The 29th Special CU-af Seminar 2021

The 29th Special CU-af Seminar 2021 August 25, 2021 14. Popova B, Kleinknecht A, Braus G. Biomolecules. 2015(5):617-34. 15. Davies S, Hallett P, Moens T, Smith G, Rocha E, Kim HT, et al. Neurobiol Dis. 2013(64). 16. Javed H, Nagoor Meeran MF, Azimullah S, Adem A, Sadek B, Ojha SK. Front Pharmacol. 2018(9):1555. 17. Cai CZ, Zhou HF, Yuan NN, Wu MY, Lee SM, Ren JY, et al. Phytomedicine. 2019(61):152842. 18. Zimmermann A, Hofer S, Pendl T, Kainz K, Madeo F, Carmona-Gutierrez D. Y. Fems Yeast Res. 2018(18). 19. Franssens V, Boelen E, Jayamani A, Vanhelmont T, Buettner S, Winderickx J. Cell Death Differ. 2009(17):746-53. 20. Petroi D, Popova B, Taheri-Talesh N, Irniger S, Shahpasandzadeh H, Zweckstetter M, et al. J Biol Chem. 2012(287):27567-79. 21. Soper JH, Kehm V, Burd CG, Bankaitis VA, Lee VM. J Mol Neurosci. 2011(43):391-405. 22. Wijayanti I, Watanabe D, Oshiro S, Takagi H. J Biochem. 2015(157):251-60. 23. Andreasson C, Ljungdahl PO. Gene Dev. 2002(24):3158-72. 24. Haitani Y, Nakata M, Sasaki T, Uchida A, Takagi H. FEMS Yeast Res. 2009(1):73-86. 25. Gray M, Piccirillo S, Honigberg SM. FEMS Microbiol Lett. 2005(248):31-6. 26. Schiestl RH, Gietz RD. Curr Genet. 1989(16):339-46. 27. Hoshikawa C, Shichiri M, Nakamori S, Takagi H. Proc Natl Acad Sci U S A. 2003(20):11505-10. 28. Bowling T, Mercer L, Don R, Jacobs R, Nare B. Int J Parasitol Drugs Drug Resist. 2012:262-70. 29. Zare M, Amin MM, Nikaeen M, Bina B, Pourzamani H, Fatehizadeh A, et al. Environ Monit Assess. 2015(5):276. 30. Travnickova E, Mikula P, Oprsal J, Bohacova M, Kubac L, Kimmer D, et al. AMB Express. 2019:1-11. 31. Hu, Q., Uversky, V.N., Huang, M., Kang, H., Xu, F., Liu, X., Lian, L., Liang, Q., Jiang, H., Liu, A., Zhang, C., Pan-Montojo, F. and Zhu, S. Biochim Biophys Acta. 2016(1862): 1883- 1890. 32. Chambers, H.F. and Sachdeva, M. J Infect Dis. 1990(161): 1170-1176. 86



Non-Invasive Blood Glucose Monitoring through Optical Fibre Technology Prattakorn METEM and Charusluk VIPHAVAKIT

The 29th Special CU-af Seminar 2021 August 25, 2021 Non-Invasive Blood Glucose Monitoring through Optical Fibre Technology Prattakorn METEM1* and Charusluk VIPHAVAKIT1* Abstract A ZnO-coated optical-fibre-based sensor for volatile organic compounds (VOCs) detection is developed in this project. A 125-micron diameter of coreless silica fibre (CSF) connected to single-mode fibre (SMF) at both ends to achieve a structure of SMF-CSF-SMF is proposed to detect VOCs biomarkers for diabetes such as isopropanol (IPA). The coreless fibre region is considered to be a sensing region where the multimode interference occurs. It undergoes surface functionalisation with ZnO nanorods prepared via hydrothermal method for the sensor to be selectively detect VOCs biomarker with electrostatic absorption. The intensity modulation responses of the ZnO coated optical sensor with vapour of IPA are analysed showing a reduction of intensity with the increase of IPA concentration, while the uncoated sensor shows no response. These results suggest that ZnO nanorod functionalisation are able to absorb IPA inside its matrices, allowing light interaction with IPA and therefore higher sensitivity, in terms of intensity modulation. 1International School of Engineering (ISE), Faculty of Engineering, Chulalongkorn University 89

The 29th Special CU-af Seminar 2021 August 25, 2021 Introduction and Objectives As invasive diagnosis is deemed as unpleasant, painful, and traumatizing, an accurate, fast-processing diagnosis sensor have attracted researchers from various fields. Numerous non-invasive diagnosis methods have been developed throughout the years, including detection of volatile organic compounds (VOCs) in exhaled breath as they contain biomarkers of some chronic diseases. Volatile organic compounds (VOCs) are organic compounds having low vapor pressure and, therefore, able to be vaporised easily at room temperature. These compounds, as a metabolic waste, are transported to the excretion organs via body fluids. One major method of excretion is exhaled breath; hence, exhaled breath contains VOCs profile corresponding to metabolism of the body[1]. Concentrations of VOCs in exhaled breath are reported to be associated with several chronic diseases and can be served as viable biomarkers. For instance, acetone and isopropanol have been widely supported as a biomarker for type 1 diabetes[2,3]. Aimed to detect a ppb-level concentration of the interested VOCs among a plethora of other abundant organic compounds, such as, carbon dioxide, nitrogen, water, and even oxygen[4], exhaled breath VOCs sensors serve as a tall challenge in terms of selectivity and sensitivity. A variety of methods has been explored in order to achieve the necessities of sensitivity and selectivity, mostly divided into 2 major methods: i) electrical-based measurement and ii) optical-based measurement. Electrical-based method revolves around measurements of resistances and potentials with a presence of the interested VOCs; for instance, chemiresistor and potentiometry sensor for detection of nitric oxide and hydrogen peroxide, respectively[5,6]. Optical-based sensors are mainly associated with absorption, transmission, and intensity measurements, which can be measured by a spectrometer[7,8]. Optical fibre sensors for VOCs sensing applications have attracted researcher due to its lightweightness, less prone to electrical noises, and possibility of multiplexing compared to its electronic counterpart[9]. Several architectures and designs have been applied for the sensing applications, in particular, singlemode-multimode-singlemode (SMS) fibre structure which has been recently explored for many biomedical applications. By using no-core fibre for multimode segment of the structure, the propagating light is allowed to interact with surrounding environment. Hence, multimode segment is served as a sensing region for the structure. On top of that, the structure is relatively easy fabrication method, and it is selected as a method of interest in this research[10]. Surface functionalisation on the sensing region of the optical fibre sensor has also been investigated in order to improve selectivity and sensitivity. Zinc oxide coated optical fibre sensor is reported to have high sensitivity and selectivity for ammonium gas and ethanol[11,12]. Gold deposited optical fibre sensor exhibits high sensitivity to methanol with a low-detection limit of 0.0001 refractive index unit[13]. The well-known effects of surface functionalisation combined with advantages of SMS optical fibre structure have intrigued researchers to design a sensor involving with this structure. Therefore, in this project, zinc-oxide-(ZnO)-nanorods-coated optical fibre sensor for volatile organic compounds detection is designed, with the optimum length of the sensor is simulated via numerical investigation, fabrication of ZnO nanorods, and characterisation of the sensor. Hence, the ultimate goal for the project is to fabricate a sensor to detect biomarkers in breath, using Zinc-oxide-(ZnO)-nanorods-coated optical fibre sensor, with 4 subobjectives. First is to design the sensor. This includes simulation of the sensor to maximise the highest 90

The 29th Special CU-af Seminar 2021 August 25, 2021 sensitivity. Second is to coat ZnO nanorods onto the optical fibre, with optimisation of the fabrication techniques, as well as characterisation of the fabricated sensor. Third is the fabrication of the sensor. Lastly, the measurement results from the sensors are studied to test their properties and sensitivity. Methods 1.Sensor Design The key specification is the sensitivity of the sensor. As biomarkers in breath has their concentrations ranged in ppm to ppb, a highly sensitive sensor must be invented. And sensitivity itself, is the constraint of designing a sensor. In order to solve the problem, optical fibre sensors are being explored for VOC sensing application, especially the singlemode-multimode-singlemode (SMS) optical fibre structure. The critical factor of using optical fibre is to let light propagating inside the optical fibre interacts with outside medium, which contains VOCs. By using no-core fibre for multimode segment of the structure, the propagating light is allowed to interact with surrounding environment. Hence, multimode segment is served as a sensing region for the structure. On top of that, the structure is relatively easy fabrication method, and it is selected as a method of interest in this research .[10] Surface functionalisation also enhance sensitivity of the sensor[10-12]. Figure 1: Design of the sensor consisting of ZnO-nanorod-coated on the coreless silica fibre which is connected to the singlemode fibre at both ends. The design is based on singlemode-multimode-singlemode (SMS) fibre structure as shown in Figure 1. It is consisted of 2 types of optical fibres, which are multimode and singlemode fibres (SMF). Singlemode fibres serve as a waveguide to deliver the light from light source to the sensor and then to the spectrometer, which records intensity spectra. The multimode fibre serves as a sensing region of the sensor. Coreless silica fibre (CSF) is multimode fibre component, which allows more light interactions at the surface of the sensor, due to the higher number of propagation modes inside the fibre, enhancing evanescent field at the interface[14,15]. As the medium has changes in concentration of VOC, light can sense the change and resulting in change of intensity spectra. This region is also coated with ZnO, as it is reported to improve sensitivity of optical sensor[11,12]. Singlemode fibre used has its core diameter of 4.2 microns and cladding diameter of 125 microns. Multimode fibre used is a coreless silica fibre (CSF) with diameter of 125 microns. ZnO nanorods are coated on the sensor due to its porosity on the surface to capture VOCs in air. The nanorods are coated onto CSF segment via hydrothermal method[16]. The sensor is assembled 91

