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Non-invasive textile based colorimetric sensor for the simultaneous detection of sweat pH and lactate
Author’s Accepted Manuscript Non-invasive textile based colorimetric sensor for the simultaneous detection of sweat pH and lactate Nadtinan Promphet, Pranee Rattanawaleedirojn, Krisana Siralertmukul, Niphaphun Soatthiyanon, Pranut Potiyaraj, Chusak Thanawattano, Juan P. Hinestroza, Nadnudda Rodthongkum www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(18)31004-X https://doi.org/10.1016/j.talanta.2018.09.086 TAL19100
To appear in: Talanta Received date: 26 May 2018 Revised date: 21 September 2018 Accepted date: 23 September 2018 Cite this article as: Nadtinan Promphet, Pranee Rattanawaleedirojn, Krisana Siralertmukul, Niphaphun Soatthiyanon, Pranut Potiyaraj, Chusak Thanawattano, Juan P. Hinestroza and Nadnudda Rodthongkum, Non-invasive textile based colorimetric sensor for the simultaneous detection of sweat pH and lactate, Talanta, https://doi.org/10.1016/j.talanta.2018.09.086 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Non-invasive textile based colorimetric sensor for the simultaneous detection of sweat pH and lactate Nadtinan Prompheta, Pranee Rattanawaleedirojnb, Krisana Siralertmukulb, Niphaphun Soatthiyanonb, Pranut Potiyarajb, Chusak Thanawattanoc, Juan P. Hinestrozad, Nadnudda Rodthongkumb,* a
Nanoscience and Technology Interdisciplinary Program, Graduate School, Chulalongkorn University, Phayathai Road, Pathumwan, Bangkok 10330, Thailand
b
Metallurgy and Materials Science Research Institute, Chulalongkorn University, Soi Chula 12, Phayathai Road, Pathumwan, Bangkok 10330, Thailand c
National Electronics and Computer Technology Center (NECTEC), Pathumthani 12120, Thailand
d
Department of Fiber Science, College of Human Ecology, Cornell University, Ithaca, New York 14850, United states *
Corresponding author: [email protected]
Abstract A non-invasive textile-based colorimetric sensor for the simultaneous detection of sweat pH and lactate was created by depositing of three different layers onto a cotton fabric: 1.) chitosan, 2.) sodium carboxymethyl cellulose, and 3.) indicator dye or lactate assay. This sensor was characterized using field emission scanning electron microscopy and fourier transform infrared spectroscopy. Then, this sensor was used to measure pH and lactate concentration using the same sweat sample. The sensing element for sweat pH was composed of a mixture of methyl orange and bromocresol green while a lactate enzymatic assay was chosen for the lactate sensor. The pH indicator gradually shifted from red to blue as the pH increased, whereas the purple color intensity increased with the concentration of lactate in the sweat. By comparing these colors with a standard calibration, this platform can be used to estimate the sweat pH (1-14) and the lactate level (0-25 mM). Fading of the colors of this sensor was prevented by using cetyltrimethylammonium bromide. The
flexibility of this textile based sensor allows it to be incorporated into sport apparels and accessories hence potentially enabling real-time and continuous monitoring of sweat pH and lactate. This non-invasive sensing platform might open a new avenue for personal health monitoring and medical diagnosis as well as for determining of the physiological conditions of endurance athletes.
Graphical abstract
Keywords: Non-invasive, textile, cotton, sweat, pH, lactate, colorimetric sensor
1.
Introduction Wearable chemical sensors can be non-invasive and provide real-time and continuous
monitoring of biometric information [1, 2]. To achieve these performance characteristics, the substrate for wearable sensors shall be biocompatible, comfortable, flexible, and stretchable. Various substrates including textile [3], tattoo [2, 4], paper [5] and stretchable elastomer [6] have been employed. Among all, a textile substrate is very attractive because it can be easily manufactured, inexpensive, compatible with human skin and easily incorporated into apparels [7] [8]. Among textiles, cotton is preferable since it is a natural fiber possessing high breathability, comfortability and compatibility with human skin with high water absorbency [9]. Sweat is a transparent biological fluid produced by sweat glands and with a slightly acidic pH of 4.0 – 6.8 for a normal human [10]. Sweat is mainly composed of water and small amounts of electrolytes (e.g. sodium, chloride, potassium, calcium), metabolites (e.g. lactate, creatinine, glucose, uric acid), small molecules (e.g. amino acid, cortisol), and proteins (e.g. interleukins, tumor necrosis factor, neuropeptide) [11, 12]. These biomarkers, can be used to estimate the hydration status [13], cystic fibrosis (CF) [14, 15], physical stress and bone mineral loss [16]. In the certain endurance sports, such as triathlon, running, cycling and boxing, real-time sweat analysis can be very important as it can reflect the physiological conditions for the athlete. Sweat pH is an important parameter as the increase of pH can indicate an enhancement of human sweat rate. Furthermore, it has been reported that sweat pH is directly related to an increase in the sodium concentration and hence indicate dehydration [17, 18]. Lactate is a direct product of anaerobic metabolism and thus an important biomarker for evaluation of performance [19]. In the anaerobic metabolic process, stored glycogen in the muscles is consumed to produce energy and lactate [2, 20]. The accumulation of lactate in the muscles causes soreness, pain and muscle fatigue [20, 21]. Sweat lactate can also provide warning signs for pressure ischemia, reflecting an insufficient oxidative metabolism [22]. Colorimetric, electrochemical, electroluminescent and ion detection techniques have been coupled with various wearable substrates to create chemical sensors with high
selectivity [2, 4, 23-25]. Among all, colorimetric technique offers several advantages because it is easier, inexpensive and the signal can be readily observed and interpreted by naked eyes. Herein, we report on two colorimetric textile-based sensors for the simultaneous detection of sweat pH and lactate. Furthermore, in order to assess the usage feasibility of this textile based sensor, we tested it on a group of human volunteers. 2.
