Nitric acid

The following reagents were used: nitric acid (H₂NO₃), sulfuric acid (H₂SO₄), monosodium dihydrogen phosphate (NaH₂PO₄), disodium hydrogen phosphate (Na₂HPO₄), potassium chloride, N-hydroxysuccinimide ester (NHS), 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC), and bovine serum albumin (BSA), all sourced from Sigma-Aldrich and Merck (Rahway, NJ, USA). Graphene nanoplatelets were obtained from XG Sciences (East Lansing, MI, USA), and the 1-Step™ Ultra TMB-ELISA substrate was procured from Thermo Fisher™ Scientific (Waltham, MA, USA). The N-protein was synthesized and purified at the Federal University of Amazonas. Additionally, a solution of 5 mmol L⁻¹ K₃Fe(CN)₆/K₄Fe(CN)₆ prepared in 0.1 mol L⁻¹ KCl was utilized as a redox probe for electrochemical measurements. All electrochemical experiments were performed at room temperature (22 ± 0.5 °C) without stirring. The Federal University of Amazonas provided human blood serum samples, and the study received approval from The Research Ethics Committee of the Federal University of Amazonas (CAAE: 34906920.4.0000.5020), adhering to Brazilian law and the Declaration of Helsinki.

All reagents were of a high analytical grade. Deionized water of level I (Millipore, Burlington, MA, USA), as defined in ISO 3696:1987 [33 ] and hydrogen peroxide (30%; Merck, Darmstadt, Germany) solutions of ultrapure grade were used. Also, concentrated nitric acid (65%; Merck, Darmstadt, Germany), first distilled in an acid distillation system (model subPUR; Milestone, Shelton, CT, USA), was used.
Working multi-element standard solutions were prepared from mono-element high purity ICP stock standards (Cu, Mn, Fe, Zn, Mg, Ca, P, Na, and K) containing 1000 mg/L of each element (Merck, Darmstadt, Germany).
Working standard solutions, sample dilutions, and blanks were prepared with a 2% concentration solution of nitric acid. A nitric acid solution (2–4%) concentration was used to wash up the ICP-OES sample introduction system.

The metal content in six regions of the sheep brain was measured using inductively coupled plasma mass-spectrometry (ICP-MS) as previously described [30] (link). Weighed tissue pieces were lyophilized, digested in 150–400 µl of 65% nitric acid (Merck, Kilsyth, Victoria, Australia) overnight, and heated for a further 20 min at 90°C. Then 400 µl of BDH prolabo 30% hydrogen peroxide (VWR, Murrarie, QLD, Australia) was added to each sample and incubated at room temperature for 30 min, then for a further 15 min at 70°C. All samples were diluted 1∶20 with 1% nitric acid before being measured in an Agilent 7700 series ICP-MS instrument (Agilent Technologies, Santa Clara, CA, USA) using a helium reaction gas cell. The instrument was calibrated using 0, 5, 10, 50, and 100 ppb of certified multi-element ICP-MS standard calibration solutions (Accustandard, New Haven, CT, USA) for a range of elements. 200 ppb of yttrium (Y89) was used as an internal control. Samples were analyzed in triplicate and median values were used for analyses. The results are expressed as micrograms of metal per gram of wet weight (µg/g).

