Emx cw epr spectrometer

EPR was employed to measure reactive oxygen species released from the nanoparticle surface, adapting a previously published method.^{9 (link)} Samples were prepared at 5% weight dispersion in 500 μL final volumes. A solution containing hydrogen peroxide at 200 mM final concentration and the spin-trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) at a final concentration of 25 mM was added to the powdered materials, mixed, and then equilibrated at room temperature for 15 min prior to measurement on a Bruker EMX CW EPR spectrometer operating at a Larmor frequency of 9.76 GHz (X-band). A flat TM₁₀₀ cell was used to hold the samples and four scans were co-added to give the final spectra. Quantification was facilitated by using 3-carboxy-PROXYL as an external standard. Double integration of the standard and sample spectra gave areas that were used to calculate the concentration of each radical species.

EPR experiments were conducted at the Ohio Advanced EPR Laboratory at Miami University. CW-EPR spectra were collected at X-band on a Bruker EMX CW-EPR spectrometer using an ER041xG microwave bridge and ER4119-HS cavity coupled with a BVT 3000 nitrogen gas temperature controller. The spin concentration for KCNE1 samples was ~80–100 μM. Each spin-labeled CW-EPR spectrum was acquired by signal averaging 10 42-s field scans with a central field of 3315 G and sweep width of 100 G, modulation frequency of 100 kHz, modulation amplitude of 1 G, and microwave power of 10 mW at 295 K. The side-chain mobility was determined by calculating the inverse central line width from each CW-EPR spectrum.
An empirical motional parameter (τ₀) was determined from the CW-EPR spectra using eq 1.^{17 (link)–20 (link)} $${\tau }_0=K\times \mathrm{\Delta }H\left[{\left(\frac{h_0}{h_{(-1)}}\right)}^{1/2}-1\right]$$ where K = 6.5 × 10⁻¹⁰ s, ΔH is the width of the center-line, and h₀ and h₋₁ are the heights of the center and high field lines, respectively.

Samples of approximately 3.5 µL volume were placed in a 0.6 mm i.d. and 0.84 o.d. quartz capillary and analyzed by ODNP, as described previously^{21 (link)}. A “pass through” NMR probe design built to fit inside a 3mm i.d. 6 mm o.d. quartz tube was used. The quartz tube was inserted into a high sensitivity microwave cavity (ER 4119HS-LC, Bruker Biospin) along with the NMR probe and sample. ODNP experiments were performed using a Bruker EMX CW EPR spectrometer and a Bruker Avance III NMR console. The samples were sealed in a capillary with a protective layer of Critoseal on the top and hot beeswax on the bottom. All ODNP measurements were performed at room temperature. The sample was irradiated with up to 6 W of microwaves at the EPR resonant frequency of the spin labels at ~10 GHz using a home-built microwave amplifier^{22 (link)}. The magnetic field was set on resonance at the central electron hyperfine transition, here at 9.8 GHz. The spin label concentration of each sample was determined from the double integral of its cw EPR spectrum measured at 1 mW irradiation power, 0.4 G modulation width. The concentration-dependent ODNP relaxation rates, k_σ, k_ρ, and k_Low, are normalized to the sample concentration derived from spin counting per integration of the cw EPR spectrum.