Department of Physics 
CHALMERS UNIVERSITY OF TECHNOLOGY 
Gothenburg, Sweden 2018 

 

 

 

 

Development of a Non-enzymatic,  

Non-invasive Sensor for Detection of 

Lactate in Sweat.  
Master’s thesis in Biotechnology 

 

ANNIE LJUNGH



  



 

 

 

 

ii 

 

Master’s thesis 2018 

 

 

 

 

 

DEVELOPMENT OF A NON-ENZYMATIC,  

NON-INVASIVE SENSOR FOR DETECTION OF 

LACTATE IN SWEAT. 
 

 

ANNIE LJUNGH 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

Department of Physics 

Division of Condensed Matter Physics 

CHALMERS UNIVERSITY OF TECHNOLOGY 

Gothenburg, Sweden 2018   



 

 

 

 

iii 

 

Development of a Non-enzymatic, Non-invasive Sensor for Detection of Lactate in Sweat. 

ANNIE LJUNGH 

 

© ANNIE LJUNGH, 2018.  

 

Supervisor: Gunnar Westman, Department of Chemistry and Chemical Engineering 

Examiner: Magnus Karlsteen, Department of Physics 

 

Master’s Thesis 2018  

Department of Physics  

Chalmers University of Technology  

SE-412 96 Gothenburg 

Telephone + 46 (0)31-772 1000  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cover: Sweet drops assembled on part of the back after training. 

 

Printed by Chalmers Printing Service 

Gothenburg, Sweden 2018  



 

 

 

 

iv 

 

Development of a non-enzymatic, non-invasive sensor for detection of lactate in sweat. 

ANNIE LJUNGH 

Department of Physics  

Chalmers University of Technology 

 

Abstract 

An athlete’s goal is to perform the best way possible and in many cases the level of lactate in blood is 

an indication of performance possibilities. Blood lactate measurement is conducted to obtain knowledge 

of the lactate threshold (LT) which is the intensity at which blood lactate starts to increase. Lactate can 

also be found in sweat and some studies have reported a correlation between lactate in sweat and blood, 

implying for the possible usage of sweat instead for these types of measurements. Using sweat result in 

non-invasive measurement, both suitable for the discomfort of the athlete tested and to not affect the 

training while testing. Most non-invasive sweat sensor are based on degrading of lactate with enzymes 

and detection with amperometric sensors. For endurance sport, such as marathon’s, an enzyme-based 

sensor can’t measure for the total performance time without losing effect.  

The aim of the thesis was to produce a sensory device for evaluating the lactate concentration in sweat. 

Molecular imprinted polymers (MIP) made of overoxidized polypyrrole (oPPy) using ammonium 

persulfate ((NH4)2S2O8) as an oxidative agent was successfully drop coated onto screen printed carbon 

electrodes (SPCE) modified with gold nanoparticles (AuNP). By evaluating the peak current observed  

by cyclic voltammogram between -0.5 V to -1.5 V at a scan rate 0.05 V/s a decreasing peak could be 

observed at -1 V to -1.5 V with a higher lactate concentration. By data analysis both with modified and 

unmodified SPCE-AuNP, showed a linear relationship between lactate concentration and peak current. 

The electrodes were used to evaluate the lactate level in a real sweat sample. The unmodified electrodes 

overestimated the lactate level with approximately 10 percent while the modified underestimated by 

approximately 30 percent. 

 

 

 

 

 

 

 

 

 

 

Key words: Lactate, Sweat, Molecular imprinted polymers, Pyrrole, Screen printed carbon electrode.  



 

 

 

 

v 

 

Acknowledgement 

This master thesis project has been conducted at the division of Condensed Matter Physics during the 

autumn of 2018. The work has been part of Chalmers sport and technology. 

First of all, I want to express gratitude towards my supervisor and examiner Magnus Karlsteen for 

helping me start the background to this work in the spring of 2017 and my supervisor Gunnar Westman 

for helping me take the project further. You both have been my guiding stars throughout this work and 

I’m truly grateful. I want to give a special thanks to Parveen Deralia for helping me in the lab and for 

all the advice. 

I want to give a big thank you to all the people at KMF, for all the laughs and interesting discussion 

during lunch. My family and friends, I’m deeply grateful for your support throughout this experience 

and for allowing me to talk about sweat with you.  Jessica Fredriksson your input and support has been 

extremely valuable for me, thank you. 

A special thanks for all persons that been helpful during the project.  

Jonathan Weidow 

Aline Maire Du Poset  

Tiina Nypelö 

Aktivitus   

 

 

Annie Ljungh, Gothenburg, December 2018  



 

 

 

 

vi 

 

Content 
List of figures ....................................................................................................................................... viii 

List of tables ........................................................................................................................................... ix 

Abbreviations .......................................................................................................................................... x 

1. Introduction ..................................................................................................................................... 1 

2. Theoretical background ....................................................................................................................... 2 

2.1 Lactate ........................................................................................................................................... 2 

2.1.1 Lactate measurement in exercise ............................................................................................ 3 

2.2 Sweat ............................................................................................................................................. 4 

2.2.1 Sweat glands ........................................................................................................................... 4 

2.2.2 Sweat rate ............................................................................................................................... 4 

2.2.3 Lactate in sweat ...................................................................................................................... 5 

2.2.4 Sweat collection ..................................................................................................................... 5 

2.3 Electrochemistry ............................................................................................................................ 6 

2.3.1 Cyclic voltammetry ................................................................................................................ 6 

2.3.2 Electrochemical cells .............................................................................................................. 7 

2.4 Selective sensor ............................................................................................................................. 7 

2.4.1 Enzyme based sensor .............................................................................................................. 7 

2.4.2 Non-enzyme based sensor ...................................................................................................... 8 

2.4.3 Modification of screen printed carbon electrodes .................................................................. 9 

2.5 Characterisation methods .............................................................................................................. 9 

2.5.1 Contact angle measurement .................................................................................................. 10 

2.5.2 Fourier transform infrared spectroscopy .............................................................................. 10 

2.5.3 Scanning electron microscopy .............................................................................................. 10 

2.5.4 Standard addition .................................................................................................................. 10 

3. Method .............................................................................................................................................. 11 

3.1 Sweat collector ............................................................................................................................ 11 

3.1.1 Test of sweat collector .......................................................................................................... 11 

3.1.2 Modification of sweat collector ............................................................................................ 11 

3.2 Sweat collection .......................................................................................................................... 12 

3.2.1 Sweat collection with pads ................................................................................................... 12 

3.2.2 Sweat collection with pipettes .............................................................................................. 12 

3.2.3 Analyse of lactate concentration in sweat. ........................................................................... 12 

3.3 Modification of SPCE ................................................................................................................. 13 

3.4 Calibration with dummy cell ....................................................................................................... 14 

3.5 Screen printed carbon electrode testing ....................................................................................... 14 



 

 

 

 

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3.5.1 Concentration curve.............................................................................................................. 15 

4. Results and discussion ....................................................................................................................... 16 

4.1 Sweat collector ............................................................................................................................ 16 

4.1.1 Sweat collector version 1...................................................................................................... 16 

4.1.2 Sweat collector version 2...................................................................................................... 16 

4.1.3 Test of modified sweat collector .......................................................................................... 17 

4.2 Sweat collection .......................................................................................................................... 17 

4.2.1 Sweat collection with pads ................................................................................................... 17 

4.2.2 Lactate value in sweat samples ............................................................................................. 18 

4.3 Modification of electrodes ........................................................................................................... 19 

4.3.1 Drop coating of electrodes .................................................................................................... 20 

4.4 Calibration with dummy cell ....................................................................................................... 21 

4.5 Concentration curves ................................................................................................................... 22 

4.5.1 SPCE with gold nanoparticles .............................................................................................. 22 

4.5.2 SPCE with gold nanoparticles and modification .................................................................. 24 

4.5.3 Sweat sensor ......................................................................................................................... 25 

5. Conclusion and future outlook .......................................................................................................... 27 

6. References ......................................................................................................................................... 28 

Appendix .................................................................................................................................................. I 

A – Contract with participants .............................................................................................................. I 

B – Design of sweat collector ............................................................................................................ VI 

C - Protocols ..................................................................................................................................... VII 

C.1 Set-up of Rodeostat before testing ......................................................................................... VII 

C.2 Calibration of Rodeostat ........................................................................................................ VII 

D – Data processing .......................................................................................................................... IX 

D.1 Data processing of calibration with dummy in MATLAB ..................................................... IX 

D.2 Data analysis of CV ................................................................................................................ IX 

D.3 Data analysis for creating concentration curves ..................................................................... XI 

D.4 Data analysis for standard addition........................................................................................ XII 

 

  



 

 

 

 

viii 

 

