Silica diatom shells tailored with Au nanoparticles enable sensitive analysis of molecules for biological, safety and environment applications
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Diatom shells are a natural, theoretically unlimited material composed of silicon dioxide, with regular patterns of pores penetrating through their surface. For their characteristics, diatom shells show promise to be used as low cost, highly efficient drug carriers, sensor devices or other micro-devices. Here, we demonstrate diatom shells functionalized with gold nanoparticles for the harvesting and detection of biological analytes (bovine serum albumin—BSA) and chemical pollutants (mineral oil) in low abundance ranges, for applications in bioengineering, medicine, safety, and pollution monitoring.
KeywordsDiatoms Frustules Sensing devices Au functionalization SERS Safety Biological sensing
Bovine serum albumin
- D24 systems
Silicon dioxide diatom shells functionalized with gold nanoparticles
Surface-enhanced Raman spectroscopy
Diatoms are unicellular algae massively present on earth with over 100,00 species distributed in aquatic (oceans, lakes, rivers) and semi-aquatic (wetlands and soils) niches. They contribute to an estimated 40–50% of the total organic material content in the oceans and to the ~ 20% of the conversion of carbon dioxide to organic compounds (i.e., photosynthesis) in the biosphere [1, 2, 3].
Diatoms are protected by a functional silicon dioxide shell (frustules) with a complex, micrometer-scale architecture and a pore size varying between species. Due to their microstructure, diatom shells show values of specific strength up to ~ 1700 kN m/kg that lay well above those of other natural cellular, composite, and silk materials including spider silk (1000 kN m/kg) [4, 5, 6, 7]. Moreover, due to the regularity and symmetry in the lattice of pores aligned on the frustule surface, often diatom frames show natural optical properties, and reveal light convergence, concentration, and trapping effects, depending on pore geometry and topology, wavelength, and the valve orientation [8, 9, 10, 11, 12].
Thus, diatoms are natural (in opposition to artificial), abundant, low cost, and easily accessible three-dimensional micro- or nanoscale structures that do not require traditional techniques of nanofabrication for their production, and, in sight of their scale, morphology, and properties thereof, display potential for being utilized as miniature sensors, drug-delivery capsules, and other micro-devices [2, 13, 14]. Nevertheless, despite this promise, there are relatively few applications of diatoms in nanotechnology [15, 16, 17], possibly because, while diatom shells represents the required support for many structures, further functionalizations (modifications) are necessary to provide these structures with the correct functions.
The device integrates different scales. (i) The sub-millimeter dimension of the shells permits system manipulation, handling, and access. (ii) The micrometer size of the pores enables molecule-harvesting, selection, and (in a more sophisticated evolutions of the device) fragmentation. (iii) The nanometer size of the Au–NPs enables control and amplification of an external electromagnetic (EM) radiation. Thus a hierarchical multi-scale architecture permits to extract-specific analytical molecular targets from a solution and characterize them using surface-enhanced Raman spectroscopy (SERS) even in very low abundance ranges. Incubation with fluorescent 50 nm microspheres (Fluoresbrite® Yellow Green Microspheres—Additional file 3) and subsequent fluorescence analysis indicates devices localization, selectivity, specificity, and absence of signal from the background (noise) (Fig. 1f).
Functionalization with Au–NPs
Simulating EM Field Around D24/Au–NPs
FEM analysis presented in Fig. 3a–c simulates the EM field in simplified 2D planar geometries—for this configuration, the EM field evolves around the Au nanoparticles on the external diatom surface. Nevertheless, in the three-dimensional scheme of Fig. 3d and other images presented in the Additional file 4, gold nanoparticles are distributed along the internal surface of the pores. This scheme, more akin to the real physical prototype, indicates that the analytes adsorbed by the D24 devices may interact with the EM field—and being detected—for any pore/analyte reciprocal localization. So, even if SERS is a short range effect and the EM field decays with a power of three of the distance from the gold nanoparticles , analyte harvesting and analyte/pore colocalization assure the sensing capabilities of the device. Further to this end, we demonstrate localization of the analyte in the pores with additional, high magnification fluorescence images of D24 systems loaded with Yellow Green 50 nm nanospheres (Additional file 3). Spatial overlap between the fluorescence signal and the D24 diatom devices and the vanishingly small background signal demonstrate that analyte uptake is highly efficient leaving no or minimal residues.
