Electromagnetic Piezoelectric Acoustic Sensor Detection of Extracellular Vesicles through Interaction with Detached Vesicle Proteins

Abstract

An electromagnetic piezoelectric acoustic sensor (EMPAS) was used to study the non-specific adsorption of human red blood cell-derived extracellular vesicle preparations. Vesicle storage history (temperature and duration) highly affected the obtained results: The signal change, namely the frequency decrease of the crystal measured at 20 °C, was negligibly small (<1 s−2) when the vesicle solutions had previously been stored at 4 °C, and was in the order of 10 s−2 when the vesicle solutions had been stored at −30 °C. Moreover, the rate of frequency decrease increased exponentially with the storage time at −30 °C. Upon a 4 °C storage period following the −30 °C storage period of the same sample, the measured frequency decrease dropped, suggesting a partial relaxation of the system. The results are explained by the disintegration of the vesicles triggered by the freeze–thaw cycle, likely due to the detachment of proteins from the vesicle surface as was proved by size-exclusion chromatography. Surface modification of the sensor crystal provided the possibility of signal enhancement, as the maximum rate of the frequency change for the same vesicle concentrations was higher on hydrophobic, octadecyl trichlorosilane–modified quartz than on hydrophilic, bare quartz. The EMPAS signal has been associated with the amount of detached proteins, which in turn is proportional to the originating vesicle concentration.

Storage conditions determine the characteristics of red blood cell derived extracellular vesicles

“…4 ). Bands corresponding to amide A, amide I and amide II vibrations (at 3288, 1654 and 1542 cm −1 , respectively) of the protein backbone usually dominate the spectra 35 , 45 . In addition to the amide bands of protein components, characteristic bands of phospholipids are also present (Table 1 ).…”

“…A spectroscopic protein-to-lipid ratio was calculated for all REV samples based on the integrated area of the amide I band (characteristic for protein content) and that of the C-H stretching region (from 3020 cm −1 to 2800 cm −1 , typical mainly for phospholipid content). The provided spectroscopic P/L proved to be discriminant for different subtypes of EVs (small vs. medium or large EVs) 35 and can also be used for assessment of REV sample purity 34 , 45 , 62 . As the amide I area is proportional to the number of peptide bonds in the proteins, the IR spectrum can be used to assess the total protein content of the REV samples, too 46 .…”

Isolation and characterization of extracellular vesicles and future directions in diagnosis and therapy

“…Similar estimates have been given by others (M. Bebelman et al, 2020 ; Verweij et al, 2018 ). The QCM has also been used to monitor EV release (from PC12 and NG108‐15 cells) after stimulation with increased potassium and by spontaneous endocytosis (Cans et al, 2001 ) or to study the absorption properties of red cell EVs (Románszki et al, 2020 ).…”

Purification of extracellular vesicles by size-exclusion chromatography using cross-linked agarose gels: the role of pore size in lipoprotein contamination

“…A spectroscopic protein-to-lipid ratio was calculated for all EV samples based on the integrated area of the amide I band (characteristic for protein content) and that of the C-H stretching region (from 3020 cm -1 to 2800 cm -1 , typical mainly for phospholipid content). The provided spectroscopic P/L proved to be discriminant for different subtypes of EVs (small vs. medium or large EVs) [19] and can also be used for the assessment of EV sample purity [20][21][22]. As the amide I area is proportional to the number of peptide bonds in the proteins, the IR spectrum can be used to assess the total protein content of the REV samples, too [23].…”