List of figures 
Figure 1, relationship between lactic acid and it’s conjugated base lactate. ........................................... 2 
Figure 2, schematic picture of the simplified pathway between the muscles and liver. .......................... 3 
Figure 3, A: illustrative picture of the skin cell with a hair coil, blood vessels and sweat gland. B: the 

parts of the sweat gland and the transport of lactate, sodium and chloride from the secretory coil and 

dermal duct (9, 21). ................................................................................................................................. 4 
Figure 4, CV made on dummy cell between -0.6 V and 1.5 V with the scan rate of 0.1 V/s. Graph show 

the change in potential over time............................................................................................................. 6 
Figure 5, picture of a screen-printed carbon electrode with gold nanoparticles from DropSens with the 

different electrodes and connections are pointed out. ............................................................................. 7 
Figure 6, showing reaction for the enzyme A: LO and B: LDH in contact with lactate, (42)................. 8 
Figure 7, illustrative picture of MIPs where a cavity is filled or unfilled depending on if the target 

molecule is present. ................................................................................................................................. 9 
Figure 8, model of sweat collector with O-ring..................................................................................... 11 
Figure 9, first version of the sweat collector. ........................................................................................ 16 
Figure 10, the second version of the sweat collector with O-ring. ........................................................ 16 
Figure 11, sweat collector ver.2 after test. A small sweat accumulation can be seen on the collector. 17 
Figure 12, graphs from standard addition for sweat collected at low (LI) and high (HI) intensity. ...... 18 
Figure 13, the synthesis process for creation of MIPs of oPPy. The different synthesis A-B had potassium 

ferrocyanide (K4Fe(CN)6·3H2O) while C-D had ammonium persulfate ((NH4)2S2O8) as an oxidative 

agent. B and D had PVA in the solution. Pictures at 1: is after addition of Py and LA solution, the 

solution is clear, 2: after 2 h, the solution has turned light pink, 3: after addition of oxidative agent, A 

and B have a yellow colour while C and D has a black colour, 4: after the synthesis was completed, all 

solutions is dark green, and 5: after decantation of supernatant, a black powder is left. ...................... 19 
Figure 14, FT-IR spectra of oPPy produced with and without PVA, of the wavenumber (cm-1) versus 

the absorbance. The background of the KBr has been subtracted. ........................................................ 20 
Figure 15, A: picture of SPCE-AuNP after addition of MIP solution, B: picture of the modified electrode 

after drying. ........................................................................................................................................... 20 
Figure 16, SEM picture of the SPCE-AuNP without and with modification. A: SPCE-AuNP at 2000 

times magnification, B: modified SPCE-AuNP at 2000 times magnification, C: SPCE-AuNP at 10000 

times magnification and D: modified SPCE-AuNP at 10000 times magnification. .............................. 21 
Figure 17, curves of the measured current compared to converted current from the calibration of 

Rodeostat via a dummy cell. ................................................................................................................. 22 
Figure 18, example of cyclic voltammogram measurement from one SPCE-AuNP with increasing 

concentration of lactic acid in 0.1 M KCl (tap water) ........................................................................... 23 
Figure 19, concentration curves obtained from measurement with different solutions. LA11-13: 0.1 M 

KCl in distilled water, LA14-16: 0.1M KCl in tap water, LA17-19: only tap water and LA20-22: 

artificial sweat. ...................................................................................................................................... 23 
Figure 20, concentration curve with test of artificial sweat samples on modified SPCE-AuNP. ......... 24 
Figure 21, cyclic voltammogram of sweat samples on A: SPCE-AuNP and B: modified SPCE-AuNP.

 ............................................................................................................................................................... 25 
Figure 22, bar plot over the observed true value of lactate in sweat sample compared with the calculated 

samples. ................................................................................................................................................. 26 
Figure B1,  design of sweat collector with place for an O-ring. ............................................................ VI 
  



 

 

 

 

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List of tables  
Table 1, definition of different concepts used for sport analysis. ............................................................ 3 
Table 2, preparation of the solutions for standard addition testing at sweat collected at low intensity. 

Water is referring to distilled water. ...................................................................................................... 12 
Table 3, preparation of the solutions for standard addition testing at sweat collected at high intensity. 

Water is referring to distilled water. ...................................................................................................... 13 
Table 4, the different chemicals used for the four different synthesis of MIP of oPPy. ....................... 13 
Table 5, dilution table for creating different concentration of lactic acid solution. .............................. 15 
Table 6, the results from the contact angle tests. The contact angle decreased with a coating of 

hemicellulose. ........................................................................................................................................ 17 
Table 7, results from calibration of dummy during 4 different current range. The slope, intersection and 

R2 value corresponding to each of the ranges........................................................................................ 21 
Table 8, slope and intersection obtained from the different concentration curves using SPCE-AuNP. 24 

  



 

 

 

 

x 

 

Abbreviations 
ACS    American Chemical Society 

AT     Anaerobic threshold 

ATP     Adenosine triphosphate 

AuNP     Gold nanoparticles  

C.E.     Counter electrode 

CV     Cyclic voltammetry 

ECP     Electronically conducting polymers 

FT-IR     Fourier transform infrared spectroscopy 

GXT     Graded exercise test 

HI    High intensity 

LDH    Lactate dehydrogenase 

LER     Lactate excretion rate 

LI    Low intensity 

LO     Lactate oxidase 

LSR     Local sweat rate 

LT     Lactate threshold 

MIP     Molecular imprinted polymers 

MLSS     Maximal lactate steady state 

OBLA     Onset of blood lactate accumulation 

oPPy     Overoxidized Polypyrrole 

PPy     Polypyrrole 

R.E.     Reference electrode 

SEM     Scanning electron microscopy 

SPCE     Screen printed carbon electrodes 

SWD     Sweet rate dependent 

TBW     Total body weight 

W.E.    Working electrode  

WBSR     Whole body sweat rate 

  



 

 

 

 

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1. Introduction 
During training many people experience ache in their muscles, this is a result of accumulated lactate in 

the muscles. For the ordinary person the ache is just a negative part of training but for elite athletes it is 

saying more about how the body is reacting to the training. An elite athlete wants to make sure that the 

training program is conducted to increase the strength and performance. A commonly used method for 

evaluating the possible performance as well as a tool for planning practice is the measurement of lactate 

concentration in blood during increasing intensity(1). This is made by taking multiple blood samples as 

the intensity of the training is increasing. Something that can be uncomfortable for the athlete and not 

letting them fully concentrate on the task of the graduating increasing exercise.  

Lactate is a product produced from the metabolism within our cells. During training the cells needs to 

produce more energy in the form of adenosine triphosphate (ATP)(2) to be able to keep performing the 

training. Because of the increasing demand of energy and the decreasing oxygen level during exercise, 

the cells have to use different pathways to keep up the energy level. When the energy is produced during 

anaerobic conditions one of the products is lactate, which starts to accumulate in the blood(3). Through 

gradually increasing the intensity of the training and measure the concentration of lactate the intensity 

where lactate starts to build up can be found using multiple different concepts(4). One of these concepts, 

the lactate threshold (LT) is the intensity of training where lactate starts to accumulate. LT is close to 

but not the anaerobic threshold (AT) which is the level where the oxygen uptake can account for the 

energy the cells need(5). 

Sweat is a biofluid used for thermoregulating the body and contains small levels of different analyte that 

can be found in the blood(6-8). Through the common discomfort of blood sampling an increasing trend 

in production of sweat sensors has started, where analytes usually evaluated from blood is evaluated in 

sweat. This allowing for a non-invasive measurement. In sweat the lactate level is an indirect indication 

of increasing physical exertion. The lactate concentration in sweat is both from the blood plasma and 

from production in the sweat glands(9). Sweat sensors used for lactate measurements have previously 

used enzymes for detection of the degrading lactate but have shown some problems regarding stability 

as enzymes is highly affected by pH and temperature(10-12). By using chemical modified electrodes, a 

non-enzymatic sensor for lactate detection in sweat has been conducted. These sensors show a better 

storage stability and is not effected in the same extent by environmental changes(13).  

The positive aspects of enzymes specificity can be mimicked in molecular imprinted polymers (MIP). 

MIPs are polymers constructed to have a cavities specific for the analyte of interest and therefore mimic 

the lock and key mechanisms found in enzymes(11). These can be produced by synthesis either by an 

oxidative agent or by potential through electropolymerization(14). During the project MIP where 

evaluated to determine if it could be a suitable solution for a non-enzymatic sweat sensor. This by 

comparing unmodified electrodes with electrodes modified with MIP. The specificity of the sensor was 

evaluated as sweat is a complex solution with a great matrix effect. 

Therefore, the aim of the project was to produce a sensory device, using a non-enzymatic solution to 

obtain quantitative measurement of lactate from sweat. The focus was placed on creating the active 

electrodes responsible for detection of lactate as well the collection of sweat. The project is focused on 

detection of lactate, this due the influence lactate concentration has on the performers of athletes.   



 

 

 

 

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2. Theoretical background 
The chapter aims to discuss the different concepts and methods used in this project, describing the use 

of lactate measurement in exercise and provides information about the bio rich fluid, sweat. Additional 

information about the concept of sweat based sensors and how these are evaluated is presented. The 

chapter also describes the basics of how sensors is constructed, tested and modified. 

