Biosensors and Bioelectronics 67 (2015) 570–575
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Chemiluminescent imaging of transpired ethanol from the palm for evaluation of alcohol metabolism Takahiro Arakawa, Kazutaka Kita, Xin Wang, Kumiko Miyajima, Koji Toma, Kohji Mitsubayashi n Department of Biomedical Devices and Instrumentation, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10, Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
art ic l e i nf o
a b s t r a c t
Article history: Received 7 June 2014 Received in revised form 18 September 2014 Accepted 21 September 2014 Available online 28 September 2014
A 2-dimensional imaging system was constructed and applied in measurements of gaseous ethanol emissions from the human palm. This imaging system measures gaseous ethanol concentrations as intensities of chemiluminescence by luminol reaction induced by alcohol oxidase and luminol–hydrogen peroxide–horseradish peroxidase system. Conversions of ethanol distributions and concentrations to 2-dimensional chemiluminescence were conducted on an enzyme-immobilized mesh substrate in a dark box, which contained a luminol solution. In order to visualize ethanol emissions from human palm skin, we developed highly sensitive and selective imaging system for transpired gaseous ethanol at sub ppmlevels. Thus, a mixture of a high-purity luminol solution of luminol sodium salt HG solution instead of standard luminol solution and an enhancer of eosin Y solution was adapted to refine the chemiluminescent intensity of the imaging system, and improved the detection limit to 3 ppm gaseous ethanol. The highly sensitive imaging allows us to successfully visualize the emissions dynamics of transdermal gaseous ethanol. The intensity of each site on the palm shows the reflection of ethanol concentrations distributions corresponding to the amount of alcohol metabolized upon consumption. This imaging system is significant and useful for the assessment of ethanol measurement of the palmar skin. & 2014 Elsevier B.V. All rights reserved.
Keywords: Transdermal emissions Imaging system Gaseous ethanol Chemiluminescence Oral alcohol administration
1. Introduction Various volatile organic compounds (VOCs) exist, such as those the transpired by humans, breath, body odor, smell of the living environment and aroma of food (Lindinger et al., 1998; Hierlemann and Gutierrez-Osuna, 2008; Shirasu and Touhara, 2011; Zhang and Li, 2010). Certain compounds, which are an indicator of disease may also be metabolized by a body, a skin, and breath (Miekisch et al., 2004). The human body emits various non-volatile and volatile molecules, depending on a person’s genetics, stress and immune status (Pandey and Kim, 2011). Human odor caused by the combined action of the skin gland and volatile organic compounds, which are regulated by human hormonal control and the bacterial population localized at skin surface (Natale et al., 2000). Metabolism of the body can be screened by analyzing a volatile human ingredient. If the volatile organic compounds of human body can be analyzed in relation to human health, a physician and a patient easily understand and evaluate the result of a diagnosis by screening human odor. n
Corresponding author. Tel.: þ 81 3 5280 8091; fax: þ 81 3 5280 8094. E-mail address: [email protected] (K. Mitsubayashi).
http://dx.doi.org/10.1016/j.bios.2014.09.045 0956-5663/& 2014 Elsevier B.V. All rights reserved.
Non-invasive diagnostic methods based on analysis of gas emitted by the body have been used. These include tests for urea expiration and tests for diagnosis of halitosis (Francesco et al., 2005; Chan et al., 2009; Natale et al., 2014). There were any problems to diagnose and monitor of emission of gas from human body because of restrictive diagnosis method. As a result, we cannot measure consecutively of expiration collection. In addition, in the bad breath diagnosis, as for the method with gas chromatography and a gas sensor, there were some problems with handiness and selectivity (Natale et al., 2014). For example, a medical doctor and a third party may directly smell and diagnose the bad breath of the patient. However, this diagnosis is subjective and inaccurate, because the sensing of bad breath depends on the olfactory senses of an individual. Since modern breath analysis started in the 1970s, gas chromatography has been used to identify more than 200 components in human exhaled breath (Lindinger et al., 1998). However, a large volume of exhaled breath was required for diagnosis and the liquid nitrogen or liquid argon was used for cryogenic pre-concentration, which was necessary for sample analysis (Philipp et al., 2004). Isotopes of 13C used in breath tests in the diagnosis of Helicobacter pylori infections, have been successfully used as diagnostic method (Romagnuolo et al., 2002). Furthermore, a gas emission from skin surface is lower
T. Arakawa et al. / Biosensors and Bioelectronics 67 (2015) 570–575
concentration than that of breath gas expiration. Thus, highly sensitive techniques for continuously and selectively measuring volatile chemicals generated by the body are essential for noninvasive diagnosis. It has been reported that the volatile chemical ingredient associated with disease and the physical physiological state including in skin gas like expiration. For example, acetone, hydrogen, alcohol was included in volatile chemical components, which was well known to researchers (Naitoh et al., 2000; Natale et al., 2000; Rock et al., 2008; Costello et al., 2014). However, sensors and measurement systems for volatiles are expensive and cumbersome to use in a medical facility. Furthermore, gas chromatographs and mass spectrometers, which are used to analyze these compounds, have demanding requirements for installation, and monitoring temporal changes in emissions of gaseous components is difficult. A bracelet-type sensor (SCRAMTM) was used to monitor ethanol concentrations in transdermal gas to measure the quantity of alcohol consumed. This sensor, which could be used in clinical and practical applications (Sakai et al., 2006; Barnett et al., 2011) was used to monitor transdermal alcohol emissions every 30 minutes for 2 weeks. Criteria for detecting alcohol from human skin for this sensor provided an accurate and unconventional approach for use with non-mandated clients. A number of biosensors have been developed for the measurement of methanol, ethanol and lower alcohols. The measurement of ethanol, in particular, is important essential for forensic science, clinical chemistry and analytical chemistry. In addition, food products, beverages and the wine industry are interested in analytical methods to control the quality and processing of the food products (Wen et al., 2007). A standard alcohol biosensor is composed of an alcohol oxidase immobilized membrane and a commercial oxygen sensor. The quantity of ethanol is determination by the change in the level of dissolved oxygen in the solution. Many sensor technologies employing enzymatic reactions have been developed, such as biochemical gas sensors (bio-sniffers) for gaseous formaldehyde and ethanol, as well as a NADH-dependent fiber-optic biosensor for the determination of gaseous components determination (Kudo et al., 2010, 2012). Also the enzyme-based chemical gas sensors are highly selective and sensitive for target chemicals. We recently obtained human body information by various kinds of methods for the treatment of a variety of diseases. Mainly, blood and urine have been used for the screening of diseases because of high reliability sample of human body. Recently, sweat, exhaled breath and saliva also used for sample for it. In this paper, we focused on the imaging of transdermal emissions of gaseous ethanol from human palms at hands with alcohol metabolism in
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human body after oral alcohol administration. This method enables non-invasive, pain-less and blood-less monitoring of information of human body. We can obtain various information of human body by analyzing the ingredient included in transpired emissions. In our previous study, we developed an imaging system of gaseous ethanol based on chemiluminescence measurement employing an EM-CCD camera for breath gaseous ethanol imaging (Wang et al., 2010; Arakawa et al., 2013a, 2013b). However, measurements of transdermal human gas through the system have not been sufficiently evaluated. Therefore, in this study, we developed a novel non-invasive temporal imaging system of transdermal gaseous ethanol to analyze alcohol metabolism in real-time. We measured the changes in chemiluminescence intensity due to emission of transdermal gaseous ethanol after alcohol administration.
