(Invited) Development of the Hydrogel-Based Biosensors for Non-Invasive Perspiration Analysis
Authors: Nagamine Kuniaki , Matsui Hiroyuki , Tokito Shizuo 
Published: December 2020

Non-invasive diagnostic sensors for biomarkers are generally required as an alternative to blood-based diagnoses for the next generation of advanced healthcare. Sweat is one of the promising bodily fluids that are non-invasively accessible source of biomarkers. However, it is difficult to collect small amount of sweat from human skin surface in a periodic or continuous manner. Whereas wearable-type of tattoo- and microfluidics-based biosensors have enabled in-situ and real-time sampling and monitoring of sweat biomarkers, it has been necessary to induce continuous perspiration under special conditions such as with exercise, in a high-temperature environment, or with the use of cholinergic agonist-assisted perspiration. These circumstances are not practical in the application of sweat sensors for daily health diagnostics.
In this presentation, we show our new perspiration sensor, a hydrogel-based touch sensor pad for the non-invasive extraction and detection of sweat components. The sensor was composed of the printed electrochemical biosensing working and planar liquid-junction Ag/AgCl reference electrodes fully covered with an agarose hydrogel including a sweat-extraction solution, Phosphate Buffer Saline (PBS). We found that biocompatible PBS with physiological osmotic pressure was suitable to continuous extraction of sweat. In this study, an enzyme-based L-lactate sensor was fabricated as a model because L-lactate is included in sweat in relatively high concentrations. L-lactate was oxidized by Lactate oxidase (LOx) to generate hydrogen peroxide, which oxidize reduced form of prussian blue (PB) immobilized on the working electrode surface. The LOx / PB-modified electrode was fabricated by screen-printing of a carbon-graphite ink including PB on an Au electrode, followed by drop-casting of a mixture of LOx with chitosan as a matrix to immobilize LOx on the electrode. The structure of the screen-printed planar liquid-junction Ag/AgCl reference electrode was composed of a silver-lead pattern, Ag/AgCl active layer, internal electrolyte layer, polyurethane-based liquid junction, and insulation layer on a substrate. Then, a silicone rubber sheet with a 6 mm diameter through-hole was attached to the electrode film as to expose the active area of working and reference electrodes to the through-hole. A low melting point agarose prepolymer solution in PBS was drop-deposited onto the through-hole in the silicone sheet, and incubated at room temperature to induce gelation.
We measured the potentiometric response of the sensor electrodes when the ball of the subject’s forefinger contacted the agarose gel surface. After 20 s of time lag for diffusion of extracted L-lactate in the gel, the potential difference started to increase, followed by approaching steady-state. This result suggested successful extraction and detection of sweat L-lactate using this device. Without LOx, no potentiometric response was observed upon touching the gel with the subject’s forefinger, suggesting that the extracted sweat compounds except for L-lactate did not affect the sensor response. This new type of touch sensor pad integrated with biosensors will enable non-invasive and daily periodic monitoring of sweat biomarkers without harsh exercise-, environmental temperature control, and cholinergic agonist-assisted perspiration. In a future study, we will apply the present sensor to the other sweat analytes such as D-glucose (diabetes biomarker), electrolytes (heat stroke biomarker), ammonium ion (anaerobic exercise biomarker), and some cytokines (immune response biomarker) to evaluate the sensor characteristics in more detail.