2.1 Lactate 

Lactate is the conjugated base of lactic acid, as one carboxy group has been deproteinised(15). In the 

body, 99% of lactic acid is reduced to lactate and hydrogen ions(16), therefore lactic acid and lactate are 

often used as synonyms within sport context. The relationship between lactic acid and lactate is 

displayed in Figure 1. Lactate is produced in muscles during anaerobic conditions from pyruvate(3). 

The accumulation of lactate in our muscles during training results in ache(3) and is a biomarker of 

fatigue(17).  

 

Figure 1, relationship between lactic acid and it’s conjugated base lactate. 

Pyruvate is produced in glycolysis, an important pathway our cells for creating energy, from glucose. A 

part of glycolysis is the production of ATP which is used as energy within the cells. Pyruvate is further 

degraded to acetyl-CoA or lactate in our cells. Acetyl-CoA is used in the Citric Acid Cycle where more 

energy in form of ATP equivalents is produced. These are then used in the electron transport chain where 

a proton gradient is made by multiple redox reactions. The proton gradient is used as energy by the 

membrane enzyme ATP synthase which produces more ATP (3).  

Muscle uses energy in form of ATP to produce movement and force, during activity, e.g. exercise, the 

ATP consumption is increasing. ATP is found in an almost constant level in the body, when it is 

hydrolysed it also needs to be resynthesized in a similar rate. During exercise the hormones produced, 

e.g. adrenaline, regulates glycolysis to increase the flux. However, when the oxygen level starts to get 

low, the pathways which needs oxygen is supressed and pathways that work in anaerobic conditions is 

used instead. Pyruvate is then converted to lactate by lactate dehydrogenase (LDH) to reoxidized NADH 

to NAD+(18), a coenzyme used in glycolysis(19). Through reverse glycolysis called gluconeogenesis 

glucose can be created from pyruvate. Lactate that is created in the muscle cells from glucose is shuttled 

to the liver via the blood where the LDH converts lactate back into pyruvate, this can be observed in 

Figure 2. During anaerobic conditions lactate starts to accumulate in the bloodstream (3).  



 

 

 

 

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Figure 2, schematic picture of the simplified pathway between the muscles and liver. 

2.1.1 Lactate measurement in exercise 

Lactate measurement can be used for multiplied purpose in a sport context, to evaluate and predict 

performance as well as a training tool(1). There are multiple concepts used for measurement of athletes 

fitness, e.g. aerobic-, anaerobic-, aerobic-anaerobic- and lactate threshold, onset of blood lactate 

accumulation (OBLA), maximal lactate steady state, individual anaerobic threshold and ventilatory 

threshold(1, 5). Some of these concepts are defined in Table 1. These different concept are ways to 

detect the intensity of exercise related to the anaerobic threshold(5). 

Table 1, definition of different concepts used for sport analysis. 

Concept Definition 

Anaerobic threshold 

(AT) 

The oxygen uptake can account for the energy 

requirement need for the high intensity, i.e. where the 

blood lactate remains at a steady state (5). 

Maximal lactate steady 

state (MLSS) 

Where there is an equilibrium between the rate of 

transport into the blood and removal of lactate. Not 

same as AT when there still is an accumulation in the 

muscles while the blood concentration is constant(5). 

Lactate threshold (LT) The intensity of exercise that result in a considerable 

increase in blood lactate during an exercise test(5). 

Onset of blood lactate 

accumulation (OBLA) 

The intensity which the lactate concentration in blood 

reached 4 mM. Based on that AT is the same as that 

concentration (5). 

 

There are multiple ways to evaluate the presented concepts, commonly used is the graded exercise test 

(GXT), where the intensity of the exercise is gradually increased in a controlled time while vital 

parameters are measured e.g. lactate concentration in blood and heart rate. The intensity is often referred 

to as the power (W) of the exercise, e.g. how fast the treadmill is going or the resistance from the cycle 

(4). The different concepts have both positive and negative aspects. OBLA ignores variability between 

individuals due to the use of a set concentration(5). The use of this concentration is due to the value 

reflecting lactate accumulation within the active muscle (1). MLSS, on the other hand, needs to use 

multiple testing but takes the variability between individuals into account. LT can be defined by multiple 

principles, as the intensity of training where the resting blood lactate level is increased by 1 mM, when 

the blood concentration starts to withdraw from the linearity as well as OBLA. These concepts are often 

close to AT but not equal to(5).   



 

 

 

 

4 

 

The concepts should be evaluated based on their context of use(5), the set-up during testing needs to be 

taken into consideration as there are multiple physical factors that can affect the result(1). Athletes want 

a high AT as this implies that the body can handle a higher intensity of exercise before the body needs 

to use anaerobic processes. As mention the use of these thresholds is often for design of training 

programs. An exercise below the intensity related to the threshold is mild, while one above is high and 

there in-between moderate(5).  

2.2 Sweat 

Sweat is used to thermoregulate the body, which is one of the fundamental activities for our bodies (6-

8). It mainly consists of water (99%) and low concentrations of analytes found in plasma, where the 

main metabolites are lactate, different amino acids and lipids(6, 20). Other biomarkers found in both 

sweat and blood includes Sodium (Na+), Chloride (Cl-), Potassium (K+) and Glucose(9). Sweat is 

dependent on multiple features, it differs between individuals and body parts(6). The different 

biomarkers have different relationship between sweat and blood. Due to the transport process the 

relationship can be passive, active or self-generating(9).  

2.2.1 Sweat glands 

Sweat is a result of the eccrine sweat glands secretion. The glands are found over large part of the body 

but with a changing density depending on the body part and individual(8). The sweat glands are located 

in the skin, see Figure 3A. The gland consists of three parts, the upper coiled duct, which ends at the 

skin surface and where the sweat is emerged, the dermal duct where some biomarkers is transported into 

sweat and the secretory coil, the base of the gland and where additional biomarkers is incorporated (9). 

This is showed in Figure 3B. 

Water is introduced to the sweat gland in the secretory coil, as well as Na+ and Cl-. Na+ and Cl- are later 

reabsorbed in the dermal duct. These ions have an active transport in the gland and are sweat rate 

dependent, a concept explained further below. In the secretory coil the transport of metabolic origin is 

also included into the fluid that is sweat. These molecules with metabolic origin e.g. lactate, is likely 

both produced in the gland and transported from the plasma(9). The transport of these molecules can be 

observed in Figure 3B. 

 

Figure 3, A: illustrative picture of the skin cell with a hair coil, blood vessels and sweat gland. B: the parts of the sweat 

gland and the transport of lactate, sodium and chloride from the secretory coil and dermal duct (9, 21). 

2.2.2 Sweat rate 

Sweat rate is the rate in which sweat is secreted. It can be calculated both from local or whole-body 

perspective, the different calculations have different aspect in regards of sweat sensing(22). It can be 

found in different units depending on the calculations, when the density of the sweat gland is taken into 



 

 

 

 

5 

 

consideration, ml/min/m2 (8) and where it is calculated based on the whole body, l/h (22). Sweat rate is 

also found to have a relationship with the exercise intensity(9, 23). 

Local sweat rate (LSR) can be measured through mass changes in a collection system over a specific 

body part.  For collection system filter paper, patches, pouches, cotton products can be used. To be sure 

that the sweat collection is representative, testing should be made when sweating reaches a steady-state. 

For avoiding changes in the sweat rate due to environmental variations, the test should be done during 

limiting time(22). Studies have shown that there is a high local sweat rate on the head, finger/hand and 

back(lumbar) of their participants (24). 

To calculate the whole body sweat rate (WBSR), the change in body mass during training can be 

evaluated. This by measuring the nude weight before and after training and correlate by the non-sweat 

body mass changes during the training e.g. consumption of food, void bladder, water consumption. 

Using nude body weight minimize the error of sweat trapped in clothes. Additional changes in mass due 

to metabolic and respiratory mass loss should be corrected to if the test was set in a challenging 

environment (high intensity or dry environment) or for a long time period (2-3h). The test should be 

under circumstances where the athlete usually exercise and the calculation can be made according to the 

Equation 1 (22). 

WBSR= 
mbefore exercise-(mafter exercise-mfluid intake during exercise-murine output during exercise)

texercise
 (22, 25)  Eq. 1 

For ions e.g, Na+ and Cl-, the sweat rate affects the reabsorption back to the sweat gland. This is for the 

body to keep its electrolyte levels(26). The concentration of these electrolytes in sweat reflect the water 

loss in blood during physical exercise. Ring M, et al, showed a significant correlation between Cl-, 

concentration and total body weight (TBW) loss during a 2 h interval training, this can be applied to Na+ 

as Na+ and Cl- is correlated. This sets a base of being able to quantify TBW loss from electrolyte 

concentrations (26). In further extent these concentrations can be used for calculation of SR as a higher 

concentration correlates to a higher sweat rate(8, 9, 27). The forearm is a suitable site for these types of 

measurement as concentration of sodium has been shown to correspond to the concentration in whole 

body sweat(22).  

2.2.3 Lactate in sweat 

Lactate can be found in a concentration of 5-60 mM in sweat(9), coming from both the blood plasma 

but as well from the eccrine gland(9, 28). The transport from plasma is not fully understood but it is 

thought to relate to the flux from the sweat gland but also passive or active transport from the plasma. 