2. Experimental 2.1. Chemicals and apparatus A system for imaging the movement of ethanol was developed as shown in Fig. 1. This imaging system consisted of an electromultiplying CCD camera (L3C95-05, pixel size: 15 35.5 μm2, image format: 768 244 pixels, spectral range: 400–1060 nm, e2v technologies limited, United Kingdom) and a video encoder (GV-MDVD3, I-O DATA, Japan). Data analysis of the recorded chemiluminescence was analyzed by Cosmos 32 software (Library Inc., Japan). All solutions were prepared in deionized distilled water obtained from a Milli-Q purification system (Millipore Co., USA). Cotton mesh substrates used for imaging (cotton: 100%, thickness: 1 mm, and interval size: 1 mm, Pip-Fujimoto Co., Japan) was evaluated for enzyme stabilization. Alcohol oxidase (AOD, E. C.1.1.3.13, A2404-1kU, 10–40 units mg 1 protein, from Pichia pastoris, Sigma-Aldrich Co., USA) and horseradish peroxidase (HRP, E. C.1.11.1.7, 169-10791, 100 units mg 1, Wako Pure Chemical Industries, Ltd., Japan), photo-crosslinkable poly(vinyl alcohol) containing stilbazolium groups (PVA-SbQ, type: SPH, 9C-10L, 10.4 wt%, Toyo Gosei Co., Ltd., Japan) were used for enzyme stabilization on the mesh substrate. A 5.0 mmol/l luminol (01253-60, Kanto Chemical Co., Inc., Japan) solution was prepared in Tris–HCl buffer solution (100 mmol/l) for measurement of chemiluminescence generated by ethanol (Wang et al., 2011). In addition, to improve the intensity of gaseous ethanol, we selected luminol sodium salt HG (5-amino-2,3-dihydro-1.4-phathalazinedione sodium salt, Lot. CDR7140, Wako Pure Chemical Industries, Ltd.) and eosin Y (Sodium Tetrabromofluorescein, Lot. CDM1099, Wako Pure
dark box AOD/HRP immobilized mesh
mass flow controller
EM-CCD
ethanol vapor 200 ml/min chemi-luminescence
gas generator
computer
video encoder
HDD recorder
Fig. 1. Schematic of the gaseous ethanol imaging system for standard gaseous ethanol. This system consisted of AOD and HRP immobilized on mesh substrate, an electromultiplying CCD camera and a standard gas generator machine.
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Chemical Industries, Ltd.). We adjusted these reagents with Tris– HCL buffer. 2.2. Principle of gaseous ethanol imaging In principle, ethanol is oxidized to acetaldehyde and hydrogen peroxide by alcohol oxidase (AOD) in the presence of oxygen. The hydrogen peroxide (HRP) reacts with the luminol solution by catalysis of HRP and resulting in the chemiluminescence. These reactions are described as follows (Fletcher et al., 2001): AOD
ethanol+O2 ⟹acetaldehyde + H2 O2 HRP
luminol +H2O2 + OH− ⟹3−aminophthate + 2H2 O+ N2 + hν
(1)
Fig. 2. Schematic view of the acrylic cell plate for imaging of transdermal ethanol from the human hand. The diameter of the hole was Ø6.0 mm, and the interval between holes was 16 mm. These holes reduced diffusion of transdermal gaseous ethanol.
