (RBCs) and leukemic cells (LCs) membranes (see Fig. 6(e)). In the case of RBCs, for example, SERS spectra were not affected by the presence of hemoglobin (Hb) but were composed of peaks only related to the membrane components (mainly lipids, proteins, and carbohydrates, as can be seen in Fig. 6(f)).

 

Fig. 6. TEM (a, scale bar: 1 $\mu$m) and AFM (b) images of a detail of a single P. multistriata valve. The main features of the valve morphology are indicated: protruded modulations (interstitiae) about 100 nm wide; alternating couples of rows of nanopores (average diameter $\simeq 70$ nm) in the striae; a vertically extruded grating made of side bridges (fibulae). Average SERS BPT spectra acquired onto (orange) and outside (red) a metalized valve (c). The Raman spectrum of pure BPT is additionally shown for comparison (blue). Simulated EF map (log 10-scale) over a detail of a metalized valve (d). The simulation has been performed over a CAD reconstruction of the valve retrieved by an AFM image. Optical image of RBCs over a single metalized valve (e). In the inset, a detail of the Raman map is superimposed to the optical image. SERS spectrum acquired at the point of contact between a single RBC and a metalized valve (f). The main contribution come from the membrane components (lipids, proteins, and carbohydrates), with no significant contribution from Hb. Reproduced with permission from Ref. [96].

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5. Diatomite nanoparticles

Diatomite nanoparticles (DNPs) are 3D porous silica nanostructures that can be easily obtained by cost-effective processes from diatomite natural deposits or freshly cultivated diatome frustules. Diatomite or diatomaceous earth (DE) is a soft and friable porous siliceous sedimentary rock, mainly formed through prolonged fossilization and accumulation of diatom remains and crushed into a fine powder [97]. It is mainly composed of largely available diatom fossils, and, therefore, its major chemical composition is amorphous silica besides various secondary and minor inorganic components (<0.1$\%$), such as alumina, calcite, iron oxide, magnesia that depend on the environmental conditions during sedimentation and the organic matter content [98]. The preparation of diatomite micro- and nano-particles from raw DE requires three main steps including crushing, purification, and size-based separation. A fine powder of diatomite with particle size of sub-micrometer to several micrometers is obtained by crushing the raw DE with a milling equipment. The result is a mixture of full DE frustules and broken fragments showing a micro and nano-scale porosity together with an hierarchical 3D nano-structured architecture inherited by the precise, genetically controlled frustule morphogenesis process. The purification steps to remove the undesired organic and inorganic impurities involve generally the treatment of the diatomite in hot-acid solutions (eg. HCl) at 75 $^{\circ }$C [99]. The incubation times, the HCl concentrations and the acid leaching treatment times are strongly dependent on the impurity content of the source of raw DE [99]. The chemical composition of diatomite particles can be analyzed by energy-dispersive X-ray spectroscopy (EDX) and Fourier transform IR (FTIR). Finally, a combination of sedimentation and filtration processes are applied to remove fractured diatom parts and obtain intact diatom micro- and nano-structures. For drug delivery applications, it is critical to further reduce and tune the size of the particles and this can be achieved by sonication process in solution [100]. All these processes are safe, reproducible and cost-effective, avoiding time-consuming and environmentally non-friendly manufacturing procedures [101].

