The 28th Special CU-af Seminar 2020

The 28th Special CU-af Seminar 2020 “Frontier Research for Sustainable Society” October 20th, 2020 Room 105, Maha-Chulalongkorn Building Chulalongkorn University

The 28th Special CU-af Seminar 2020 October 20, 2020 MESSAGE from PRESIDENT, CHULALONGKORN UNIVERSITY Professor Bundhit Eua-Arporn, Ph.D. It is my great pleasure to welcome all of you to Chulalongkorn University and to this 28th Special Chulalongkorn University and the Asahi Glass Foundation seminar 2020. Our university has taken pride in a very successful record in our research performance throughout these years. However, Chulalongkorn University cannot achieve these goals without long supports from our precious partners like the Asahi Glass Foundation. For nearly forty years, the university has been receiving continuous and generous support from the Asahi Glass Foundation of Japan, enabling us to promote various impactful research works especially in the areas of engineering, medical sciences, health sciences, environmental sciences as well as information technology. Research and development is one of the major factors in the development of higher educa- tion and the country nowadays, as we are gearing towards a knowledge-based global community and creative economy. Therefore, the research for new knowledge and technological advance- ment is indispensable and must be carried out in view of particular needs and practical uses on both national and global levels. It should also be emphasized here that research in university can both enhance and enrich our teaching to be more up-to-date and relevant to the contemporary and sustainable society. It also helps in building the capacity of our student and staff for this fast changing world. This seminar is organized to showcase the research projects supported by the Asahi Glass Foundation and demonstrates our strong commitment to be a research university who create knowledge and innovations for sustainable society. It also allows researchers to publicise their findings and to exchange ideas among distinguished audience. I am confident that this kind of academic exchange will greatly contribute to the existing body of knowledge and enhance the atmosphere of intellectual spirit. I would like to take this opportunity to express my warmest thanks to the Asahi Glass Foundation for the generous support for nearly four decades. It remains in my firm believe that the relationship between us will be even stronger in the future. May I take this opportunity to declare the 28th Special Seminar open. I would like to express my best wishes for the success of this event and hope that the seminar will achieve all of its goals. I

The 28th Special CU-af Seminar 2020 October 20, 2020 CONGRATULATORY ADDRESS from CHAIRMAN, THE ASAHI GLASS FOUNDATION Mr.Takuya Shimamura Thank you very much for your kind introduction. Good morning, everyone. My name is Takuya Shimamura, I am Chairman of the Asahi Glass Foundation and CEO of AGC Inc. It is both honor and pleasure for me to be this ceremony, as a representative of Asahi Glass Foundation in Japan. Firstly, please allow me to extend my sincere gratitude to Professor Bundhit Eua-arporn, President of the University, Professor Chakkaphan Sutthirat, Vice President, our distinguished guests and the members hosting today’s ceremony. Thank you very much. Secondly, let me introduce Asahi Glass Foundation briefly. It was established in 1933 to celebrate the 25th anniversary of the founding of Asahi Glass Co., Ltd. Asahi Glass Co., Ltd. was renamed the AGC Inc. in 2018. Now we provide 3 major programs such as the Research Grant Program, Scholarship Program and Commendation Program named “Blue Planet Prize”. In addition, we have publicized the annual survey on the global environment named “Environment Doomsday Clock”. In terms of the relationship between AGC Inc. and Thailand, In1964, AGC began to produce flat glass in Thailand, and expanding its business to automotive glass, chemicals and electric materials. Today total annual sales revenue of group is approximately 44 billion THB 1.4 Billion USD in Thailand. And many alumni of the Chulalongkorn University have joined AGC Flat Glass, Thailand, AGC Chemicals, Thailand and so on. In 1982, Asahi Glass Foundation initiated our grant program with Chulalongkorn University. This is the 39th grant presentation ceremony. So AGC Inc, and Asahi Glass Foundation have a long history with Thailand and Chulalongkorn University as well. We have supported more than 290 grant themes and contributed approximately 2.4 M USD from the beginning. I believe these researches can contribute enough for the development and prosperity of Thailand. We wish our support can be helpful for your researchers. As one of other major activities of Foundation, we provide “Blue Planet Prize”, from 1992, which has become internationally recognized award successfully. The foundation has awarded the Blue Planet Prize to two people who have made outstanding contributes towards solving global environment issues with a certificate of merit, commemorative trophy and 50 million Japanese yen or 480,000 Usd per each in prize money. Prof. Jared Diamond and Prof. Eric Lambin were winners in 2019. And Prof. David Tilman from USA and Dr. Simon Stuart from UK this year. Please visit our website, which explains the winner’s achievement in detail. And you can also reach the names and research themes of this year’s new grantees and the research reports of the past year’s grantees. As you know, our environmental situation is getting worse every year. Humankind has sacrificed its environment in exchange for its prosperity. That is the reason why we established the “Blue Planet Prize”. I hope the winner of this prize from Thailand will be awarded someday. I sincerely hope all of researchers here can contribute to our precious Blue planet through your researches. II