(2)
These enzymes from AOD and HRP immobilized substrate were prepared with PVA-SbQ and used for gaseous ethanol imaging. For fabrication of the enzyme-immobilized substrate, AOD and HRP were dissolved in phosphate buffer solution (PB, 0.1 mmol/l, pH7.5), mixed with PVA-SbQ in a volume/weight ratio of 1:2. The enzymes/PVA-SbQ mixture was coated onto the mesh substrate, spread and then cured for 3 h at 4 °C in a dark box. The substrate was cured under low power ultraviolet (UV) irradiation for 5 min. A standard gaseous ethanol was supplied at a flow rate of 200 ml/min for 20 s from a gas generator unit, which was also used a standard gas generator for calibration purposes. 2.3. Patch test for imaging of human skin An enzyme of acetaldehyde type 2 deficiency is involved in the metabolism of acetaldehyde, which is a toxic metabolite of ethanol. The skin patch test for ethanol was performed following the method reported (Higuchi et al., 1987). A patch plaster of lint pads on an adhesive tape was prepared, 100 μL of 70% ethanol was placed on one of the lint pads, and as a control, and the same volume of distilled water was put on another pad. The patches were taped to the inner surface of the arm for 7 min and then removed slowly. Patch areas that showed erythema after removal corresponded to a positive test result for reduced ALDH 2 activity (Wang et al., 2011). In this experiment, an ALDH 2 negative volunteer was selected for the further experiments after oral administration of alcohol. 2.4. Imaging and measurement of transpired ethanol Experiments on the palm were carried out to evaluate gas transpired from human skin (Zhang and Li, 2010). Therefore, we improved the gaseous ethanol imaging system to the transdermal ethanol imaging system from the human palm. Imaging of ethanol released from the palm part was carried out after drinking. Due to monitor a wide skin area for the skin gas measurement, an acrylic cell plate was indispensable part for retaining gap between the skin side and the enzyme mesh for quantitative measurement. We manufactured the pattern plate, which formed the cell of the same shape into dots form covering the enzyme immobilization mesh. The enzyme immobilized mesh had same size of square pattern at a square 10 mm on a side; interval of squares was 16 mm. The circular cell (5 mm depth, 6 mm diameter, 141.4 mm3 capacity) plate of the same dot pattern to control diffusion of the transdermal gas with the enzyme square mesh of the palm shape was shown in Fig. 2. The mesh with square patterns was soaked in 5 mmol/l luminol of high grade and 3.0 μmol/l eosin Y solutions. And palm part of the volunteer adheres to a cell plate after the drinking and recorded the emission of light with the outbreak of
the skin gas by an EM-CCD camera. Fig. 3 shows a schematic view of the measurement system for transdermal ethanol. For measurement of transdermal ethanol from the palm, gaseous ethanol samples were collected after oral administration of alcohol and measured authorizing by the Human Investigations Committee of Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (authorization code: 2012-06, permitted since 26th March, 2013), acting up to Declaration of Helsinki. Healthy volunteers aged from 20 to 30 years old, with height between 150 and 170 cm and with body weight from 50 to 80 kg, were asked to refrain from consuming alcohol, smoking cigarettes, or using other drugs that might alter alcohol metabolism for 72 h before the experiment. After fasting for at least 4 h, a volunteer was given an alcoholic drink (0.4 g per kg body weight) within 15 min. All volunteers sat on a chair during sample collection.
3. Results and discussion 3.1. Imaging and quantitative analysis of standard gaseous ethanol measurement AOD and HRP immobilized mesh substrate was saturated with concentration-adjusted luminol solution or luminol sodium salt, and installed in a dark box for chemiluminescence measurement. To enhance the sensitivity of the system, 0.1 mmol/l and pH10.1 Tris–HCl buffer solution was selected and used to measure standard gaseous ethanol. We evaluated the ultraviolet (UV) irradiation time for 5 min for immobilization of enzyme on mesh substrate using photo-cross linkable photopolymer PVA-SbQ under low power UV light. The coefficient of variation of peak chemiluminescence emission was 3.8% of 5 min UV irradiation. The immobilized enzymes of AOD and HRP were treated with low damage to the functionalization of the enzyme proteins. More than 10 min UV light irradiation caused damaged to enzymes, consequently, chemiluminescent intensity became weak with a low coefficient of variation. The chemiluminescence intensity was evaluated by measuring the response to various concentrations of gaseous ethanol introduced into the system. Injection of standard gaseous ethanol was at a flow rate of 200 ml/min for 10 s. Each injected sample had a volume of approximately 33 ml. The chemiluminescence average intensity was increased following to injections of standard gaseous ethanol, the chemiluminescence peaks appeared in 30 s and gradually decreased until 60 s at 100 ppm gaseous ethanol. This decrease is an improvement over results achieved in a previous study (Wang et al., 2011), as it reduced the time of diagnosis and burden for the participants. The concentration of gaseous ethanol was calibrated from 50 to 350 ppm with a correlation coefficient of 0.998 (Fig.4). The
T. Arakawa et al. / Biosensors and Bioelectronics 67 (2015) 570–575
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alcohol intake 0.4 g alcohol / 1 kg BW
cell plate dark box
AOD/HRP mesh
250
chemi-luminescence
fixture EM-CCD infrared camera stage
Fig. 3. Schematic view of transdermal ethanol measurement system. AOD and HRP immobilization mesh substrate, an acrylic cell plate and a fixture was used in combination for imaging of gaseous ethanol from a palm. Alcohol intake condition of 0.4 g alcohol drink per 1 kg body weight was selected. The human palm was placed on the combination of enzyme mesh, and recorded using an electro-multiplying CCD camera during 90 min.
35 30
luminol
25
peak of average intensity
peak of average intensity
40
20 L-HG + EY 15 10 5
10 8 6 4 2 0 0
0 0
100
200
300
20 40 60 80 100 ethanol vapor (ppm)
400
ethanol vapor (ppm) Fig. 4. Calibration curve for the visualization system with luminol or luminol HG and eosin Y solution for standard gaseous ethanol.
chemiluminescence intensity increased rapidly following the injection of gaseous ethanol using a standard gas generator. 3.2. Improvement of chemiluminescence sensitivity of the imaging system In order to improve the sensitivity of the imaging system, concentration of luminol high grade (L-HG) of high-purity luminol solution and eosin Y as an enhancer were optimized in the gaseous ethanol imaging system at the concentrations of 5 mmol/ l and 3.1 10 3 mmol/l, respectively (Figs. 2 and 3). The average intensity of L-HG was achieved 5 times more than one of luminol. The detection limit of gaseous ethanol was defined 10 ppm. The Emission of light strength rose in 3.0 10 3 mmol/l and the emission was amplified 5-folds more than without eosin Y. The detection limit of low concentration gaseous ethanol using L-HG and eosin Y was improved to 3 ppm at the optimized condition. Fig. 4 shows the calibration curve of this system for gaseous ethanol measurement with L-HG and eosin Y. The chemiluminescence intensities were related to the concentrations of gaseous ethanol from 3 to 150 ppm with a correlation coefficient of
Fig. 5. Chemiluminescence image corresponding to transdermal gaseous ethanol on the palm using gaseous ethanol imaging system. (a) 37 min and (b) 48 min after alcohol administration.