SERS Analysis of BSA in Solution
Here, we assess the ability of D24 devices to operate as molecular harvesting agents and sensing devices in biological systems. We incubated D24 devices in a solution containing bovine serum albumin (BSA) in a 10−16 M concentration, with a relative abundance of 1 mg of D24 devices in 1 ml of solution. The complex network of openings that penetrates into the diatom represents a filter that can absorb the molecules with a hydrodynamic diameter smaller than the pore size. Considering that, for the present configuration, the average pore size is nearly 200 nm, BSA proteins with a characteristic length size of ~ 6 nm  would easily accumulate within the pore matrix. After 10 min from incubation, D24 systems were separated and extracted from the initial solution through sedimentation. D24 devices containing BSA were positioned on the stage of a Renishaw inVia micro-Raman microscope for analysis.
Raman and SERS peaks of BSA in the 800–1800 cm−1 range, and tentative assignment of peaks
Raman peak BSA
SERS peak BSA
Phenylalanine (Phe) ring breathing
Carbon skeleton stretching modes at 1509 cm−1
Trp - Phe
SERS Analysis of Mineral Oil
D24 devices were demonstrated in the analysis and detection of mineral oil in increasingly low dilution factors. Mineral oil is a by-product of the distillation of petroleum to produce gasoline. It contains light mixtures of higher alkanes with the paraffins of this mineral oil ranging from about C18 to C40: it roughly corresponds to the composition of base oil for manufacturing lubricating or hydraulic oils . In a recent commentary , it is recommended to reduce the exposition to mineral oil to levels lower than ~ 50 mg/kg, i.e., 50 ppm. Analysis of mineral oil (m.o.) and related products is therefore of interest in environmental pollution and food safety. m.o was examined using Raman spectroscopy following the methods described in the previous BSA analysis.
The described scheme allows analyte capture, localization, sequestration, and detection. Analytes adsorbed by porous D24 diatoms can be easily collected, manipulated, separated, and aliquoted into samples. Each sample is constituted by (i) functionalized diatoms loaded with (ii) specific analytes. Thus a sample is combination of the analyte and the device necessary to detect it. Different aliquots can be processed using simple Raman set-ups, stored for future analysis, perhaps kept in a refrigerator or a freezer for long periods of time. Thus, D24 systems are a hybrid device which operates in symbiosis with the target molecules that one wants to analyze. While traditional SERS substrates or metal nanoparticles have been heretofore used in isolation, and interaction between the SERS substrate and the analyte takes place in intermittent episodes and is often limited at the time of the measurement, D24 systems integrate the sensor and the target molecule in an individual device that is multifunctional, manageable, and portable. Moreover, differently from traditional SERS substrates, D24 systems are sufficiently small to act as tracers. Released in the microcirculation circuits, D24 systems will be transported through the arteries, arterioles, and microvessels of the living tissue, interact with blood and the waste product of cells, internalize analytes, peptides and biomarkers, and operate analysis of biomolecules with elevated spatial and temporal resolution. Analytical maps of biomolecules may be, in turn, associated to individual cancer risk, pathological risk, or physiological state of a patient to support medical decisions and plan interventions.
Notice that the idea of using diatoms as microcapsules for sensing is not completely new. Nevertheless, previously reported works depart from our analysis to an extent that depends on individual contributions as explained in the subsequent section.
In reference , Ren and colleagues used simulations to explore the electric field enhancements generated by plasmonic nanoparticles coated on the surface of diatom skeletal shells. Then, they prepared SERS substrates by assembling silver nanoparticles on diatom surfaces. While similar devices achieve excellent sensing enhancement factors, diatoms are immobilized on a substrate, and may not be freely administrated in the microcirculation, within biological fluids or solutions, biological compartments, aqueducts, channels, seawater, ocean streams and currents, for biological or technical applications.