The 29th Special CU-af Seminar 2021 August 25, 2021 by spicing singlemode fibre, the ZnO-nanorod-coated CSF, and another singlemode fibre together. One end of the singlemode fibre is then connected to the light source, while the other end is connected to the spectrometer for spectrum measurement. 2.Numerical investigation and simulation The singlemode-multimode-singlemode (SMS) fibre structure is illustrated in Figure 1. It is composed of a multimode fibre (MMF) segment inserted in between two segments of singlemode (SMF) fibres. The theories necessary for the simulations, propagation mode calculation and multimode interference theory is covered, as well as calculation of medium’s refractive index calculation. 2.1 Propagation mode calculation In optical fibre, electric field at a specific position inside the fibre can be calculated from the equation (1)[14] assuming on-axis coupling, (1) where r is radial position of an optical fibre. α is a radius of propagating region. Jl and Kl are lth order of Bessel function of the first kind and lth order modified Bessel function of the second kind, while A and C are fitting constants. Due to circular symmetry of light propagating through the singlemode fibre, it can be assumed that the modes in the SMS fibre structure is radially symmetrical, and that lth order Bessel functions are assumed to be 0th order only. Therefore, (1) can be rewritten as (2) where u and w can be defined as (3) (4) Here, k0 is a wavenumber of a light in a vacuum. ncore and ncladding are refractive indexes of core and cladding of the fibre, respectively. For the coreless silica fibre (CSF), ncore is the refractive index of the glass fibre region, and ncladding is substituted with effective index of the surrounding medium. neff is an effective index of the whole optical fibre system. Since the electric field must be continuous for all of domain r, we obtain 2 boundary conditions from (2) which are, 92

The 29th Special CU-af Seminar 2021 August 25, 2021 (5) (6) and thus, (8) (7) Through (7) and (8), the effective indexes can be calculated. After substitution of effective indexes into (2), intensity of electric fields propagating through an optical fibre is obtained. 2.2 Multimode interference and reimaging distance When the light is coupled from SMF into MMF, the multiple-mode light propagation is occurred in the MMF. These excited multimodes of propagation can interfere with each other, which is defined as multimode interference. The interference governs the intensity at the output of the SMS structure, which is defined by coupling efficiency, η .[15] The coupling efficiency determines the loss of the intensity at the output compared with the input and can be calculated from where (9) (10) (11) (12) (13) (14) 93

The 29th Special CU-af Seminar 2021 August 25, 2021 Denoted as j or h is a parameter of jth or hth propagation mode of the output. ãj and aj represent modified expansion coefficient and expansion coefficient of the jth mode of propagation, respectively. The two parameters show how the power of the field expanded between input field (Es) and output field (Rj) when there are two optical fibres connected. Representing wave number of the light in propagating z-direction, β is called propagating constant and is a product of the wave number in vacuum, k0, and effective index, neff, of the jth mode. The equation (9) provides a relationship between coupling efficiency, η, and propagation length, z. The propagation length that yields the maximum coupling efficiency is defined as reimaging distance, which is used as the length of the no-core fibre for further calculation of the 2.3 Sensor fabrication ZnO nanorods are coated on to CSF (Thorslab) via hydrothermal method[16]. Briefly, the fibres are cut into a specific length and have their jacket removed by optical fibre stripper or by sonicating with acetone. Then, the samples are placed on a hotplate set at 80 oC. Zinc acetate in ethanol solution is dropped onto the samples ten times as shown in Figure 2(a). Next, the seeded fibres are annealed at 250 oC for 1 hour. After removing the samples from the oven, it is ready to grow ZnO nanorods. They are put into test tubes filled with zinc nitrate and hexamine in water solution Figure 2(b) and then subjected to 90 oC heat in an oven. Time of the growth is varied and optimised in order to attain highest sensor sensitivity. Lastly, they are annealed at 350 oC for 1 hour. After obtaining ZnO-nanorods coated CSF, CSF segment is connected to two singlemode fibre (Thorslab) segments by a splicer (Sumitomo Electric) to obtain the sensor. Figure 2: Experimental method for coating ZnO nanorods onto CSF. (a) ZnO seeding procedure, showing the dropping of zinc acetate in ethanol onto heated CSFs. (b) ZnO nanorod growing procedure, showing set up made of glass slides used to suspend CSFs in the zinc nitrate in hexamine solution. 2.4 Sensitivity Measurement The experimental setup aims for detection of isopropanol vapor using the ZnO-coated SMS sensor is shown in Figure 3(a). It is composed of a visible light source (Ocean Optics), the sensor chamber, and a spectrometer (Compact CCD Spectrometers CCS100, Thorlabs). Inside the chamber, isopropanol (IPA) reservoir is inserted along with the sensor as shown in 94

The 29th Special CU-af Seminar 2021 August 25, 2021 Figure 3(b). When the light is coupled from the light source into the sensor, the change in the refractive index inside the chamber due to the vaporisation of IPA resulting in the change in light spectra overtime. The output intensity spectra are detected by the spectrometer. For measurement of sensor’s sensitivity, firstly, 3 ml of IPA was poured into the IPA reservoir and placed inside the chamber, and the lid was closed. Then, the light source is turned on and intensity spectra were recorded every 1 minute for 30 minutes using integration time of 2500 ms. The experiment is repeated 3 times to study the repeatability and consistency of the sensor. After that, the obtained data was smoothed, processed, and visualized in Matlab. Figure 3: (a) Actual experimental setup, including a visible light source, the test chamber, and a spectrometer. (b) The plastic chamber is composed of the sensor and isopropanol (IPA) reservoir. Results and Discussion Simulation results The result of the relationship between propagation distance (length of the sensor) and coupling efficiency, using the wavelength of 633 nm and volume fraction of ZnO of 0.05 is shown in Figure 4. The graph is similar to a reported research[15]. Reimaging distance is calculated to be 14.28 cm. However, at this length, it is not practical for fabricating the sensor and hence, the length of 5.5, 10.2, and 10.3 cm with acceptable coupling efficiency of -12.56, -8.38, -9.88 dB are selected instead. Figure 4: Graph of coupling efficiency (dB) and propagation distance (micron) with the length of 5.5 cm, 10.2 cm, 10.3 cm, and reimaging distance indicated. 95

The 29th Special CU-af Seminar 2021 August 25, 2021 Sensor fabrication ZnO nanorods are successfully fabricated on the CSF with growth time of 3, 4, 5, and 7 hours. The nanorods are observed from SEM (Quanta FE SEM) as shown in Figure 5. At 3 hours, nanorods are started to grow on the surface of CSF. While 4 hours and 5 hours show longer nanorods, they lack uniformity and there are the signs of nanorod fragmentations. And at 7 hours, the nanorods collapsed completely. Also, the longer the time of hydrothermal growth, the resulting fibres become more fragile for fibre splicing procedure. Therefore, 3-hour growth time of the ZnO nanorod is selected to practically fabricate the sensor. Figure 5: SEM micrographs of ZnO nanorods on CSF with growth time of (a) 3 hours (b) 4 hours (c) 5 hours and (d) 7 hours. Three sensors are successfully fabricated with ZnO-nanorod-coated CSF with the length of 5.5, 10.3, and 10.2 cm. Also, an uncoated CSF with the length of 6 cm is also fabricated as a reference as shown in Figure 6. Figure 6: Fabricated sensors with the sensor length of (a) 5.5 cm (b) 10.3 cm (c) 10.2 cm, and (d) the reference with the length of CSF segment of 6.0 cm. 96

The 29th Special CU-af Seminar 2021 August 25, 2021 Sensitivity measurement The three intensity spectra of different trial at different time are averaged to obtain average intensity spectra as shown in Figure 7. The intensity spectra of 5.5-cm sensor show spectra peak at around 0.11, while for 10.2 and 10.3 cm long sensors have their intensity peak at 0.44 and 0.31, respectively. These results are in accordance with the simulation showing a higher coupling efficiency, and therefore, higher intensity at the sensors’ length of 10.2 and 10.3. Figure 7: Intensity spectra after 0, 6, 12, 18, 24, and 30 minutes, respectively for the sensor with the length of a) 5.5 cm b) 10.3 cm c) 10.2 cm, and d) the reference with the length of CSF segment of 6.0 cm. In terms of intensity modulation, average intensity around the peak wavelength at bandwidth of 16 nm is plotted with time of experiment as shown in Figure 8. The reference shows no trend in intensity changes when the time increases, while all ZnO-nanorod-coated sensors exhibit a drop in intensity. These results are in accordance with waveguide theory, as when the concentration of isopropanol increases overtime, the refractive indexes also increase. The increase in refractive index resulting in fewer mode propagations to be guided inside the CSF, hence, the output intensity is decreased. It is also shown that ZnO nanorods are able to absorb the isopropanol inside its matrices as the uncoated sensor does not exhibit any change in terms of intensity modulation. The results suggest that ZnO nanorods are able to enhance the sensitivity of the sensor, in terms of intensity modulation. 97

The 29th Special CU-af Seminar 2021 August 25, 2021 Figure 8: Average intensity of the peaks with bandwidth of 16 nm after 0, 6, 12, 18, 24, 30 minutes, respectively for the sensor with the length of a) 5.5 cm b) 10.3 cm c) 10.2 cm, and d) the reference with the length of CSF segment of 6.0 cm. Conclusion A ZnO-nanorod-coated optical-fibre-based sensor is successfully designed by numerical investigation and the reimaging distance is calculated to be 14.28 cm. However, the length is unpractical for sensor fabrication; and hence, sensors are fabricated with sensing region of 5.5, 10.2, and 10.3 cm instead. In terms of ZnO hydrothermal growth, the nanorods start to grow and coat on CSF after 3 hours of hydrothermal process. Intensity spectra are recorded, and intensity modulation is analysed. Intensity modulation exhibits a decrease in average intensity. The results shows that ZnO-nanorods are able to absorb IPA inside their matrices to allow direct light interaction with IPA. The sensitivity of the sensor can be further improved by optimising the length of the sensing region and nanorod growth time. Also, the surface functionalisation has to be further studied for a better selectivity such as, the coating of gold nanoparticles and/or other porous materials. 98

