Icap q icp ms

The cellular Zn²⁺ and Ni²⁺ content of K. pneumoniae was carried out according to the method described by Maunders et al.^{66 (link)} with some modifications. An overnight culture of K. pneumoniae was diluted 1/200 in LB medium (5 mL) and was either untreated or treated with 0.2 mM AMA or Zn-AMA. The cells were grown for 16 h at 37 °C, harvested by centrifugation, and washed twice with PBS containing 5 mM EDTA and twice with ultra-pure water. The cell pellet was subsequently digested in 0.25 mL 65% HNO₃ at 80 °C for 30 minutes in borosilicate glass tubes using a water bath. The samples were diluted to 2% HNO₃ and filtered through a 0.45 μm syringe filter. ⁶⁰Ni and ⁶⁶Zn were quantified using a Thermo iCAP Q ICP-MS.

Whole blood (0.5 ml) and organ (∼0.2 g) samples were digested by concentrated nitric acid (1 ml, 67% v/v, VWR Chemicals, PA, USA) in an 85°C hot water bath for 3 h. During acid digestion, hydrogen peroxide (1 ml, Merck, Germany) was carefully added to each sample to facilitate oxidation. After complete digestion, all samples were transferred to a volumetric flask and diluted to a final volume of 20 ml by using double deionized water. Analyses of arsenic (As) and mercury (Hg) were conducted by inductive coupled plasma–mass spectrometry (ICP-MS) (Thermo Scientific iCAP-Q ICP-MS). The quality of data was checked by the analysis of recovery rate using standard reference materials (bovine liver, NIST SRM 1577c, Sigma-Aldrich, MO, USA).

2.8-L shake flasks were cleaned for Ln contamination as described above. Cultures of wild-type M. extorquens AM1 harboring pNG284 were grown with methanol and 20 μM LaCl₃ to OD₆₀₀ ~6. XoxF1 was purified by IMAC and processed, including determination of metal content by ICP-MS and PQQ content by UV-visible spectroscopy, as reported^{5 (link)}. The absorbance spectrum of the enzyme was determined in a 1-cm-path-length cuvette at room temperature using a UV-2600 UV-Vis spectrophotometer (Shimadzu, Columbia, MD). PQQ concentration and content were calculated using the molar absorption coefficient of 9,620 M⁻¹·cm^{−1 32 (link)}. Ln content was determined by ICP-MS at the Michigan State University Laser Ablation ICP-MS Facility using a Thermo Fisher Scientific ICAP Q ICP-MS (Waltham, MA) in collision cell mode.

Concentrations of four elements (Al, Pb, As, and Cd) in the digested sample solutions (n = 10) were measured by ICP-MS (iCAP Q, Thermo Fisher Scientific, Waltham, MA, USA). Samples were analyzed in duplicate washed and unwashed portions. iCAPQ ICP-MS Thermo Scientific operating conditions are as follows: power = 1550 W; cool gas flow = 14.1 L/min; nebulizer gas flow = 0.94 L/min; auxiliary gas flow = 0.79 L/min; dwell time = 0.01 s; peristaltic pump speed = 40 rpm; total time for each sample measurement = 3 min.

The metal contents in the LpNASAT sample were measured using Thermo Scientific iCAP Q ICP-MS. The purified protein and buffer solution were heated at 65 °C in 2 M HNO3 for 20 min, kept at room temperature overnight, and centrifuged at 14,000× g for 20 min. The metal concentrations of the common transition metals (Cu, Ge, Mg, Mn, Ni, Fe, Zn, Co and Ru) in the supernatant were analyzed by ICP-MS. All samples were measured in three replicates.

After being individually ground and sieved, 0.2-g samples of roots, stems, and leaves were digested with concentrated sulfuric acid (H₂SO₄, AR, 98%) and hydrogen peroxide (H₂O₂, GR, ≥30%). From the resulting digestion and after the addition of 100 mL of deionized H₂O, N and P concentrations were obtained with an Auto Analyzer 3 (AA3) continuous flow analyzer (SEAL Analytical, Norderstedt, Germany), while the K concentration was analyzed by a flame photometer (M410; Sherwood Scientific Ltd., Cambridge, United Kingdom). Other 0.1-g samples were digested with nitric acid (HNO₃, AR, 65%) using the microwave reaction system (Multiwave PRO; Anton Paar GmbH, Graz, Austria). Elemental analyses of S, Ca, Mg, Fe, Mn, Cu, Zn, B, Al, Cr, Ni, As, Mo, Pb, and Cd were performed by inductively coupled plasma-atomic emission spectroscopy (iCAP Q ICP-MS; Thermo Fisher Scientific Co., Waltham, MA, United States).

For assessment of uranium concentration in the soluble and particulate fractions within the replicate microcosms, 2 ml of culture at each time point were filtered through 0.025 μm filters (Millipore Corp, Billerica, MA, USA) in an anaerobic chamber (Coy Laboratory Products, Grass Lakes, MI, USA). This soluble fraction (filtrate) was acidified by addition of 0.2 ml nitric acid (70%) and stored in glass vials until analysis. The particulate fraction (filter and the uranium adhering to the walls of the culture vials) were collected by dissolving the filter in 3 ml nitric acid and heating 250°C for 3 hours, or by rinsing the empty culture vials with 3 ml of nitric acid and vortexing for 2 minutes. The uranium concentration was determined by iCAPQ ICP-MS (Thermo Fisher Scientific Inc., Waltham, MA, USA) using Indium as an internal standard. Three iterations per sample were analyzed and RSD percentages averaged 3.6%. A commercial standard (High Purity Standards, Charleston, SC, USA) was used to generate a standard curve for uranium mass based on the instrument signal (counts per second; r² = 0.9955).