In reference , Chen and colleagues pressed Au nanoparticle-coated diatomaceous earth into hard, button-like millimeter tablets. Then, they used these SERS tablet devices to analyze the chemical composition of eccrine sweat in latent fingerprints, that is a brilliant, very practical application of the device in medicine. Yet, it is specific and the analysis is still executed at a macroscale level.
In reference , the group led by Luca De Stefano functionalized diatom frustule with Au nanoparticles using electroless deposition. Then, they tested the device using p-Mercaptoaniline (pMA). pMA can form self-assembled monolayer on metal surfaces and is therefore used as a surface probe molecule in SERS. Electroless deposition is an assessed technique for the synthesis of gold nanoparticles on an autocatalytic surface that allows attaining high control on the size and density of the particles [18, 19, 32]. Differently from this approach, we used here a photo-deposition process that is more direct, faster, and requires no or minimal sample treatment compared to electroless deposition. Nevertheless, the approach proposed by De Stefano is promising and deserves to be verified even further in biological or environmental applications.
We developed ways to modify economic, easily accessible, and abundant diatomaceous earth to obtain miniature sensor devices where the pores of the diatom shells have the ability to capture molecules in solution, and the gold nanoparticles amplify the spectroscopy signal of several order of magnitude to reveal molecules in otherwise unattainable low abundance ranges. We demonstrated similar D24 devices in the analysis of biological BSA proteins in solution and for the detection of traces of mineral oil in a binary emulsion with water. In both cases, we revealed target molecules in low dilutions down to 10−16 M for BSA and 50 ppm for mineral oil. The devices may find applications in analytical chemistry, the surveillance and assessment of biological risk, food safety, contaminant monitoring, and surveillance in seawaters, aqueducts, and drinking water.
Scanning Electron Microscopy of Samples
Silica shells functionalized with Au nanoparticles (D24 systems) were dispersed directly on a carbon adhesive tape for scanning electron microscopy (SEM) imaging. Samples were imaged using a Zeiss Auriga Compact FE-SEM equipped with InLens secondary-electron detector, for morphological imaging, and anular Back Scattering detector, for Z-contrast imaging (in order to highlight the presence of Au).
Characteristics of Diatomaceous Earth Used in This Study
Diatom material used in this study was a food grade, high-quality diatomaceous earth provided by Perma-Guard (Perma-Guard Europe Sollaris Sp. z o.o., Otwock, Poland) as a 1 kg free sample of Fossil Shell Flour®. Its current market price is ~ 16 euro per 1 kg. It is composed of cylindrical shells of extinct freshwater diatom Melosira preicelanica. Its main fraction constitutes amorphic silica (up to 94%), followed by smectites (~ 3%), kaolinite (~ 2%), feldspars (~ 1%), calcite (> 1%), and quartz (> 1%). Grinding in a low-speed hammer mill was performed to homogenize the particle size. Main properties (including physical and optical properties) of Fossil Shell Flour® are median particle size: 10 μm, mesh screen residue: 2%; refractive index: 1.43; oil absorption: 120%; brightness (green filter): 85; specific gravity: 2.2; surface area: 44.2 m2/g; pH: 8.0; total volume of pores: 0.132 cm3/g; micropores (< 20 Å): 14%; mesopores (20–500 Å): 65%.
Fluorescence Analysis of D24 Systems
D24 systems were incubated with Fluoresbrite® Yellow Green Microspheres with a diameter of 50 nm for 10 min, with a ratio D24 : fluorescent particles = 1 : 10. Then, we collected D24 systems from the solution and placed them on the optical stage of an inverted Leica TCS-SP2® laser scanning confocal microscopy system. All measurements were performed using a ArUv laser. The pinhole (80 μm) and laser power (80% power) were maintained throughout each experiment. Yellow fluorescence (similar to FITC) was excited using a λ1 = 441 nm excitation line and confocal images were collected at the maximum of emission λ2 = 485 nm using × 10/20 objectives. Images were acquired over a region of interest of 975 × 750 μm2 and were averaged over four lines and ten frames to improve quality and reduce noise. Images were digitalized into 1280 × 960 pixels.