By using these protocols, 3D nano-patterned silica structures can be easily prepared in the size range of 100-400 nm revealing unique properties, such as high chemical and mechanical stability, biocompatibility, non toxicity, high surface area and thermal resistance making them an intriguing material for several applications ranging from filtration to pharmaceutic [99]. The nanostructure is characterized by high porosity, allowing for the immobilization of target molecules not only on the external surface of the substrate but also inside of the pores, which enables the loading of large amounts of sensing molecules. Indeed, by soaking the DNPs into a solution of selected molecules allows for the physi- or chemisorption of the molecules onto the surface while concentration gradient-driven diffusion enables their subsequent release. The amorphous silica nature of DNPs provides high levels of free reactive hydroxyl groups (-OH) that can be exploited for the chemical modification (with groups such as -NH$_2$, -COOH, -SH, and -CHO) allowing the selective binding with biomolecules (eg. enzymes, proteins, antibodies, peptides, DNA, aptamers). Furthermore, diatom amorphous silica is categorized by the International Agency for Research on Cancer (IARC) as “not classifiable as to its carcinogenicity to humans" and is approved by the Food and Drug Administration (FDA) for food and pharmaceutical applications. Several different in vitro and in vivo studies have demonstrated the non-toxicity and high biocompatibility of DNPs [102–105]. The ability to be functionalized with different components, together with a high drug-loading capacity and biocompatibility, make DNPs one of the most potential materials for biosensing and theranostics.

5.1 Plasmonic assisted-diatomite nanoparticles for drug delivery

The feasibility of loading diatomite microcapsules with poorly water-soluble drugs (Indomethacin, a nonsteoridal nati-inflammatory drug) with a loading capacity of about 22 wt$\%$ (percentage by weight) has been demonstrated for the first time by Losic et al. in 2011 [106]. Few years later, Zhang et al. used diatomite for release of mesalamine and prednisone in the gastro-intestinal tract [107]. The developed bare diatoms showed low cytotoxicity at a concentration of up to 1000 $\mu$g/mL on cancer cell lines (Caco-2, HT-29 and HT-116) and, at the same time, promoted intracellular permeation enhancement of the drugs.

More recently, Terracciano et al. developed DNPs modified with a dual-biofunctionalization method by polyethylene glycol (PEG) coverage and cell-penetrating peptide (CPP) bioconjugation for enhancing their intracellular uptake in cancer cells [108]. The surface modification improved the in vitro biocompatibility, indeed the modified DNPs showed high hemocompatibility after 48 h of incubation with erythrocytes, and negligible cytotoxicity after incubation with MCF-7 and MDA-MB-231 breast cancer cells for 24 h, at concentration up to 200 $\mu$g/mL. Moreover, the dual surface modification of DNPs improved both the loading of a poorly water-soluble anticancer drug, Sorafenib, with a loading capacity up to 22 wt$\%$, and also enhanced the drug release profiles in aqueous solutions. The biocompatibility of the system was further tested in-vivo in Hydra. Several parameters, including Hydra morphology and growing rate, cells apoptosis, genotoxic endpoints and genetic analysis confirmed the biosafety of this material at high concentrations (up to 3.5 g/L for 72 h), opening the way to new applications in nanomedicine [105].

The main limitations of the developed nanocarriers is that the dynamics of the uptake cannot be easily visualized and the drug release cannot be evaluated in living cells [109]. The use of fluorescence labels could modify the size and chemical properties of the NPs perturbing the study of their intracellular delivery and properties. On the other hand, TEM imaging offers high resolution, down to cellular organelle scale, but it is a time consuming and destructive imaging approach.

Plasmonic NPs-decorated diatomite biosilica allow a direct visualization of the nanovectors in living cells and can be additionally used for studying the drug release in real-time. Recently, Rea et al. proposed polyethylene glycol (PEG)-modified diatomite NPs (PEG-DNPs) decorated with gold NPs (AuNPs) by one-pot liquid-phase synthesis for application in medicine [102]. Figure 7(a) shows the TEM images of the developed diatomite NPs. The presence of AuNPs does not alter the loading capacity of the DNPs as well as the pore availability, as investigated by nitrogen adsorption/desorption isotherm analysis (Fig. 7(b,c)). The materials showed good stability, efficient uptake in human cervix epithelioid carcinoma (HeLa) cells up to 48 h, very low in-vitro cytotoxicity with concentrations of about 400 $\mu$g/mL for 72 h (Fig. 7(d)).