Experimental
2.1 Materials and reagents Scoured and bleached knitted cotton fabrics with an area weight of 163 g/m2 was supplied by Sinsaenee Co., Ltd (Bangkok, Thailand). Screen-printed ink (Expantex ink) from Chiyaboon brother group Co., Ltd (Bangkok, Thailand). Candle wax from Somjai Co., Ltd (Bangkok, Thailand). Chitosan from squid pen (degree of deacetylation (DD) = 85%, Mw = 800,000) was obtained from Biolifeland (Bangkok, Thailand). Sodium carboxymethylcellulose (NaCMC) (Mw 250,000), phosphate buffer saline (PBS), bromocresol green (BCG), methyl orange (MO), cetyltrimethylammonium bromide (CTAB), lactate oxidase (LOx) (from Aerococcus), Horseradish peroxidase (HRP), 4aminoantipyrin
(4-AAP)
and
N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline,
sodium salt, dihydrate (TOOS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Aluminium chloride (AlCl3) was obtained from Ajax Finechem (New South Wales, Australia). Acetic acid and citric acid were purchased from Carlo Erba reagent (Milano, Italy). All chemicals were used as their purchased forms. 2.2 Preparation of textile based colorimetric sensor Figure 1a shows the sweat pH and lactate colorimetric sensor pattern and Figure 1b shows the fabrication process. Initially, 2% w/v chitosan was dissolved in 1% v/v acetic acid under continuous magnetic stirring and then 5%w/w citric acid was added to the chitosan solution. This solution was used to coat the cotton substrate via padding. The wet cotton fabric was dried at 100 ˚C for 180 sec and it was cured at 80 ˚C for 30 sec. For the second layer, the detection area of the sweat pH sensor was fabricated via screen-printing
using a mixture of two indicator dyes: bromocresol green (BCG) and methyl orange (MO). BCG and MO were dissolved in 5 % ethanol and deionized water, respectively. Then, 0.5%w/v BCG and 0.5%w/v MO were mixed at 1:1 ratio and then 0.5%w/v CTAB was added into the mixed indicator. The as-prepared indicator-CTAB solution was used to dissolve 2.5%w/v of NaCMC. Next, 5%w/w citric acid was added to the NaCMC-CTABindicator solution and screen-printed on the cotton substrate which was previously coated with chitosan. The substrate was cured at 170 ˚C for 150 sec. For the third layer, 2.5%w/v of NaCMC was dissolved in deionized water and used to coat onto the NaCMC-CTABindicator/ chitosan coated cotton substrate. The substrate was immersed into 0.25%w/v AlCl3 to create a NaCMC hydrogel structure via chemical crosslinking. After assembly of the three layers, the detection area for pH was soaked in deionized water at least 3 times to eliminate the excess of indicator dye and of AlCl3 and the sensor was dried at 100 ˚C for 300 sec. A hydrophobic material was screen-printed onto the coated textile to guarantee channel separation between the pH and the lactate detection zones. To fabricate the lactate detection area, 50 µL of lactate assay freshly prepared was used. This lactate assay solution contains a 1:1:1:1 mixture of 1 mg/mL HRP, 50 mM 4-AAP, 10 mM TOOS and 50 unit/ mL LOx [26]. For the application of the sensors with the volunteers, Expantex ink was screenprinted on the front of the sensor to separate the pH and lactate detection channel and candle wax was applied on the back side of the sensor to prevent the leaching of the indicator dye and lactate assay during usage.
Figure 1. Schematic illustration of a colorimetric sensor pattern design (a) and fabrication process of textile based colorimetric sensor for the simultaneous detection of sweat pH and lactate (b).
2.3 Characterization of textile based colorimetric sensor The surface morphology of the textile-based colorimetric sensor was investigated by using a JSM-7610F field emission scanning electron microscope (FESEM) with energy dispersive spectroscopy capabilities (Japan Electron Optics Laboratory Co., Ltd, Japan). Fourier transform infrared (FTIR) spectroscopy (PerkinElmer Spectrum One LabX, USA) was carried out in a range of 400-4000 cm-1 to investigate the chemical modification of cotton substrates. The color changes of textile-based colorimetric sensor were captured and extracted by using a camera and a portable spectrophotometer (Datacolor CHECK3, Datacolor, USA), respectively. 2.4 Characterization of sensor color using a colorimeter To prepare the standard calibration charts of pH and lactate, phosphate buffer saline (PBS) with varying pH values (pH of 1.0 to 14.0) and different lactate concentrations were prepared. The standard solutions were dropped in the pH and the lactate detection areas and the color change was captured using a camera. For the pH calibration chart, the color was quantified using a portable spectrophotometer (Datacolor CHECK3, Datacolor, USA), and the color changes were represented in the L*, a*, b* space. For the semi-quantitative analysis of lactate, the color change was analyzed by using an Image J software (National Institute of Health, USA) to measure the mean grey color intensity and quantify the lactate concentration level. 2.5 Simultaneous detection of sweat pH and lactate on volunteers The textile-based colorimetric sensor was combined with transporeTM plaster and directly patched on the skin of the volunteers before exercise. After exercise, a spectrometer was used to analyze the pH and lactate concentration.
