High-Throughput Silica Nanoparticle Detection for Quality Control of Complex Early Life Nutrition Food Matrices

The addition of nanomaterials to improve product properties has become a matter of course for many commodities: e.g., detergents, cosmetics, and food products. While this practice improves product characteristics, the increasing exposure and potential impact of nanomaterials (<100 nm) raise concerns regarding both the human body and the environment. Special attention should be taken for vulnerable individuals such as those who are ill, elder, or newborns. But detecting and quantifying nanoparticles in complex food matrices like early life nutrition (ELN) poses a significant challenge due to the presence of additional particles, emulsion-droplets, or micelles. There is a pressing demand for standardized protocols for nanoparticle quantification and the specification of “nanoparticle-free” formulations. To address this, silica nanoparticles (SiNPs), commonly used as anticaking agents (AA) in processed food, were employed as a model system to establish characterization methods with different levels of accuracy and sensitivity versus speed, sample handling, and automatization. Different acid treatments were applied for sample digestion, followed by size exclusion chromatography. Morphology, size, and number of NPs were measured by transmission electron microscopy, and the amount of Si was determined by microwave plasma atomic emission spectrometry. This successfully enabled distinguishing SiNP content in ELN food formulations with 2–4% AA from AA-free formulations and sorting SiNPs with diameters of 20, 50, and 80 nm. Moreover, the study revealed the significant influence of the ELN matrix on sample preparation, separation, and characterization steps, necessitating method adaptations compared to the reference (SiNP in water). In the future, we expect these methods to be implemented in standard quality control of formulation processes, which demand high-throughput analysis and automated evaluation.

Transmission electron microscopy images of DSM Premix with and without AA (20 mg/mL in MQ water) taken on Formvar coated copper grids with a ZEISS EM900 at 50kV.Left image: 2µL of the 10x diluted premix with AA solution was dried on the grid and measured at 7kx.Right image: 2µl of Premix without AA solution incubated on the grid for 2 min until the drop was removed with a dust-free paper tissue.The image was taken at 12kx.

Figure SI- 1 .
Figure SI-1.Transmission electron microscopy image of Tixosil 0.1mg/mL in MQ-water, dried on a Formvar coated copper grid.Measured with a ZEISS EM900, 50kV with a Magnification of 3000x (left) and 85000x (right).

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Figure SI-2.Transmission electron microscopy images of DSM Premix with and without AA (20 mg/mL in MQ water) taken on Formvar coated copper grids with a ZEISS EM900 at 50kV.Left image: 2µL of the 10x diluted premix with AA solution was dried on the grid and measured at 7kx.Right image: 2µl of Premix without AA solution incubated on the grid for 2 min until the drop was removed with a dust-free paper tissue.The image was taken at 12kx.

Figure SI- 3 .
Figure SI-3.Total Silicon concentration of the DMF digested samples measured with MP-AES (right) and ICP-MS (left).

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Figure SI-4.a) Comparison of intensity measured at 251.66 nm (silicon peak) of different concentrated silicon AAS standard solution (blue) versus SiNP standard solutions (red) measured by the MP-AES.Samples were diluted in 0.1% nitric acid solution.b) The resolved MP-AES intensities of the SiNP standard solutions with diameters of 20, 50 and 80 nm.

Figure SI- 5 .
Figure SI-5.Atomic percentage (at%) of the Silicon 2p peak of Premix with and without AA and on Premix sample with only few AA measured by X-ray photo electron spectrometry.Powders were measured with a PHI 5800 spectrometer equipped with a Mg twin anode at 10kV & 10 mA, peak area were evaluated using CasaXPS.Each sample was measured twice A&B.

Figure SI- 6 .Figure
Figure SI-6.Silicon concentration in SEC fraction of SiNPs with different diameters (20, 50 and 80nm) and Si AAS Standard run on a Superose 6 GL30/100 column and analyzed by MP-AES.The column did not separate the different sized SiNP References.

Figure SI- 9 :
Figure SI-9: Transmission electron microscopy image of Si AAS standard ((NH4)2SiF6Solution 1000mg/L) after SEC with Sepharose 4B column.The fraction of the Ammonium Fluorosilicate solution was dried on 200 Mesh, Copper Formvar TEM grid and washed twice with MQ water.The black square is a remaining salt crystal.

Figure SI- 10
Figure SI-10 Size exclusion chromatography of the Fluorescence marker PDMPO on a Sepharose 4B column run with 0.05%SDS/PBS (grey) or NaOH 0.5M gradient method.

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Figure SI-11 a) Silicon concentrations in SEC fractions of SiNP 20nm at concentrations from 0.05-1.25mg/mL(relish) and the Silicon AAS standard at a concentration of 0.1mg/mL (gray).b) Standard curve of SiNP 20nm area under the peak versus its theoretical concentrations.

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Figure SI-12: a) Photograph of Sepharose 4B Column (left) next to Superose 6 (GL30/100) column (right).b) Silicon concentration in SEC fractions of SiNP 20 nm 2.5mg/mL measured by MP-AES and transmission electron microscope images (at magnification of 30kx) of three fractions.c)Silicon concentration in SEC fractions of SiNP 50nm (1.25-5 mg/mL) measured by MP-AES and transmission electron microscope images (at magnification of 30kx).d) Silicon concentration in SEC fractions of SiNP 80nm (0.625-5mg/mL) measured by MP-AES and transmission electron microscope images (at magnification of 30kx).

Figure SI- 13 :
Figure SI-13: Transmission electron microscopy image of the SEC fraction nr.43 of Form 1 digested by microwave, measured on a Copper/Formvar grid at 50kx with a Zeiss EM900

Figure SI- 15 :
Figure SI-15: Size exclusion chromatogram of DSM premix sample without AA spiked with 1mg/mL 20 & 50nm SiNP standard samples (black) and the DSM premix sample without AA (grey).TEM images were taken from Fraction 9(left &middle) and 43(right) image, at 50kx with a Zeiss EM900

Figure SI- 17 :
Figure SI-17: Transmission electron microscopy images of the standard SiNPs with 80, 50 and 20nm diameter (left to right column), at a magnification of 50kx pure (top row), at 50kx (middle row) and at 250kx (lowest row), after the microwave digestion with a Mars6 Microwave from CEM Corporation.Images were taken with a Zeiss EM900 on 200mesh carbon coated formvar copper grids after neutralisation.

Figure SI- 18 :
Figure SI-18: Cross contaminated fraction nr.14 of a SEC run of SiNP standard with 20 nm diameter in PBS with 0.05% SDS as an eluent and Sepharose4B column.The column was not rinsed prior the run and the other standard SiNPs with 50 and 80nm diameter were run before.The cross contamination with the larger SiNPs is evident.(SS278-7)