X-Ray Photoelectron Spectroscopy Analysis of Samples
X-ray photoelectron spectra were recorded on a X-ray photoelectron spectroscopy (XPS) Versa Probe II (PHI, Chanassen US) by large area analysis mode where the mono-chromate Al anodic beam of 100 μm, 100 W power, normal to the surface, is rastered over an area of 1400 × 300 μm2 with the analyzer at 45° with respect to the sample surface. Survey spectra were acquired with an accumulation time at least of 20 min at high pass energy (187 keV) while high-resolution spectra of the elements of interests where acquired at 11.7 keV with same power and 0.1 eV resolution. Spectra were analyzed by Multipack (PHI, Chanassen USA) software and all the peaks were referenced to the adventious carbon peaks C1s at 284.8 eV binding energy.
Simulating the Electromagnetic Field Within the D24 Systems
In order to calculate numerically the electric field profile throughout the decorated structure, a finite element method (FEM) 3D model has been developed employing the commercial software COMSOL Multiphysics 5.3. Simulations have been conducted on a single cubic unit cell where 125 particles have been placed on the surface of the patterned dielectric surface. The overall optical response has been investigated as a function of the incident angle of the electromagnetic field which was approximated as a TM linearly polarized plane wave (Additional file 4). The periodicity of the system was taken into account by applying Floquet boundary conditions on the lateral sides of the unit cell, perpendicular to the plane of incidence; subsequently, results have been periodically extended to visualize the diatom array (Additional file 4). A wavelength of λ = 633.0 nm was set. Power of the incident radiation was arbitrarily chosen as P inc = 1 W, the unit cell area equals 3.9 × 10−13 m2 and the resulting intensity is I = 2.5 × 10−8 W/cm2 (notice that intensity dependant non-linearity is here neglected). Regarding the materials, the diatom was optically described as a dielectric with refractive index ndiatom = 1.3, whereas the surrounding environment is air with nair = 1. Gold nanoparticles, modeled as perfect spheres with a diameter d = 20 nm, were modeled following the dielectric formulation reported in . The geometrical domain has been discretized using tetrahedral elements. Maximum size of the mesh element has been chosen as 1/5 of the effective wavelength value that had to be resolved in each domain, depending on its refractive index. The minimum mesh element was set to r/1.5, r = 10 nm is the radius of each nano-sphere. Maxwell equations have been numerically solved within the unit cell by placing perfectly matched layers at the top and the bottom of the structure, in order to avoid unphysical reflections at the boundaries of the domain. In addition, the electromagnetic field symmetry has been exploited to reduce the computational effort of the simulation. As a result, equations are solved for a half of a diatom only, and perfect magnetic conductor boundary conditions have been imposed to the lateral sides of the unit cell, parallel to the plane of incidence, coherently with the polarization of the incident field.
Raman Analysis of Samples
D24 devices containing BSA were positioned on the stage of a Renishaw inVia micro-Raman microscope for analysis. Samples were analyzed using × 20/50 objectives of a Leica microscope. Raman spectra were excited by the 633.0 nm line of an HeNe laser in backscattering geometry and acquired with a CCD with 1024 × 1024 pixels. Laser power was adjusted as 0.18 mW and maintained constant throughout the whole measurements. Interferograms were recorded with an integration time of 20 s. Each spectrum was base line corrected with a second degree polynomial function. Raman maps were performed with a step size of 400 and 600 nm in the x and y axes direction.
This work has been partially funded from the Italian Minister of Health under the project “Cancer biomarker detection using micro-structured/super-hydrophobic surfaces and advanced spectroscopy techniques” (Project n. GR-2010-2320665).
Availability of Data and Materials
The datasets generated during and/or analyzed during the current study are available from the corresponding author on request.
VO performed Raman and SERS analysis of biological samples and mineral oil. MV and DC modified, functionalized, and acquired SEM images of the diatom shells with Au NPs. MLC supervised the experimental part of the work. RM conceived the idea and helped in writing the manuscript. AA and AS performed numerical simulations and helped in writing the manuscript. EB performed XPS analysis of samples and helped in writing the manuscript. NC, MC, EDF, and FA commented on the manuscript and helped in interpreting results. FG conceived the idea, supervised and coordinated the overall activity, and wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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