2.6 Testing of the color fastness of the textile based colorimetric sensor The color fastness of the textile based colorimetric sensor was examined by soaking the as-prepared sensor in PBS solution (pH 7.4) for 0, 30, and 60 minutes. The color fade of textile based colorimetric sensor was observed and captured with a colorimeter.
3.
Results and discussions
3.1 Physical characterization of different layers of modified cotton Photographs showing the appearance of the different layers of cotton-based sensors are shown in Figure 2. Figure (2a) shows a chitosan/cotton substrate (2b), a NaCMCCTAB-indicator dyes/chitosan/cotton substrate (2c), a NaCMC hydrogel/ NaCMC-CTABindicator dyes/chitosan/cotton substrate (2d), and (2e). NaCMC hydrogel/NaCMC-CTABindicator dyes/ chitosan/cotton substrate with the hydrophobic barrier edge. The original cotton substrate shows a bright white color (Figure 2a) which after the coating of chitosan becomes light yellow (Figure 2b). The chitosan film was coated as a first layer to serve as a dye fixator due to the interaction between positively charged amino groups of chitosan and negatively charged groups of the indicator dyes [27-29]. The presence of the cationic surfactant (CTAB) in the second layer shows a blue-green color (Figure 2c) and facilitates the adsorption and fixation of the indicator dyes on the textile substrate. The NaCMC hydrogel was added to form a hydrogel network on the top of the sensor and it can be seen in a yellow-green color (Figure 2d). The hydrogel layer was used for enzyme entrapment and to provide long-term stability of the lactate oxidase [30]. This hydrogel also facilitated the sweat absorption onto the sensor surface, and automatically pre-concentrated the analyte solution. Figure 2e shows the hydrophobic barrier boundary edge on the textile based sensor for prevention of analyte solution leakage.
Figure 2. Photographs of unmodified cotton substrate (a), chitosan/cotton substrate (b), NaCMC-CTAB-indicator dyes/chitosan/cotton substrate (c), NaCMC hydrogel/NaCMCCTAB-indicator dyes/chitosan/ cotton substrate (d) and NaCMC hydrogel/NaCMC-CTABindicator dyes/ chitosan/cotton substrate with the hydrophobic barrier edge (e). To examine each modified cotton layer, FESEM was used. Figure 3 shows FESEM images of cotton substrate (a), chitosan/cotton substrate (b), NaCMC-CTAB-indicator dyes/chitosan/cotton
substrate
(c) and NaCMC
hydrogel/NaCMC-CTAB-indicator
dyes/chitosan/ cotton substrate (d), respectively. The surface morphology of the cotton substrate shows a smooth fiber surface with an average diameter of 12.7±2.5 µm (Figure 3a). For the chitosan coated cotton substrate, (Figure 3b) the higher roughness which is due to a thin film of chitosan layer. The coating of NaCMC- CTAB-indicator dyes on the chitosan/cotton substrate (Figure 3c) shows a thicker coated film on the cotton with random aggregation which possibly originated when the negatively charged indicator dyes selectively bind to the oppositely charged groups on the surface of the cotton fibers. A FESEM image of NaCMC hydrogel coated cotton substrate (Figure 3d) reveals a thicker NaCMC hydrogel film with small cracking surface at the thick coated area.
Figure 3. FESEM images of cotton substrate (a), chitosan/cotton substrate (b), NaCMCCTAB-indicator dyes/ chitosan substrate (c) and NaCMC hydrogel/ NaCMC-CTABindicator dyes/ chitosan/cotton substrate (d). 3.2 Chemical characterization of the sensor layers A FTIR spectrometer was used to characterize each layer of cotton in a range of 400 – 4000 cm-1 as shown in Figure 4. The FTIR spectrum of the unmodified cotton substrate shows the characteristic peaks of cellulose, the main component of cotton, at 3297, 2899 and 1640 cm-1 corresponding to the O-H, C-H and CH2 vibrations [31]. The FTIR spectrum of the chitosan coated cotton shows peaks at 3334 related to N-H vibrations [29]. For NaCMC-CTAB-indicator dyes/ chitosan and NaCMC hydrogel/ NaCMC-CTAB-indicator
dyes/ chitosan coated cotton substrate, the spectra were very similar to the chitosan coated cotton substrate.
Figure 4. FTIR spectra of different layers of coated cotton substrates.
3.3 Sensor performance testing 3.3.1 Standard color chart of textile based pH sensor A mixed indicator dye containing methyl orange (MO) and bromocresol green (BCG) was selected to indicate the color change as defined by their pKa values [32]. In this study, the color change of pH sensor can be clearly observed by naked eye and it can be divided into 7 domains including pH 1, pH 2, pH 3-6, pH 7, pH 8-10, pH 11-13 and pH
14 depending on the change of the slope of Figure 5b. This means that this sensor can distinguish the change of pH over a typical pH range for human sweat (pH 4.0 – 7.0) [10, 17, 18, 33]. The change of color was quantified by using L* a* b* model as shown in Figure 5b. The value of L* represents brightness (+ = lighter, - = darker) while the a* and b* represent the distance along the red-green (+ = redder, - = greener) and blue-yellow (+ = yellower, - = bluer) axis, respectively [34].
