Non-Invasive Sensor for Quantitative Determination of Lactate in Sweat

Lactic acid is a key biomarker of anaerobic respiration and descriptor of an individual’s health state.  Currently, lactate monitoring is performed though continuous invasive blood sampling and sample processing which is inconvenient and unpractical when trying to obtain real time measurements of an active individual.  Non-invasive sensing methods are needed for monitoring human performance in sports, military and health care fields.

Lactate is found in various bodily fluids including sweat and a correlation between blood and sweat lactate concentration has been determined (1). Hence, monitoring sweat lactate levels is a good noninvasive alternative to blood sampling methods.

Here we report the development, characterization and optimization of an amperometric lactate sensor based on a lactate dehydrogenase and carbon nanotube chitosan electrode coupling system previously developed by our group (3).  Due to the high overpotential of NADH oxidation, the electrocatalyst, polymethylene green (PMG) was electrochemically deposited onto the surface of multi-walled carbon nanotube (CNT) material called Bucky paper for NAD⁺ regeneration.  Lactate dehydrogenase was immobilized onto the Bucky paper-PMG electrode with a chitosan-based carbon nanotubes (CNT) mixture. The bioelectrodes were tested in a standard three-electrode polycarbonate cell hardware consisting of enzymatic working electrode, Ag/AgCl reference and platinum wire counter electrode and operated in a chronoamperometric regime.

Sweat composition varies between individuals and different parts of the human body resulting in variations in pH, salt concentration (related to conductivity,) and lactate levels before and after exercise  with rates of release decreasing over time. In order to address some of these issues, calibration curves at various buffer pHs and buffer concentrations were created (Fig 1).  The variation of pH of solutions from 5 to 7 showed an increase in bioelectrode response. The slope of the current/lactate concentration linear dependence was strongly influenced by the solution pH with low pH leading to decreased sensitivity most likely as a result of influenced enzyme activity.  A similar trend was observed when the concentration of the electrolyte was varied from 0.01 to 0.245 M. The slope of the calibration curve was strongly influenced by the solution conductivity with low values leading to decreased reproducibility and sensitivity. In all cases the electrode response was linear with lactate concentration allowing one to tailor the system based on individual needs.

An artificial sweat was prepared, composed of NaCl, urea, pH 6.5, glucose, NAD⁺, and having a conductivity of 16.6 mS/cm. As it was expected the initial tests of the LDH-electrode with the artificial sweat showed lower sensitivity most likely due to low conductivity of the solution, the generated current form the lactate oxidation followed linear dependence from lactate concentration (data not shown). The strong dependence of the slope of the sensor calibration curve from the electrolyte conductivity and pH can be minimized by buffering the sweat, which in real conditions is performed by impregnation of the sensor sweat collector with a buffer salts.

A prototype of a patch lactate sensor was developed composed of a LDH-working electrode, a Ag/AgCl reference electrode and carbon yarn counter electrode. The three electrodes were placed on a medical adhesive and covered with a bandage, preventing a contact between electrodes and the skin and at the same time adsorbing and collecting the sweat. The designed patch sensor displayed an increase in current density with increasing lactate concentration with high sensitivity.

To avoid artificial introduction of NAD⁺ in the sweat, a method for NAD⁺immobilization of the electrode surface will be implemented along with encapsulation of the enzyme into silica gel matrix, which improve the linearity of the response and prolong the life -time of the sensor.

The proposed amperometric enzyme electrode coupling approach along with optimization experiments provides the opportunity to monitor noninvasively and in real-time sweat lactate concentrations.

(1) Sakharov, D.A. et al., 2010. Relationship between lactate concentrations in active muscle sweat and whole blood. Bull. Exp. Biol. Med.. 150 (1) 83-5.

(2)   Nikolaus, N., and Strehlitz B., 2008. Review. Amperometric lactate biosensors and their application in (sports) medicine, for life quality and wellbeing. Microchim Acta. 160, 15-55.

(3). Narvaez Villarrubia C.W. et al., 2011. Biofuel cell anodes integrating NAD⁺-dependent enzymes and multiwalled carbón nanotube papers. ACS Appl. Mater Interfaces. 2011, 3(7), p. 2402-9 (7)