0.993. The maximum intensity of more than 200 ppm was too high (higher than 256) for accurate and quantitative measurement with the CCD camera. As a result, the calibration range
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shifted to a lower concentration. The imaging system with L-HG and eosin Y could thus be highly sensitive in the detection of gaseous ethanol.
3.3. Selectivity of the imaging system The selectivity of the imaging system was evaluated with other chemical substances (methanol, acetaldehyde, trimethylamine, acetone, methyl mercaptan, and dimethyl sulfide) in combination with ethanol from human body and breath. The lower molecular weight alcohols, such as methanol were catalyzed by AOD. However, the concentration of methanol in the human body is too low to be detected. Therefore, AOD showed the highest activity with ethanol. This system detected the ethanol selectively in measurement of transdermal ethanol after alcohol drinking.
The imaging system for gaseous ethanol and the highly sensitive imaging using high-purity luminol and eosin Y were used for analysis of standard gaseous ethanol. These were also used to measure levels of transdermal ethanol transpired from palm after alcohol administration. To measure low concentrations of transdermal ethanol in the palm, we fabricated and arranged an acrylic cell plate with the same interval of dots patterns between enzyme mesh and the volunteer’s hand. AOD and HRP were immobilized at different square size pattern of 1.5, 6.0, 10, and 20 mm. We evaluated the variation coefficient of chemiluminescent intensity of the each dot pattern applying by standard gaseous ethanol. The variation coefficient of each dot, 1.5 and 6.0 mm was higher 10%, 10 and 20 mm were 9.6% and 2.7%, respectively. The dots pattern of a
b
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3.4. Imaging of transpired ethanol from the palm
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Fig. 6. Time–intensity plots of the chemiluminescence image of transdermal gaseous ethanol on the palm. (a) Fore finger, (b) center of the palm, (c) middle finger and (d) ring finger.
T. Arakawa et al. / Biosensors and Bioelectronics 67 (2015) 570–575
10 mm squares was used to evaluate the emission distribution of transdermal gas. The enzyme mesh was placed on a dot pattern of 10 mm squares forming the shape of a hand (Supplemental Fig. 1), the numbers of dot patterns were 58 on the enzyme mesh. The optimized condition of eosin Y as a sensitizer in the L-HG solution for low concentration of emissions of skin gaseous ethanol was carried out for skin gas imaging. A volunteer's hand was placed stably on the acrylic cell plate after alcohol administration. Chemiluminescence depended on gaseous ethanol emissions from the palm skin and evaluated using a highly sensitive imaging system. Fig. 5 shows the chemiluminescence pattern corresponding to transdermal gaseous ethanol on the palm, (a) 37 min after drinking and (b) 48 min after drinking. Supplementary movie 1 shows the changes in temporal and spatial distributions of chemiluminescence upon 15 to 110 min after drinking. As a result of the experiments, chemiluminescence intensity following emissions of transdermal gaseous ethanol rapidly increased from the palm part, which was confirmed from drinking 35 min later. All of emission points gradually decreased until 110 min. This time-dependent change in average intensity of the palm was nearly identical to emission time of transdermal ethanol in another research (Hawthorne and Wojcik, 2006). Fig. 6 shows time–intensity plots of chemiluminescence image of transdermal gaseous ethanol on the palm, (a) fore finger, (b) center of palm, (c) middle finger and (d) ring finger from 15 to 110 min after drinking. These time–intensity changes and the quantity of emission of the transdermal gaseous ethanol were different for every enzyme pattern in the palm. That is, the distributions of released ethanol gas differed over various parts of the palm, suggesting that such distributions varied with alcohol metabolism. Especially, the ethanol transpired from the area of the fore finger and middle finger rapidly increased. Furthermore, evaluation the temperature distribution of the palm was recorded by infrared camera (TP-L0225UN, Chino-Corp. Co. Ltd.). The high temperature area of the hand was related to area of emissions of gaseous ethanol. This phenomenon might be related to release of ethanol facilitated by elevation in temperature and diaphoretic skin after drinking. Consequently, the imaging system enabled visualization of distribution, developmental distribution, and temporal change in concentrations of transdermal gaseous ethanol. Supplementary material related to this article can be found online at http://dx.doi.org/10.1016/j.bios.2014.09.045.
4. Conclusions A 2-dimensional gaseous ethanol imaging system has been developed and demonstrated using a hydrogen peroxide–luminol– horseradish peroxidase system with high-purity luminol solution of luminol HG and eosin Y as a chemiluminescence enhancer. This system measures ethanol concentrations as intensities of chemiluminescence by luminol reaction. The imaging of gaseous ethanol was achieved at 3 ppm at optimal concentration of luminol HG solution and eosin Y. We used this high-sensitivity imaging system to measure transdermal ethanol on the palm of volunteer who consumed alcohol. The maximum concentration of emission of gaseous ethanol was observed over 40 min. Concentrations of gaseous ethanol peaked at 35 min and then gradually decreased until the end of sample collection at 110 min. Ethanol metabolism in the body proceeded, reducing transdermal emissions of ethanol
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from the palm, as confirmed by images showing variations in concentrations and distributions of ethanol in the palm. In the future, more sensitive and non-invasive imaging system would allow us to improve transdermal analysis of the human body and may thereby simplify screening for disease.
Acknowledgments This study was supported in part by Japan Society for the Promotion of Science (JSPS KAKENHI 24560512 and 24650078) and Ministry of Education, Culture, Sports, Science and Technology (MEXT) Special Funds for “Education and Research Advanced Research Program in Sensing Biology”.