The 29th Special CU-af Seminar 2021 August 25, 2021 References 1. Haick, H., et al., Assessment, origin, and implementation of breath volatile cancer markers. 2014. 43(5): p. 1423-1449. 2. Wang, Z. and C.J.J.o.b.r. Wang, Is breath acetone a biomarker of diabetes? A historical review on breath acetone measurements. 2013. 7(3): p. 037109. 3. Rudnicka, J., et al., Determination of volatile organic compounds as biomarkers of lung cancer by SPME–GC–TOF/MS and chemometrics. 2011. 879(30): p. 3360-3366. 4. Di Natale, C., et al., Solid-state gas sensors for breath analysis: A review. 2014. 824: p. 1-17. 5. Pantalei, S., et al., Improving sensing features of a nanocomposite PEDOT: PSS sensor for NO breath monitoring. 2013. 179: p. 87-94. 6. Komkova, M.A., et al., Hydrogen peroxide detection in wet air with a prussian blue based solid salt bridged three electrode system. 2013. 85(5): p. 2574-2577. 7. McCurdy, M., Y. Bakhirkin, and F.J.A.P.B. Tittel, Quantum cascade laser-based integrated cavity output spectroscopy of exhaled nitric oxide. 2006. 85(2): p. 445-452. 8. Mitsubayashi, K., et al., Optical bio-sniffer for methyl mercaptan in halitosis. 2006. 573: p. 75-80. 9. Elosua, C., et al., Volatile organic compound optical fiber sensors: A review. 2006. 6(11): p. 1440-1465. 10. Lan, X., et al., Fiber ring laser interrogated zeolite-coated singlemode-multimode-singlemode structure for trace chemical detection. 2012. 37(11): p. 1998-2000. 11. Renganathan, B., et al., Nanocrystalline ZnO coated fiber optic sensor for ammonia gas detection. 2011. 43(8): p. 1398-1404. 12. Wen, X., et al., ZnO-coated SMS structure interrogated by a fiber ring laser for chemical sensing. 2014. 25(11): p. 114002. 13. Mitsushio, M., et al., Construction and evaluation of a gold-deposited optical fiber sensor system for measurements of refractive indices of alcohols. 2004. 111(2-3): p. 252-259. 14. Kawano, K. and T. Kitoh, Introduction to optical waveguide analysis. 2004: Wiley Online Library. 15. Mohammed, W.S., P.W. Smith, and X.J.O.l. Gu, All-fiber multimode interference bandpass filter. 2006. 31(17): p. 2547-2549. 16. Sani, E. and A.J.O.M. Dell’Oro, Spectral optical constants of ethanol and isopropanol from ultraviolet to far infrared. 2016. 60: p. 137-141. 99

Non-enzymatic Electrochemical Detection of Cholesterol using the β-Cyclodextrin Modified 3D Paper-based Device Sudkate Chaiyo

The 29th Special CU-af Seminar 2021 August 25, 2021 Non-enzymatic Electrochemical Detection of Cholesterol using the β-Cyclodextrin Modified 3D Paper-based Device Sudkate Chaiyo1* Abstract Cholesterol (Chol) can cause several diseases, such as heart attack, atherosclerosis, and stroke, thus, the development of cholesterol biosensor is greatly required. Herein, we introduce for the first time a 3D electrochemical paper-based analytical devices (3D-ePADs) for the non-enzymatic detection of cholesterol by modifying a cellulose filter paper with β-cyclodextrin (β-CD). The modifying β-CD-paper was characterized by fourier-transform infrared spectroscopy (FTIR), energy dispersive X-ray spectroscopy (EDX), and scanning electron microscopy (SEM). The 3D-ePADs comprises two components: an origami folding paper (oPAD) and an insert pad (iPAD). This 3D-ePADs eliminates the undesirable procedure of multiple-step in a complex assay. Under the optimal experimental conditions and using differential pulse voltammetry (DPV) as transduction technique the sensor was able to detect cholesterol level ranges from 0.1 µM to 1000 μM, with a detection limit of 30 nM. Specificity of the developed 3D-ePAD sensor towards target analyte (cholesterol) was confirmed in the presence of common interfering species. The applicability of proposed 3D-ePAD sensor was also demonstrated for cholesterol determination in human serum samples with good recovery results (98–105%) and maximum RSD (relative standard deviation) of 2.8%. Graphical abstract 1The institute of biotechnology and genetic engineering, Chulalongkorn University 101

The 29th Special CU-af Seminar 2021 August 25, 2021 Introduction and Objectives Cholesterol is an essential component of the mammalian cell membrane and is a precursor of bile acid and steroid hormones. However, abnormal cholesterol levels are related to several diseases, such as hypertension, coronary heart disease, arteriosclerosis, brain thrombosis, lipid metabolism dysfunction and myocardial infarction[1]. Thus, the monitoring of the cholesterol level is very important for disease control and prevention. Various analytical strategies for cholesterol assays have been developed that can be sub-classified into two groups, including a chemical and an enzymatic method[2], [3], [4], [5]. Currently, the enzymatic method has received greater attention for cholesterol analysis because it provides greater sensitivity and selectivity than the chemical method. The well-known principle of an enzyme-based cholesterol sensor is typically based on the reaction between cholesterol and the cholesterol oxidase (ChOx) enzyme. Free cholesterol is oxidized by cholesterol oxidase to produce 4-cholestene-3-one and hydrogen peroxide (H2O2), and the H2O2 generated can be used for indirect quantification of cholesterol[6], [7], [8]. Detection selectivity in most of these methods relies on the use of cholesterol selective enzymes which are expensive and prone to denaturation. As an alternative for simple and cost-effective methods, the optical sensors are highly appreciable whereas an electrochemical non-enzymatic sensing process has an ample scope for better sensitivity. It is well known that β-cyclodextrin (β-CD) is a cyclic oligosaccharide consisting of β-glucopyranose units. The internal cavity is lined with C3H and C5H hydrogen and ether-like oxygen that provide a hydrophobic environment, whereas the external faces of the cyclodextrin molecule are hydrophilic. Due to its ability to encapsulate hydrophobic compounds, this internal cavity of β-CD allows hydrophobic cholesterol molecules to be soluble in aqueous solutions[9]. β-CDs have a high affinity for sterols as compared to other lipids in vitro[10], which make these compounds quite effective in modifying cholesterol metabolism. Agnihotri and coworker[11] have developed an electrochemical method of cholesterol detection using β-CD functionalized graphene. The detection limit of cholesterol is achieved as low as 1 µM. But this sensing platforms provide good performance for cholesterol detection, limitations remain in terms of time consumption, detection performance and big instruments. To overcome this drawback, it is necessary to design and fabricate a new cholesterol sensor that offers simple fabrication, and a short analysis time. The first microfluidic paper-based analytical device (μPADs) was proposed by Whitesides’ group[12]. This platform has gained more attention in use because of its natural abundance, inexpensiveness, flexibility, disposability, and low sample-reagent usage[13-15]. These relevant properties make μPADs attractive and simple platforms for point-of-care monitoring (POCT). Based on these platforms, the origami paper-based system was developed. Origami is the art of paper folding, which originated from Japan[16]. Moreover, the flexibility of the μPADs was fabricated by folding into a desired configuration[17]. Therefore, versatile origami paper-based analytical devices (oPAD) have been reported for various cancer marker detections[18, 19]. In addition, the oPAD can be designed to integrate the electrode modifications and analytical measurements in a single device[20]. Also, the paper device or filter paper can immobilize various substances for increased specificity and sensitivity. Ares and coworker[21] have designed a cellulosic filter paper functionalized with β-CD for the removal, and subsequent analysis, of common pharmaceuticals from water and saline media. This research has successfully immobilized β-CD on filter paper, which is an easy method and few steps. 102

The 29th Special CU-af Seminar 2021 August 25, 2021 Herein, we engineered a new novel design: a 3D electrochemical paper-based analytical devices (3D-ePAD) based on the sliding strip concept. Unlike the other work described above, this platform demonstrates a device that allows reagents to be stored in insert PAD (iPAD) and transported sequentially to the detection zone of origami PAD (oPAD). Only a single introduction of a carrier buffer sufficiently runs a completed assay; therefore, minimal manipulation of solutions is needed by the user. This simple type of platform can be applied to multiple types of electrochemical assays while maintaining simplicity. In this work, for the first time, a non-enzymatic electrochemical 3D-ePAD using modifying a cellulose filter paper with β-CD was developed and applied for the determination of cholesterol. Objectives • To design and fabricate 3D paper-based sensor coupled with electrochemical transduction for non-enzymatic cholesterol detection. • To immobilize β-cyclodextrin on cellulose filter paper for non-enzymatic cholesterol detection. • To optimize the preparation parameters for producing the highest sensitivity for selective detection of cholesterol. • To apply the proposed sensor for the determination of cholesterol in blood samples. Methods Materials, reagents, and equipment Conductive graphene ink was purchased from Serve science company (Bangkok, Thailand). Conductive ink of silver/silver chloride (Ag/AgCl) was purchased from SunChemical (Bath, UK). β-cyclodextrin (β-CD), NaOH, Cholesterol, NaCl, Whatman chromatography paper #1 (58 cm × 60 cm), uric acid, Chloroacetic acid, Carbodiimide, and potassium hexacyanoferrate (III) (K3[Fe(CN)6]) were purchased from Sigma–Aldrich (Buchs, Switzerland). All reagents were of analytical grade, and were used without further purification. All solutions were prepared using ultra-purified water (>18 MΩ cm) refined by a cartridge purification system (Millipore, UK). β-CD grafting onto the surface of paper was monitored by Fourier transform infrared spectroscopy (FTIR). FTIR measurements were performed by Bio-Rad model 400 using KBr as background over the range of 4000–400 cm−1. Fabrication of 3D-ePAD The 3D-ePAD consists of two components including a origami pad (oPAD) for a β-CD modified cellulose filter paper, and an insert pad (iPAD) for multiple step of cholesterol detection. The device design was created using the Adobe Illustrator program (version 23.0.4). A wax-printing method was used to create a hydrophobic barrier. For device fabrication, the designed pattern was printed onto filter paper (Whatman No. 1) and then placed in oven at 150 °C for 60 s for wax penetrating trough the paper. The oPAD was divided into three main parts: the β-CD modified paper zone, sample and running buffer introduction zone, and waste reservoir as shown in Figure 1A. The iPAD consists of two parts: part A and B as a mirror symmetry. Each of part comprises three components: the sampling zone, washing zone, and detection zone as illustrate in Figure 1B. Graphene conductive ink as working electrode was screen-printed at the front side of detection zone (part A), while the counter electrode as graphene conductive ink and reference electrode as Ag/AgCl ink were screen-printed at the back side of 103