Lactate is sweet rate dependent (SWD) and can therefore be found in higher concentration in sweat 

compared to blood. However the level of lactate is an indirect indicator of physical effort(9). Due to the 

complexity regarding SWD there are different studies both arguing for and against if lactate in sweat 

correlate to blood and exercise intensity. Changes in concentration is dependent on the parameters used, 

testing site and test conditions(7).   

Sakharov D. A. et al. found a relationship between lactate in sweat and blood, by evaluating the 

percentual increment of lactate above the working muscle(28). Buono M. J, et al. suggested an additional 

way of analysing lactate concentrations in sweat, using lactate excretion rate (LER) (nmol/cm2/min) 

instead of concentration of lactate. LER is defined as the sweat lactate concentration and multiplied with 

the sweat rate at the test site. Taking LSR into account minimizing the error a dilution effect as the 

intensity of the exercise increases (23).  

2.2.4 Sweat collection 

Using sweat as a biofluid for sensing allows for the sensor to come close to the newly produced liquid(9). 

As discussed previously there are multiple ways to collect sweat for measurement of e.g. LSR. In 

addition, there are sweat collection system using the sweat glands itself as a hydraulic pump. Liu et al, 



 

 

 

 

6 

 

created a sweat collector by 3D printing a round collector with a curvature of 1 mm sagitta and a hole 

(d=2mm) touching the skin. The sweat was transported through the hole to a tube where they used the 

sweat for conductive measurement(29). Sweat collectors can be placed on multiple body parts e.g. 

forearm(29) and under the chest(26). 

2.3 Electrochemistry 

Electrochemistry involves the relationship between electrical and chemical reactions. When conducting 

electrochemical measurement a potentiostats is used to control the electrochemical cells(30). 

2.3.1 Cyclic voltammetry 

Cyclic voltammetry (CV) is a widely used electrochemical method(30) that can be used to get a 

fingerprint of chemical reactions conducted at the surface of an electrode. Just with a few calculations a 

lot of information regarding the measurement can be obtained, e.g. thermodynamic and kinetic 

parameters(31). The analyte will move to the electrodes surface by convention, migration and diffusion. 

Through experimental setup the convention and migration movement can be kept to a minimum. The 

convention movement is the result of e.g. stirring or vibrations, migration is due to electrodes moving 

due to an electric field(30).  

The method consists of a triangular change in potential(32), from the initial potential a linear increase 

of potential is made until reaching the switching potential, where the potential is switched and decreased 

to the starting value(31). The rate of the potential changes from the initial to the final potential is called 

the sweep rate(31) or scan rate(30), this variable controls the time for the experiment(31, 32). This result 

in a potential profile as seen in Figure 4. The molecules charge changes depending on the potential. The 

diffusion layer is depending on the number of molecules with a changed charge that can be found at the 

electrodes surface. If the potential is scanned negatively positively charged ions will be reduced to its 

neutral form. The more molecules that have been reduced create a larger diffusion layer and make it 

harder for the non-reduced molecules to reach the electrode surface(30). In addition, the diffusion layer 

depends on the scan rate, a high rate results in higher currents as an result of a smaller diffusion layer(30). 

 

Figure 4, CV made on dummy cell between -0.6 V and 1.5 V with the scan rate of 0.1 V/s. Graph show the change in potential 

over time. 

From a CV scan the relationship between the potential (E) and the resulting current (i) (30, 32) is of 

interest. During scanning when the potential is reduced it is called negative or cathodic direction, the 

peak current in this area is referred to as ip,c. On the contrary when the potential is increased it is called 

positive or anodic direction, where the peak current is ip,a (30). These peaks together with the equivalent 



 

 

 

 

7 

 

peak potential is important parameters obtain from the test(32). The peak current can be used to find a 

relationship with concentration(33). 

The experimental parameters for CV measurement include the scan rate, but also information about the 

solvents used as well as the electrodes. The analyte needs to come in contact with the surface of the 

electrode and this is made by the supporting electrolyte. A supporting electrolyte reduces the solutions 

resistances which helps the movement of the electrodes. The supporting electrolyte is a salt dissolved in 

a solvent suitable for the experiment conditions. A high level of electrolyte maximizes the movement of 

analyte through migration to the electrodes(30). 

2.3.2 Electrochemical cells 

There are multiple electrode setups, often used is a three-electrode setup, containing of a working 

electrode (W.E.), counter electrode (C.E.) and reference electrode (R.E.). The working electrode is 

where the main electrochemical reaction happens, the surface should be well defined as the 

measurements depends on the surface interaction with the molecules. The reference electrode is used as 

a reference point during electrochemical measurement, therefore, the applied potential is often referred 

versus the reference used. The counter electrode is used to complete the electrical circuit. Therefore the 

current can be measured between the W.E. and C.E. (30).  

Screen printed electrodes 

The use of screen printed electrodes allows for a minimization of the setup for electrochemical 

measurements, require less maintenance and no need for large sample volumes(33, 34). DropSens 

provides screen printed carbon electrodes (SPCE) made with a three-electrode configuration. The 

different electrodes are printed onto a ceramic substrate, with a W.E. and C.E. of carbon, pseudo R.E. 

and electric contacts of silver. Over these an insulated layer is printed leaving only a volume of 50 µL 

uncovered, called the working area (33). A SPCE can be seen in Figure 5. 

 

Figure 5, picture of a screen-printed carbon electrode with gold nanoparticles from DropSens with the different electrodes 

and connections are pointed out. 

2.4 Selective sensor 

To minimize the interference from the complex solutions, sensors can be made to be selective for the 

analyte of interest. There are both enzymatic and non-enzymatic selective sensors and they have 

different advantages depending on the area of use. 

2.4.1 Enzyme based sensor 

Most non-invasive sweat sensors are based on degrading of lactate with enzyme (35-41) and detection 

often by amperometric sensors. The amperometric sensor measure the change in hydrogen ions and the 

signal can be converted to a lactate concentration. Two types of enzymes can be used for these types of 



 

 

 

 

8 

 

measurement, lactate oxidase (LO) and LDH(42). The mechanisms of the breakdown can be shown in 

Figure 6. 

 

Figure 6, showing reaction for the enzyme A: LO and B: LDH in contact with lactate, (42).  

The usage of enzymes provides a specific and sensitive method, however there are some negative 

aspects. Enzymes are very sensitive to different conditions as heat and pH(10-12), the change of 

conditions can result in inactivation of the enzyme, and therefore a non-functional sensor. During 

training, the temperature increases which impact the characteristics of sweat, meaning usage of the 

sensor can affect the enzyme negatively. Enzyme based sensors have a restricted effective time, meaning 

that the usage of these will be limited to two hours (38) and that is not enough time for a long-distance 

sport. In addition, the sensors need to be stored in the cold to not loose sensitivity during long 

storage(38).  

2.4.2 Non-enzyme based sensor 

A non-enzymatic sensor needs to be able to have similar specificity as the enzymatic sensor while 

providing better storage and stability over time.  

Molecular imprinted polymers 

A possible alternative to enzymes is molecularly imprinted polymers (MIP) that mimic the lock and key 

mechanism of enzymes. MIPs mimic enzymes active site by being synthesis to form cavities matching 

the analyte of interest, therefore making them specific(11). The target can bound back to the MIPs and 

result in a change that can be picked up depending on sensor. In comparison with enzymes MIPs are 

more stable for extreme pH, temperature and pressure(43). 

The target, often referred to as template in the synthesis process, should not be affected by the 

polymerization and temperature changes. The template binds to functional monomers to create the 

complex which later create the cavities (44). These functional monomers is to enhance the selectivity of 

the target(12). For use in electrochemical measurement, it is suitable to use monomers that can form 

non-conducting films or conducting films(14). Cross-linkers is then used to stabilize the polymer matrix 

created around the template and functional polymers. (44). The function of MIPs can be observed in 

Figure 7. When modifying an electrode with MIPs the thickness of the layer is important, a too thick 

layer can negatively affect the sensing capability while a too thin layer does not provide the desired 

specificity and stability(12).  



 

 

 

 

9 

 

 

Figure 7, illustrative picture of MIPs where a cavity is filled or unfilled depending on if the target molecule is present.  

By using MIPs for the sensor similar features as with enzymes can be achieved. A molecular imprinted 

sensor have good limit of detection and because the sensory system can be small it is suitable for smaller 

sensory as handhold devices(11). Previously studies have used this technique to detect lactate in sweat 

(13).  

2.4.3 Modification of screen printed carbon electrodes 

SPCE can be modified both with biological samples e.g. strings of nucleic acids and proteins as well as 

non-biological samples. The use of these modified electrodes is wide, electrodes have been specified to 

e.g. ethanol(33) glucose(12) and lactate(13). In addition, gold nanoparticles (AuNP) are often 

electrodeposited on SPCE to increase the stability and sensitivity. This due to gold’s electrocatalytic 

activities and compatibility with multiple biomolecules (45).  