The 28th Special CU-af Seminar 2020 October 20, 2020 In closing my speech, I’d like to express my deepest appreciation to Professor Bundhit Eua-arporn, Professor Chakkaphan Sutthirat and all members concerned kindly supported the seminar, and award ceremony as well. As closing my speech, I’d like to express the followings: I firmly believe in for all of you today, Your future is bright. Your potential is unlimited. And your possibilities are endless. Thank you very much. Takuya Shimamura Chairman of Asahi Glass Foundation October 20th, 2020 III

The 28th Special CU-af Seminar 2020 October 20, 2020 REPORT ADDRESS by VICE PRESIDENT, CHULALONGKORN UNIVERSITY Professor Chakkaphan Sutthirat, Ph.D Since Chulalongkorn University received the first support from theAsahi Glass Foundation for research back in 1982, the Asahi Glass Foundation has continuously supported the university for almost 40 years. Initially, this support was focused on research in the field of engineering. Subsequently, it has expanded to other vital areas, including medical science, pharmaceutical sciences, environmental sciences, and information technology. Up to now, more than 290 research projects have been made possible through the generous support of the Asahi Glass Foundation. The findings of these research projects are compiled and published with the support of the Asahi Glass Foundation for dissemination to various agencies worldwide. Moreover, the seminars that have been organized regularly over the past 27 years based on the research findings have provided avenues for the exchange of knowledge and expertise among academics, external organizations, and agencies in both the governmental and private sectors. It is hoped that these seminars will promote the application of the research in the industrial sector as well. Amid the outbreak of SARS-CoV-19, or COVID-19, when the lockdown was announced, It was distressing to think that this meaningful and impactful seminar would not be able to be held this year. However, despite the international travel restrictions, the excellent cooperation between the Asahi Glass Foundation, Japan and Chulalongkorn University has ensured that participants would be able to participate seamlessly in the seminar both onsite and via an online meeting platform. We are proud that this special seminar in the year 2020 marks the 28th such gathering. We are particularly honored by the presence of Professor Dr. Suttichai Assabumrun- grat, the keynote speaker, to present the topic of “Process Intensification and Multifunctional Reactors for Supporting Thailand’s Transformation to a Bio-Circular-Green (BCG) Economy”. After his lecture, ten research papers will be presented, of which seven are from the Faculty of Science, two are from the Faculty of Engineering, and one is from the Metallurgy and Materials Science Research Institute. May I take this opportunity to express our sincere appreciation to the Asahi Glass Foun- dation, Japan, for their continuous support of research projects at Chulalongkorn University. We also thank all our researchers who have worked with strong commitment on their research projects, which will help enrich their teaching and their societies as well as our country. IV

The 28th Special CU-af Seminar 2020 October 20, 2020 TABLE OF CONTENTS Page I Message form President, Chulalongkorn University II Congratulatory Address form Chairman, The Asahi Glass Foundation IV Report Address by Vice President, Chulalongkorn University V Schedule Keynote Lecture 1 Process Intensification and Multifunctional Reactors for Supporting Thailand’s Transformation to Bio - Circular - Green (BCG) Economy Prof. Suttichai Assabumrungrat, Ph.D. Department of Chemical Engineering, Faculty of Engineering Invited Papers 5 Session 1: Biotechnology and Environmental Research 21 1. Poly (Lactic Acid) /Cellulose Green Composites 35 for Automotive Applications 45 Dr. Chuanchom Aumnate and Niphaphun Soatthiyanon 55 2. Optical fiber sensor based on surface plasmon resonance or sensitive and selective detection of Atrazine herbicides Assoc. Prof. Dr. Pakorn Varanusupakul 3. Evaluation of in vitro biocompatibility and efficiency of novel bioactive calcium silicate glass-Thai silk fibroin hybrid scaffolds for bone tissue engineering Dr. Peerapat Thongnuek 4. Synthesis and Applications of Citronella Oil Nanoemulsion Assoc. Prof. Dr. Kawee Srikulkit 5.Sequencing and Characterization of a hypovirulence dsRNA virus from an oomycete Dr.Thanyanuch Kriangripipat Session 2: Research on Energy & Industrial Development 71 1. Removal of Polycyclic Aromatic Hydrocarbons (PAHs) 87 in Waste Tire Pyrolysis Oil via Catalytic Hydrogenation Assoc. Prof. Dr. Napida Hinchiranan 2. Development of natural rubber composite for carbon dioxide adsorbent material Assoc. Prof. Dr. Sirilux Poompradub V