The 29th Special CU-af Seminar 2021 August 25, 2021 detection zone (part B). After that, the iPAD was dried in the oven at 55 °C for 1 hr. Next, the mediator as 10 μL of 50.0 mM [Fe(CN)6]3–/4– was applied in the detection zone for the current response in the label-free system. Figure 1 : Design and composition of 3D-ePAD Modification of β-cyclodextrin on oPAD According to the previous method of β-CD preparation in the literature by Furusaki et al, 100 g β-CD was firstly treated as carboxymethyl-β-CD by mixing with 93 g NaOH in 16.3%v/v chloroacetic acid (270 mL) and sonicated for 30 min. After that, carboxymethyl-β-CD was precipitated with methanol and then dried at 40 °C at the room temperature. To prepare a β-CD on oPAD, 10 mg mL-1 carboxymethyl-β-CD was firstly functionalized by adding 25 mg mL-1 of carbodiimide solution containing 0.003M phosphate buffer saline (pH 6) and sonicated to complete reaction for 90 min. The functionalized carboxymethyl-β-CD was dropped on oPAD and subsequently incubated for 1 hr to covalently immobilize onto PAD via substitution of carboxymethyl-β-CD substituent reacted with the hydroxy group (−OH) of cellulose on the PAD surface. 3D-ePAD operation The 3D-ePAD was designed to integrate three main steps for the determination of cholesterol using β-CD modified on PAD via the collecting sample, the washing step, and analysis step into a single device. The 3D-ePAD consists of 2 parts: oPAD for β-CD modified on PAD and iPAD for running multistep in this device. For the device assembly, the folding oPAD was operated as shown in Figure 2A, and then inserted on the backward part of iPAD as shown in Figure 2B. After that, the sample inlet zone on oPAD was fold to iPAD as a first layer. The final assembled 3D-ePAD is illustrated in Figure 2C. At this stage, the device was ready to use. Figure 2: Operation of the 3D PAD 104

The 29th Special CU-af Seminar 2021 August 25, 2021 For the operation of the 3D-PAD, 3D-ePAD was covered with the punched acrylic sheets for an ease of moving the iPAD part in this developed device (as shows in Figure 3.). First, the sample was introduced on the inlet zone and incubated for 30 min at room temperature. At this point, the sample flowed through to β-CD PAD layer. Subsequently, the target analyte was immobilized into β-CD. After reaction completed, the iPAD was slide upward such that the washing zone was located underneath and coincided with β-CD-PAD layer of the oPAD. The running buffer as 0.003 M phosphate buffer saline pH 6 was applied to remove unbound cholesterol for 5 min. After washing step completed, the iPAD was slide upward again. At this stage, the three electrodes system was located between the target analyte immobilized on β-CD PAD layer containing the residue of buffer solution. Afterward, the current response will be measured using the differential pulse voltametric (DPV) technique. Figure 3: Electrochemical procedure on 3D-ePAD for non-enzymatic cholesterol detection Electrochemical detection For electrochemical detection step, a voltammetric experiment was performed using PGSTAT 128 N AutoLab potentiostat (Metrohm, Switzerland) controlled by corresponding software (Nova 2.0). After collecting and washing sample step, differential pulse voltammetry (DPV) was used by a potential scan from -0.3 to 0.3 V vs Ag/AgCl ink. For characterization, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were performed. CV was scan from -0.3 to 0.3 V vs Ag/AgCl ink at scan rate of 0.1 V/s. EIS was performed by applying 0.1 V of AC potential through a frequency range over 0.1-100000 Hz. Results and Discussion Synthesis and characterization of β-CD-PAD β-CD on PAD was synthesized using two-steps (carbodiimide method). The first step involves the formation of carboxyl group onto β-CD by reacting chloroacetic acid with β-CD in the alkaline condition. In the second step, carboxymethyl-β-CD was covalently bonded onto the cellulose on the PAD surface via carbodiimide activation. The binding carboxymethyl-β-CD on PAD was confirmed by FTIR spectroscopy. Figure 4 shows the FTIR spectra of bare paper and carboxymethyl-β-CD on PAD in the 400–4000 cm−1 wave number range. The spectrum of carboxymethyl-β-CD shows the characteristic peaks at 945, 1030, 1157 and 1704 cm−1. 105

The 29th Special CU-af Seminar 2021 August 25, 2021 The peak at 945 cm−1 is due to the R-1,4-bond skeleton vibration of β-CD, and the peaks at 1030 and 1157 cm−1 corresponded to the antisymmetric glycosidic νa (C–O–C) vibrations and coupled ν(C–C/C–O) stretch vibration. The peak at 1704 cm−1 corresponds to carbonyl group (=CO) stretching which confirms the incorporation of the carboxymethyl group (–COOCH3) into β-CD molecule. So, it can be concluded that carboxymethyl-β-CD has been grafted successfully on the surface of cellulose on the PAD surface. Figure 4: FTIR spectra of the 3D-ePAD (a) bare paper and (b) β-CD on paper Furthermore, EIS and CV were employed to electrochemically characterize the successful fabrication of the cholesterol sensor in a step-by-step fashion. To get a Nyquist plot, the negative imaginary impedance – Z” is plotted versus the real part of the impedance Z’. In the EIS study, the semicircular diameters represent the electron-transfer resistance (Ret) at the transducer interface. As displayed in Figure B, the functionalized β-CD on PAD (β-CD on paper, red line: b) offered a small semicircle with an Ret of 95.5 Ω compared to the bare paper (bare paper, black line: a) that exhibited a nonlinearity with a high Ret of 185 Ω at high frequencies since the oxidation reaction caused a rough and scaly residue PAD surface. The oxidation process not only introduces the higher number of oxygen functional groups, but also develops the hydrophilic properties and the electrochemical properties of the PAD surface. However, the Ret value remarkably increased from 458 Ω (cholesterol = 0.1 mM) to 687 Ω (cholesterol = 0.5 mM) after the addition of the cholesterol target molecule (β-CD on paper + Chol, blue ang green line: c and d). This Ret increment can be described by the shielding ability of the complexes (β-CD with cholesterol) that has been created on the PAD surface, resulting in a charge-transfer resistance. The evidence here supports the successful construction of the cholesterol sensor. For the CV investigation, the obtained results were like those studied by EIS, indicating the successful fabrication of the cholesterol sensor (Figure 5A). 106

The 29th Special CU-af Seminar 2021 August 25, 2021 Figure 5: Nyquist impedance (A) and cyclic voltammograms (B) comparison of the bare paper (a), β-CD on paper (b), β-CD with cholesterol 0.1 mM on paper (c), and β-CD with cholesterol 0.5 mM on paper (c) . The reaction solution was 10 mM [Fe(CN)6]3-/4- in 0.1 M KCl solution, scan rate: 100 mV s-1. Electrochemical measurement of cholesterol The electrochemical measurement of cholesterol was completed by differential pulse voltammetry (DPV) using the redox [Fe(CN)6]3/4− mediate signal response as an indicator. In this work, a specific β-CD was covalently anchored onto the PAD surface. cholesterol target was assigned to bind with the immobilized on the β-CD–paper surface where the efficiency of binding depends on the size of target molecule. After the reaction stops, different types of complexes on the PAD surface generate different shielding ability, resulting in the detected signal. The higher concentration of cholesterol in the sample, the lower signal of redox species appears as shown in Figure 6. When the prepared sensor was exposed to cholesterol solution, a significant decrease in the [Fe(CN)6]3−/4− current response was observed. The results confirm that the fabricated sensor effectively detected cholesterol. The apparent increment in the signal response can be explained by the low shielding ability of the inherently nonconductive complexes (β-CD-Chol) on the PAD surface (compared with the absence of cholesterol, resulting in a lowly responsive signal. Figure 6: DPV responses of the cholesterol and the graphical comparison of the electron transfer capability at the paper surface, with and without the target cholesterol 107

The 29th Special CU-af Seminar 2021 August 25, 2021 The effect of β-CD concentrations The effect of the β-CD concentration was first examined. The concentrations ranging from 10 to 200 ng mL-1 were examined using the following conditions: 0.50 mM cholesterol, and 30 min incubation time. The results revealed that the current response gradually decreased with increasing β-CD concentration, reached an optimal value, and plateaued at 100 ng mL−1. This result can be interpreted as the oversaturation of the PAD surface, and therefore, there were a limited number of complexes (β-CD-cholesterol) that could be established on the PAD surface. However, at high concentrations of β-CD (β-CD >150 ng mL−1), the detected signal tended to decrease. This result indicates that a greater number of β-CD were immobilized onto the PAD surface, resulting in a higher shielding ability and increased signal. Consequently, 100 ng mL−1 of β-CD was selected for the subsequent experiments. After achieving an appropriate condition for the β-CD, the incubation time was then investigated, and it was discovered that 30 min was an appropriate time for this parameter. Analytical performance of the proposed cholesterol biosensor The calibration curve was constructed by plotting the current response signal of [Fe(CN)6]3−/4− obtained from DPV at different concentrations of cholesterol ranging from 0 µM to 1000 µM (Figure 7A). As expected, a lower [Fe(CN)6]3−/4− signal was detected when higher cholesterol concentrations were applied (Figure 7A), and it was discovered that the plot between the current response and Log concentration of cholesterol was linear over a range from 0.1 to 1000 µM (R2 = 0.9901) (Figure 7C). From the calculation, the values of 0.05 and 0.1 µM were achieved for the limit of detection (LOD) (3SDb/m) and quantitation (LOQ) (10SDb/m), respectively. The analytical performance of this proposed method and other previously reported cholesterol sensors is tabulated in Table 1. Figure 7: Representative DPV after addition of the target cholesterol (A). Plot of current response vs. the concentrations of cholesterol (B) and plot of current response vs. log concentrations of cholesterol (B) 108

The 29th Special CU-af Seminar 2021 August 25, 2021 Table 1: Comparison of analytical performances of different non-enzyme sensors for the determination of cholesterol. Specificity of cholesterol electrochemical sensor The specificity is a key factor while developing a sensor device for any target analyte. The developed sensor device should not interact or generate response for other interfering molecules. The blood is a complex matrix composed of different other species which can interfere in cholesterol detection. Therefore, the developed sensor device was evaluated by using three commonly interfering compounds in blood serum including BSA, glucose, glycine, uric acid, ascorbic acid, NaCl, KCl, MgCl2 and carbohydrates at a concentration of 100 μM along with the same concentration of analyte (cholesterol). β-CD on paper were exposed to above mentioned interfering agents. Figure 8 shows the changes in response of this sensor after incubation with interference and cholesterol. The change in response for non-specific compounds are much lower than that for cholesterol. These results illustrate negligible effects of interfering compounds on cholesterol detection, and the proposed sensor has sufficient specificity to cholesterol. Figure 8: Comparative interference studies using different species in the developed cholesterol detection method, 109