The MIPs can be integrated to the electrode by different methods, e.g. drop-coating and 

electropolymerization (14). Drop coating is made by dispersion of a solution with the chosen molecules 

on the working electrode and then left to dry(33). Electropolymerization uses the potential from the 

electrode to build the polymer structure onto the electrode(46). This speeds up the time of an otherwise 

long process(14). For verifying that there is polymers left after cleaning on the modified electrode, a CV 

measurement is often made. This to see how a modification change the cyclic voltammogram e.g. 

characterization of the sensor probe(12) as well as determination of the new surface(33). 

Often used for sensors is electronically conducting polymers (ECP) due to their electronic properties. 

An example of ECP is polypyrrole (PPy) synthesised by oxidation through an oxidative agent. During 

synthesis of MIPs a problem is often related to the removal of template before usage. When synthesising 

MIPs using ECP overoxidation of the polymer structure cope with this problem. Overoxidized 

Polypyrrole (oPPy) is an ECP used in multiple MIPs for chemical based sensors. Pyrrole is a common 

used functional monomer during productions of MIPs(47). PPy has been used previously for production 

of MIP due to its stability both over time and during different pH. As the oxidation of PPy to oPPy is 

irreversible and related to the high stability(48). Using a one-step preparation technique MIP imprinted 

with lactic acid of oPPy has been synthesis for used for detection of amino acids. By different oxidative 

agent as well as additional polyvinylpyrrolidone (PVP) to they were able to produced MIP of oPPy 

colloids(49). 

2.5 Characterisation methods 

For characterisation multiple methods can be used. To evaluate the change of surface behaviour for 

sweat collector contact angle measurement can be used. Chemical characterisation of the modified 

material can be observed by analysing the surface as well as the chemical bonds, which can be made 



 

 

 

 

10 

 

using contact angle measurement, SEM and FT-IR. For a complex matrix, standard addition is a method 

to evaluate the concentration of an analyte taking the matrix into consideration. 

2.5.1 Contact angle measurement  

To investigate the character of a surface, surface contact angle can be measured. This by measuring the 

angle between the liquid and solid when a droplet of liquid is placed onto the surface. A lower contact 

angle is corresponding to a more hydrophilic surface(50).  

2.5.2 Fourier transform infrared spectroscopy 

Fourier transform infrared spectroscopy (FT-IR) gives insight in the functional groups of the molecules, 

as it detects the properties of the bonds within the molecules. The infrared energy able to pass through 

the sample is plotted against the wavelength (often showed in wavenumber(cm-1), the number of 

wavelengths in one centimetre). The peaks observed in the spectrum correlate to a bond within the 

molecule(51).  

2.5.3 Scanning electron microscopy 

Scanning electron microscopy (SEM) is a method for detection the topology of a surface. This by 

scanning every position of a x-y grid made over the sample. Each placement of the grid is hit by the 

electron beam and analysis of the scattering from the point is evaluated. Depending on the relative 

strength of the measured signal the part of the grid is varying from black to white(52).    

2.5.4 Standard addition 

When the matrix effects of a sample are specific for the sample standard addition is a method where the 

matrix effect is taken into account. In standard addition the concentration of an analyte (X) is evaluated 

dependent on a known added standard (S). A known volume (V0) of the unknown sample is diluted in 

increasing volume (VS) of a known standard solution to a set volume (V). The different dilution is then 

evaluated, and the response is the total response from both the unknown and known sample. The 

relationship between the concentration and response (I) from the initial (i) compared to the finale (f) are 

equal(53) as shown in Equation 2.  

[𝑋]𝑖

[𝑆]𝑓+[𝑋]𝑓
=  

𝐼𝑋

𝐼𝑆+𝑋
 Eq. 2 

[𝑋]𝑓 =  [𝑋]𝑖 (
𝑉0

𝑉
) , [𝑆]𝑓 =  [𝑆]𝑖 (

𝑉𝑠

𝑉
)  Eq. 3-4 

 

Equation 2-4 can be further elaborated to Equation 5, from this the concentration of the sample ([X]i) 

in the solution can be calculated as the x-intercept, i.e. when the y-axis is equal to zero(53).  

𝐼𝑆+𝑋 (
𝑉0

𝑉
) =  𝐼𝑋 +

𝐼𝑋

[𝑋]𝑖
[𝑆]𝑖 (

𝑉𝑠

𝑉
)                 {

𝑥 − 𝑎𝑥𝑖𝑠 =  [𝑆]𝑖 (
𝑉𝑠

𝑉
)

𝑦 − 𝑎𝑥𝑖𝑠 =  𝐼𝑆+𝑋 (
𝑉0

𝑉
)
 Eq. 5 

 

 

  



 

 

 

 

11 

 

3. Method 
Multiple aspects of a sweat sensor have been evaluated. Firstly, sweat collection system, how to take 

the newly created liquid to the sensor. The modification of screen printed carbon electrodes and how 

they affected the analysis of lactate in sweat. Multiple concentration curves were conducted with 

increasing matrix complexity, to be able to create a reference from the observed peak current during 

cyclic voltammogram. Finally, using the obtained modification and concentration curve a pure sweat 

sample was evaluated to observed which method came closest to the true value found in the sweat 

sample. Before testing with a person all participants had to sign a consent form and health survey found 

in Appendix A – Contract with participants. 

3.1 Sweat collector 

A sweat collector was 3D-printed in PLA (Polylactic acid) by knowledgeable personal according to the 

design shown in Figure 8. The collector was printed in two different sizes, on smaller (2.9x0.4x2 cm) 

and one larger (5.5x0.4x4.2 cm) with an O-ring. The design of the larger collector can be seen in 

Appendix B – Design of sweat collector. 

 

Figure 8, model of sweat collector with O-ring. 

3.1.1 Test of sweat collector 

Test of the sweat collector’s function was made by a participant cycling with the goal to effectively start 

sweating. The sweat collector was secured with an elastic band around the upper arm of the participant. 

The heart rate and cadence were measured using Garmin 735 XT with effect pedals and heart rate 

monitoring belt under the chest. During the exercise the change in physical appearance (e.g. sweating) 

and if the participant started to feel fatigue was observed. The sweat collector was observed if sweat 

was participated through the column. After the test the sweat collector was removed and observed 

underneath. 

3.1.2 Modification of sweat collector 

To further develop the sweat collector, a modification of the surface was made through coating. A small 

volume of newly synthesised hemicellulose was dissolved in water during stirring and heating overnight. 

The solution was then distributed over a flat PLA surface and left to dry for 3 days. Contact angle 

measurement was conducted by analysing the angle between a water droplet and the uncoated/coated 

PLA surface using an Attension Theta optical tensiometer by KSV instrument. The mean contact angle 

between the uncoated or coated PLA and water was compared.  

  



 

 

 

 

12 

 

3.2 Sweat collection 

Sweat was collected using two different methods, the first using absorptive pads and the second using 

pipettes. The collected sweat was from a male 50-55 years old with good physic. 

3.2.1 Sweat collection with pads 

Sweat was collected simultaneously as a lactate threshold measurement where the subject conducted a 

GXT on bicycle.  Sterile pads were attached to the participants leg, neck and forearm with medical tape. 

Before application of the pads the body part was cleaned with alcogel. The pads on the leg was attached 

before the test and left during the entire testing period. Pads on the neck and forearm was changed 

approximately every three minutes to correspond to the blood samples taken for lactate threshold 

measuring. The pads were removed and stored in plastic containers sealed with parafilm in 

approximately 4º C until extraction. In total 13 samples were collected, one from the leg and six samples 

each from the neck and forearm. The pads were centrifuged 3000 rpm for a total of 30 min before 

additional centrifuge in 10 min at 3500 rpm in tubes with filter to extract the liquid from the pad.  

3.2.2 Sweat collection with pipettes 

During a training session sweat was collected from the back by pipettes. The participated cycled for 1.5 

h in a low intensity pace before doing additional cycling for 1 h in a high intensity pace. The pace was 

set according to participant’s anaerobic threshold. After the first part of the session approximately 0.5 

ml of sweat was collected by a pipette and transferred to an Eppendorf tube. Approximately 10 minutes 

before the participant stop the session the existing sweat on the back was dried of with a sterile pad, 

after the session was done approximately 0.5 ml of sweat was again collected into a new Eppendorf 

tube. The Eppendorf tubes were closed and covered in parafilm and instantly chilled. The samples were 

kept below 0º C until testing.  

3.2.3 Analyse of lactate concentration in sweat. 

Using the standard addition method, the collected sweat was analysed to find the concentration of lactate 

in the sweat. For samples collected during low intensity exercise the sample was diluted according to 

Table 2, while sample collected during high intensity was diluted according to Table 3. The standard 

solution was 1 M of lactic acid solution created by diluting American Chemical Society (ACS) graded 

Lactic acid (85%) solution with distilled water. 

Table 2, preparation of the solutions for standard addition testing at sweat collected at low intensity. Water is referring to 

distilled water. 

 

V0 

(µl) 

V 

(µl) 

[S]I 

(M) 

Vs 

(µl) 

Vwater 

(µl) 

Blank 0 500 1 0 500 

1 50 500 1 0 450 

2 50 500 1 25 425 

3 50 500 1 50 400 

4 50 500 1 100 350 

5 50 500 1 150 300 

6 50 500 1 300 150 
  



 

 

 

 

13 

 

Table 3, preparation of the solutions for standard addition testing at sweat collected at high intensity. Water is referring to  

distilled water. 