 

Fig. 7. (a) TEM images of bare DNPs and DNPs decorated with AuNPs. (b) Nitrogen adsorption/desorption isotherms and pore size distributions measured for PEG-DNPs and (c) PEG-DNPs@AuNPs. (d) Confocal laser scanning microscopy images of PEG-DNPs@AuNPs (100 $\mu$g/ml) labelled with Alexa Fluor 488 (green) internalized into HeLa cells at 6 and 48 h time points. Cell nuclei and membranes were stained with Hoechst (blue) and WGA-Alexa Fluor 555 (red), respectively. Scale bars = 20 $\mu$m. (a) Reproduced with permission from Ref. [109]. (b-d) Reproduced with permission from Ref. [102].

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Furthermore, the strong Raman enhancement of molecules close to AuNPs (EF=10$^5$) can be used for label-free intracellular drug monitoring without the use of any fluorophore or external marker, avoiding fluorescence-quenching issues [110]. Indeed, the same group demonstrated that the hybrid nanosystem constituted by DNPs decorated with AuNPs and capped with a gelatin (Gel) layer can provide efficient Galunisertib (LY) delivery in living colorectal cancer cells (CRCs) and local quantification of the drug release with attogram-scale resolution (see Fig. 8). With this nanosystem, the loading capacity can be tuned by varying the amount of gelatin in the external shell rather than the mass of DNPs. At the same time, the gelatin shell allows for the modulation of the LY release via pH-dependent degradation of the polymer chains. Indeed, at physiological pH (7.4), the gelatin layer is compact and the drug is retained in the system. In presence of cancer cells, where the pH is acidic, the gelatin shell is biodegraded and the LY release promoted. The monitoring of LY internalization and release is made possible by the presence of the AuNPs providing a highly sensitive SERS readout of LY with non-invasive laser illumination exciting their plasmonic resonance. The incorporation of LY into the DNP-AuNPs system and the enhancement of the LY signal were preliminary evaluated by studying and comparing the Raman and SERS LY spectra. The SERS spectra were well reproducible and the intensities on a selected nanocomplex and on different clusters could vary up to about 10$\%$ showing a really good inter-sample reproducibility (Fig. 8(a)). The LY release from the DNP-AuNPs-LY@Gel nanosystem was preliminary investigated in in-vitro experiments with two different pH environments, 5.5 and 7.4, via HPLC and SERS spectroscopy (see Fig. 8(c)). The amount of LY released from the carrier was quantified via SERS according to the nanosystem loading capacity calculated by HPLC. After 24 h, a very small LY release (about 4$\%$) was observed at pH 7.4 (the SERS LY signal is almost constant), and a slow release (about 60$\%$) detected after 48 h. On the other hand, at pH 5.5, mimicking the cancer cell microenvironment, the LY release became faster. After 24h, a great amount of LY (about 50$\%$) was already released, corresponding to about 0.125 fg per nanocomplex unit, reaching about 90$\%$ after 48 h. The same analysis has been performed in parallel by HPLC analysis, confirming that the loaded anticancer drug was efficiently released in acidic conditions [110]. The local real-time sensing of the LY release in living CRCs is provided by analysing the LY SERS signal as a function of the incubation time up to 48 h with an unprecedented resolution of 7.5 × 10$^{-18}$g, as reported in Fig. 8(d). The biocompatibility was demonstrated in CRCs, showing that the DNP-AuNPs-LY@Gel treatment was safe up to a concentration of 50 $\mu$g/mL. Crucially, the release of LY from the DNP-AuNPs-LY@Gel complex in CRCs inhibits their proliferation and induces the reversion to a normal phenotype with greater efficiency compared to the free drug, as demonstrated by analysing the expression level of specific genes (E- CADHERIN, SNAIL-1, and TWIST-1). The DNP-AuNPs-LY@Gel antimetastatic effect was further demonstrated by confocal microscopy, revealing that the shape of CTCs efficiently turned from mesenchymal (elongated shape) to epithelial-like phenotype (rounded shape) in the presence of the multi-functional platform. These studies showed that the LY loading by the developed nanoplatform can help lowering the amount of drug required to inhibit the metastatic process in cancer cells, reducing the formation of drug-related toxic metabolites [110]. The LSPS response of the hybrid system can be additionally used to monitor the gelatin shell thickness, degradation, and consequent LY release [111]. By measuring the localized surface plasmon resonance red-shifts of AuNPs due to the presence of the gelatin, it is possible to estimate optically the shell thickness as well as to monitor its degradation. Indeed, the authors additionally demonstrated that the nanosystem loading capacity can be tuned and controlled by varying the gelatin thickness [111].