Appendix A. Suplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.09.045.
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Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Chemiluminescent imaging of transpired ethanol from the palm for evaluation of alcohol metabolism Takahiro Arakawa, Kazutaka Kita, Xin Wang, Kumiko Miyajima, Koji Toma, Kohji Mitsubayashi n Department of Biomedical Devices and Instrumentation, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10, Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
art ic l e i nf o
a b s t r a c t
Article history: Received 7 June 2014 Received in revised form 18 September 2014 Accepted 21 September 2014 Available online 28 September 2014
A 2-dimensional imaging system was constructed and applied in measurements of gaseous ethanol emissions from the human palm. This imaging system measures gaseous ethanol concentrations as intensities of chemiluminescence by luminol reaction induced by alcohol oxidase and luminol–hydrogen peroxide–horseradish peroxidase system. Conversions of ethanol distributions and concentrations to 2-dimensional chemiluminescence were conducted on an enzyme-immobilized mesh substrate in a dark box, which contained a luminol solution. In order to visualize ethanol emissions from human palm skin, we developed highly sensitive and selective imaging system for transpired gaseous ethanol at sub ppmlevels. Thus, a mixture of a high-purity luminol solution of luminol sodium salt HG solution instead of standard luminol solution and an enhancer of eosin Y solution was adapted to refine the chemiluminescent intensity of the imaging system, and improved the detection limit to 3 ppm gaseous ethanol. The highly sensitive imaging allows us to successfully visualize the emissions dynamics of transdermal gaseous ethanol. The intensity of each site on the palm shows the reflection of ethanol concentrations distributions corresponding to the amount of alcohol metabolized upon consumption. This imaging system is significant and useful for the assessment of ethanol measurement of the palmar skin. & 2014 Elsevier B.V. All rights reserved.
Keywords: Transdermal emissions Imaging system Gaseous ethanol Chemiluminescence Oral alcohol administration
1. Introduction Various volatile organic compounds (VOCs) exist, such as those the transpired by humans, breath, body odor, smell of the living environment and aroma of food (Lindinger et al., 1998; Hierlemann and Gutierrez-Osuna, 2008; Shirasu and Touhara, 2011; Zhang and Li, 2010). Certain compounds, which are an indicator of disease may also be metabolized by a body, a skin, and breath (Miekisch et al., 2004). The human body emits various non-volatile and volatile molecules, depending on a person’s genetics, stress and immune status (Pandey and Kim, 2011). Human odor caused by the combined action of the skin gland and volatile organic compounds, which are regulated by human hormonal control and the bacterial population localized at skin surface (Natale et al., 2000). Metabolism of the body can be screened by analyzing a volatile human ingredient. If the volatile organic compounds of human body can be analyzed in relation to human health, a physician and a patient easily understand and evaluate the result of a diagnosis by screening human odor. n
Corresponding author. Tel.: þ 81 3 5280 8091; fax: þ 81 3 5280 8094. E-mail address: [email protected] (K. Mitsubayashi).
http://dx.doi.org/10.1016/j.bios.2014.09.045 0956-5663/& 2014 Elsevier B.V. All rights reserved.
Non-invasive diagnostic methods based on analysis of gas emitted by the body have been used. These include tests for urea expiration and tests for diagnosis of halitosis (Francesco et al., 2005; Chan et al., 2009; Natale et al., 2014). There were any problems to diagnose and monitor of emission of gas from human body because of restrictive diagnosis method. As a result, we cannot measure consecutively of expiration collection. In addition, in the bad breath diagnosis, as for the method with gas chromatography and a gas sensor, there were some problems with handiness and selectivity (Natale et al., 2014). For example, a medical doctor and a third party may directly smell and diagnose the bad breath of the patient. However, this diagnosis is subjective and inaccurate, because the sensing of bad breath depends on the olfactory senses of an individual. Since modern breath analysis started in the 1970s, gas chromatography has been used to identify more than 200 components in human exhaled breath (Lindinger et al., 1998). However, a large volume of exhaled breath was required for diagnosis and the liquid nitrogen or liquid argon was used for cryogenic pre-concentration, which was necessary for sample analysis (Philipp et al., 2004). Isotopes of 13C used in breath tests in the diagnosis of Helicobacter pylori infections, have been successfully used as diagnostic method (Romagnuolo et al., 2002). Furthermore, a gas emission from skin surface is lower
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concentration than that of breath gas expiration. Thus, highly sensitive techniques for continuously and selectively measuring volatile chemicals generated by the body are essential for noninvasive diagnosis. It has been reported that the volatile chemical ingredient associated with disease and the physical physiological state including in skin gas like expiration. For example, acetone, hydrogen, alcohol was included in volatile chemical components, which was well known to researchers (Naitoh et al., 2000; Natale et al., 2000; Rock et al., 2008; Costello et al., 2014). However, sensors and measurement systems for volatiles are expensive and cumbersome to use in a medical facility. Furthermore, gas chromatographs and mass spectrometers, which are used to analyze these compounds, have demanding requirements for installation, and monitoring temporal changes in emissions of gaseous components is difficult. A bracelet-type sensor (SCRAMTM) was used to monitor ethanol concentrations in transdermal gas to measure the quantity of alcohol consumed. This sensor, which could be used in clinical and practical applications (Sakai et al., 2006; Barnett et al., 2011) was used to monitor transdermal alcohol emissions every 30 minutes for 2 weeks. Criteria for detecting alcohol from human skin for this sensor provided an accurate and unconventional approach for use with non-mandated clients. A number of biosensors have been developed for the measurement of methanol, ethanol and lower alcohols. The measurement of ethanol, in particular, is important essential for forensic science, clinical chemistry and analytical chemistry. In addition, food products, beverages and the wine industry are interested in analytical methods to control the quality and processing of the food products (Wen et al., 2007). A standard alcohol biosensor is composed of an alcohol oxidase immobilized membrane and a commercial oxygen sensor. The quantity of ethanol is determination by the change in the level of dissolved oxygen in the solution. Many sensor technologies employing enzymatic reactions have been developed, such as biochemical gas sensors (bio-sniffers) for gaseous formaldehyde and ethanol, as well as a NADH-dependent fiber-optic biosensor for the determination of gaseous components determination (Kudo et al., 2010, 2012). Also the enzyme-based chemical gas sensors are highly selective and sensitive for target chemicals. We recently obtained human body information by various kinds of methods for the treatment of a variety of diseases. Mainly, blood and urine have been used for the screening of diseases because of high reliability sample of human body. Recently, sweat, exhaled breath and saliva also used for sample for it. In this paper, we focused on the imaging of transdermal emissions of gaseous ethanol from human palms at hands with alcohol metabolism in
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human body after oral alcohol administration. This method enables non-invasive, pain-less and blood-less monitoring of information of human body. We can obtain various information of human body by analyzing the ingredient included in transpired emissions. In our previous study, we developed an imaging system of gaseous ethanol based on chemiluminescence measurement employing an EM-CCD camera for breath gaseous ethanol imaging (Wang et al., 2010; Arakawa et al., 2013a, 2013b). However, measurements of transdermal human gas through the system have not been sufficiently evaluated. Therefore, in this study, we developed a novel non-invasive temporal imaging system of transdermal gaseous ethanol to analyze alcohol metabolism in real-time. We measured the changes in chemiluminescence intensity due to emission of transdermal gaseous ethanol after alcohol administration.