 

V0 

(µl) 

V 

(µl) 

[S]I 

(M) 

Vs 

(µl) 

Vwater 

(µl) 

Blank 0 500 1 0 500 

1 50 500 1 0 450 

2 50 500 1 50 400 

3 50 500 1 150 300 

4 50 500 1 300 150 

5 50 500 1 350 100 

6 50 500 1 450 0 

 

The samples were further diluted by 10 (50 µl of solution in 450 µl of distilled water) to obtain a response 

within the limits of the system. This was included in the calculations of the true value of lactate within 

the sample. The samples were tested as described in 3.5.1 Concentration curve and analysed according 

to Appendix D.2 Data analysis of CV and D.4 Data analysis for standard addition. 

3.3 Modification of SPCE 

Based on previously reported one step preparation of MIP of oPPy (49), four different synthesis were 

made, exact volumes can be observed in Table 4. All containing pyrrole and ACS graded lactic acid 

(85%), two with poly(vinyl alcohol) (PVA) and with either potassium ferrocyanide (K4Fe(CN)6·3H2O) 

or ammonium persulfate ((NH4)2S2O8) as an oxidative agent. 0.0086 g of PVA (99%) was dissolved in 

distilled water during heating (60º C) and occasional stirring. Additional distilled water was added 

during the heating due to water losses. After 2 h most of the PVA had dissolved but some small 

undissolved parts could still be found in the solution.  

Table 4, the different chemicals used for the four different synthesis of MIP of oPPy. 

 A B C D 

Py 0.108 

g 

0.107 

g 

0.103 

g 

0.1 g 

LA 85% 0.2 ml 0.2 ml 0.2 ml 0.2 ml 

PVA solution - 1.5 ml - 1.5 ml 

Distilled H2O 10 ml 8.5 ml 10 ml 8.5 ml 

K4Fe(CN)6·3H2O 0.4226 

g 

0.4226 

g 

- - 

(NH4)2S2O8 - - 0.2284 

g 

0.2283 

g 

 

Pyrrole and lactic acid were first dissolved in room temperature distilled water creating a clear solution. 

The solution was left for 30 min before adding the PVA solution. After 2 hours the solution had turned 

lightly pink. The solutions were placed on a magnetic stirrer before the oxidative agent was added. In 

the solutions containing ammonium persulfate there was an instant precipitate resulting in that the 

solution becoming black whereas there was no noticeable product in the solutions containing potassium 

ferrocyanide. The pH was modified to pH 4 with 0.1M NaOH(aq). The synthesis was then carried out 

in room temperature for 16.5 hours. After synthesis all solutions had a precipitate and had turned dark 

green.  



 

 

 

 

14 

 

After synthesis the solution was centrifuged twice for 15 min and 4300 rpm. The solutions were decanted 

before 30 ml of distilled water was added and additional centrifuge for 15 min and 4300 rpm was 

conducted. The solutions were decanted again before addition of 5ml of distilled water, final centrifuge 

of 15 min and 4300 rpm (twice for the solution containing ammonium persulfate due to the precipitate 

still dissolved in the supernatant). The solutions were decanted carefully using a pipette and the pellets 

was left to dry overnight in room temperature. The black powder was then additional dried in 40º C for 

1.5 h. 0.006 g of powder was then diluted in 2 ml of distilled water to create a polymer solution. The 

SPCE-AuNP was then drop-coated with the polymer solution and dried in room temperature for 3 days. 

The powder was analysed by FT-IR using a Spectrum One FT-IR Spectrometer (Perkin Elmer 

instruments). 600 mg of dried potassium bromide (KBr) was grinded together with three equal parts of 

the sample powder (approximately 6 mg in total). The powder was then divided into three and pressed 

into pellets used for analyses. The analyse was conducted with 16 scans with a 4 cm-1 resolution from 

4000-400 cm-1, and background scan together with scan of pure KBr pellet as reference. Both 

unmodified SPCE-AuNP and the modified SPCE-AuNP were imaging using SEM on a LEO Ultra 55 

(Gemini). 

3.4 Calibration with dummy cell  

For the electrochemical measurement an open source potentiostats(54), Rodeostat (IO Rodeo), was used 

together with screen printed carbon electrodes with gold nanoparticles (DRP-110GNP, DropSens). 

These were connected with an adapter (B, IO Rodeo) and the potentiostat was connected to a computer 

where the provided Rodeostat software app was used. The Rodeostat was set up according to protocol 

that can be found in Appendix C.1 Set-up of Rodeostat before testing. 

Calibration of the Rodeostat was made following the protocol that can be found in Appendix C.2 

Calibration of Rodeostat, based on the method suggested in a google document(55) from IO Rodeo. The 

potentiostat was connected to the computer and a 50 kΩ resistor. A cyclic voltammogram was taken 

between -0.6 V and 1.5 V with the scan rate of 0.1 V/s, for each of the available current range. The data 

was processed according to the code specified in Appendix D.1 Data processing of calibration with 

dummy, the applied potential was converted to current using ohm’s law as the resistant of the dummy 

cell was known. The converted current was plotted against the obtain current from the test and a fitted 

line was obtained.  

3.5 Screen printed carbon electrode testing 

In total four different types of solutions were used for measurement with increasing complexity, 0.1 M 

potassium chloride (KCl) in distilled water or tap water, only tap water or artificial sweat. Tap water 

was used without any further modification. A simple artificial sweat was made containing Na+, Cl- and 

K+ and glucose according to previously artificial sweat formulation (56). In an Erlenmeyer flask 

containing 50 ml of heated water (approximately 37º C) 0.0031 g of Glucose, 0.0456 g of KCL and 

0.0099 g of Sodium chloride (NaCl) were added and stirred during heating. The pH was checked to be 

in-between 5-6 before filling the flask to 100 ml. This resulted in a concentration of 6.1 mM K+, 1.7 mM 

Na+, 7.8 mM Cl- and 0.17 mM Glucose, which is in the range of concentration found in sweat. The 

solution was then left to cool done to room temperature before used for lactic acid stock solutions.  

From a stock solution of 1 M lactic acid a 0.1 M solution was made, this was further dilute according 

to Table 5 to create multiple samples of known lactic acid concentrations.  

  



 

 

 

 

15 

 

Table 5, dilution table for creating different concentration of lactic acid solution. 

Finale lactic acid 

concentration [mM] 

Diluted from what 

solution [mM] 

Volume of lactic 

acid solution[µl] 

Volume of 

solvent [µl] 

Total volume 

[µl] 

2.5 25 100 900 1000 

5 25 200 800 1000 

10 100 100 900 1000 

15 100 150 850 1000 

20 100 200 800 1000 

25 100 250 750 1000 

30 100 300 700 1000 

60 100 600 400 1000 

 

3.5.1 Concentration curve 

To create a concentration curve the electrode was inserted into the adapter and connected to the 

Rodeostat. 50 µl of the known lactate acid concentration was dispersed over the working area, making 

sure to cover the R.E. Cyclic voltammogram was taken between -0.5 V to -1.5 V with the scan rate of 

0.05 V/s for each of the concentration and background scan. Between each measurement the electrode 

was rinsed with distilled water and dried carefully with a paper towel. Measurement was done in triplets 

by using three different electrodes. The current obtained from the measurements was corrected 

according to the calibration curve, the current was then background subtracted and filtered before 

analysed for local minimum and maximum. The mean of the peak currents was then used for linear 

regression and creation of a concentration curve. One-sided ANOVA testing was made to observe if 

there were any large differences between the electrodes. This was processed according to code that can 

be found in appendix D.2 Data analysis of CV and D.3 Data analysis for creating concentration curves. 

For the unmodified electrodes all different solutions were used and for the modified electrodes only 

artificial sweat was used for scanning concentration curve. Both electrodes where used for real sweat 

samples. For the modified electrodes only one scan was made per concentration and all scans was 

conducted on different electrodes.  



 

 

 

 

16 

 

4. Results and discussion 
The results obtained by testing of the sweat collector, sweat collection, modification of electrodes and 

the finale sweat sensor will be presented and evaluated. 

4.1 Sweat collector  

Two types of sweat collectors were 3D printed, one smaller and one bigger version. They were tested 

so observe is sweat could be collected through the column during exercise. 

4.1.1 Sweat collector version 1 

The first version of the sweat collector with the size of 2.9x0.4x2 cm can be observed Figure 9. 

 

Figure 9, first version of the sweat collector. 

During testing of the sweat collector no sweat was observed to pass through the column. After the 

exercise when the collector was removed the elastic band used to keep the collector in place was wet 

and on the inside of the collector sweat drops could be observed. Due to the accumulation of sweat 

overall on the body, this could be the effect of the collector not sitting tight enough for the sweat gland 

to fully work as pumps.  

4.1.2 Sweat collector version 2 

The second version of the sweat collector was made in larger dimensions (5.5x0.4x4.2 cm) and with 

packing in the form of an O-ring to provide a seal between the skin and the sweat collector. The second 

version of the sweat collector can be observed in Figure 10. 

