Trace Analysis of Biological Thiols in Exhaled Breath Using Gold Nanoparticle Microcolumns

Researchers at the Institute of Analytical Chemistry of the Czech Academy of Sciences and Masaryk University in Brno have developed a method to concentrate biological thiols from exhaled breath condensate (EBC) without centrifugation. 

The study, by Jiří Volánek, Dana Strouhalová and Petr Kubáň, is published in Analytica Chimica Acta. 

The team fixed gold nanoparticles onto a strong anion exchange sorbent, packed it into disposable microcolumns and measured the extracted homocysteine, cysteine and glutathione by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Detection limits reached 0.04 nM for cysteine and 0.05 nM for homocysteine.

Why thiols in breath are hard to measure

Homocysteine, cysteine and glutathione take part in redox regulation, enzyme activity and antioxidant defence. The paper cites links between thiols and Parkinson's disease, Alzheimer's disease and cystic fibrosis, among others. This study develops an analytical method, though, not a diagnostic test.

EBC can be collected non-invasively. In this study, the researchers used a custom-built, cooled three-tube sampler. The resulting samples are highly dilute, with thiols present at low nanomolar concentrations or below. 

Gold binds thiols strongly, so colloidal gold nanoparticles are an established way to pull them out. But the usual protocol needs two rounds of centrifugation, with manual handling at each step, and the centrifuge limits how much sample can be processed.

Gold nanoparticles in a column

Citrate-stabilised gold nanoparticles carry a negative surface charge, so they hold onto a strong anion exchange (SAX) material by electrostatic attraction. The coated sorbent goes into microcolumns with a void volume of about 10 µL, and between 1 and 10 mL of EBC is drawn through by peristaltic pump. Thiols bind to the gold as the sample passes. Dithiothreitol (DTT) then releases them into 15 µL, and they are derivatised with naphthalene-2,3-dicarboxaldehyde and cyanide so that they fluoresce.

Of six commercial solid-phase extraction materials tested, weak anion exchange and aminopropyl phases took up slightly more gold but released impurities that disturbed the electropherograms. SAX gave clean ones.

Unexpectedly, columns only partly coated with gold gave markedly higher thiol recoveries than fully saturated ones. The authors say the mechanism is not yet fully understood. They suggest that the greater gold loading in saturated columns may consume more of the DTT, leaving less available to release the captured thiols. 

Raising the DTT concentration improved recoveries from the partly coated columns, which they describe as indirect support for that idea.

What the method achieved

Preconcentration factors were 57.3 for homocysteine and 44.0 for cysteine, but only 8.8 for glutathione. Detection limits were 0.05 nM, 0.04 nM and 0.58 nM respectively. 

Across five separately prepared columns, migration-time RSDs stayed below 1.8% and peak-area RSDs below 18%, indicating good repeatability across the preparation and analysis procedure.

The detection limits are not the amounts found in breath. Applied to native EBC, the method measured all three thiols in three samples from one person, collected weeks to months apart. Values ranged from 0.27 nM (homocysteine and cysteine, first sample) to 7.42 nM (glutathione, third sample). 

Absolute concentrations varied between collections, although the relative proportions of the three thiols were broadly similar. The authors call this a proof of concept needing a larger cohort to validate it.

The separation stage and the platinum electrodes

The microcolumn does the extraction and preconcentration; a CE-LIF instrument built in-house does the separation and detection. 

The platinum wire, supplied by Advent Research Materials, belongs to the second stage.

The instrument applied 18 kV across a 50 cm fused silica capillary through two platinum wire electrodes, each 0.5 mm in diameter and 3 cm long, placed in plastic vials of 10 mM borate background electrolyte at pH 10. 

The reported detection limits reflect the combined analytical method, including preconcentration, derivatisation and CE-LIF analysis. The study does not assess the contribution of the electrode material separately.

What follows is general background from other studies; this paper did not examine its electrodes. In CE, ions carry the current through the electrolyte, while at each electrode surface charge passes by electron-transfer reactions. 

In aqueous systems, these reactions can include water oxidation at the anode and water reduction at the cathode, producing local changes in electrolyte pH. Macka, Andersson and Haddad measured this in 1998 and found that the pH shift reaching the capillary depended on how near the capillary end sat to the electrode and on how well the electrolyte was buffered [1]. 

Platinum has good chemical stability and is widely used in electrochemistry, but it is not immune to dissolution. Studies in acidic electrolytes show that its dissolution is potential-dependent and associated with the formation and subsequent reduction of surface oxide [2]. 

Those conditions differ from the alkaline borate electrolyte used in this study, so the findings provide general background rather than evidence of how the CE electrodes performed.

Where this leaves the method

The microcolumn replaces two centrifugation steps with a single flow-through step and allows EBC sample volumes of 1 to 10 mL to be processed. 

The authors see scope for automating it and for applying the same column to other small thiol-containing analytes. The much lower preconcentration factor for glutathione and the single-person sample set show the validation work still ahead.

Title: Preconcentration of biological thiols from large volume of exhaled breath condensate using microcolumns with immobilized gold nanoparticles
Journal: Analytica Chimica Acta, Volume 1421 (2026), article 346030
Authors: Jiří Volánek, Dana Strouhalová, Petr Kubáň
Published online: 1 August 2026 (open access, CC BY 4.0)
DOI: 10.1016/j.aca.2026.346030

Abstract: A novel microcolumn-based preconcentration strategy for the selective extraction of biological thiols from exhaled breath condensate (EBC) was developed using gold nanoparticles (AuNPs) immobilized on strong anion exchange (SAX) solid-phase extraction material. The approach combines…

Background sources for the platinum section
[1] M. Macka, P. Andersson and P. R. Haddad, "Changes in electrolyte pH due to electrolysis during capillary zone electrophoresis", Analytical Chemistry 70 (1998) 743–749. DOI: 10.1021/ac970428p
[2] A. A. Topalov, S. Cherevko, A. R. Zeradjanin, J. C. Meier, I. Katsounaros and K. J. J. Mayrhofer, "Towards a comprehensive understanding of platinum dissolution in acidic media", Chemical Science 5 (2014) 631–638. DOI: 10.1039/C3SC52411F