The 28th Special CU-af Seminar 2020 103 117 October 20, 2020 133 3. Efficiency and photostability of visible-light driven metal-doped NaTaO3 149 photocatalysts for environmental purification and clean energy production Assoc. Prof. Dr. Pornapa Sujaridworakun 4. Intelligent Monitoring and Estimation of Surface Roughness and Straightness in CNC Turning Prof. Dr. Somkiat Tangjitsitcharoen 5. Investigation of Defect States from Radiative Emissions in Culn1-xGaxSe2 / Cu(In1-xGax)3Se5 Bi-Layer Systems by Photoluminescence Technique Asst. Prof. Dr. Sojipong Chatraphorn Appendix VI

The 28th Special CU-af Seminar 2020 October 20, 2020 The 28th Special CU-af Seminar 2020 “Frontier research for sustainable society” October 20, 2020 Room 105, Maha-Chulalongkorn Building Chulalongkorn University 09.00 – 09.30 Registration 09.30 – 10.00 Opening Ceremony (Room 105) • Welcome Remark and Report Prof. Chakkaphan Sutthirat, Ph.D. Vice President, Chulalongkorn University • Opening Address Prof. Bundhit Eua-arporn, Ph.D. President, Chulalongkorn University • Congratulatory Address Mr. Takuya Shimamura Chairman, The Asahi Glass Foundation, Japan • 2020 Grant Presentation Prof. Bundhit Eua-arporn, Ph.D. President, Chulalongkorn University Grantees (1) Asst. Prof. Dr. Juthamas Ratanavaraporn Faculty of Engineering (2) Dr. Wilailuck Niyommaneerat Environmental Research Institute (3) Dr. Sira Sriwasdi Faculty of Medicine (4) Assoc. Prof. Dr. Pitsanupong Kanjanapayont Faculty of Science (5) Asst. Prof. Dr. Numpon Insin Faculty of Science (6) Dr. Sudkate Chaiyo Institute of Biotechnology and Genetic Engineering (7) Asst. Prof. Dr. Asada Leelahavanichkul Faculty of Medicine (8) Dr. Sirichai Leelachao Faculty of Engineering (9) Dr. Nithiwach Nawaukkaratharnant Metallurgy and Materials Science Research Institute VII

The 28th Special CU-af Seminar 2020 October 20, 2020 10.00 – 10.20 Keynote Lecture Process Intensification and Multifunctional Reactors for Supporting Thailand’s Transformation to Bio - Circular - Green (BCG) Economy Prof. Suttichai Assabumrungrat, Ph.D. Department of Chemical Engineering, Faculty of Engineering 10.20 – 10.30 Group Photo Session 10.30 – 10.45 Coffee Break 10.45 – 12.00 Concurrent Sessions Session 1: Biotechnology and Environmental Research Session 2: Research on Energy & Industrial Development 12.00 –13.00 Networking Lunch (Room 108) Concurrent Sessions Session 1: Biotechnology and Environmental Research (Room 105) Chairperson: Asst. Prof. Dr. Juthamas Ratanavaraporn Biomedical Engineering Program, Faculty of Engineering Co-chairperson: Dr. Wilailuck Niyommaneerat Research Affairs, Environmental Research Institute 10.45 – 11.00 Poly(Lactic Acid)/Cellulose Green Composites for Automotive Applications Dr.Chuanchom Aumnate Metallurgy and Materials Science Research Institute 11.00 – 11.15 Optical fiber sensor based on surface plasmon resonance for sensitive and selective detection of Atrazine herbicides Assoc. Prof. Dr. Pakorn Varanusupakul Department of Chemistry, Faculty of Science 11.15 – 11.30 Evaluation of in vitro biocompatibility and efficiency of novel bioactive calcium silicate glass-Thai silk fibroin hybrid scaffolds for bone tissue engineering Dr. Peerapat Thongnuek Biomedical Engineering Program, Faculty of Engineering 11.30 – 11.45 Synthesis and Applications of Citronella Oil Nanoemulsion Assoc. Prof. Dr. Kawee Srikulkit Department of Material Science, Faculty of Science 11.45 – 12.00 Sequencing and Characterization of a hypovirulence dsRNA virus from an oomycete Dr.Thanyanuch Kriangripipat Department of Microbiology, Faculty of Science Session 2: Research on Energy & Industrial Development (Room 102) Chairperson: Assoc. Prof. Dr. Pitsanupong Kanjanapayont Department of Geology, Faculty of Science Co-chairperson: Asst. Prof. Dr. Numpon Insin Department of Chemistry, Faculty of Science VIII

