Measurement of Human and Bovine Exhaled Breath Condensate pH Using Polyaniline-Modified Flexible Inkjet-Printed Nanocarbon Electrodes

Abstract

The collection, processing, and electrochemical analysis of exhaled breath condensate (EBC) from healthy human and animal subjects is reported on. EBC is a biospecimen potentially rich in biomarkers of respiratory disease. The EBC pH was analyzed potentiometrically using a disposable polyaniline (PANI)-modified inkjet-printed (IJP) carbon electrode. Comparison measurements were performed using a commercial screen-printed carbon (SPC) electrode. The PANI-modified electrodes exhibited reproducible and near-Nernstian responses for pH values between 2 and 9 with slopes from -50 to -60 mV/dec. The PANI-modified IJP carbon electrode exhibited a faster response time and superior reproducibility to the modified SPC electrode. In proof-of-concept studies, the healthy human EBC pH was found to be 6.57 ± 0.09 and the healthy bovine EBC pH was 5.9 ± 0.2. All pH determined using the PANI-modified electrodes were in good agreement with the pH determined using a micro glass pH electrode. An RTube device was used to collect EBC from humans while a modified device was used to collect EBC from calves in the field. EBC volumes of 0.5-2 mL for 5-6 min of tidal breathing were collected from healthy animals. The pH of EBC from healthy calves (17 animals) depends on their age from 1 to 9 weeks with values ranging from 5.3 to 7.2. A distinct alkaline shift was observed for many animals around 20 days of age. The bovine EBC pH also depends on the ambient temperature and humidity at the time of collection. The results indicate that the PANI-modified IJP carbon electrodes outperform commercial SPC and provide reproducible and accurate measurement of pH across various biospecimen types.

Figure 1

A calf, with excessive nasal discharge, is suspected of being ill with BRD.

Figure 2

Processing steps for PANI electrodeposition on (a) screen-printed (SPC) and (b) IJP electrodes from a solution of 0.1 M aniline in 0.1 M H₂SO₄.

Figure 3

Apparatus used to collect the EBC biospecimens from calves in the field.

Figure 4

Representative (a) chronoamperometric i–t curves for the electrodeposition of PANI by constant potential electrolysis in 0.1 M aniline dissolved in 0.1 M H₂SO₄ and (b) cyclic voltammetric i–E curves for PANI-modified PA/CNT-IJP (black) and PA/Gr-IJP (red) electrodes in 0.1 M H₂SO₄. Comparison measurements for the SPC electrodes are also presented (blue). Reference = Ag/AgCl (3 M KCl) for the IJP electrodes and a quasi-Ag/AgCl for the SPC electrode. Application of +1.0 V for 90 s. Scan rate = 50 mV s^–1.

Figure 5

Scanning electron micrographs of a representative PA/CNT-IJP electrode (a) before and (b) after modification with PANI, and a representative PA/Gr-IJP electrode (c) before and (d) after modification with PANI. For comparison, scanning electron micrographs are presented for a representative SPC electrode (e) before and (f) after modification with PANI. All micrographs were collected using secondary electron mode and are presented at 10,000× magnification.

Figure 6

Potential vs time measurements in standard 0.01 M BR buffer solutions ranging between pH 2.3–9.0 using multiple (a) PANI/PA/CNT-IJP and (b) PANI/PA/Gr-IJP electrodes. Comparison measurements with the PANI/SPC electrodes are also presented (c). Corresponding potential vs pH response curves are displayed on the right. Data are presented in the response curves as mean ± std. dev. values for N = 3 sensors of each type in all four buffer solutions.

Figure 7

Repeatability tests in buffer solutions of different pH without any cleaning step between each measurement for (a) PANI/PA/CNT-IJP, (b) PANI/PA/Gr-IJP and (c) PANI/SPC electrodes. Plots of the equilibrium potential vs time during contact with the different buffer solutions are presented.

Figure 8

Influence of 38 μM of Na⁺, 9 μM of Ca²⁺ and 11 μM of K⁺ on the pH determination of a 0.01 M BR buffer at pH 6.40 using the PANI/SPC and PANI/PA/Gr-IJP electrodes. Data are presented as mean ± std. dev. for N = 3 electrodes of each type. The PANI/PA/Gr-IJP electrode pH results in the presence of cations were not statistically different to the control as assessed a two-tailed, paired student’s t-test with a difference assessed at p-value ≤0.05.

Figure 9

(a) EBC pH data for three healthy female calves as a function of age obtained using PANI/PA/Gr-IJP electrodes. pH data are also presented for the same biospecimens obtained using a standard micro glass pH electrode. Data are plotted as the average ± std. dev. for the three calves examined. (b) Plot showing the aggregate pH data for multiple healthy (6 animals, 27 measurements) and ill (14 animals) female calves obtained with the PANI/PA/Gr-IJP electrodes.

Figure 10

EBC pH measurement data for multiple healthy female calves (17 animals, 79 measurements) at different time points aggregated as a function of the outside (a) ambient temperature (AT) and (b) absolute humidity (AH) collected from each animal. The pH values were determined using the micro glass pH electrode. The Spearman coefficients (r_s) for the pH vs AT and AH correlations were significant at p < 1 × 10^–5 and p < 4 × 10^–4, respectively.

Figure 11

(a) pH measurement data for EBC biospecimens from four healthy calves before and after multiple freeze–thaw cycles over a 7 day period and (b) the time dependence of human EBC pH change during exposure to the ambient atmosphere. The inset shows the human EBC pH values between 2 and 30 min. The pH values were determined using the micro glass pH electrode. Data are presented as mean ± std. dev. for N = 4 EBC biospecimens from healthy animals.