The 29th Special CU-af Seminar 2021 August 25, 2021 Application of the sensor The above results indicate that the β-CD-3D-ePADs is feasible for the determination of cholesterol in human blood. Comparative results were obtained by a commercial cholesterol meter (The EasyTouch® GCHb). The results are summarized in Table 2. Recoveries of cholesterol were found in the range of 98.86–105.35% with RSDs from 1.59–2.86%. The results are in good agreement with those obtained with a commercial cholesterol meter; the paired t-test at the 95% confidence level did not show any significant difference (the calculated t value of 1.7089 is significantly below the critical t of 2.1318 with 4 degrees of freedom). The test also did not show any significant difference for each type of sample. Hence, the β-CD-3D-ePADs can be used as a suitable material for the detection of cholesterol in human serum samples. Table 2: Determination of cholesterol in normal blood serum samples (all measurements in triplicate). Conclusion In the present work, we developed a novel planform 3D-ePADs sensor for cholesterol detection using β-CD modified on cellulose filter paper. This 3D-ePADs eliminates the undesirable procedure of multiple-step in a complex assay. The sensor system showed good sensitivity and reproducibility ranges from 0.1 to 1000 μM with 50 nM LOD. Furthermore, the developed 3D-ePADs sensor does not require any antibody or enzyme in recognition process and still, can detect cholesterol efficiently in nano molar range with prominent selectivity in the presence of interfering species. Thus, 3D-ePAD sensor based on β-CD with mentioned unique features is the best alternative of cholesterol enzymatic sensor due to its low cost, easy fabrication, and robustness. References 1. The expert panel, Arch. Intern. Med. 148 (1988) 36-69. 2. V. Raj, T. Johnson, K. Joseph, Biosens. Bioelectron. 60 (2014) 191-194. 3. J. Zhang, W. Wang, S. Chen, Y. Ruo, X. Zhong, X. Wu, Biosens. Bioelectron. 57 (2014) 71-76. 4. M.B. Gholivand, M. Khodadadian, Biosens. Bioelectron. 53 (2014) 472-478. 5. R. Ahmad, N. Tripathy, S.H. Kim, A. Umar, A. Al-Hajry, Y.-B. Hahn, Electrochem. Commun. 38 (2014) 4-7. 6. J. Shen, C.-C. Liu, Sens. Actuators B 120 (2007) 417-425. 7. N. Ruecha, W. Siangproh, O. Chailapakul, Talanta 84 (2011) 1323-1328. 8. M. Zhang, R. Yuan, Y. Chai, S. Chen, H. Zhong, C. Wang, Y. Cheng, Biosens. Bioelectron. 110

The 29th Special CU-af Seminar 2021 August 25, 2021 32 (2012) 288-292. 9. J. Pitha, T.Irie, P.B. Sklar, J.S. Nye, Life Sciences, 43 (1988) 493-502. 10. T. Irie, K. Fukunaga, J.Pitha, J. Pharma Scienc, 81 (1992) 521-523, 11. N. Agnihotri, A. D. Chowdhury, A. De, Biosens. Bioelectron., 63 (2015) 212-217, 12. A.W. Martinez, S.T. Phillips, G.M. Whitesides, E. Carrilho, Anal Chem, 82(1): (2010) 3–10. 13. M. Srisa-Art, K.E. Boehle, B.J. Geiss, C.S. Henry, Anal Chem, 90 (2018) 1035–1043. 14. A.C. Glavan, J. Niu, Z. Chen, F. Güder, C-M. Cheng, D. Liu, G.M. Whitesides, Anal Chem, 88 (2016) 725–731. 15. A.W. Martinez, S.T. Phillips, E. Carrilho, S.W. Thomas, H. Sindi, G.M. Whitesides, Anal Chem, 80 (2008) 3699–3707. 16. H. Liu, R.M. Crooks, J Am Chem Soc, 133 (2011) 17564–17566. 17. C. Renault, M.J. Anderson, R.M. Crooks, J Am Chem Soc, 136 (2014) 4616–4623. 18. L. Li, W. Li, H. Yang, C. Ma, J. Yu, M. Yan, X. Song, Electrochim Acta, 120 (2014) 102–109. 19. C. Ma, W. Li, Q. Kong, H. Yang, Z. Bian, X. Song, J. Yu, M. Yan, Biosens Bioelectron, 63 (2015) 7–13. 20. K. Pungjunun, S. Chaiyo, I. Jantrahong, S. Nantaphol, W. Siangproh, O. Chailapakul, Microchim Acta, 185(2018) 324. 21. A. M. Ares, R. Muiño, A. Costoya, R. A. Lorenzo, A. Concheiro, A.M. Carro, C. Alvarez-Lorenzo, Carbohydrate Polymers, 220 (2019) 43-52. 22. M. Saha, S. Das, J. Nanostruct. Chem., 4 (2014), p. 94. 23. Y.J. Lee, J.Y. Park,Biosens. Bioelectron., 26 (2010), pp. 1353-1358. 24. Li Ya, H. Bai, Q. Liu, J. Bao, M. Han, Zh Dai,Biosens. Bioelectron., 25 (2010), pp. 2356-2360. 25. J. Yang, H. Lee, M. Cho, J. Nam, Y. Lee Sens. Actuators B, 171–172 (2012), pp. 374-379. 26. H.S. Yoon, S.J. Lee, Y.J. Park, Sensors (2014), pp. 347-350. 27. N. Joshi, K. Rawat, P.R. Solanki, H.B. Bohidar,Biochem. Eng. J., 102 (2015), pp. 69-73. 28. N. Joshi, A. Sharma, K. Ashokan, K. Rawat, D. Kanjilal, RSC Adv., 6 (27) (2016), pp. 22664-22672. 29. Yu Tong, H. Li, H. Guan, J. Zhao, S. Majeed, S. Anjum, F. Liang, G. Xu, Biosens. Bioelectron., 47 (2013), pp. 553-558. 111

Coal Combustion Product Utilization for Degraded Soil Improvement in Nan Province Kreangkrai MANEEINTR Pimsiri TIYAYON Nghia Trung PHAN and Pinyo MEECHUMNA

The 29th Special CU-af Seminar 2021 August 25, 2021 Coal Combustion Product Utilization for Degraded Soil Improvement in Nan Province Kreangkrai MANEEINTR1* Pimsiri TIYAYON1* Nghia Trung PHAN1* and Pinyo MEECHUMNA1* Abstract Bottom ash applied in many industries can be used in agriculture to improve soil quality by adjusting the pH in soil and provide some nutrients. Moreover, soil degradation is a major problem in agricultural countries. Soil degradation is the decline in soil quality due to the improper land use. In Thailand, soil degradation is a serious problem in agriculture in the northern mountainous areas. Therefore, the objective of this research is to apply the bottom ash to improve soil quality of degraded soil in Nan province, Thailand and to investigate the effects of amount of bottom ash from 0-30% by weight on pH, electrical conductivity, bulk density and soil texture. Also, corn is grown in the real field environment with bottom ash to see the quality of products. From the results, with higher amount of bottom ash, the quality of degraded soil has been improved especially pH and soil texture. The bulk density of soil decreases at all rates of bottom ash which is favorable for plant growth. This research is applied to improve the yield for plant growing. The results show the improvement of product quality, including the corn height, the size and completeness of corn products. 1Carbon Capture, Storage and Utilization Research Group, Department of Mining and Petroleum Engineering Faculty of Engineering, Chulalongkorn University Bangkok, Thailand 113

The 29th Special CU-af Seminar 2021 August 25, 2021 Introduction and Objectives Nowadays, an economic growth and the rising world population are leading to an increase in the global energy consumption. The coal-fired power plants still play an important role to supply the global energy. Furthermore, a large amount of waste like bottom as is generated from the coal-fired power plants and becomes the major environmental concerns. However, it is used in many applications like cement industry, concrete and landfill as presented in Figure 1. In agriculture, the bottom ash can be used as well. However, the utilization rate is only 0.1% [1]. Due to its properties, bottom ash can replace lime to increase pH value of soil[2]. Also, it can be applied to improve soil texture, enhance water holding capacity and air content as well as supply some necessary mineral ingredients of most soil types; thus increasing quantity and quality of peanut[3]. Bottom ash also can be used as soil amendment in heavy clay soil that can help increase soil workability and porosity, improve crop yield as well as has less impact on environment[4]. Since bottom ash is a light-weight material, it can mix with soil to provide light-weight media for plants of the green roofs[4]. It can be seen that the potential use of the bottom ash in agriculture is very high. However, from most previous work, bottom ash is applied in normal soil to estimate the change in soil properties. In addition, the properties of bottom ash greatly depend on coal sources as well as technology that used in coal-fired power plant. If the utilization rate in agriculture of bottom ash can be increased, it can help to reduce the environmental problems and the operation cost of coal-fired power plants in developing countries as well as agriculture countries. Figure 1: Utilization of coal waste in various sectors [1] In addition, another huge problem in agriculture is the soil degradation defined as a change in the soil health status resulting in a diminished capacity of the ecosystem to provide goods and services for its beneficiaries[5]. Soil degradation involves in salinity, loss of organic matter, fertility decline, soil acidic or alkalinity etc. The main causes of soil 114

The 29th Special CU-af Seminar 2021 August 25, 2021 degradation are human activities such as overuse of pesticide, herbicide, and fertilizer, deforestation, expansion of cultivated areas. Soil degradation decreases soil quality, as well as reduces yield and quality of crops. In Thailand, soil degradation is a serious problem in agriculture in the northern mountainous areas, including Nan province due to human activities such as clearing land for agricultural practices, deforestation by people, exploitation of marginal soils under inadequate soil management practice[6], expansion of cultivated areas, landslide, flooding and agrochemicals[7]. Figure 2 and 3 present the deforestation and land use for corn growing in Nan province. Objective. The objectives of this study are to apply and evaluate the bottom ash from Mae-Moh power plant to improve the quality of degraded soil in Nan province and to determine the optimum ratio of bottom ash-soil combinations as well as to estimate potential use in agriculture of bottom ash from Mae-Moh power plant for degraded soil in Nan. This study is expected that bottom ash can be applied in agriculture to improve soil quality, reduce environmental impacts, and improve profitability of bottom ash; thus contributing to the economic and social development of Thailand as well as developing countries in the region. In addition, the cost of fertilizer consumption and waste-management can be decreased. Furthermore, effective land use and friendly environment of waste reduction as well as deforestation can be lower. Figure 2: The deforestation to prepare the land for corn growing. Figure 3: Land use for corn growing. 115

