Invenio spectrometer

The FTIR spectra of the samples were measured using the Bruker INVENIO^® Spectrometer (Bruker Optics, Leipzig, Germany), which was equipped with a high-throughput extension HTS-XT for precise analysis. The OPUS v8.5 software was employed to control the data acquisition process. Spectra were acquired in transmission mode, covering a range of 4000–600 cm⁻¹ (4 cm⁻¹ resolution). To establish a baseline, a background spectrum of the empty 96-well Silicon plate was collected before each sample measurement. The background scans were repeated 40 times. Subsequently, the 5 replicate spectra of samples were averaged and baseline corrected using the Unscrambler^TM v11 software from Camo Analytics AS (Oslo, Norway).

FTIR measurements were carried out on a Bruker INVENIO^® Spectrometer (Bruker Optics, Germany) equipped with a high-throughput extension HTS-XT. Samples were measured as thin films on 96-well Si plates with 5 replicate measurements for each sample. Data acquisition was controlled by OPUS v8.5 software. Spectra were collected in transmission mode in the range 4000–600 cm⁻¹ with a resolution of 4 cm⁻¹. Before each sample measurement, a background spectrum of the empty Si plate was collected; the number of scans for both background and sample was 40. In order to achieve optimal signal response, a sample volume of 6 μL per well for CH-2% AA and 2 μL for CH-1%AA-1%CA was used. For CH-2%CA, CH-4%CA, and CH-6%CA the volume was 2 μL after diluting with H₂O 1:1 to avoid detector saturation. Baseline correction and averaging of the five replicate spectra were performed with UnscramblerTM v11 (Camo Analytics AS, Oslo, Norway) software.

In order to reveal possible changes in the HRP secondary structure after the incubation of its solution in the center of the half-sphere, the attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) method was employed. HRP solutions, incubated either in the center of the half-sphere or at a 2 m distance from the half-sphere (control solution), were analyzed with an INVENIO spectrometer (Bruker Scientific LLC, Billerica, MA, USA). The ATR-FTIR measurements were performed in the following way: 12 µL of the analyzed 10⁻⁴ M HRP solution in 2 mM PBSD buffer were placed into the measuring cell of the spectrometer. Therefore, a high (10⁻⁴ M) protein concentration was used due to the sensitivity limitations of the spectrometer employed. Each experiment was carried out with three samples, and for each sample, the measurements were performed twice. The number of technical replicate scans in each experiment was 120. Data, obtained in the ATR-FTIR measurements, were presented in standard form provided by the spectrometer operation software. In order to account for the contribution of the PBSD buffer to the resulting spectra, blank measurements with pure protein-free buffer were performed prior to the experiments with the HRP solutions within the same spectral range. The so-obtained ATR-FTIR spectrum of the buffer was subtracted from that of the protein solutions.