The 28th Special CU-af Seminar 2020 October 20, 2020 10.45 – 11.00 Removal of Polycyclic Aromatic Hydrocarbons (PAHs) in Waste Tire Pyrolysis Oil via Catalytic Hydrogenation Assoc. Prof. Dr. Napida Hinchiranan Department of Chemical Technology, Faculty of Science 11.00 – 11.15 Development of natural rubber composite for carbon dioxide adsorbent material Assoc. Prof. Dr. Sirilux Poompradub Department of Chemical Technology, Faculty of Science 11.15 – 11.30 Efficiency and photostability of visible-light driven metal-doped NaTaO3 photocatalysts for environmental purification and clean energy production Assoc. Prof. Dr. Pornapa Sujaridworakun Department of Materials Science, Faculty of Science 11.30 – 11.45 Intelligent Monitoring and Estimation of Surface Roughness and Straightness in CNC Turning Prof. Dr. Somkiat Tangjitsitcharoen Department of Industrial Engineering, Faculty of Engineering 11.45 – 12.00 Investigation of Defect States from Radiative Emissions in Culn1-xGaxSe2 / Cu(In1-xGax)3Se5 Bi-Layer Systems by Photoluminescence Technique Asst. Prof. Dr. Sojipong Chatraphorn Department of Physics, Faculty of Science 12.00 – 13.00 Lunch (Room 108) IX

Keynote Lecture Process Intensification and Multifunctional Reactors for Supporting Thailand’s Transformation to Bio - Circular - Green (BCG) Economy Prof. Suttichai Assabumrungrat, Ph.D.



The 28th Special CU-af Seminar 2020 October 20, 2020 Keynote Lecture Process Intensification and Multifunctional Reactors for Supporting Thailand’s Transformation to Bio - Circular - Green (BCG) Economy Prof. Suttichai Assabumrungrat, Ph.D.1,2,* Abstract The concept of Bio – Circular – Green (BCG) economy model according to the Thailand 4.0 policy offers many promising benefits such as being a sustainable growth based on less dependency of fossil-based resources. It provides a new opportunity for industry, research, policy and financing stakeholders to work together to create new values for the country balancing between the economy, society, and environment. However, the transformation of existing chemical and petrochemical industries to those based on the BCG economy model is of great challenge. Generally, a bio-based feedstock is complex in nature and requires additional pretreatment steps or even some major changes of existing process units, and therefore the bio-based process is typically not really competitive compared to the existing one. Process intensification and multifunctional reactors could play an important role to achieve a substantially more efficient technology with cleaner and safer operation, and consequently allow the bio-based process to become more competitive. This presentation will provide some examples of development of process intensification and multifunctional reactors for some applications such as biodiesel production using several multifunctional reactors such as spinning disc reactor, tube-in-tube reactor and reactive distillation, and hydrogen production via sorption-enhanced reaction and chemical looping reaction. The presentation also includes a systematic methodology for designing an integrated bio-based process with pulp and paper industry. 1Center of Excellence in Catalysis and Catalytic Reaction Engineering, Department of Chemical Engineering, Faculty of Engineering Chulalongkorn University, Bangkok 10330, Thailand 2Bio-Circular-Green-economy Technology & Engineering Center, BCGeTEC, Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok, Thailand 10330 *e-mail: [email protected] 1



