Rapid Measurement of Lactate in the Exhaled Breath Condensate: Biosensor Optimization and In-Human Proof of Concept

Abstract

Lactate concentration is of increasing interest as a diagnostic for sepsis, septic shock, and trauma. Compared with the traditional blood sample media, the exhaled breath condensate (EBC) has the advantages of non-invasiveness and higher user acceptance. An amperometric biosensor was developed and its application in EBC lactate detection was investigated in this paper. The sensor was modified with PEDOT:PSS-PB, and two different lactate oxidases (LODs). A rotating disk electrode and Koutecky-Levich analysis were applied for the kinetics analysis and gel optimization. The optimized gel formulation was then tested on disposable screen-printed sensors. The disposable sensors exhibited good performance and presented a high stability for both LOD modifications. Finally, human EBC analysis was conducted from a healthy subject at rest and after 30 min of intense aerobic cycling exercise. The sensor coulometric measurements showed good agreement with fluorometric and triple quadrupole liquid chromatography mass spectrometry reference methods. The EBC lactate concentration increased from 22.5 μM (at rest) to 28.0 μM (after 30 min of cycling) and dropped back to 5.3 μM after 60 min of rest.

Keywords: Prussian blue; clinical diagnostics; exhaled breath condensate; lactate biosensor; lactate oxidase; non-invasive detection.

Figure 1

(A) Reaction scheme of the lactate sensor. (B) Blank disposable sensor (L), with PEODT:PSS-PB and LOD modifications (R). (C) EBC collection device (from Respire Diagnostics).^,

Figure 2

(A) Koutecky–Levich plot for the RDE modified with PEDOT:PSS-PB and Sekisui LOD for lactate concentrations between 50 and 250 μM. (B) Slope of the K–L intercept fit against LOD concentration for lactate measurements and (C) slope of the K–L intercept fit against LOD concentration for H₂O₂ measurements.

Figure 3

(A) Coulometry calibration measured using the electrochemical sensor with PEDOT:PSS-PB-LOD modification, against the Lineweaver–Burk plot for fluorimetry calibration using the Amplex red and HRP assays. (B) 2D Bland Altman analysis comparing electrochemical sensor results and the Amplex Red assay results (n = 5). (C) Comparison of the sensor average sensitivity (± sd, n = 3) with Sekisui LOD and LOD-N118 gel modifications at day 0, 7, and 60. (D) Sensor and fluorometric data for EBC lactate concentrations measured from a healthy subject at rest, directly after exercise, and after 45 min of rest. (E) 3D Bland–Altman analysis comparing sensor and fluorometric results. (F) EBC lactate measurements from a healthy volunteer at rest using a sensor, TQ LC/MS, and fluorometry.