Figure 5. Standard color charts of the textile based pH sensor (a) and L* a* b* color model of pH in 0.1 M PBS solution (b). 3.3.2 Standard color calibration of textile based lactate sensor The intensity of color in the sensor visibly increases as a direct function of lactate concentration as shown in Figure 6a. It should be noted that the lactate concentration in human sweat depends on the metabolism of person [35]. This sensor can be used to differentiate between low (< 10 mM) and high (≥ 12.5 mM) lactate levels in the human sweat [35]. Since this sensor was specially designed for sport application, to protect the muscles soreness, pain, cramp during the exercise, 12.5 mM of lactate was selected as a
threshold level that will remind the sport persons to recognize the abnormality of health status and stop their sport activity in time. This approach might be very useful for determining of the physiological conditions of endurance athletes in the future.
Figure 6. Standard color charts of textile based lactate sensor in the concentration range of 0, 0.5, 2.5, 12.5 and 25 mM (a) and the plot of lactate concentration versus the mean grey color intensity (b). 3.3.3 On-body trials Initially, this sensor was placed on three different positions on the volunteer body including upper arm, lower back and abdomen and the results for both pH and lactate obtained from these areas were not significantly different. Thus, we finally selected the
abdomen position for this sensor because it is the most convenient area on the body for our application purpose, which is integration of this textile based sensor into the sport clothes. This textile-based colorimetric sensor was applied to three human volunteers before asking them to perform physical exercise. The sensor was directly patched on the abdomen of the volunteers (n ≥ 3) before start jogging. After 30 min, the sensor patch was collected, and the color was compared with the standard color chart. The color of both detection areas (pH and lactate) changed after exercise. The color of sweat pH detection area shifted into green as seen in Figure 5a. This result indicates that the sweat pH values of all volunteers range between 8 and 10, and the shift in pH was caused by the higher sweat rate and metabolism rate induced by the physical exercise. For lactate, the intensity of the color increased but that increase depends on the personal metabolism and fitness level of each individual. After 30 minutes of jogging, volunteer 1 had a lactate level approximately 13 mM while volunteers 2 and 3 had the highest lactate concentration, which are approximately 60 and 64 mM, respectively (Figure 7) compared with the standard chart of lactate shown in Figure 6a-b. These results suggest that volunteers 2 and 3 would have muscle fatigue due to the excess amount of lactate in the muscle. These semi-qualitative experiments were performed as proof-of-concept to evaluate the body’s response during exercise.
Figure 7. The textile-based colorimetric sensor tested on three human volunteers after 30 minutes of jogging.
3.3.4 Testing of the color fade of textile based colorimetric sensor The color fastness against sweat during exercise, indicating the durability of the sensor, is an important factor to verify that this sensor is able to be used with performance apparels. For example, if an athlete has a high sweat rate, the sweat may wash out the sensing elements during the exercise leading to the color fade and error in color readout signal. To solve this problem, the addition of CTAB, a common and inexpensive surfactant into the mixture of indicator dyes was carried out, and then the sensors were immersed in a PBS solution at a pH of 7.4. The results indicate that the pH sensing element of the sensor without CTAB was washed out after 30 min, whereas the pH sensing of the sensor with CTAB remained on the textile sensor after 60 min of immersion (Figure 8). Herein, CTAB enhances the interaction between its cations and anions present in indicator dyes. This interaction leads to the incorporation of indicator dye molecules into the micellar structures hence increasing the durability of this sensor [36]. Furthermore, CTAB can maintain the textile flexibility, breathability and comfortability without causing allergy and irritation to the human skin.
Figure 8. Photographs of textile based colorimetric sensors without CTAB (A) and with CTAB(B) after soaking in PBS solution (pH 7.4) for 0, 30, and 60 min. 4.
Conclusions A non-invasive textile based colorimetric sensor for the simultaneous assessment of
sweat pH and lactate level was firstly created. The incorporation of a mixture of MO and BCG indicator dyes and lactate enzymatic assay into cotton substrate was capable of
differentiating the change of sweat pH and lactate levels in human sweat via both naked eye and spectrophotometer. This sensor was successfully applied to three human volunteers before they went for a 30-min jogging and the sensor was able to differentiate the fitness and potential muscle fatigue of these volunteers. Due to the textile nature, these sensors have a high potential to be incorporated in items such as bedsheets, pajamas, shirts, tights, wristbands, headbands, and integrated with wearable device for real time monitoring of human health and athletic performance in a non-intrusive manner in the future.
Acknowledgements Nadtinan Promphet gratefully acknowledges the financial support from the Overseas Research Experience Scholarship for Graduate Student and the 90th Anniversary of Chulalongkorn University, Rachadapisek Sompote Fund. This research was funded by the Ratchadapisek Sompoch Endowment Fund (2017), Chulalongkorn University (761002).
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Highlights
Non-invasive textile-based colorimetric sensor for simultaneous detection of sweat pH and lactate was created.
This sensor was simplily fabricated by coating of cotton with chitosan, sodium carboxymethyl cellulose, and indicator dye or lactate assay.