2. Experimental 2.1. Chemicals and apparatus A system for imaging the movement of ethanol was developed as shown in Fig. 1. This imaging system consisted of an electromultiplying CCD camera (L3C95-05, pixel size: 15 35.5 μm2, image format: 768 244 pixels, spectral range: 400–1060 nm, e2v technologies limited, United Kingdom) and a video encoder (GV-MDVD3, I-O DATA, Japan). Data analysis of the recorded chemiluminescence was analyzed by Cosmos 32 software (Library Inc., Japan). All solutions were prepared in deionized distilled water obtained from a Milli-Q purification system (Millipore Co., USA). Cotton mesh substrates used for imaging (cotton: 100%, thickness: 1 mm, and interval size: 1 mm, Pip-Fujimoto Co., Japan) was evaluated for enzyme stabilization. Alcohol oxidase (AOD, E. C.1.1.3.13, A2404-1kU, 10–40 units mg 1 protein, from Pichia pastoris, Sigma-Aldrich Co., USA) and horseradish peroxidase (HRP, E. C.1.11.1.7, 169-10791, 100 units mg 1, Wako Pure Chemical Industries, Ltd., Japan), photo-crosslinkable poly(vinyl alcohol) containing stilbazolium groups (PVA-SbQ, type: SPH, 9C-10L, 10.4 wt%, Toyo Gosei Co., Ltd., Japan) were used for enzyme stabilization on the mesh substrate. A 5.0 mmol/l luminol (01253-60, Kanto Chemical Co., Inc., Japan) solution was prepared in Tris–HCl buffer solution (100 mmol/l) for measurement of chemiluminescence generated by ethanol (Wang et al., 2011). In addition, to improve the intensity of gaseous ethanol, we selected luminol sodium salt HG (5-amino-2,3-dihydro-1.4-phathalazinedione sodium salt, Lot. CDR7140, Wako Pure Chemical Industries, Ltd.) and eosin Y (Sodium Tetrabromofluorescein, Lot. CDM1099, Wako Pure
dark box AOD/HRP immobilized mesh
mass flow controller
EM-CCD
ethanol vapor 200 ml/min chemi-luminescence
gas generator
computer
video encoder
HDD recorder
Fig. 1. Schematic of the gaseous ethanol imaging system for standard gaseous ethanol. This system consisted of AOD and HRP immobilized on mesh substrate, an electromultiplying CCD camera and a standard gas generator machine.
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Chemical Industries, Ltd.). We adjusted these reagents with Tris– HCL buffer. 2.2. Principle of gaseous ethanol imaging In principle, ethanol is oxidized to acetaldehyde and hydrogen peroxide by alcohol oxidase (AOD) in the presence of oxygen. The hydrogen peroxide (HRP) reacts with the luminol solution by catalysis of HRP and resulting in the chemiluminescence. These reactions are described as follows (Fletcher et al., 2001): AOD
ethanol+O2 ⟹acetaldehyde + H2 O2 HRP
luminol +H2O2 + OH− ⟹3−aminophthate + 2H2 O+ N2 + hν
(1)
Fig. 2. Schematic view of the acrylic cell plate for imaging of transdermal ethanol from the human hand. The diameter of the hole was Ø6.0 mm, and the interval between holes was 16 mm. These holes reduced diffusion of transdermal gaseous ethanol.