The 29th Special CU-af Seminar 2021 August 25, 2021 Soil properties. The following are some main properties of soil that can be improved when applying coal ash as a soil amendment. 1. Soil texture. Soil texture reflects the particle size distribution of soil. It is an important parameter affecting the water content ability and draining speed of soil. For example, clay soils do not drain well but they hold water well. In contrast, sandy soils have quick water drainage and do not hold water well. Loam and other soils within sand and clay ranges have varied characteristics based upon the size of the particles. Soil texture affects to plant growth indirectly. For example, it controls the pore space of soil that affects the movement of water, air, and temperature in soil, which in turn, affecting to plant growth. Moreover, soil texture influences the available water in the soil, which directly affects to plant growth in soil. The available water in the soil is the difference between the maximum water content in soil (field capacity) and the amount of water that cannot be extracted by the plant (permanent wilting point). It also depends on the soil texture and soil organic matter content. 2. Soil pH, ECse. Soil pH is known as the acidity of soil represented by the negative logarithm of the hydrogen ion concentration in soil. It is divided into three ranges. They are acidic (pH 1-6) neutral (pH 7), and alkaline (pH 8-14). Soil pH significantly affects the solubility of the nutrients in the soil so that it also affects proper plant growth and development. Different types of plant grow well over different ranges of soil pH values. Therefore, soil pH is a useful parameter to choose the type of crop suitable for soil. Depending on coal source, coal ash can be acidic or alkaline that is useful to buffer the soil pH. The addition of fly ash in strip mine soils help to neutralize acid soil and enhance plant growth[4]. Fly ash may neutralize soil acidity and increase crop yield[5]. From Wright[6], bottom ash is used to amend soil. The research confirms that bottom ash can be used to alternate for lime in agriculture. Furthermore, salinity is a critical parameter that affects the productivity of crop. In general, almost of crops are sensitive to salinity because of the high concentration of salt in soil. A high soil salinity can slow the plant growth such as shorter stature, smaller leaves, and sometimes fewer leaves[7]. The soil salinity is estimated from the electrical conductivity (EC) of a soil solution. Nowadays, ECse is considered as the most dilute soil solution concentration, which plants can be encountered in the field. It is usually used to relate plant response to the soil salinity. Therefore, this study uses ECse as a parameter to explain the relation between plant growth and soil salinity. A high ECse soil restricts water uptake by plant roots, even if the soil has high water content. 3. Bulk density Bulk density is a parameter used to indicate the compaction of soil. The higher the bulk density soil is the lower the soil porosity and the higher the soil compaction. Bulk density influences the root growth, available water in soil, soil porosity, nutrients for the plant growth, and soil microorganism activity. These affect the plant productivity. If bulk density increases, the available water capacity of soil reduces. 116

The 29th Special CU-af Seminar 2021 August 25, 2021 Bulk density is the proportion of dry soil mass in a given volume typically expressed in grams/cm3. The main factors affecting bulk density are soil texture, soil organic matter, and the density of soil mineral. Additionally, bulk density increases in the deeper layers of soil since it is more compacted, less root grows, and less pore space in there as compared with surface layers. Experiment Materials Bottom ash is obtained from the Mae-Moh power plant, Lampang province and degraded soil is provided from Nan province, Thailand. Bottom ash will be mixed with degraded soil at certain ratio from 0-30% by weight of bottom ash. Equipment X-ray fluorescence (XRF) is used to determine the composition of soil, bottom ash and fertilizer. This study used benchtop pH/water quality analyzer from Laqua Model F-74 to test pH, of materials with an accuracy of ±0.001. Also, dry sieve and wet sieve are conducted to analyze soil texture of materials. Sieves number used are ranging from number 4 to 325. The bulk density is calculated as the mass of dry soil in a given cylinder volume. Experimental procedure There are two parts of this study. For Part 1, the study is to measure the soil qualities like the bulk density, pH EC and soil texture of the mixtures ranging from 0 to 30% wt bottom ash. The results of the experiment will indicate how bottom ash can improve soil properties. For Part 2, the experiment is performed by growing corn in the bucket to control the factors for plant growing. For this experiment, coal-waste are mixed with soil and fertilizer to grow corn. The original soil without coal ash as well as soil with fertilizer are performed and compared the results. Moreover, the study is evaluated the effect of bottom ash as a soil amendment on plant growth which are recorded properly every weeks. 1. Soil texture measurement To determine the soil texture, the percentage of clay, silt and sand in soil and combinations are measured by wet sieve and hydrometer experiment, following the American Society for Testing and Materials (ASTM- D422-63) standard[8]. Based on ASTM standard, 100 grams of sample is putted in 500 ml cylinder. At the same time, place 125 ml of 40g/l sodium hexametaphosphate solution into the cylinder that contains the sample, then stir the sample until it is thoroughly wetted and soak it at least 16 hours. After 16 hours, place the solution into sedimentation cylinder and add distilled water until total volume is 1000 mL and cover the end of cylinder, turn it upside down and back for 1 minute. Read from hydrometer and measure temperature of solution at the following intervals of time from 0 to 1440 mins. After 24 hours, transfer solution to the set sieve No.40 to 200 and wash with water until cylinder is clear. After that, dry the particles retained on sieve No.40 to No.200 in the oven for 24 hours, then weigh of each portion. 117

The 29th Special CU-af Seminar 2021 August 25, 2021 2. pH and EC measurement The pH and EC value of all samples are measured by using Benchtop pH/Water Quality Analyzer LAQUA F-74. To prepare solution the measurement, 10 grams of sample is dissolved with 10 ml of distilled water for pH measurement. Also, for EC measurement, 15 grams of sample is dissolved in 15 ml of distilled water. The solution is shaken for about 2-3 minutes, then leave it to settle for 2 minutes. The pH/Water Quality Analyzer is used to measure pH and EC value of the samples. The procedure is repeated for three times to get the average value of pH and EC. 3. Bulk density measurement From Tan[9], the equipment used to measure the bulk density of samples is scale, oven and 100 mL graduated cylinder. The procedure is that the sample is added into the cylinder and compacts the samples by tapping the bottom of the cylinder. Keep tapping and filling the sample until 100 mL of cylinder is filled. Weigh and record it of the cylinder contain sample. Repeat all procedure for three times to get the average value of the bulk density. 4. Plant growing In this study, based on the results of Part 1, the practical combination of the bottom ash at 10% and 20% by weight is considered as optimum ratios combinations and is applied in the real field for the study in Part 2. Corn is selected to grow in the plastic containers at the agricultural learning center, School of Agricultural Resources Chulalongkorn University (CUSAR) at Nan, Thailand to evaluate the effects of bottom ash and FGD gypsum on plants growth. There are 10 treatments applied including 5 treatments without fertilizer (S100, S90:BA10, S80:BA20, S90:FGD10 and S80:FGD20) and 5 treatments mixed with fertilizer (S100:F, S90:BA10:F, S80:BA20:F, S90:FGD10:F and S80:FGD20:F) to evaluate the effect on corn growth of bottom ash, with and without fertilizer. To increase the reliability of this study, there will be 3 replications for each treatment. Therefore, corn would be grown in 30 treatment plots. The height of corn tree as well as the size and completeness of corn product will be recorded and compared. Due to the low soil quality, the fertilizer is required to add in the soil sample as well. It is added for 50 g in soil every week for 6 weeks. The total amount of fertilizer is 6% by weight. The samples with and without fertilizer are studied to compare the results of the quality of the corn tree and products. Results and Discussion Physicochemical properties of bottom ash Bottom ash is considered as strong alkaline. Based on the sample analysis, the main parts of bottom ash are the oxides of silicone (Si), calcium (Ca), aluminum (Al) and iron (Fe) for 27.0%, 23.9%, 11.8% and 12.0%, respectively. Moreover, other components are different essential elements, including both macronutrients phosphorus (P), potassium (K), calcium (Ca), sulfur (S) and micronutrients Zinc (Zn), iron (Fe), copper (Cu) for plant growth. These nutrients can be added to soils when natural concentrations are deficient for plant growth. 1. Effect of bottom ash on soil texture Soil texture is one of the most important soil properties. It affects other soil properties such as water holding capacity, soil structure, soil compaction; thus influencing plant growing. 118

The 29th Special CU-af Seminar 2021 August 25, 2021 Each type of plant will be suitable with different soil texture. However, loam is considered as an ideal soil for sufficient vegetative growth, root development[10]. From this study, the effects of amount of bottom ash on soil texture are presented in Figure 4 and Figure 5. Figure 4 presents the particle size distribution curve to determine percentage of sand, silt and clay in soil, bottom ash and combinations. Also, soil texture of combinations are classified by plotting percentage of sand, silt and clay on soil texture triangle graph as shown on Figure 5. From Figure 5, the degraded soil in Nan is classified as clay, which contains 60% of clay, 20% of silt and 20% of sand. Bottom ash from Mae-Moh power plant is sand, which contains 98.5% of sand and 1.5% of silt. The results show that bottom ash can reduce the clay content in the soil and increase the sand content. By the increasing percentage of bottom ash in the combinations, the soil texture has the tendency to be sand. Therefore, changing the texture of soil from clay to clay loam, with most ratios of bottom ash are applied, except combination of 5% bottom ash and 95% of soil. Consequently, bottom ash is considered to be helpful for soil texture improvement. Figure 4: Particle size distribution curve of soil, bottom ash and combinations Figure 5: The change in soil texture when mixing with bottom ash 2. Effect of bottom ash on pH. pH is the chemical characteristics of the soil that affects the availability of plant nutrients and plant growth. Most plants can grow best in soil with a slightly acid reaction[9]. Soil in Nan is acidic with pH=5.664 and bottom ash from Mae-Moh power plant is strongly alkaline with pH=9.740. According to the results, bottom ash can increase soil pH as shown in Figure 6. The mixtures of bottom ash from 5% to 25% are considered the appropriate ratios for the pH improvement of soil, which increase the soil pH from 5.664 to 7.115. At the 30% bottom ash, soil becomes relatively alkaline and it is not good for plant growth. 119

The 29th Special CU-af Seminar 2021 August 25, 2021 Figure 6: pH measurement of soil, bottom ash and combinations. 2. Effect of bottom ash on pH. pH is the chemical characteristics of the soil that affects the availability of plant nutrients and plant growth. Most plants can grow best in soil with a slightly acid reaction[9]. Soil in Nan is acidic with pH=5.664 and bottom ash from Mae-Moh power plant is strongly alkaline with pH=9.740. According to the results, bottom ash can increase soil pH as shown in Figure 6. The mixtures of bottom ash from 5% to 25% are considered the appropriate ratios for the pH improvement of soil, which increase the soil pH from 5.664 to 7.115. At the 30% bottom ash, soil becomes relatively alkaline and it is not good for plant growth. 3. Effect of bottom ash on electrical conductivity (EC). Soil The electrical conductivity (EC) is also the important soil property. It is an indirect measure of the total salt concentration in soil, or salinity. It influences crop yields, crop suitability, plant nutrient availability, and activity of soil microorganisms. Practically, it is difficult to measure directly EC value of soil. Therefore EC (1:5) is considered as an alternative method to measure the EC value of the soil. However, to predict the plant response, EC (1:5) must be converted to the EC of a saturated extract (ECSE) by the equation as shown below[11]. According to the classification of crop tolerance to salinity of Maas and Hoffman[12], most plants cannot grow with ECSE higher than 32 dS/m. From the results, bottom ash increases ECSE value of soil at all ratios as shown in Figure 7. Although, ECSE value of bottom ash is very high (34.72 dS/m), ECSE value is still suitable for plant grow when bottom ash is mixed with soil. ECSE (dS/m) = EC (1:5) (dS/m) x Conversion Factor Where the Conversion Factor is derived from clay content. Figure 7: Electrical conductivity of saturated extract value of soil, bottom ash and combinations. 120