The sensor was capable of differentiating changes of sweat pH and lactate levels in human sweat via both naked eye with high durability.
This sensor was successfully applied for simultaneous detection of sweat pH and lactate in human volunteers.
PII: DOI: Reference:
S0039-9140(18)31004-X https://doi.org/10.1016/j.talanta.2018.09.086 TAL19100
To appear in: Talanta Received date: 26 May 2018 Revised date: 21 September 2018 Accepted date: 23 September 2018 Cite this article as: Nadtinan Promphet, Pranee Rattanawaleedirojn, Krisana Siralertmukul, Niphaphun Soatthiyanon, Pranut Potiyaraj, Chusak Thanawattano, Juan P. Hinestroza and Nadnudda Rodthongkum, Non-invasive textile based colorimetric sensor for the simultaneous detection of sweat pH and lactate, Talanta, https://doi.org/10.1016/j.talanta.2018.09.086 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Non-invasive textile based colorimetric sensor for the simultaneous detection of sweat pH and lactate Nadtinan Prompheta, Pranee Rattanawaleedirojnb, Krisana Siralertmukulb, Niphaphun Soatthiyanonb, Pranut Potiyarajb, Chusak Thanawattanoc, Juan P. Hinestrozad, Nadnudda Rodthongkumb,* a
Nanoscience and Technology Interdisciplinary Program, Graduate School, Chulalongkorn University, Phayathai Road, Pathumwan, Bangkok 10330, Thailand
b
Metallurgy and Materials Science Research Institute, Chulalongkorn University, Soi Chula 12, Phayathai Road, Pathumwan, Bangkok 10330, Thailand c
National Electronics and Computer Technology Center (NECTEC), Pathumthani 12120, Thailand
d
Department of Fiber Science, College of Human Ecology, Cornell University, Ithaca, New York 14850, United states *
Corresponding author: [email protected]
Abstract A non-invasive textile-based colorimetric sensor for the simultaneous detection of sweat pH and lactate was created by depositing of three different layers onto a cotton fabric: 1.) chitosan, 2.) sodium carboxymethyl cellulose, and 3.) indicator dye or lactate assay. This sensor was characterized using field emission scanning electron microscopy and fourier transform infrared spectroscopy. Then, this sensor was used to measure pH and lactate concentration using the same sweat sample. The sensing element for sweat pH was composed of a mixture of methyl orange and bromocresol green while a lactate enzymatic assay was chosen for the lactate sensor. The pH indicator gradually shifted from red to blue as the pH increased, whereas the purple color intensity increased with the concentration of lactate in the sweat. By comparing these colors with a standard calibration, this platform can be used to estimate the sweat pH (1-14) and the lactate level (0-25 mM). Fading of the colors of this sensor was prevented by using cetyltrimethylammonium bromide. The
flexibility of this textile based sensor allows it to be incorporated into sport apparels and accessories hence potentially enabling real-time and continuous monitoring of sweat pH and lactate. This non-invasive sensing platform might open a new avenue for personal health monitoring and medical diagnosis as well as for determining of the physiological conditions of endurance athletes.
Graphical abstract
Keywords: Non-invasive, textile, cotton, sweat, pH, lactate, colorimetric sensor
1.
Introduction Wearable chemical sensors can be non-invasive and provide real-time and continuous
monitoring of biometric information [1, 2]. To achieve these performance characteristics, the substrate for wearable sensors shall be biocompatible, comfortable, flexible, and stretchable. Various substrates including textile [3], tattoo [2, 4], paper [5] and stretchable elastomer [6] have been employed. Among all, a textile substrate is very attractive because it can be easily manufactured, inexpensive, compatible with human skin and easily incorporated into apparels [7] [8]. Among textiles, cotton is preferable since it is a natural fiber possessing high breathability, comfortability and compatibility with human skin with high water absorbency [9]. Sweat is a transparent biological fluid produced by sweat glands and with a slightly acidic pH of 4.0 – 6.8 for a normal human [10]. Sweat is mainly composed of water and small amounts of electrolytes (e.g. sodium, chloride, potassium, calcium), metabolites (e.g. lactate, creatinine, glucose, uric acid), small molecules (e.g. amino acid, cortisol), and proteins (e.g. interleukins, tumor necrosis factor, neuropeptide) [11, 12]. These biomarkers, can be used to estimate the hydration status [13], cystic fibrosis (CF) [14, 15], physical stress and bone mineral loss [16]. In the certain endurance sports, such as triathlon, running, cycling and boxing, real-time sweat analysis can be very important as it can reflect the physiological conditions for the athlete. Sweat pH is an important parameter as the increase of pH can indicate an enhancement of human sweat rate. Furthermore, it has been reported that sweat pH is directly related to an increase in the sodium concentration and hence indicate dehydration [17, 18]. Lactate is a direct product of anaerobic metabolism and thus an important biomarker for evaluation of performance [19]. In the anaerobic metabolic process, stored glycogen in the muscles is consumed to produce energy and lactate [2, 20]. The accumulation of lactate in the muscles causes soreness, pain and muscle fatigue [20, 21]. Sweat lactate can also provide warning signs for pressure ischemia, reflecting an insufficient oxidative metabolism [22]. Colorimetric, electrochemical, electroluminescent and ion detection techniques have been coupled with various wearable substrates to create chemical sensors with high
selectivity [2, 4, 23-25]. Among all, colorimetric technique offers several advantages because it is easier, inexpensive and the signal can be readily observed and interpreted by naked eyes. Herein, we report on two colorimetric textile-based sensors for the simultaneous detection of sweat pH and lactate. Furthermore, in order to assess the usage feasibility of this textile based sensor, we tested it on a group of human volunteers. 2.