Materials: Ammonium hydroxide aqueous solution (Fluka, 25%), N_α-benzoyl-D,L-arginin-4-nitroanilide hydrochloride (BAPNA) (Sigma, 98%), benzylamine (BzA) (Janssen Chimica), 2,2´-bipyridine (bpy) (Aldrich, 99%), citric acid monohydrate (Grüssing GmbH, 99,5%), (4-(chloromethyl)phenyl)trimethoxysilane (CPS) (ABCR, 95%), copper(I) bromide (CuBr) (Aldrich, 98%), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (ABCR, 98%), N-hydroxy succinimide (NHS, Fluka), iron(III) chloride hexahydrate, iron(II) chloride tetrahydrate (Fluka, 98%), ninhydrin (Riedel-de-Haen), oligo(ethylene glycol) methylether methacrylate (OEGMA, Aldrich, M_n = 290 g·mol⁻¹), 2-(2-methoxyethoxy)ethyl methacrylate (MEEMA), porcine pancreas trypsin type IX-S (Aldrich), tetramethylammonium hydroxide aqueous solution (25%) were used as received without further purification. Ethanol, diethyl ether and acetone were purified by distillation before use. Dimethyl sulfoxide (DMSO; min. 99.5%, Riedel-de-Haen) was distilled under reduced pressure from calcium hydride and stored under argon and molecular sieve (3A). HEPES buffer was prepared from 11 mM HEPES (Sigma), 140 mM NaCl (Merck), 4 mM KCl (Merck), 10 mM D(+)-glucose, and dissolved in deionized water. 2-Methoxyethyl methacrylate (MEMA, Aldrich, 99%) was distilled under reduced pressure and stored under argon. Nitric acid (conc., p.a., Merck) was diluted with distilled water resulting in a 2 N solution. Succinimidyl methacrylate (SIMA) was synthesized by a method by Gatz et al [26 ,60 (link)].
Synthesis and stabilization of Fe₃O₄ nanoparticles: The synthesis of magnetite nanoparticles on the gram scale was carried out by alkaline precipitation of iron(III) and iron(II) chloride following a method of Cabuil and Massart and is described in detail elsewhere [43 ]. For stabilization, the freshly synthesized nanoparticles were stirred with 420 mL 2 N Nitric acid for 5 min. After washing with distilled water, 90 mL 0.01 N citric acid (CA) was added to the nanoparticles and stirred for 5 min. The particles were magnetically separated from the supernatant and 15 mL of tetramethyl ammonium hydroxide aqueous solution was added to obtain 3.32 g magnetic nanoparticles Fe₃O₄@CA in 92 mL of a stable dispersion at pH 8–9 (yield: 42.5%).
The Fe₃O₄ content µ(Fe₃O₄) in dispersion and the magnetic core diameter d_c were determined via VSM (µ(Fe₃O₄) = 2.55 mass%, d_c = 11.7 nm). DLS: d_h,n = 14.3 nm (25 °C in H₂O). FT-IR (Diamond): ν (cm⁻¹) = 2357, 2335 (C-N), 1247 (OH), 1098 (C-O), 1080 (OH).
Surface modification of Fe₃O₄ nanoparticles: For the immobilization of initiator sites on the particle surface of Fe₃O₄@CA, the dispersion was diluted with ethanol to a mass content of 1.0 g·l⁻¹, and 1.80 mmol CPS per gram of Fe₃O₄ was added. After stirring for 24 h at ambient temperature, ethanol was removed under reduced pressure at 40 °C and the particles were washed with ethanol/acetone (1:1) five times. The particles were then redispersed in DMSO, resulting in a Fe₃O₄ content µ(Fe₃O₄) of 6.44 mass % (VSM) in dispersion (yield: 46.4%). The magnetic core diameter d_c was measured to be 11.1 nm (VSM). The functionalization degree of CPS was determined by EA to be 0.87 mmol CTS on 1.94 g Fe₃O₄@CPS. FT-IR (Diamond): ν (cm⁻¹) = 2357, 2335 (C–N), 1241 (OH), 1115 (Si–O), 1011, 948 (Si–C).
Surface-initiated ATRP of functional polymer shells: The obtained CPS coated particles served as a macroinitiator for the following ATRP. The synthesis of Fe₃O₄@P(O₁₀₀) is described, representatively. Therefore 6 mL of the DMSO-based particle dispersion (0.65 g Fe₃O₄@CPS) was mixed with 5 mL of a DMSO solution of 37.3 mg (0.26 mmol) CuBr and 101 mg (0.65 mmol) bpy. The polymerization was started by adding 5.83 mmol of the monomer (here: OEGMA). The mixture was stirred for 24 h at ambient temperature. The obtained viscous magnetic fluid was diluted with 10 ml DMSO to the final ferrofluid. The Fe₃O₄ content µ(Fe₃O₄) in dispersion and the magnetic core diameter d_c were determined via VSM. The polymer content χ_Pol in the dried particles was obtained from EA and TGA.