 

Figure 10, the second version of the sweat collector with O-ring. 

The second version of the sweat collector showed more promising results. After testing a clear sweat 

accumulation could be seen under the collector as seen in Figure 11, but no sweat was transported 



 

 

 

 

17 

 

through the entire collector. There was a clear mark on the participants arm of the O-ring suggesting 

that the collector was sitting tight and allowing the sweat to only transport within the collector. The 

increased size of the collector could be the result of this as it may take a longer period of time to fill the 

collector, so the glands can work as pumps. 

 

Figure 11, sweat collector ver.2 after test. A small sweat accumulation can be seen on the collector. 

4.1.3 Test of modified sweat collector 

By coating the material, the contact angle decreases as can be observed in Table 6, this indicates that 

the surface became more hydrophilic. As a more hydrophilic surface would imply that the surface would 

be better at driving the sweat forward using capillary force. 

Table 6, the results from the contact angle tests. The contact angle decreased with a coating of hemicellulose. 

PLA 

62.26 ± 5.15 (5) mean degree ± SD (n) 

PLA with hemicellulose 

44.35 ± 14.08 (6) mean degree ± SD (n) 

 

4.2 Sweat collection 

Sweat was collected using two different collection methods for the aim of using it for lactate 

determination using the standard addition method. 

4.2.1 Sweat collection with pads 

During testing the participant showed a large sweat accumulation and that resulted in that the tape release 

from the skin in the neck. After centrifuging no accumulation of liquid could be observed in the tubes. 

There are multiple probable causes of this, the adsorption power of the pads was too good or that not 

enough sweat was collected during the short intervals. This can be the indication why the pads did not 

become saturated during testing and no fog was observed in the tubes. Due to the failed extraction no 

results could be obtained from this collection. Further test could be done on the headspace of the pads 

via HPLC to evaluate the concentrations.  



 

 

 

 

18 

 

4.2.2 Lactate value in sweat samples 

Sweat collection using the second method resulted in approximately 0.5 ml of sweat that could be used 

for further testing. Using the accumulated sweat through this method the value of lactate within the 

sweat sample could be evaluated using standard addition. The standard addition curves can be seen in 

Figure 12 and through calculation the level of lactate was 14.32 and 7.81 mM for low- and high intensity, 

respectively. The decrease of concentration between low and high intensity may be a result of the 

dilution effect as sweat rate is affected by the intensity. Due to that SR was not determined at the time 

of testing the LER could not be calculated. For further sweat testing, SR should be a collected.  

 

Figure 12, graphs from standard addition for sweat collected at low (LI) and high (HI) intensity.  

  



 

 

 

 

19 

 

4.3 Modification of electrodes 

During the synthesis of oPPy the solution underwent multiple colour changes from a clear solution to a 

black powder as observed in Figure 13. Through the separation process not enough powder was formed 

from the solution using potassium ferrocyanide as oxidative agent. This indicated that there was a too 

low concentration of oxidative agents in these solutions. Further testing with different concentration of 

oxidative agent can be done to evaluate the concentration that results in a higher yield.  

 

Figure 13, the synthesis process for creation of MIPs of oPPy. The different synthesis A-B had potassium ferrocyanide 

(K4Fe(CN)6·3H2O) while C-D had ammonium persulfate ((NH4)2S2O8) as an oxidative agent. B and D had PVA in the 

solution. Pictures at 1: is after addition of Py and LA solution, the solution is clear, 2: after 2 h, the solution has turned light 

pink, 3: after addition of oxidative agent, A and B have a yellow colour while C and D has a black colour, 4: after the 

synthesis was completed, all solutions is dark green, and 5: after decantation of supernatant, a black powder is left. 

The synthesis using ammonium persulfate as oxidative agent resulted in a higher volume of powder, 

which was analysed using FT-IR to evaluate the level of lactic acid left within the polymer network. A 

lower level of lactic acid results in more empty cavities. The spectra can be observed in Figure 14. For 

PPy the peak at 1558 cm-1 is correlated to the C=C bond(49). The C=O and C-O bond in lactic acid is 

observed at 1651 and 1292 cm-1 respectively(49). There is a negative trend at approximately 1651 cm-1 

and a peak can be observed at 1294 cm-1 correlating to lactic acid. The peak at 1294cm-1 is larger for the 

oPPy produced with PVA, this may be a result of the C-O bond in the PVA molecule. Overall there is 

no large difference between the polymers, but further testing was conducted using the PVA free oPPy 

as it may be the one with most cavities.  



 

 

 

 

20 

 

 

Figure 14, FT-IR spectra of oPPy produced with and without PVA, of the wavenumber (cm-1) versus the absorbance. The 

background of the KBr has been subtracted. 

4.3.1 Drop coating of electrodes 

After drying a black layer could be observed on the modified SPCE-AuNP as shown in Figure 15, as 

further shown in Figure 16 the polymer network can be shown compared with the unmodified electrode. 

This indicated that there is a layer of MIPs on the surface of the electrode. 

 

Figure 15, A: picture of SPCE-AuNP after addition of MIP solution, B: picture of the modified electrode after drying. 



 

 

 

 

21 

 

 

Figure 16, SEM picture of the SPCE-AuNP without and with modification. A: SPCE-AuNP at 2000 times magnification, B: 

modified SPCE-AuNP at 2000 times magnification, C: SPCE-AuNP at 10000 times magnification and D: modified SPCE-

AuNP at 10000 times magnification. 

4.4 Calibration with dummy cell  

From the cyclic voltammogram of the dummy cell at different current ranges, the slopes and intersection 

of the linear regression can be seen in Table 7. The low R2 values for the current rang ±1 µA and ±10 

µA may be as the current was greater than the limits and therefore some flat lines can be found in these 

curves, as shown in Figure 17. 

Table 7, results from calibration of dummy during 4 different current range. The slope, intersection and R2 value 

corresponding to each of the ranges. 

Current 

range (µA) Slope Intersection R2 

±1 10.9 4.43 0.64 

±10 1.56 2.39 0.83 

±100 0.99 -0.84 0.99 

±1000 0.99 -7.78 0.99 



 

 

 

 

22 

 

 

Figure 17, curves of the measured current compared to converted current from the calibration of Rodeostat via a dummy 

cell. 

4.5 Concentration curves 

By using the different solutions of known lactic acid content, concentration curves could be obtained.  

4.5.1 SPCE with gold nanoparticles 

From each of the electrodes a cyclic voltammogram for each of the concentrations could be obtained. 

This can be seen in Figure 18, where the local minimum between -1.1 V and -1.5 V is correlating to the 

lactic acid concentration. The peak current is decreasing with the increasing lactic acid concentration 

which is further evaluated to create concentration curves. 



 

 

 

 

23 

 

 

Figure 18, example of cyclic voltammogram measurement from one SPCE-AuNP with increasing concentration of lactic acid 

in 0.1 M KCl (tap water)  

The linear regression obtained from concentration curves are displayed in Figure 19. Where there is a 

clear indication that the R2 is decreasing in combination with an increase in the complexity of the 

solution used, with the exception of the artificial sweat measurement. This can be due to multiple 

parameters such as there being very low concentrations in the artificial sweat and only a few of the many 

analytes found in sweat and therefore being mostly distilled water.  

 

Figure 19, concentration curves obtained from measurement with different solutions. LA11-13: 0.1 M KCl in distilled water, 

LA14-16: 0.1M KCl in tap water, LA17-19: only tap water and LA20-22: artificial sweat. 



 

 

 

 

24 

 

Further comparison between the different solutions can be observed in Table 8, There are no significant 

differences between the different solutions, the slope from each concentration curve is within the same 

range and there are very small difference between the different R2 values. However, the standard 

derivation is smaller for the more complex solutions. 

Table 8, slope and intersection obtained from the different concentration curves using SPCE-AuNP. 

Solution 
Lactic acid 

range 

Slope 

[µA/mM] 

Intersection 

[µA] 
R2 

0.1 M KCl in 

distilled water 
2.5 - 30 mM -11.34 -13.05 0.966 

0.1 M KCl in 

tap water 
2.5 - 60 mM -8.96 -11.93 0.965 

Tap water 2.5 - 60 mM -8.50 -31.67 0.938 

Artificial 

sweat 
2.5 - 60 mM -11.60  -31.23  0.984  

 

4.5.2 SPCE with gold nanoparticles and modification 

Because of the few available modified electrodes only one scan per concentration could be made, which 

increases the uncertainty of the concentration curve. In addition, the electrodes could not be reused so 

each measurement was conducted on a new electrode further increasing the error. With this in mind, the 

results from the concentration curve, shown in Figure 20, is similar to the result from the unmodified 

electrodes tested with artificial sweat. 

 

Figure 20, concentration curve with test of artificial sweat samples on modified SPCE-AuNP. 