Poly (Lactic Acid) /Cellulose Green Composites for Automotive Applications Dr. Chuanchom Aumnate and Niphaphun Soatthiyanon



The 28th Special CU-af Seminar 2020 October 20, 2020 Poly (Lactic Acid) /Cellulose Green Composites for Automotive Applications Dr. Chuanchom Aumnate and Niphaphun Soatthiyanon Abstract Biocomposites materials containing cellulose fibers are gaining increasing interest. In this study, poly(lactic acid)/extracted kenaf cellulose biocomposites were fabricated via melt- compounding process and then manufactured by 3D printing process to optimize the potentially used in automotive applications. Eco-friendly extracted kenaf cellulose fibers from locally grown kenaf plants were prepared and used as reinforcement for such biocomposites to improve their properties, in particular, the mechanical performance. The compatibilizers (PE-g-MA and PP-g-MA) and the plasticizer (Triacetin) were also incorporated into the biocomposites to improve their properties. The effects of extracted kenaf cellulose fibers loading, the addition of the compatibilizers and plasticizer on the mechanical, thermal and morphological properties, also the water absorption of the biocomposites, were examined. Metallurgy and Materials Science Research Institute Chulalongkorn University Bangkok, Thailand 5

The 28th Special CU-af Seminar 2020 October 20, 2020 Introduction and Objectives Currently, environmental issues and problems such as waste disposal and pollution, are much concerned. The use of environmentally friendly materials is attempted in many industries, such as the automotive industry. In the automotive industry, not only the environmental impact is considered but also reducing the weight of vehicles. Thus, the use of various bioplastics and biocomposites has increased and has been continually developed [1]. Poly(lactic acid) or PLA is a biodegradable thermoplastic polymer that is considered to be used as an alternative material for automotive components. This is attributed to its advantages, including renewability, rigidity, high strength, low density (1.25 g/cm3) and low CO2 emission. PLA is an aliphatic polyester derived by direct polycondensation of the lactic acid, which can be obtained from the fermentation of agricultural products, such as corn, sugar beet, and wheat and sugarcane. Also, PLA can be obtained from ring-opening polymerization of the cyclic lactide. Generally, PLA can be processed by several techniques such as film/sheet casting, extrusion, injection molding, blow molding, thermoforming, foaming, fiber spinning and electrospinning. PLA has Young’s modulus of 3.6 GPa, the tensile strength of 70 MPa, the elongation at break of 2.4%, the flexural strength of 98 MPa, the impact strength of 16.5 kJ/m2, and the notched impact strength of 3.3 kJ/m2. In addition, PLA has better mechanical properties than both poly(ethylene terephthalate) (PET) and polypropylene (PP), which are commonly used in automobiles. In recent years, PLA has been used in diverse components (door trims, racing seats, air filter parts, and spare tire cover) by several automobile manufactures, such as Toyota, Ford and Mazda. Additionally, the use of renewable fibers or natural fibers as reinforcements has increased for interior components of several vehicles to produce cost-effective composites [1-8]. However, PLA has poor toughness (low elongation at break and impact strength) due to high glass transition temperature (55oC). Hence, PLA is brittle. Plasticizers, such as polyethylene glycol (PEG), glucosemonoesters and partial fatty acid, have been used to solve this problem [3,6]. Fabricating PLA composites with natural fibers is an alternative route to overcome the brittleness of PLA. Natural fiber is a natural polymer containing cellulose as the main component. It is biodegradable, biocompatible, renewable and inexpensive. In general, the performance of such composites is based on properties of natural fibers used as reinforcements [1]. There is the number of publications on PLA/natural fiber composites, in which the mechanical properties have been reported. For example, Anuar et al. (2012) prepared PLA/ kenaf fiber composites with 0, 5, 10, 15 and 20 wt% reinforcement loading using co-rotating twin-screw extrusion and followed by injection molding. The average length and diameter of the kenaf fibers used were 2954 and 98 µm, respectively. Hence, the aspect ratio of the kenaf fibers was 30. The tensile and flexural moduli of the composites increased with increased fiber loading, while the tensile strength decreased with 5-15 wt% fiber contents. However, the addition of 20 wt% kenaf fibers improved the tensile strength (approximately 18%) as compared to the neat PLA. With increasing the fiber content, the specific tensile and flexural properties of the composites were enhancing while weakening the impact strength [9]. Besides, Yussuf et al. (2010) also fabricated PLA/kenaf (PLA-K) and PLA/rice husk (PLA-RH) composites with 20 wt% reinforcement using twin-screw extrusion followed by injection molding. It was reported that both kenaf and rice husk resulted in a decrease in the flexural strength and impact strength of the neat PLA. This is attributed to poor interfacial addition between the reinforcements and the PLA matrix. The effects of reinforcement addition were dominant for the PLA-K composites [10]. Moreover, Yu et al. (2009) suggested that incorporating 30 wt% of natural fibers, ramie and jute, could improve the mechanical properties of the PLA composites [11]. Additionally, 6