Experimental
2.1 Materials and reagents Scoured and bleached knitted cotton fabrics with an area weight of 163 g/m2 was supplied by Sinsaenee Co., Ltd (Bangkok, Thailand). Screen-printed ink (Expantex ink) from Chiyaboon brother group Co., Ltd (Bangkok, Thailand). Candle wax from Somjai Co., Ltd (Bangkok, Thailand). Chitosan from squid pen (degree of deacetylation (DD) = 85%, Mw = 800,000) was obtained from Biolifeland (Bangkok, Thailand). Sodium carboxymethylcellulose (NaCMC) (Mw 250,000), phosphate buffer saline (PBS), bromocresol green (BCG), methyl orange (MO), cetyltrimethylammonium bromide (CTAB), lactate oxidase (LOx) (from Aerococcus), Horseradish peroxidase (HRP), 4aminoantipyrin
(4-AAP)
and
N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline,
sodium salt, dihydrate (TOOS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Aluminium chloride (AlCl3) was obtained from Ajax Finechem (New South Wales, Australia). Acetic acid and citric acid were purchased from Carlo Erba reagent (Milano, Italy). All chemicals were used as their purchased forms. 2.2 Preparation of textile based colorimetric sensor Figure 1a shows the sweat pH and lactate colorimetric sensor pattern and Figure 1b shows the fabrication process. Initially, 2% w/v chitosan was dissolved in 1% v/v acetic acid under continuous magnetic stirring and then 5%w/w citric acid was added to the chitosan solution. This solution was used to coat the cotton substrate via padding. The wet cotton fabric was dried at 100 ˚C for 180 sec and it was cured at 80 ˚C for 30 sec. For the second layer, the detection area of the sweat pH sensor was fabricated via screen-printing
using a mixture of two indicator dyes: bromocresol green (BCG) and methyl orange (MO). BCG and MO were dissolved in 5 % ethanol and deionized water, respectively. Then, 0.5%w/v BCG and 0.5%w/v MO were mixed at 1:1 ratio and then 0.5%w/v CTAB was added into the mixed indicator. The as-prepared indicator-CTAB solution was used to dissolve 2.5%w/v of NaCMC. Next, 5%w/w citric acid was added to the NaCMC-CTABindicator solution and screen-printed on the cotton substrate which was previously coated with chitosan. The substrate was cured at 170 ˚C for 150 sec. For the third layer, 2.5%w/v of NaCMC was dissolved in deionized water and used to coat onto the NaCMC-CTABindicator/ chitosan coated cotton substrate. The substrate was immersed into 0.25%w/v AlCl3 to create a NaCMC hydrogel structure via chemical crosslinking. After assembly of the three layers, the detection area for pH was soaked in deionized water at least 3 times to eliminate the excess of indicator dye and of AlCl3 and the sensor was dried at 100 ˚C for 300 sec. A hydrophobic material was screen-printed onto the coated textile to guarantee channel separation between the pH and the lactate detection zones. To fabricate the lactate detection area, 50 µL of lactate assay freshly prepared was used. This lactate assay solution contains a 1:1:1:1 mixture of 1 mg/mL HRP, 50 mM 4-AAP, 10 mM TOOS and 50 unit/ mL LOx [26]. For the application of the sensors with the volunteers, Expantex ink was screenprinted on the front of the sensor to separate the pH and lactate detection channel and candle wax was applied on the back side of the sensor to prevent the leaching of the indicator dye and lactate assay during usage.
Figure 1. Schematic illustration of a colorimetric sensor pattern design (a) and fabrication process of textile based colorimetric sensor for the simultaneous detection of sweat pH and lactate (b).
2.3 Characterization of textile based colorimetric sensor The surface morphology of the textile-based colorimetric sensor was investigated by using a JSM-7610F field emission scanning electron microscope (FESEM) with energy dispersive spectroscopy capabilities (Japan Electron Optics Laboratory Co., Ltd, Japan). Fourier transform infrared (FTIR) spectroscopy (PerkinElmer Spectrum One LabX, USA) was carried out in a range of 400-4000 cm-1 to investigate the chemical modification of cotton substrates. The color changes of textile-based colorimetric sensor were captured and extracted by using a camera and a portable spectrophotometer (Datacolor CHECK3, Datacolor, USA), respectively. 2.4 Characterization of sensor color using a colorimeter To prepare the standard calibration charts of pH and lactate, phosphate buffer saline (PBS) with varying pH values (pH of 1.0 to 14.0) and different lactate concentrations were prepared. The standard solutions were dropped in the pH and the lactate detection areas and the color change was captured using a camera. For the pH calibration chart, the color was quantified using a portable spectrophotometer (Datacolor CHECK3, Datacolor, USA), and the color changes were represented in the L*, a*, b* space. For the semi-quantitative analysis of lactate, the color change was analyzed by using an Image J software (National Institute of Health, USA) to measure the mean grey color intensity and quantify the lactate concentration level. 2.5 Simultaneous detection of sweat pH and lactate on volunteers The textile-based colorimetric sensor was combined with transporeTM plaster and directly patched on the skin of the volunteers before exercise. After exercise, a spectrometer was used to analyze the pH and lactate concentration.