 

Fig. 8. (a) Raman spectrum of LY (black line). Experiments were carried out with laser wavelength 638 nm, laser power 20 mW, microscope objective 60X, integration time 1 s. The SERS spectrum of LY from 500 $\mu$g/ml of the complex DNP-AuNPs-LY@Gel (blue line), the background signals from the DNP-AuNPs@Gel (red line) and the DNP-AuNPs alone (green line) were measured. Laser power: 1 mW. All the SERS and Raman spectra were rescaled to a common laser power of 1 mW so that the intensities could be directly compared. Spectra are offset for clarity. (b) Optical image and Raman mapping images showing the internalization of DNP-AuNPs-LY@Gel (50 $\mu$g/ml) into CRC cells after 0, 18, and 24 h of incubation. (Scale bar = 10 $\mu$m). (c) Time-dependent LY SERS intensity from the DNP-AuNPs-LY@Gel complex and LY mass on the nanovector in PBS buffer at pH7.4 and pH 5.5. (d) Time-dependent LY SERS signal from the DNP-AuNPs-LY@Gel complex in living CRC cells. Reproduced with permission from Ref. [110].

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5.2 Diatomite for biosensing

The combination of metallic nanoparticles (NPs) located near or inside the periodic nanopores of diatomite can form hybrid photonic-plasmonic modes. Furthermore, diatomite can be easily spin-coated to form an uniform film on a substrate. Both properties can be used to realize a low-cost lab-on-chip device allowing ultra-sensitive SERS detection of the target molecules, as demonstrated by Kong et al. [112]. DNPs decorated with 60 nm AuNPs has been used for both separating and detecting small molecules from mixture samples with ultra-high detection sensitivity down to 1 ppm [112]. As shown in the scheme of Fig. 9(a), the diatomite was spin-coated on a glass slide working as the stationary phase on the chromatography plate. The liquid sample was spotted on the bottom of the chromatography plate and the chromatography was performed in a developing chamber with the optimal eluent chosen through screening various conditions. The separated analyte spots were located under the UV illumination and visualized by iodine colorimetry. After deposition of AuNPs onto chromatography plate, the NPs were distributed on the surface of diatomite allowing an EF of the Raman signal of about 10$^6$. Therefore, the developed hybrid plasmonic-diatomite biosilica has been used as the matrix for on-chip chromatography, as well as the SERS substrate for separating and detecting Phenethylamine (PEA) from plasma. PEA and substituted PEAs are a class of molecules affecting the central nervous system that recently have caused attention by the U.S. Department of Justice for forensic toxicology issues. Interestingly, the intensity and the density of hot-spots on the diatomite/silica substrate are enhanced by the coupling between LSPs and GMRs due to photonic crystal structure of the diatomite and these strong hot-spots are crucial to detect low level of analyte molecules. Figure 9(b) shows the SERS spectra of human plasma with different concentrations of PEA (10, 100, 1000 ppm) separated by diatomite chromatography plates. The presence of the diatomite and AuNPs allowed the separation of PEA from the plasma and provided additional Raman enhancement to improve the SERS sensitivity, resulting in more than 10$\times$ better LOD than commercial chromatography plates [112]. The same lab-on-chip device has been used for detection of different molecules of biomedical interest as, for instance, phenethylamine and miR21cDNA in human plasma with unprecedented sensitivity and specificity [112].