(2)
These enzymes from AOD and HRP immobilized substrate were prepared with PVA-SbQ and used for gaseous ethanol imaging. For fabrication of the enzyme-immobilized substrate, AOD and HRP were dissolved in phosphate buffer solution (PB, 0.1 mmol/l, pH7.5), mixed with PVA-SbQ in a volume/weight ratio of 1:2. The enzymes/PVA-SbQ mixture was coated onto the mesh substrate, spread and then cured for 3 h at 4 °C in a dark box. The substrate was cured under low power ultraviolet (UV) irradiation for 5 min. A standard gaseous ethanol was supplied at a flow rate of 200 ml/min for 20 s from a gas generator unit, which was also used a standard gas generator for calibration purposes. 2.3. Patch test for imaging of human skin An enzyme of acetaldehyde type 2 deficiency is involved in the metabolism of acetaldehyde, which is a toxic metabolite of ethanol. The skin patch test for ethanol was performed following the method reported (Higuchi et al., 1987). A patch plaster of lint pads on an adhesive tape was prepared, 100 μL of 70% ethanol was placed on one of the lint pads, and as a control, and the same volume of distilled water was put on another pad. The patches were taped to the inner surface of the arm for 7 min and then removed slowly. Patch areas that showed erythema after removal corresponded to a positive test result for reduced ALDH 2 activity (Wang et al., 2011). In this experiment, an ALDH 2 negative volunteer was selected for the further experiments after oral administration of alcohol. 2.4. Imaging and measurement of transpired ethanol Experiments on the palm were carried out to evaluate gas transpired from human skin (Zhang and Li, 2010). Therefore, we improved the gaseous ethanol imaging system to the transdermal ethanol imaging system from the human palm. Imaging of ethanol released from the palm part was carried out after drinking. Due to monitor a wide skin area for the skin gas measurement, an acrylic cell plate was indispensable part for retaining gap between the skin side and the enzyme mesh for quantitative measurement. We manufactured the pattern plate, which formed the cell of the same shape into dots form covering the enzyme immobilization mesh. The enzyme immobilized mesh had same size of square pattern at a square 10 mm on a side; interval of squares was 16 mm. The circular cell (5 mm depth, 6 mm diameter, 141.4 mm3 capacity) plate of the same dot pattern to control diffusion of the transdermal gas with the enzyme square mesh of the palm shape was shown in Fig. 2. The mesh with square patterns was soaked in 5 mmol/l luminol of high grade and 3.0 μmol/l eosin Y solutions. And palm part of the volunteer adheres to a cell plate after the drinking and recorded the emission of light with the outbreak of
the skin gas by an EM-CCD camera. Fig. 3 shows a schematic view of the measurement system for transdermal ethanol. For measurement of transdermal ethanol from the palm, gaseous ethanol samples were collected after oral administration of alcohol and measured authorizing by the Human Investigations Committee of Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (authorization code: 2012-06, permitted since 26th March, 2013), acting up to Declaration of Helsinki. Healthy volunteers aged from 20 to 30 years old, with height between 150 and 170 cm and with body weight from 50 to 80 kg, were asked to refrain from consuming alcohol, smoking cigarettes, or using other drugs that might alter alcohol metabolism for 72 h before the experiment. After fasting for at least 4 h, a volunteer was given an alcoholic drink (0.4 g per kg body weight) within 15 min. All volunteers sat on a chair during sample collection.
3. Results and discussion 3.1. Imaging and quantitative analysis of standard gaseous ethanol measurement AOD and HRP immobilized mesh substrate was saturated with concentration-adjusted luminol solution or luminol sodium salt, and installed in a dark box for chemiluminescence measurement. To enhance the sensitivity of the system, 0.1 mmol/l and pH10.1 Tris–HCl buffer solution was selected and used to measure standard gaseous ethanol. We evaluated the ultraviolet (UV) irradiation time for 5 min for immobilization of enzyme on mesh substrate using photo-cross linkable photopolymer PVA-SbQ under low power UV light. The coefficient of variation of peak chemiluminescence emission was 3.8% of 5 min UV irradiation. The immobilized enzymes of AOD and HRP were treated with low damage to the functionalization of the enzyme proteins. More than 10 min UV light irradiation caused damaged to enzymes, consequently, chemiluminescent intensity became weak with a low coefficient of variation. The chemiluminescence intensity was evaluated by measuring the response to various concentrations of gaseous ethanol introduced into the system. Injection of standard gaseous ethanol was at a flow rate of 200 ml/min for 10 s. Each injected sample had a volume of approximately 33 ml. The chemiluminescence average intensity was increased following to injections of standard gaseous ethanol, the chemiluminescence peaks appeared in 30 s and gradually decreased until 60 s at 100 ppm gaseous ethanol. This decrease is an improvement over results achieved in a previous study (Wang et al., 2011), as it reduced the time of diagnosis and burden for the participants. The concentration of gaseous ethanol was calibrated from 50 to 350 ppm with a correlation coefficient of 0.998 (Fig.4). The
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alcohol intake 0.4 g alcohol / 1 kg BW
cell plate dark box
AOD/HRP mesh
250
chemi-luminescence
fixture EM-CCD infrared camera stage
Fig. 3. Schematic view of transdermal ethanol measurement system. AOD and HRP immobilization mesh substrate, an acrylic cell plate and a fixture was used in combination for imaging of gaseous ethanol from a palm. Alcohol intake condition of 0.4 g alcohol drink per 1 kg body weight was selected. The human palm was placed on the combination of enzyme mesh, and recorded using an electro-multiplying CCD camera during 90 min.
35 30
luminol
25
peak of average intensity
peak of average intensity
40
20 L-HG + EY 15 10 5
10 8 6 4 2 0 0
0 0
100
200
300
20 40 60 80 100 ethanol vapor (ppm)
400
ethanol vapor (ppm) Fig. 4. Calibration curve for the visualization system with luminol or luminol HG and eosin Y solution for standard gaseous ethanol.
chemiluminescence intensity increased rapidly following the injection of gaseous ethanol using a standard gas generator. 3.2. Improvement of chemiluminescence sensitivity of the imaging system In order to improve the sensitivity of the imaging system, concentration of luminol high grade (L-HG) of high-purity luminol solution and eosin Y as an enhancer were optimized in the gaseous ethanol imaging system at the concentrations of 5 mmol/ l and 3.1 10 3 mmol/l, respectively (Figs. 2 and 3). The average intensity of L-HG was achieved 5 times more than one of luminol. The detection limit of gaseous ethanol was defined 10 ppm. The Emission of light strength rose in 3.0 10 3 mmol/l and the emission was amplified 5-folds more than without eosin Y. The detection limit of low concentration gaseous ethanol using L-HG and eosin Y was improved to 3 ppm at the optimized condition. Fig. 4 shows the calibration curve of this system for gaseous ethanol measurement with L-HG and eosin Y. The chemiluminescence intensities were related to the concentrations of gaseous ethanol from 3 to 150 ppm with a correlation coefficient of
Fig. 5. Chemiluminescence image corresponding to transdermal gaseous ethanol on the palm using gaseous ethanol imaging system. (a) 37 min and (b) 48 min after alcohol administration.
