An NAD+-dependent enzymatic sensor with biofuel cell power source system for non-invasive monitoring of lactate in sweat was designed, developed, and tested. The sensor component, based on lactate dehydrogenase, showed linear current response with increasing lactate concentrations with limits of detection from 5 to 100 mM lactate and sensitivity of 0.2 µA.mM−1 in the presence of target analyte. In addition to the sensor patch a power source was also designed, developed and tested. The power source was a biofuel cell designed to oxidize glucose via glucose oxidase. The biofuel cell showed excellent performance, achieving over 80 mA at 0.4 V (16 mW) in a footprint of 3.5 × 3.5 × 0.7 cm. Furthermore, in order to couple the sensor to the power source, system electronic components were designed and fabricated. These consisted of an energy harvester (EH) and a micropotentiostat (MP). The EH was employed for harvesting power provided by the biofuel cell as well as up-converting the voltage to 3.0 V needed for the operation of the MP. The sensor was attached to MP for chronoamperometric detection of lactate. The Sensor Patch System was demonstrated under laboratory conditions.

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) Figure 1

Non-invasive monitoring of biomarkers in biological fluids through the use of wearable sensors gained significant interest in the past several decades [1].  Research efforts have been focused on the development of chemical and biochemical sensors for a range of metabolites, including glucose, lactate, ethanol, trace metals and various ions, targeting several bodily fluids such as tears, saliva, and sweat [2].  Work presented here will discuss the development of a sensor system, capable of real-time monitoring of lactate levels in sweat.  Such a device is desired in order to correlate increasing levels of lactate to fatigue levels of a person performing physically demanding activities [3].  Most existing systems employ traditional batteries as power sources for sensors.  The system described here utilizes a light weight biofuel cell as the power source giving a complete bio-friendly sensor system. The system is comprised of three main elements: a biosensor, a biofuel cell, and electronics interface.  The biosensor detects lactate levels in sweat via enzymatic reaction of Lactate Dehydrogenase (LDH).  Sensor patch features a three electrode design assembled onto generic athletic tape and covered with medical gauze, providing both flexibility and sweat collection capability.  LDH is immobilized at the working electrode via conductive carbon-based ink.  The electrode surface is then further modified by vapor-deposited tetramethyl orthosilicate (TMOS) coating in order to provide greater stability to the sensor against temperature and pH fluctuations.  Sensor calibration time period was relatively quick (5 minutes) and response was immediate with addition of lactate.  Test results for the sensor patch showed an open circuit potential of ~0.06V vs. Ag/AgCl and an equilibrated current of approximately 30µA when held at 0.3V vs. Ag/AgCl.  The patch also demonstrated a sensitivity of 0.2µA/mM lactate when tested in a range of 5-100mM lactate. A glucose-based biofuel cell provides the power to operate the biosensor.  The anode, driving glucose oxidation, is comprised of immobilized Glucose Dehydrogenase (GDH) on a high surface area carbon felt (CF) electrode.  Prior to enzyme immobilization, CF electrode surface was modified with electrochemically deposited polymethylene green (PMG) as well as chemically tethered Nicotinamide Adenine Dinucleotide (NAD).  An oxygen-reducing cathode was employed as counter electrode.  The biofuel cell generated an open circuit potential of 0.7V vs. Ag/AgCl, a total current of 81.0mA, and total power of 16.7mW. The patch sensor is coupled to the biofuel cell via external electrical components: an energy harvester and a micropotentiostat.  The energy harvester component is connected to the biofuel cell. Its primary function is to continuously provide stable output voltage to the micropotentiostat.  Thus it is constantly drawing power from the biofuel cell and up-converting the voltage from 0.7 to 3V.  The patch sensor is connected to the micropotentiostat.  A constant potential of 0.3V vs. Ag/AgCl is needed to operate the sensor, which is supplied by the micropotentiostat.  Additionally, it is used to provide a read-out of an electrical signal, i.e. current, generated during sensor operation.  System performance was demonstrated, employing artificial sweat.  Results showed linear sensor response with increasing lactate content of the artificial sweat solution.  [1] A. J. Bandodkar and J. Wang, "Non-Invasive Wearable Electrochemical sensors: A Review," Trends in Biotechnology, vol. 32, no. 7, pp. 363-371, 2014. [2] J. Kim, W. R. de Araujo, I. A. Samek, A. J. Bandodkar, W. Jia, B. Brunetti, T. R. Paixao and J. Wang, "Wearable temporary tattoo sensor for real-time trace metal monitoring in human sweat," Electrochemistry Communications, vol. 51, pp. 41-45, 2015. [3] S. Jadoon, S. Karim, M. R. Akram, A. K. Khan, M. A. Zia, A. R. Siddqi and G. Murtaza, "recent Developments in Sweat Analysis and Its Applications," International Journal of Analytical Chemistry, pp. 1-7, 2015. Figure 1