The 29th Special CU-af Seminar 2021 August 25, 2021 4. Effect of bottom ash on bulk density. Bulk density is an important physical characteristic of soil because it affects the root growth. Bulk density is measured to evaluate the compaction of soil. The soil with high bulk density can limit root growth, associated plant nutrient and water uptake. Like soil texture, each soil will have a different ideal bulk density for plant growth and threshold of bulk density value that restricts root growth. In this study, the soil sample is clay. According to United States Department of Agriculture (USDA), the ideal bulk density for root growth in clay or clay loam soil is lower than 1.10 g/cm3 and the threshold bulk density is higher than 1.47 g/cm3 for clay and 1.5 g/cm3 for clay loam, respectively[13]. From the experimental results, the application of bottom ash can decrease bulk density of soil as shown in Figure 4, bottom ash with 1.184 g/cm3 bulk density can reduce bulk density of soil from 1.270 g/cm3 to 1.210 g/cm3. Although, bulk density of all mixtures higher than ideal bulk density for plant growth, it is still lower than that to restrict root growth. Figure 8: Bulk density experiment result of soil, bottom ash and combinations. 5. Effect of Bottom Ash on Plant Growth. This study also investigate the effect of bottom ash on the growth rate of plants, including measuring plant height, diameter and weight of the corn products for many months. The growth of corn, the size and weight of corn product obtained are shown in Table 2 and Figure 9 and 11. The application of bottom ash from 0% to 20% wt. provides the height of plant higher than the original acidic soil sample. For this case, the mixture of acidic soil, bottom ash and fertilizer can improve the soil properties. From the results, it can be seen that with the mixture of bottom ash, the quality of soil is improved except at 20% BA. However, if the mixture is added with 50 g of fertilizer every weeks for 6 weeks, the soil quality becomes higher compared to other conditions. The reasons may be assumed that at 20% BA, the EC is higher compared to the results at 10% BA with and without fertilizer. Furthermore, with the fertilizer, the quality of product is much improved for all cases. Therefore, adding fertilizer with small amount is required to improve soil quality and plant growth. In addition, based on the root system, the lateral roots and fibrous roots from the mixture with fertilizer have longer and more expanded roots compared to the original soil or soil with only fertilizer. Therefore, the plant can take more nutrients in the soil easily. 121

The 29th Special CU-af Seminar 2021 August 25, 2021 Table 2: Quality of corn product in the corn crop in the buckets Figure 9: Comparison of corn height grown in the buckets Figure 10: Comparison of corn products grown in the buckets 122

The 29th Special CU-af Seminar 2021 August 25, 2021 Figure 11: Comparison of corn root system grown in the buckets From the results of this study, it can be used in the real application because the bottom ash is intended to improve the quality degraded soil in Nan province, Thailand for corn growing and to determine the optimum ratio of bottom ash and soil combinations as well as the conditions that fit well with corn growth. Moreover, the properties of soil after applying the bottom ash can be used for various types of crops in that province. Conclusion From this study, the effects of bottom ash on soil properties are investigated in various mixing ratios from 0% to 30% by weight. The results present that the application of bottom ash can improve soil texture from clay to clay loam at ratio from 10% to 30% by weight, adjust the pH value of soil to fit well for soil amendment and decrease the bulk density of soil at all ratios to have more space for air in soil. For the electrical conductivity (EC), the bottom ash cannot improve EC of soil, but EC value after bottom ash applied are still in the range that is suitable for plant growth at all ratios. In addition, the application of bottom ash can improve qualities of degraded soil such as soil texture, bulk density, pH value that help soil function fit well with various crops. For the quality of corn after mixed with bottom, it is obvious that the quality of soil with bottom and fertilizer has been improved and the corn product including root system is much better compared with original soil. From this research, it is also helpful for environment on waste management and cost reduction on waste disposal and effective land use. Furthermore, the application of the bottom ash at 10% and 20% by weight is considered as optimum ratio bottom ash-soil combinations and can be applied in the real field for further study of soil property improvement. 123

The 29th Special CU-af Seminar 2021 August 25, 2021 References 1. https://www.acaa-usa.org/Portals/9/Files/PDFs/2016-Survey-Results.pdf. 2. Korcak R F 1998 Agricultural Uses of Municipal Animal and Industrial Byproducts USDA-ARS, Conservation Research Report vol 44, ed Wright R J (Washington DC: Agricultural Research Service) chapter 6 pp. 103-119. 3. Wearing, C., Birch C.J. and Nairn J.D., Developments in Chemical Engineering and Mineral Processing 2004(12): 531-543. 4. Ramme B. and Tharaniyil M., Coal Combustion Products Utilization Handbook, 3rd ed., We Energies 2013. 5. http://www.fao.org/soils-portal/soil-degradation-restoration/en/. 6. Aumtong S and Magid J 2006 Proc Int. Symp. Towards Sustainable Livelihoods and Ecosystems in Mountainous (Chiang Mai, Thailand) p. 7 7. Baicha W 2016 Geography and Natural Resources 37 87. 8. American Society for Testing and Materials (ASTM) 1998 Standard Test Method for Particle-Size Analysis of Soil D422-63 9. Tan K H 2005 Soil Sampling, Preparation, and Analysis vol 2 (Georgia: CRC press) chapter 8-9 pp. 175 10. Abdulazeez A 2017 J. Agriculture and Veterinary Science 10 70. 11. http://downloads.backpaddock.com.au/SoilMate_Info_Library/SoilMate_NutriFacts/ SOIL_ELECELECTR CONDUCTIVITY ECS06.pdf 12. http://library.wur.nl/WebQuery/wurpubs/fulltext/409817 13. https://www.nrcs.usda.gov/Internet/FSE_DOCUMENTS/nrcs142p2_053260.pdf 124



Application of EBSD to petroleum related strike-slip zones in Thailand Pitsanupong KANJANAPAYONT

The 29th Special CU-af Seminar 2021 August 25, 2021 Application of EBSD to petroleum related strike-slip zones in Thailand Pitsanupong KANJANAPAYONT1* Abstract There are over 60 basins have a petroleum potential in Thailand, and some of these basins have operated for petroleum production. These north-south Cenozoic basins are formed by movement on the north-south trending normal faults, which proposed to be related to movement of strike-slip zones including the Three Pagodas shear zone in Western Thailand. The 9 samples of quartz mylonites within the northwest-southeast trending Three Pagodas shear zone in Kanchanaburi province, Western Thailand were selected for crystallographic preferred orientation (CPO) analysis to understanding the structural evolution of the petroleum related strike-slip zone. The result indicates that the average kinematic vorticity number (Wk) is 0.93 ±0.05, which implied that the quartz mylonites within this strike-slip shear zone have a simple shear component dominantly. This strike-slip shear zone, which characterized the sinistral simple shear, has a significant to be related with the Cenozoic petroleum basin forming. 1Department of Geology, Faculty of Science Chulalongkorn University Bangkok, Thailand 127

The 29th Special CU-af Seminar 2021 August 25, 2021 Introduction and Objectives In Thailand, over 60 basins have a petroleum potential, and some of these basins have operated for petroleum production for a long time (Department of Mineral Resources, 2014). These north-south basins are formed by movement on north-south trending normal faults, which are linked to strike-slip zones since the Cenozoic (Polachan et al., 1991). The strike-slip zones are generally orientated either northeast-southwest or northwest- southeast from northern through southern Thailand (Kanjanapayont, 2015). Previous studies of the geology of the strike-slip zones and their deformation history were limited and emphasized on the sinistral strike-slip movements (e.g. Lacassin et al., 1993, 1997). These previous studies are based on observations of a few outcrops and there is no clear observational evidence to link to the observed movements and basin formation. The study area is located at the NW–SE striking Three Pagodas shear zone in Kanchanaburi province, Western Thailand (Figure 1). The mylonites of this shear zone is 250 km long and 25 km wide. They extend from the Three Pagodas Pass on Thai–Myanmar border towards central Thailand (Kanjanapayont, 2015). The strike–slip zone cross cuts the sedimentary units of the Palaeozoic (Silurian–Permian) and Mesozoic sedimentary units, and Cambrian–Ordovician low–metamorphic units (Department of Mineral Resources, 1982). The high-grade metamorphic rocks within the ductile shear zone were named as “Thabsila gneiss” or “Thabsila metamorphic complex” in the Kanchanaburi province, Western Thailand (Nantasin et al., 2012). It crops out as a lenticular slice of the high–grade metamorphic rocks. These rocks have been inferred as Precambrian basement rock (Bunopas, 1981; Department of Mineral Resources ,1982). Based on lithology and structural features, the Thabsila gneiss is shown as mylonites comprising of four units: (1)Unit A: marble, mica schist, fine-grained biotite gneiss, and quartzite, (2)Unit B: mylonitic gneiss and mylonite, (3)Unit C: calc–silicate, and (4)Unit D: biotite gneiss, orthogneiss, and sillimanite gneiss (Nantasin et al., 2012). Petrology and mineral chemistry reveal that the Thabsila gneiss was formed during single metamorphic event at upper amphibolite to lower granulite facies conditions, and experienced constrictional strain with a sinistral shearing during exhumation (Nantasin et al., 2012). Previous studies of 40Ar/ 39Ar dating were indicated that the deformation of the Three Pagodas and the Mae Ping shear zones terminated around 30.5 Ma (Oligocene) before experienced rapid cooling around 23 Ma (Oligocene–Miocene) (Lacassin et al., 1997). Sinistral shearing occurred after the collision of India and Asia and later rotated and pushed Indochina southeastward leading the formation the South China Sea (Lacassin et al., 1993, 1997). The zircon rims U–Pb ages at 51–57 Ma suggest that the peak upper amphibolite facies metamorphism occurred during the early collision between India and Asia in the Paleocene– Eocene (Nantasin et al., 2012). The biotite Rb–Sr ages of 32–36 Ma (Nantasin et al., 2012), biotite K–Ar ages of 33–36 Ma (Bunopas, 1981), and biotite 40Ar/ 39Ar age of 33 Ma. (Lacassin et al., 1997) imply that the cooling of the basement rocks was in the Eocene–Oligocene. 2–dimensional strain analysis reveals that the averaged strain ratio (Rs) for the lower greenschist facies increment of XZ–plane is Rs = 1.60–1.97 with the average kinematic vorticity number of 0.90 ±0.07 by using the Fry’s method (Kanjanapayont et al., 2018). It showed that a simple shear component with a small pure shear component in the Three Pagodas shear zone (Kanjanapayont et al., 2018). The analysis of crystallographic preferred orientation (CPO) is an effective classical tool to quantify rock microstructure and deformation (e.g. Bauer et al., 2018; Elyaszadeh et al., 2018; Nagaya et al., 2017; Sivand et al., 2021; Xypolias et al., 2018). This study aims to 128