 

 

 

 

25 

 

4.5.3 Sweat sensor 

By scanning real sweat samples the lactate concentration could be evaluated. For the modified electrodes 

there was no reusability so different electrodes was used for each measurement (Blank, LI and HI) while 

the unmodified was reused for all three measurements. As seen in Figure 21, a difference can be 

observed between the samples taken during high intensity training compared to the one at low intensity 

training. Lower peak current (at -1.4 to -1.5 V) can be observed for the high intensity exercise compared 

to at low intensity.  

 

Figure 21, cyclic voltammogram of sweat samples on A: SPCE-AuNP and B: modified SPCE-AuNP. 

Using the equation obtained by the concentration curves displayed in Table 8 and Figure 20, the lactate 

concentration can be evaluated. The results of this compared to the true value from the standard addition 

measurement can be observed in Figure 22. 



 

 

 

 

26 

 

 

Figure 22, bar plot over the observed true value of lactate in sweat sample compared with the calculated samples. 

The least difference can be found between the true value and the value calculated from the test of 

artificial sweat on SPCE-AuNP. There is a 12.75 and 9.21 percent difference for the sample during low 

intensity and high intensity respectively. The non-modified electrodes showed in general a higher 

concentration than the true value while the modified showed a lower value. As discussed previously a 

too thick layer of polymer on the electrode can result in a negative effect of the sensing capability. The 

low value from the modified electrodes can be a result of a too thick layer of oPPy, not fully allowing 

the change of lactate to reach the electrode surface properly. More data is needed to be collected to get 

a more statistical foundation of the measurements. 

 

 

 

  



 

 

 

 

27 

 

5. Conclusion and future outlook 

The aim of the project was to produce a non-enzymatic sensory device for lactate measurement in sweat, 

which was succeeded. As shown, there is a great possibility in measuring the lactate concentration of a 

sweat sample using the suggested method. However, there are some additional modification needed 

before a product can be produced and used for these types of measurement. 

The transportation of the newly produced biofluid of interest to the sensors was investigated using a 

sweat collector within this project. A collector made of a less stiff material with a coating would possibly 

make the transport better and would be a contributed factor to possibly have a more continuous 

measurement. Because of the small sample volume of sweat needed for lactate concentration evaluation, 

a test protocol similar to those used for blood lactate measurements should be possible. This as the 

measurement with electrodes is both quick and easy.  

The modification of the electrodes showed to underestimate the lactate value and could not be reused. 

Through a more elaborate study it would be possible to examine more monomers that could be used for 

the modification as well as finding the optimal conditions for production. However, using a more 

complex artificial sweat for calculation of concentration curves would give a more exact results of the 

estimated lactate level. A more complex solution would come closer to sweat and also take into 

consideration how the level amino acids and other organic compounds affects the scans. For a product 

the electrodes should be produced on a flexible surface to be able to put close to the produced sweat. 

Lactate is sweat rate dependent there is a need for measuring the local sweat rate (LSR) at the site where 

the sweat is tested to further use lactate excretion rate (LER) as the measurement instead. There are 

multiple ways of doing this, one way would be to evaluate the Cl- concentration. The relationship 

between Cl- and sweat rate makes it possible to calculate the sweat rate from the concentration. The 

sweat rate can then be used for calculation of LER. The relationship between these parameters needs 

then to be evaluated further. Another possibility would be to use a complementary technique e.g. FT-IR 

for evaluating the content of lactate in sweat, to evaluate the dilution factor further. This by correlating 

the complementary technique with the suggested method. Other parameters that need to be evaluated 

further is how the response from the sensor is affected by the outer environment such as temperature 

and pH. These will be affected during exercise and should be taken into consideration when conducting 

a more complex model for sweat sensing. Further research is also needed to fully understand the 

correlation between the concentration of lactate in blood and sweat to be able to use this sensor for 

lactate threshold evaluation as there is both studies agreeing and disagreeing with this possible 

correlation. With that in mind there is a great possibility for further evaluation of non-enzymatic based 

sweat sensor with a quick response and accurate evaluation of sweat samples as shown in this report.   

  



 

 

 

 

28 

 

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https://blog.iorodeo.com/rodeostat-software/


 

 

 

 

I 

 

Appendix 

A – Contract with participants 

Information regarding study, Development of a Non-

enzymatic, Non-invasive Sensor for Detection of Lactate in 

Sweat. 

Background and aim 

During physical exercise the body needs to produce more energy. If there is a shortage of oxygen for 

the muscles the body needs to find an alternative route for energy production. When there is no oxygen 

the process is called anaerobe and when the muscles start to work anaerobe, lactate starts to accumulate. 

Lactate is a biomarker for fatigue in muscles.  

For many athletes it is important to know at what intensity the anaerobic threshold is, i.e. the highest 

intensity where the oxygen can account for the energy requirement. Lactate is measured in blood during 

increasing exercise intensity to evaluate at which intensity it starts to accumulate. Taking blood sample 

may be a discomfort for the athlete as well as shifting their focus during training. Some research has 

been conducted in sweat sensing where sweat is used instead of blood for measurement of these kinds. 

Most non-invasive sweat sensor is based on degrading of lactate with enzymes. For endurance sport, 

such as marathon’s, an enzyme-based sensor can’t measurer for the total performance time without 

losing effect. Due to the increasing interest in non-invasive measuring methods this work aim to create 

the base for a non-enzymatic and non-invasive sensor for lactate detection. This by evaluating sensory 

alternatives and how it reacts to a complex fluid like sweat. Therefore, the aim of the project is to produce 

a sensory device, using a non-enzymatic solution to obtain quantitative measurement of lactate from 

sweat.  

Study implementation and sampling 

The study wants to follow you during your ordinary training routine and during same prerequisites of 

lactate threshold measurement as you are used to. The main difference is the use of an additional 

sampling during these occasions depending on the test protocol. Additional information from health 

survey as well as weight in connection with exercise will be collected.  

The study aims to test two different concepts. The first being a sweat collector to easily collect sweat 

that is accumulated during physical exercise. This will be tested by attaching the sweat collector to you 

during a standard exercise session. Your performance in heart rate, exercise intensity, observed physical 

changes during testing will be evaluated.  

The second is collection of sweat during similar prerequisites as lactate threshold testing. The sweat will 

be collected and used for detection of lactate by the produced sensor. The data obtained during lactate 

threshold tests will be compared to the data obtained from your sweat samples to be able to see if there 

is an indication of correlation between your blood and sweat lactate values. All information will be 

handled in a safe manner. 

After documentation of your samples, they will be discarded and not traceable back to you. 



 

 

 

 

II 

 

Follow up after the studies implementation 

You will be able to take part of all the results obtained by your samples. 

Risks 

There is no risk associated with sweat sampling for you both in short and long time.  

Advantages of being part of the study 

Due to your patriation in this study you are part of the research for a possible future sweat sensor. The 

results of the study may be used for further work within the area and provide a greater understanding to 

how sweat and blood correlate.  

Data management and privacy 

The data collected during this study will be processed and documented. Your name and personal 

information will be changed to a code during data processing for your security. The key to the code is 

only available for the responsible person of the study. If results from the study is published the results 

will be coded. Your information will be handled according to GDPR. 

Compensation 

Participation in the study is not compensated. 

Voluntary 

Your participant in the study is voluntary and you can cancel your participation without any explanation. 

Additional information 

For additional information regarding the study the following person can be contacted 

 

Annie Ljungh 

Responsible for the project 

Phone:  

Email:  

 

 

 

 

 

 



 

 

 

 

III 

 

 

The undersigned confirms that the active has read and understood the document as well as 

approves the terms in the document 

I voluntarily take responsibility for all known and unknown risks, even if they are caused by negligence. 

 

I will voluntarily and truthfully answer questions concerning my health and medical history that may 

affect activity or cause health risks. 

 

I agree that my results are filed and may be used in research which results in articles published in local 

and international press. 

 

I hereby voluntarily take responsibility for my practice and hereby revoke the persons working with this 

project, the Department of Physics and Chalmers University of Technology from responsibility for my 

safety and health. I hereby disclaim my right to claim compensation, damages or legal liability for bodily 

or material damage or loss even if damage is caused by gross negligence or misconduct. 

 

I have read this disclaimer and fully understand its content. I understand that I give up my legal 

rights by signing the agreement and I do this with free will without compulsion from others 

 

Signature       

  Date:     

 

Clarification of signature:      

     

 

Minor (Age under 18) 

Guardian signature     

  Date:     

 

Clarification of signature:      

     

  



 

 

 

 

IV 

 

Health survey 

Name:     

Day of birth (YYYYMMDD):  Age:    

Phone number:                                 Email:     

Sport:  Comment:      

How would you define your fitness level?    

Have you interrupted training / competition in the last two to three weeks? YES NO 

If yes, why: 

  

 

Do you have/ have had any injury that you believe may affect your performance?        YES  NO 

If yes, which: 

  

 

Do you have /have had any respiratory or cardiovascular disease (e.g. asthma)? YES  NO 

If yes, what/ when: 

  

 

Do you have /have had any other known disease? YES  NO 

If yes, what/ when: 

  

 

Do you use any prescription drugs? YES  NO 

If yes, state which: 

  

 

How long time have you been healthy since your last cold? 

  

 

Have you trained and trained at high intensity sinc