Enhancing Electrospray Ionization Mass Spectrometry with Nanobubbles

A study by a team from Western Michigan University investigated the improvements that could be gained in electrospray ionization mass spectrometry (ESI-MS) through the introduction of nanobubbles (NBs). Their findings were published in the journal Analytical Chemistry (1).

Microbubble or nanobubble can assist with localized drug delivery 3d rendering: © Love Employee - stock.adobe.com

Nanobubbles (NBs) are tiny gas-filled cavities in solution that can remain stable for extended periods. Unlike microbubbles, which coalesce and burst quickly, NBs persist because of their high internal pressure and surface electrical charge. This makes them particularly attractive for a wide range of scientific applications, from water treatment (2) to drug delivery (3).

The study utilized three methods to generate NBs: i) Tesla valve flow regime switching; ii) pressure cycling; and iii) sonication. The team explored the effects on ESI-MS, with their experiments demonstrating that the addition of carbon dioxide (CO₂) or nitrogen (N₂) NBs to electrospray solvents leads to substantial improvements in ionization efficiency. By introducing NBs into spray solutions the signal response of various analytes, including caffeine, hydrocortisone, ibuprofen, and proteins such as cytochrome c, was enhanced. This increase was dependent on factors such as analyte identity, solvent composition, NB gas type, and the method of NB generation.

For example, caffeine’s signal nearly doubled in the presence of CO₂ NBs, while hydrocortisone saw a 3.5-fold increase. Ibuprofen, analyzed in negative ion mode, exhibited a 3.7-fold improvement. The signal enhancement was even more pronounced when using ammonium bicarbonate as an additive, with caffeine showing a 9-fold increase when both CO₂ NBs and ammonium bicarbonate were used.

Notably, NBs also contributed to reducing ion suppression effects, a common challenge in ESI-MS. The researchers observed that the presence of NBs helped maintain the signal intensity of Rhodamine 6G despite increasing caffeine concentrations, effectively mitigating ion suppression and improving quantitative accuracy.

In addition to small molecules, the study demonstrated the benefits of NBs in protein analysis. When cytochrome c was analyzed using CO₂ NB-enriched solvents, the signal intensity increased by a factor of 18.7. Moreover, the average charge state of the protein shifted from +8.1 to +9.6, suggesting partial unfolding at the gas-liquid interface created by the NBs. This finding supports the hypothesis that NBs increase the available hydrophobic interface, facilitating protein unfolding and improving ionization efficiency.

Nanobubbles also played a role in accelerating microdroplet reactions, a growing area of interest in mass spectrometry. The reaction between N,N-dibutyl-1,3-propane diamine and CO₂ showed significant conversion rate improvements when NBs were present. With N₂ NBs, the reaction rate doubled compared to standard conditions, and with CO₂ NBs, the conversion rate was further enhanced.

The incorporation of NBs into ESI-MS workflows presents several advantages, namely, enhanced sensitivity, reduced ion suppression, faster reaction kinetics, and more efficient protein ionization. Given these benefits, nanobubbles could become a valuable tool for improving mass spectrometric analyses across various applications, including pharmaceuticals, environmental testing, and biomolecular research.

References

(1) Joseph, G.; Binny, B.; Venter, A. R. Nanobubbles in Electrospray Ionization Mass Spectrometry. Anal. Chem.2025. DOI: 10.1021/acs.analchem.4c06040

(2) Jia, M.; Farid, M. U.; Kharraz, J. A.; et al. 2023, Nanobubbles in Water and Wastewater Treatment Systems: Small Bubbles Making Big Difference. Water Res. 2023, 245, 120613. DOI: 10.1016/j.watres.2023.120613

(3) Jin, J.; Yang, L.; Chen, F.; Gu, N. Drug Delivery System Based on Nanobubbles. Interdiscip. Mater. 2022, 1 (4), 471−494. DOI: 10.1002/idm2.12050