Particle transfer to water/buffer: The DMSO-based particle dispersion was added dropwise to diethyl ether (Et₂O). The precipitate was washed five times with Et₂O/Acetone (1:1) and was redispersed in distilled water or buffer to obtain an aqueous magnetic fluid.
Immobilization of trypsin: 30 mg trypsin was dissolved in 6 mL HEPES buffer and mixed with 6 mL of a HEPES buffer-based Fe₃O₄@P(O₈₅S₁₅) particle dispersion (µ(FeO_x) = 0.15 mass %). In order to allow reactivation of active ester functions that may have hydrolyzed during storage, 6 mL of 2.21 μM EDC/NHS solution was added. The binding reaction was carried out for 6 h at ambient temperature on a shaker. The obtained trypsin functionalized particles were separated and washed carefully with water to remove any residues of free trypsin, and redispersed in HEPES buffer.
Determination of immobilized enzyme kinetics and activity: BAPNA was used as the model substrate. Four HEPES buffered BAPNA solutions with concentrations between 2.0 mM and 0.5 mM, and a 6.0 μM trypsin solution were prepared and tempered to the desired temperature. The respective BAPNA solution was added to a cuvette and mixed with 100 μL of FeO_x@POEGMA-trypsin nanoparticle dispersion or with 50 μL trypsin solution. The cuvette was placed into the spectrophotometer and tempered. Starting with the addition of the enzyme, the change in absorption at 410 nm was detected over a period of up to 20 min by UV–vis spectroscopy.
Analytic methods and instrumentation: ATR-IR spectra were measured on a Nicolet 6700 spectrometer. Elemental analyses were performed on a Perkin-Elmer 2400 CHN analyzer. The organic content was calculated through C content. For TGA, a Netzsch STA 449c in a He atmosphere was used with a heating rate of 10 K·min⁻¹ between 30 and 600 °C. Gel permeation chromatography (GPC) elugrams were collected on THF (300 × 8 mm² MZ Gel Sdplus columns, Waters 410 RI-detector) relative to polystyrene standards. NMR spectroscopy was performed on a Bruker DRX500 at 500 MHz and ambient temperature. DLS experiments and zeta potential measurements were performed on a Malvern Zetasizer Nano ZS at 25 °C. The particle size distribution was derived from a deconvolution of the measured intensity autocorrelation function of the sample by the general purpose mode (non-negative least-squares) algorithm included in the DTS software. Each experiment was performed at least three times. Cloud point photometry of aqueous particle dispersions was performed on a Tepper TP1 cloud point photometer at 1 K·min⁻¹ in HEPES buffer. From the turning point of the turbidity curves, the cloud point temperature T_c was obtained. Vibrating sample magnetization (VSM) measurements were implemented on an ADE Magnetics vibrating sample magnetometer EV7. Induction heating experiments were performed on a Hüttinger HF generator Axio 5/450T equipped with a copper inductor (l = 50 mm, dI = 35 mm, n = 5), and operating at 250 kHz and at a magnetic field of 31.5 kA·m⁻¹. The experiments were performed in a vacuum-isolated glass sample container. Different samples with varying magnetite concentrations μ(Fe₃O₄) of Fe₃O₄@P(O₁₀₀)-based magnetic fluid in water were exposed to the oscillating magnetic field. Via a fiber-optical sensor the fluid temperature T was measured against time t. For UV–vis spectroscopy, a Nicolet UV 540 spectroscope, a Unicam UV 500 or a Perkin Elmer Lambda19 with a thermostat Colora NBDS was used. Differential scanning calorimetry thermograms were collected on a Mettler-Toledo DSC 822^e at 5 K·min⁻¹. TEM pictures were taken on a Hitachi H 600.