ATR-IR spectroscopy

Infrared spectra were acquired using both laboratory Cary 630 (Fig 1a) and portable 4500 series (Fig 1b) attenuated total reflectance mid-infrared spectrometers (Agilent Technologies, Dansbury, Connecticut, USA). The laboratory Cary 630 spectrometer is interfaced with a customized triple-reflection diamond ATR sampling accessory with a 1-mm diameter sampling surface, and a 200 μm active area providing a 2 μm depth of penetration for IR energy at 1700 cm^-1. However, the portable 4500 series spectrometer is interfaced with a triple-reflection diamond crystal ATR accessory with a 2-mm diameter sampling surface and a 200 μm active area providing a 6 μm effective penetration depth for IR energy at 1700 cm^-1.

(a) laboratory Cary 630 and (b) portable 4500 series (Agilent Technologies, Dansbury, Connecticut, USA).

Thawed serum samples were diluted (1:1) with deionized sterile water and vortexed at a maximum of 2700 rpm for 10 s. From each diluted sample a 5 μL aliquot was placed onto the diamond crystal of the spectrometer and dried by a stream of air from a domestic hair dryer. Samples were fully dried within 3–5 min, forming a thin film on the crystal. The IR spectra were then collected using Agilent MicroLab PC software (Agilent Technologies, Dansbury, Connecticut, USA). At a spectral resolution of 8 cm^-1, 512 scans were co-added to optimize the signal to noise ratio over the wavenumber range of 4000–650 cm^-1. Two independent spectra were collected from each serum sample with each spectrometer. The spectrometer crystal was cleaned with 100% ethanol and a laboratory wipe between each sample application and a new background spectrum (I₀) was performed before applying the next serum sample. A total of 350 (175 x 2) ATR-IR spectra were collected from each spectrometer, saved in spectrum (SPC) format, and then converted into printable (PRN) formatted data using GRAMS software (GRAMS/AI version 7.02, Thermo Fisher Scientific). The PRN format spectral data were imported into MATLAB^® (version R2016a, MathWorks, Natick, MA) for data analyses.