The 29th Special CU-af Seminar 2021 August 25, 2021 apply the CPO technique to the strike-slip zones in Thailand for understanding the structural evolution of the area where related to the petroleum basin forming. The result is not only used for improvement the petroleum model in Thailand, but also can apply to the other prospects related with the strike-slip systems. Figure 1: Geological map of the Three Pagodas shear zone (modified after Department of Mineral Resources, 1982). Methods Field observation was performed around the Three Pagodas strike-slip shear zone in Kanchanaburi province, Western Thailand to collecting rock samples and geological data such as the orientations of foliation and lineation. Geological data were processed and plotted in the stereographic net. Rock samples were prepared for the thin sections before quartz CPO analysis. Due to COVID-19 pandemic, electron backscatter diffraction (EBSD) analysis for CPO at the University of Tokyo, Japan cannot access, a universal stage on an optical microscope for CPO at the Department of Geology, Faculty of Science, Chulalongkorn University, Thailand was took place to achieve the objective. The thin sections were measured under the optical microscope for δ, which is angle between the main foliation and the oblique grain shape fabric. The β, angle between the main foliation and the perpendicular the central girdle segment of quartz c-axis fabric, was derived from the CPO analysis. Both δ and β are applied for the kinematic vorticity number (Wk) in two-dimensional flow. The Cosine of θ = δ+β between the two eigenvectors gives the mean kinematic vorticity number (Wk) (Bobyarchick, 1986; Passchier, 1986; Wallis, 1995). An estimate of kinematic vorticity number (Wk) can be obtained from both δ and β angles by using the equation: 129

The 29th Special CU-af Seminar 2021 August 25, 2021 cos θ = Wk (1) Pure shear described by Wk = 0, while simple shear is described by Wk = 1. General shear is the intermediate between pure and simple shear (Passchier and Trouw, 2005). Results and Discussion The 9 samples including TP01A, TP02A, TP03A, TP06A, TP07A, TP10A, TP10B, TP12A, TP12B of quart mylonites in the Three Pagodas shear zone were selected for CPO analysis. The sample locations were selected by the best quality of exposure. They widespread through the shear zone. All outcrop exposures show the strong NW-SE foliation with the moderate to steep dipping in the NE and SW (Figure 2). The stretching lineation clearly oriented with sub-horizontal in the NW and SE directions (Figure 2). Figure 2: Quartz mylonite samples from the Three Pagodas shear zone; (a) sample TP01A, (b) sample TP02A, (c) sample TP03A, (d) sample TP06A, (e) sample TP10A, (f) sample TP12A 130

The 29th Special CU-af Seminar 2021 August 25, 2021 Under the microscope, the quartz mylonite samples from the Three Pagodas shear zone show the major composition of quartz and feldspar. The minor composition is typically composed of chlorite, biotite, muscovite, rock fragments and opaque minerals. Grain shape is generally oblique to the main filiation in the same direction. The angle between the main foliation and the oblique grain shape fabric, δ, of quartz mylonite samples of this strike-slip shear zone is range from 12° to 24°. The samples of TP01A, TP02A, TP03A has a small number of 12° and 13°, while the samples of TP06A, TP07A, TP10A, TP10B, TP12A, TP12B are greater from 19° to 24° (Figure 3). Figure 3: Angle between the main foliation and the oblique grain shape fabric under plane polarized light of quartz mylonite samples from the Three Pagodas shear zone; (a) sample TP01A, (b) sample TP02A, (c) sample TP03A, (d) sample TP07A, (e) sample TP10A, (f) sample TP10B 131

The 29th Special CU-af Seminar 2021 August 25, 2021 The angle between the main foliation and the perpendicular the central girdle segment of quartz c-axis fabric, β, of all sample is range from 10° to 20°. The samples of TP01A, TP02A, TP03A, TP06A, TP07A, TP10A, TP10B are 17° to 20°, and the samples of TP12A and TP12B are 10° (Figure 4). Figure 4: Stereographic plots of quart c-axis fabrics of quartz mylonite samples from the Three Pagodas shear zone with the angle between the main foliation and the perpendicular the central girdle segment of quartz c-axis fabric. 132

The 29th Special CU-af Seminar 2021 August 25, 2021 Both δ and β are summarized in Table 1. The kinematic vorticity number (Wk) of the quartz mylonite samples from the Three Pagodas shear zone in Kanchanaburi province, Western Thailand ranges from 0.88-0.99. An average of the kinematic vorticity number (Wk) is 0.93 ±0.5. Simple shear deformation is represented by the samples TP06A, TP07A and TP10A which have described by Wk = 1. Table 1: Summarized data from quartz mylonite samples in the Three Pagodas shear zone. The summation of δ and β angles and kinematic vorticity nsiummpbleersh(Weakr) indicate that the Three Pagodas shear zone mainly deformed in the sinistral environment (Figure 5). This result concords to other shear zones nearby including the Mae Ping shear zone in Northern Thailand (Ponmanee et al., 2016), the Three Pagodas shear zone itself (Kanjanapayont et al., 2018), and the Khlong Marui shear zone in Southern Thailand (Kanjanapayont et al., 2012). Considering to the model of the Cenozoic basins related strike-slip systems (Polachan et al., 1991) and the previous geochronological works (Bunopas, 1981, Lacassin et al., 1997, Nantasin et al., 2012), the strike-slip systems in Thailand were coincided with the Cenozoic basins. Then, the Three Pagodas and adjacent shear zones may influence the process of petroleum basin forming during the Cenozoic. Figure 5: Map showing the kinematic vorticity number (Wk) of the sinistral Three Pagodas shear zone, which related to the petroleum Cenozoic basins (gray). 133

The 29th Special CU-af Seminar 2021 August 25, 2021 Conclusion The crystallographic preferred orientation (CPO) analysis was applied in quartz mylonite samples from the Three Pagodas shear zone in Kanchanaburi province, Western Thailand. The 9 samples are TP01A, TP02A, TP03A, TP06A, TP07A, TP10A, TP10B, TP12A, TP12B. The angle between the main foliation and the oblique grain shape fabric, δ, is range from 12° to 24°, and the angle between the main foliation and the perpendicular the central girdle segment of quartz c-axis fabric, β, is range from 10° to 20°. The kinematic vorticity number (Wk) ranges from 0.88-0.99 with an average of 0.93 ±0.5. This result shows that the quartz mylonites within the Three Pagodas shear zone in Kanchanaburi province, Western Thailand dominate a simple shear component. The sinistral simple shearing of the strike-slip Three Pagoda shear zone has a significant to be related with the Cenozoic petroleum basin forming. References 1. Bauer, H., Rogowitz, A., Grasemann, B., Decker, K., Geology, 2018, 46(4): 375-378. 2. Bobyarchick, A.R., Tectonophysics, 1986, 122: 35-51. 3. Bunopas, S., PhD Thesis, Victoria University of Wellington, Victoria, New Zealand, 1981, 810 pp. 4. Department of Mineral Resources, Geological map of Thailand, 1982, scale 1:1,000,000. 5. Department of Mineral Resources, Geology of Thailand, 2014, 511 p. 6. Elyaszadeh, R., Prior, D.J., Sarkarinejad, K., Mansouri, H., J. Struct. Geol., 2018, 107: 38-52. 7. Kanjanapayont, P., In: Mulchrone, K.F., Mukherjee, S. (Eds.), Ductile Shear Zones: From Micro- to Macro-scales, 2015, 250-269. 8. Kanjanapayont, P., Grasemann, B., Edwards, M.A., and Fritz, H., J. Struct. Geol., 2012, 35: 17–27. 9. Kanjanapayont, P., Ponmanee, P., Grasemann, B., Klötzli, U., and Nantasin, P., Austrian J. Earth Sci., 2018, 111(2): 171–179. 10. Lacassin, R., Leloup, P.H., and Tapponnier, P., J. Struct. Geol., 1993, 15: 677-692. 11. Lacassin, R., Maluski, H., Leloup, P.H., Tapponnier, P., Hinthong, C., Siribhakdi, K., Chuaviroj, S., and Charoenpravat, A., J. Geophys. Res., 1997, 102: 10,013-10,037. 12. Nagaya, T., Wallis, S. R., Seto, Y., Miyake, A., Soda, Y., Uehara, S., and Matsumoto, M., J. Struct. Geol., 2017, 95: 127-141. 13. Nantasin, P., Hauzenberger, C., Liu, X., Krenn, K., Dong, Y., Thöni, M., and Wathanakul, P., J. Asian Earth Sci., 2012, 60: 68–87. 14. Passchier, C.W., Earth Planet. Sci. Lett., 1986, 77: 70-80. 15. Passchier, C.W., and Trouw, R.A.J.,. Microtectonics, 2nd, 2005, 366 pp. 16. Polachan, S., Pradidtan, S., Tongtaow, C., Janmaha, S., Intarawijitr, K., and Sangsuwan, C., Mar. Pet. Geol., 1991, 8: 84-97. 17. Ponmanee, P., Kanajanapayont, P., Grasemann, B., Klötzli, U., and Choowong, M., Austrian J. Earth Sci., 2016, 109(2): 233–240. 18. Sivand, S.M., Faghih,A., Keshavarz, S., and Soleimani, M., J. Struct. Geol., 2021, 143: 104270. 19. Wallis, S.R., J. Struct. Geol., 1995. 17: 1077-1093. 20. Xypolias, P., Gerogiannis, N., Chatzaras, V., Papapavlou, K., Kruckenberg, S.C., Aravadinou, E., and Michels, Z., J. Struct. Geol., 2018, 115: 61-81. 134