Accred. Qual. Assur., 2014, Springer Verlag Berlin Heidelberg

Feasibility of the development of reference materials for the detection of Ag nanoparticles in food: neat dispersions and spiked chicken meat

Ringo Grombe, Günter Allmaier, Jean Charoud-Got, AgnieszkaDudkiewicz, HåkanEmteborg, Thilo Hofmann, Erik Huusfeldt Larsen, Angela Lehner, MeritxellLlinàs, KatrinLoeschner, Kristian Mølhave, Ruud J Peters, John Seghers, ConxitaSolans, Frank von der Kammer, Stephan Wagner, Stefan Weigel, Thomas PJLinsinger*

Instrument parameters and sample preparation for the veracious analytical techniques used

1.1Dynamic light scattering

For homogeneity and stability testing liquid viscosity was set to 0.89 mPa•s (25 °C) and the liquid refractive index were set to 1.33 (25 °C). The AgNP refractive index and absorption was set to 0.14 and 3.99, respectively.

1.2Inductively coupled plasma optical emission spectrometry (ICP-OES) on the silver dispersions

Three subsamples of about 0.3 g were taken from each ampoule of the AgNP dispersions NanoLyse03 and NanoLyse04 tested. Each subsample was weighed into a 10 mL polypropene vessel and 1 mL of concentrated nitric acid was added. The solutions changed from brown to colorless and were left to stand for at least 30 min. Samples were then diluted gravimetrically to total masses of about 10 g with ultrapure water (MilliQ system, Millipore, Molsheim, France, 18 Mcm). ICP-OES measurements were made using an external calibration with five standards in the range 4 to 8 µg/mL, to bracket the expected concentration of the diluted samples of 6µg/mL. The calibration was checked by measurement of an independently prepared control standard of 6 µg/mL silver before and after the sample measurement sequence.

1.3ICPMS on ultrafiltrates

Samples were measured in a sequence with a calibration blank and a standard measured between each 6 samples. All blanks were < 0.02µg/L. Sub-samples of 0.45µL were pipetted into 50 mL poly(propylene) vessels containing 1.5mL of concentrated nitric acid (>60% suprapure, Merck, Darmstadt, Germany). The solutions were then diluted gravimetrically to about 45g with ultrapure water (MilliQ, 18 Mcm, Millipore, Framingham, MA, USA).). The results were obtained from three samples subjected to two consecutive measurements.

1.4TRANSMISSION ELECTRON MICROSCOPY (TEM) ON DISPERSIONS AND SPIKED CHICKEN MEAT

Aqueous neat suspensions of NanoLyse03 and NanoLyse04 (approximately 10 mL) were sonicated for 1 min at 100 W using sonication probe (Sonicator, Ultrasonic Processor, Misonix, Farmingdale, NY, USA). Then both suspensions were diluted with borate buffer (0.05 mol/LH3BO3, 0.05 mol/LKCl, 0.004 mol/LNaOH) at pH 8.0 (BB8.0) in ratio 1:799 and sonicated again at the same conditions. During sonication vials with the samples were partly immersed in the water with ice to prevent the sample from overheating. 0.5 g of the diluted sample was then transferred to the polyallomer centrifuge tube for a Beckman Xl-100 SW40Ti swing bucket rotor (Beckman, Brea, CA, USA). The tubes were then filled with BB8.0 up to the desirable volume (2-3 mm below the tube edge according to the manufacturer’s specification) and weight. Then two formvar-carbon coated TEM grids (Agar Scientific, Stansted, United Kingdom) were placed floating on the liquid. In order to provide flat support for the grids all tubes were equipped with a solid filler placed at the tube’s bottom, which was made out of Agar 100 resin (Agar Scientific, Stansted, United Kingdom). Prepared samples were centrifuged at 100000 g at 20 °C for 60 min. These conditions allowed, according to Stoke’s law, to sediment silver particles down to the diameter of 4 nm. No additional drying was required, as the TEM grids are hydrophobic, leaving a dry surface after pipetting off the water.

The AgNP spiked chicken meat samples were prepared by homogenizing the sample in the BB8.0 in ratio 1:399. To suspend NanoLyse13 and NanoLyse14 in the BB8.0, pastes were first added to the BB8.0 using a potter type of homogenizer and subsequently sonicated at the same conditions as the particle suspensions. The mixtures were then prepared for the centrifugation on the TEM grids in the same way as specified for the particle suspensions.

1.5Gas-Phase Electrophoretic Molecular Mobility Analysis (GEMMA) on dispersions

The electrospray unit was run in the positive ion mode with a mixture of 0.5 L/min compressed air supplied by a table top compressor (Dürr-Technik, Bietigheim-Bissingen, Germany) and 0.1 L/min CO2 (99.995%, Air Liquide, Schwechat, Austria) and an applied voltage of 2.5 kV resulting in an electrical current of 360-460 nA. A cone tipped (angle 75º) fused silica capillary with 40 µm inner diameter was used and the pressure difference along the capillary was 4 psid. The nanoDMA was run with a sheath flow of 3 L/min and the condensation particle counter used n-butanol in the high flow mode. Samples were diluted in 20mmol/LCH3COONH4 directly before measurement.

1.6SINGLE-PARTICLE ICPMS (SP-ICPMS) ON SPIKED CHICKEN MEAT

Laboratory 1: 200 mg sample were weighed into a 12 mL polyethylene test tube. 3.0 mL buffer (10 mmol/LTris, 1 % Triton X, 1 mmol/LCa acetate) was added and the sample was vortexed. 1 mL protease K solution (approximately 22 U/mL) was added and the mixture was incubated for 3 h at 37°C. The sample was vortexted and the supernatant was transferred into a 20 mL screw cap vial. The digests were diluted 1:100000 (NanoLyse13) or 1:500000 (NanoLyse14) and were quantified by sp-ICPMS on a Thermo Scientific X series 2 instrument equipped with a Burgener Mira Mist Nebulizer (PEEK) and a quartz spray chamber. Dwell time was set to 3 ms and each measurement lasted for one minute; gas flows were 13 L/min, 0.7 L/min and 1.0 L/min for plasma, auxiliary and nebulizer respectively and the RF power was set to 1404 W.

Laboratory 2: 5 mL Protease K from Engyodontium album (Sigma-Aldrich St. Louis, MO, USA) in buffer (50 mmol/LNH4HCO3; pH=7.4; 45 mg/mL SDS; 0.2 mg/mL NaN3) were added to 0.25 g meat paste. The mixture was incubated under continuous stirring at 37 °C until no more meat fibers were visible (approx.. 40 min). 10 mL sample were injected into an AF4 system and quantified using a Thermo Scientific iCAP Q ICP-MS (Thermo Fisher Scientific GmbH, Bremen, Germany) and an Agilent Micro Flow nebulizer and a Scott double pass spray chamber. Dwell time was set to 3 ms; gas flows were 14 L/min, 0.96 L/min and 0.80 L/min for plasma, auxiliary and nebulizer respectively and the RF power was set to 1550 W.