 

Fig. 9. (a) schematic representation of the on-chip chromatography-SERS detection of target molecules from mixtures based on plasmonic NPs-decorated diatomite biosilica (b) SERS spectra of human plasma with different concentrations of PEA separated by diatomite chromatography plates. Reproduced with permission from Ref. [112]. (c) Schematic illustration of on-chip chromatography-SERS biosensing using the microfluidic diatomite analytical devices. (d) SERS spectra of human plasma with different concentrations of cocaine separated by microfluidic diatomite analytical devices and the SERS intensity as a function of logarithm scale Cocaine concentration in Plasma. Reproduced with permission from Ref. [113].

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By further combining the highly porous photonic crystal properties of the diatomite with microfluidics, Kong et al. demonstrated that is possible to realize lab-on-a-chip devices to detect illicit drugs [113]. The microfluidic diatomite analytical devices were fabricated via cost-effective and robust procedures and a schematic is reported in Fig. 9(c). The glass slide was covered by an adhesive tape, then 400 $\mu$m wide channels were cut by a razor blade. The diatomite was spin-coated on glass, and finally the channels with 400×30 $\mu$m cross section were fabricated by gently removing the tape (tape-stripping method). The microfluidic diatomite device effectively increases the concentration of target molecules at the sensor surface that can simultaneously separate small molecules from the complex background and acquire the SERS spectra of the target chemicals with high specificity after the deposition of plasmonic nanoparticles [113]. Figure 9(d) reports the SERS spectra of cocaine at different concentrations (from 10 ppb to 100 ppm) in human plasma. With the proposed device, the limit of detection was of 10 ppb, which is better than many traditional analytical methods.

The versatility of the developed system was further demonstrated by monitoring histamine at trace levels in food products as salmon and tuna [114]. Indeed, the proposed lab-on-a-chip photonic crystal can be used as cheap, robust, sensitive and portable food sensing platform for monitoring harmful ingredients in food products.

Another example of food safety application of diatomite based sensors has been proposed by Ma et al. [115]. Diatomite and plasmonic nanoparticles are the heart of the colorimetric sensor for quantitative analysis of pesticides in tea developed in [115]. A porous chitosan/partially reduced graphene oxide/diatomite (CS/prGO/DM) composite was synthesized via a hydrothermal treatment and the relative concentrations of diatomite, chitosan and graphene oxide were experimentally evaluated for efficient adsorption of tea interferents and to optimize the signal of the analyte molecules. The pesticides contain sulfuric groups, which have a strong tendency to bind with gold surfaces through Au-S bonds. These strong interactions induce a reduction of the distance between AuNPs and consequently the AuNPs aggregation that is accompanied by a blue shift in the color of the tea solution. With the help of UV-vis spectroscopy and smartphone assisted RGB value analysis, the proposed sensing platform was used for detecting phosalone and thiram with detection limit of 90 nM and 13.8 nM in a complex tea matrix, respectively. Diatomite-based materials for on site colorimetric quantitative detection are attractive for several applications including bioanalysis and food sensing.

6. Conclusions

The potentiality of plasmonic-based sensing platforms for biochemical and biomedical applications has been well established in recent years. Still, the fabrication of complex, two- and three-dimensional metallic nanostructures can be accomplished only by means of expensive nano-lithographic techniques, making difficult their production on a large scale for routine practices. On the other hand, living organisms able to synthesize ordered nanostructures as their constituent parts can represent a natural, low-cost alternative source of materials which can easily undergo metallization processes. In particular, a single diatom culture can be viewed as a living nanofactory able to give rise to elaborate dielectric nano-architectures self-replicating at high repetition rates and with noticeable reproducibility, this process being under the control of a complex, finely-tuned genetic mechanism refined through tens of millions of years of evolution. Proper metallization of intact diatom frustules or the retrieval of diatomite/metallic NPs hybrids allow obtaining efficient coupling of the plasmonic field with external optical radiation. The present review article represents an attempt to give a complete, wide overview on diatom biosilica metallization techniques and on the applications of metallized diatom fustules and diatomite in environmental, food safety, biochemical, and biomedical sensing and in cutting-edge theranostic experimentations.