2.6 Testing of the color fastness of the textile based colorimetric sensor The color fastness of the textile based colorimetric sensor was examined by soaking the as-prepared sensor in PBS solution (pH 7.4) for 0, 30, and 60 minutes. The color fade of textile based colorimetric sensor was observed and captured with a colorimeter.
3.
Results and discussions
3.1 Physical characterization of different layers of modified cotton Photographs showing the appearance of the different layers of cotton-based sensors are shown in Figure 2. Figure (2a) shows a chitosan/cotton substrate (2b), a NaCMCCTAB-indicator dyes/chitosan/cotton substrate (2c), a NaCMC hydrogel/ NaCMC-CTABindicator dyes/chitosan/cotton substrate (2d), and (2e). NaCMC hydrogel/NaCMC-CTABindicator dyes/ chitosan/cotton substrate with the hydrophobic barrier edge. The original cotton substrate shows a bright white color (Figure 2a) which after the coating of chitosan becomes light yellow (Figure 2b). The chitosan film was coated as a first layer to serve as a dye fixator due to the interaction between positively charged amino groups of chitosan and negatively charged groups of the indicator dyes [27-29]. The presence of the cationic surfactant (CTAB) in the second layer shows a blue-green color (Figure 2c) and facilitates the adsorption and fixation of the indicator dyes on the textile substrate. The NaCMC hydrogel was added to form a hydrogel network on the top of the sensor and it can be seen in a yellow-green color (Figure 2d). The hydrogel layer was used for enzyme entrapment and to provide long-term stability of the lactate oxidase [30]. This hydrogel also facilitated the sweat absorption onto the sensor surface, and automatically pre-concentrated the analyte solution. Figure 2e shows the hydrophobic barrier boundary edge on the textile based sensor for prevention of analyte solution leakage.
Figure 2. Photographs of unmodified cotton substrate (a), chitosan/cotton substrate (b), NaCMC-CTAB-indicator dyes/chitosan/cotton substrate (c), NaCMC hydrogel/NaCMCCTAB-indicator dyes/chitosan/ cotton substrate (d) and NaCMC hydrogel/NaCMC-CTABindicator dyes/ chitosan/cotton substrate with the hydrophobic barrier edge (e). To examine each modified cotton layer, FESEM was used. Figure 3 shows FESEM images of cotton substrate (a), chitosan/cotton substrate (b), NaCMC-CTAB-indicator dyes/chitosan/cotton
substrate
(c) and NaCMC
hydrogel/NaCMC-CTAB-indicator
dyes/chitosan/ cotton substrate (d), respectively. The surface morphology of the cotton substrate shows a smooth fiber surface with an average diameter of 12.7±2.5 µm (Figure 3a). For the chitosan coated cotton substrate, (Figure 3b) the higher roughness which is due to a thin film of chitosan layer. The coating of NaCMC- CTAB-indicator dyes on the chitosan/cotton substrate (Figure 3c) shows a thicker coated film on the cotton with random aggregation which possibly originated when the negatively charged indicator dyes selectively bind to the oppositely charged groups on the surface of the cotton fibers. A FESEM image of NaCMC hydrogel coated cotton substrate (Figure 3d) reveals a thicker NaCMC hydrogel film with small cracking surface at the thick coated area.
Figure 3. FESEM images of cotton substrate (a), chitosan/cotton substrate (b), NaCMCCTAB-indicator dyes/ chitosan substrate (c) and NaCMC hydrogel/ NaCMC-CTABindicator dyes/ chitosan/cotton substrate (d). 3.2 Chemical characterization of the sensor layers A FTIR spectrometer was used to characterize each layer of cotton in a range of 400 – 4000 cm-1 as shown in Figure 4. The FTIR spectrum of the unmodified cotton substrate shows the characteristic peaks of cellulose, the main component of cotton, at 3297, 2899 and 1640 cm-1 corresponding to the O-H, C-H and CH2 vibrations [31]. The FTIR spectrum of the chitosan coated cotton shows peaks at 3334 related to N-H vibrations [29]. For NaCMC-CTAB-indicator dyes/ chitosan and NaCMC hydrogel/ NaCMC-CTAB-indicator
dyes/ chitosan coated cotton substrate, the spectra were very similar to the chitosan coated cotton substrate.
Figure 4. FTIR spectra of different layers of coated cotton substrates.
3.3 Sensor performance testing 3.3.1 Standard color chart of textile based pH sensor A mixed indicator dye containing methyl orange (MO) and bromocresol green (BCG) was selected to indicate the color change as defined by their pKa values [32]. In this study, the color change of pH sensor can be clearly observed by naked eye and it can be divided into 7 domains including pH 1, pH 2, pH 3-6, pH 7, pH 8-10, pH 11-13 and pH
14 depending on the change of the slope of Figure 5b. This means that this sensor can distinguish the change of pH over a typical pH range for human sweat (pH 4.0 – 7.0) [10, 17, 18, 33]. The change of color was quantified by using L* a* b* model as shown in Figure 5b. The value of L* represents brightness (+ = lighter, - = darker) while the a* and b* represent the distance along the red-green (+ = redder, - = greener) and blue-yellow (+ = yellower, - = bluer) axis, respectively [34].