0.993. The maximum intensity of more than 200 ppm was too high (higher than 256) for accurate and quantitative measurement with the CCD camera. As a result, the calibration range
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shifted to a lower concentration. The imaging system with L-HG and eosin Y could thus be highly sensitive in the detection of gaseous ethanol.
3.3. Selectivity of the imaging system The selectivity of the imaging system was evaluated with other chemical substances (methanol, acetaldehyde, trimethylamine, acetone, methyl mercaptan, and dimethyl sulfide) in combination with ethanol from human body and breath. The lower molecular weight alcohols, such as methanol were catalyzed by AOD. However, the concentration of methanol in the human body is too low to be detected. Therefore, AOD showed the highest activity with ethanol. This system detected the ethanol selectively in measurement of transdermal ethanol after alcohol drinking.
The imaging system for gaseous ethanol and the highly sensitive imaging using high-purity luminol and eosin Y were used for analysis of standard gaseous ethanol. These were also used to measure levels of transdermal ethanol transpired from palm after alcohol administration. To measure low concentrations of transdermal ethanol in the palm, we fabricated and arranged an acrylic cell plate with the same interval of dots patterns between enzyme mesh and the volunteer’s hand. AOD and HRP were immobilized at different square size pattern of 1.5, 6.0, 10, and 20 mm. We evaluated the variation coefficient of chemiluminescent intensity of the each dot pattern applying by standard gaseous ethanol. The variation coefficient of each dot, 1.5 and 6.0 mm was higher 10%, 10 and 20 mm were 9.6% and 2.7%, respectively. The dots pattern of a
b
16
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8 6 4
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3.4. Imaging of transpired ethanol from the palm
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Fig. 6. Time–intensity plots of the chemiluminescence image of transdermal gaseous ethanol on the palm. (a) Fore finger, (b) center of the palm, (c) middle finger and (d) ring finger.
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10 mm squares was used to evaluate the emission distribution of transdermal gas. The enzyme mesh was placed on a dot pattern of 10 mm squares forming the shape of a hand (Supplemental Fig. 1), the numbers of dot patterns were 58 on the enzyme mesh. The optimized condition of eosin Y as a sensitizer in the L-HG solution for low concentration of emissions of skin gaseous ethanol was carried out for skin gas imaging. A volunteer's hand was placed stably on the acrylic cell plate after alcohol administration. Chemiluminescence depended on gaseous ethanol emissions from the palm skin and evaluated using a highly sensitive imaging system. Fig. 5 shows the chemiluminescence pattern corresponding to transdermal gaseous ethanol on the palm, (a) 37 min after drinking and (b) 48 min after drinking. Supplementary movie 1 shows the changes in temporal and spatial distributions of chemiluminescence upon 15 to 110 min after drinking. As a result of the experiments, chemiluminescence intensity following emissions of transdermal gaseous ethanol rapidly increased from the palm part, which was confirmed from drinking 35 min later. All of emission points gradually decreased until 110 min. This time-dependent change in average intensity of the palm was nearly identical to emission time of transdermal ethanol in another research (Hawthorne and Wojcik, 2006). Fig. 6 shows time–intensity plots of chemiluminescence image of transdermal gaseous ethanol on the palm, (a) fore finger, (b) center of palm, (c) middle finger and (d) ring finger from 15 to 110 min after drinking. These time–intensity changes and the quantity of emission of the transdermal gaseous ethanol were different for every enzyme pattern in the palm. That is, the distributions of released ethanol gas differed over various parts of the palm, suggesting that such distributions varied with alcohol metabolism. Especially, the ethanol transpired from the area of the fore finger and middle finger rapidly increased. Furthermore, evaluation the temperature distribution of the palm was recorded by infrared camera (TP-L0225UN, Chino-Corp. Co. Ltd.). The high temperature area of the hand was related to area of emissions of gaseous ethanol. This phenomenon might be related to release of ethanol facilitated by elevation in temperature and diaphoretic skin after drinking. Consequently, the imaging system enabled visualization of distribution, developmental distribution, and temporal change in concentrations of transdermal gaseous ethanol. Supplementary material related to this article can be found online at http://dx.doi.org/10.1016/j.bios.2014.09.045.
4. Conclusions A 2-dimensional gaseous ethanol imaging system has been developed and demonstrated using a hydrogen peroxide–luminol– horseradish peroxidase system with high-purity luminol solution of luminol HG and eosin Y as a chemiluminescence enhancer. This system measures ethanol concentrations as intensities of chemiluminescence by luminol reaction. The imaging of gaseous ethanol was achieved at 3 ppm at optimal concentration of luminol HG solution and eosin Y. We used this high-sensitivity imaging system to measure transdermal ethanol on the palm of volunteer who consumed alcohol. The maximum concentration of emission of gaseous ethanol was observed over 40 min. Concentrations of gaseous ethanol peaked at 35 min and then gradually decreased until the end of sample collection at 110 min. Ethanol metabolism in the body proceeded, reducing transdermal emissions of ethanol
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from the palm, as confirmed by images showing variations in concentrations and distributions of ethanol in the palm. In the future, more sensitive and non-invasive imaging system would allow us to improve transdermal analysis of the human body and may thereby simplify screening for disease.
Acknowledgments This study was supported in part by Japan Society for the Promotion of Science (JSPS KAKENHI 24560512 and 24650078) and Ministry of Education, Culture, Sports, Science and Technology (MEXT) Special Funds for “Education and Research Advanced Research Program in Sensing Biology”.
Appendix A. Suplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.09.045.
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