Figure 5. Standard color charts of the textile based pH sensor (a) and L* a* b* color model of pH in 0.1 M PBS solution (b). 3.3.2 Standard color calibration of textile based lactate sensor The intensity of color in the sensor visibly increases as a direct function of lactate concentration as shown in Figure 6a. It should be noted that the lactate concentration in human sweat depends on the metabolism of person [35]. This sensor can be used to differentiate between low (< 10 mM) and high (≥ 12.5 mM) lactate levels in the human sweat [35]. Since this sensor was specially designed for sport application, to protect the muscles soreness, pain, cramp during the exercise, 12.5 mM of lactate was selected as a
threshold level that will remind the sport persons to recognize the abnormality of health status and stop their sport activity in time. This approach might be very useful for determining of the physiological conditions of endurance athletes in the future.
Figure 6. Standard color charts of textile based lactate sensor in the concentration range of 0, 0.5, 2.5, 12.5 and 25 mM (a) and the plot of lactate concentration versus the mean grey color intensity (b). 3.3.3 On-body trials Initially, this sensor was placed on three different positions on the volunteer body including upper arm, lower back and abdomen and the results for both pH and lactate obtained from these areas were not significantly different. Thus, we finally selected the
abdomen position for this sensor because it is the most convenient area on the body for our application purpose, which is integration of this textile based sensor into the sport clothes. This textile-based colorimetric sensor was applied to three human volunteers before asking them to perform physical exercise. The sensor was directly patched on the abdomen of the volunteers (n ≥ 3) before start jogging. After 30 min, the sensor patch was collected, and the color was compared with the standard color chart. The color of both detection areas (pH and lactate) changed after exercise. The color of sweat pH detection area shifted into green as seen in Figure 5a. This result indicates that the sweat pH values of all volunteers range between 8 and 10, and the shift in pH was caused by the higher sweat rate and metabolism rate induced by the physical exercise. For lactate, the intensity of the color increased but that increase depends on the personal metabolism and fitness level of each individual. After 30 minutes of jogging, volunteer 1 had a lactate level approximately 13 mM while volunteers 2 and 3 had the highest lactate concentration, which are approximately 60 and 64 mM, respectively (Figure 7) compared with the standard chart of lactate shown in Figure 6a-b. These results suggest that volunteers 2 and 3 would have muscle fatigue due to the excess amount of lactate in the muscle. These semi-qualitative experiments were performed as proof-of-concept to evaluate the body’s response during exercise.
Figure 7. The textile-based colorimetric sensor tested on three human volunteers after 30 minutes of jogging.
3.3.4 Testing of the color fade of textile based colorimetric sensor The color fastness against sweat during exercise, indicating the durability of the sensor, is an important factor to verify that this sensor is able to be used with performance apparels. For example, if an athlete has a high sweat rate, the sweat may wash out the sensing elements during the exercise leading to the color fade and error in color readout signal. To solve this problem, the addition of CTAB, a common and inexpensive surfactant into the mixture of indicator dyes was carried out, and then the sensors were immersed in a PBS solution at a pH of 7.4. The results indicate that the pH sensing element of the sensor without CTAB was washed out after 30 min, whereas the pH sensing of the sensor with CTAB remained on the textile sensor after 60 min of immersion (Figure 8). Herein, CTAB enhances the interaction between its cations and anions present in indicator dyes. This interaction leads to the incorporation of indicator dye molecules into the micellar structures hence increasing the durability of this sensor [36]. Furthermore, CTAB can maintain the textile flexibility, breathability and comfortability without causing allergy and irritation to the human skin.
Figure 8. Photographs of textile based colorimetric sensors without CTAB (A) and with CTAB(B) after soaking in PBS solution (pH 7.4) for 0, 30, and 60 min. 4.
Conclusions A non-invasive textile based colorimetric sensor for the simultaneous assessment of
sweat pH and lactate level was firstly created. The incorporation of a mixture of MO and BCG indicator dyes and lactate enzymatic assay into cotton substrate was capable of
differentiating the change of sweat pH and lactate levels in human sweat via both naked eye and spectrophotometer. This sensor was successfully applied to three human volunteers before they went for a 30-min jogging and the sensor was able to differentiate the fitness and potential muscle fatigue of these volunteers. Due to the textile nature, these sensors have a high potential to be incorporated in items such as bedsheets, pajamas, shirts, tights, wristbands, headbands, and integrated with wearable device for real time monitoring of human health and athletic performance in a non-intrusive manner in the future.
Acknowledgements Nadtinan Promphet gratefully acknowledges the financial support from the Overseas Research Experience Scholarship for Graduate Student and the 90th Anniversary of Chulalongkorn University, Rachadapisek Sompote Fund. This research was funded by the Ratchadapisek Sompoch Endowment Fund (2017), Chulalongkorn University (761002).
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Highlights
Non-invasive textile-based colorimetric sensor for simultaneous detection of sweat pH and lactate was created.
This sensor was simplily fabricated by coating of cotton with chitosan, sodium carboxymethyl cellulose, and indicator dye or lactate assay.
The sensor was capable of differentiating changes of sweat pH and lactate levels in human sweat via both naked eye with high durability.
This sensor was successfully applied for simultaneous detection of sweat pH and lactate in human volunteers.