The 28th Special CU-af Seminar 2020 October 20, 2020 the effects of different natural fibers on the properties of the PLA composites have been studied. Graupner et al. (2009) fabricated the PLA composites with 40 wt% loading of various natural fibers, including cotton fibers, hemp fiber bundles and kenaf fiber bundles. It was reported that the PLA/hemp composite had the highest tensile strength and Young’s modulus followed by the PLA/kenaf and PLA/cotton composites. However, the PLA/cotton composites gave the highest values of the elongation at break and Charpy impact strength as compared to those of the others [12]. For polymer/natural fiber composites, good interfacial adhesion between natural fibers and the polymer matrix is an essential parameter that affects the composite properties, especially mechanical properties. To improve such interfacial adhesion, Razak et al. (2014) modified kenaf fibers using bleaching treatment and examined the effects of the bleached kenaf fibers on the mechanical properties of the PLA/kenaf fiber composites. It was found that the tensile and flexural strengths, the elongation at break, and the impact strength of the composites were lower than those of the neat PLA, whereas the tensile and flexural moduli of the composites improved. It was also reported that the tensile and flexural strengths of the composites increased with increasing the fiber contents up to 30 wt%, while the elongation at break and the impact strength of the composites decreased. Moreover, the bleached kenaf fiber successfully offered higher mechanical properties as compared to the unbleached kenaf fiber, which could be attributed to the good interfacial adhesion between the bleached kenaf fiber and the PLA matrix [7]. Furthermore, several additives such as compatibilizers, flexibilizers, plasticizers, impact modifiers and coupling agents have been used to improve the properties of polymer composites. Serizawa et al. (2006) investigated the flexural properties, impact strength and distortion temperatures of the PLA/kenaf fiber composites and the effects of a flexibilizer. The contents of the kenaf fibers used were varied from 10 to 20 wt%. The flexural modulus and the distortion temperature under a 1.8 MPa load of the composites increased with increasing the fiber loading, while the flexural strength and impact strength of the composites decreased. The addition of the flexibilizer , a copolymer of lactic acid and aliphatic polyester (20 wt%), improved the composites’ impact strength; however, the heat resistance of the composites was reduced [13]. In addition, Ibrahim et al. (2010) studied the effects of the plasticizer, which was triacetin (glycerol triacetate), on the mechanical properties of the PLA/kenaf bast fiber composites. It was found that the tensile strength and modulus of the composites increased when the 5 and 10 wt% triacetin were added. However, a 15 wt% triacetin loading reduced the tensile strength and modulus of the composites due to weak interfacial adhesion between the kenaf fiber and the PLA matrix. Also, the elongation at break of the composites increased with increasing triacetin contents. This is because the segmental mobility of the PLA chain increased [14]. Taib et al. (2014) also examined the effects of an impact modifier, ethylene acrylate copolymer, on the mechanical properties of the PLA/kenaf bast fiber composites with a 40 wt% fiber loading. The composites were fabricated using internal mixing followed by compression molding. It was found that the notched impact strength and the elongation at break of the composites increased with increasing the impact modifier contents; however, the tensile strength and modulus decreased [15]. Not only to natural, but also cellulose has been used to enhance the performance of PLA composites. Penjumras et al. (2015) examined the mechanical properties and water absorption of the durian rind cellulose reinforced PLA biocomposites with 25 and 35 wt% reinforcement loading. These biocomposites were also fabricated using internal mixing and hot pressing. It was found that Young’s modulus, impact strength and water absorption of the composites increased 7

The 28th Special CU-af Seminar 2020 October 20, 2020 with increased cellulose content. The higher cellulose loading led to faster water absorption, which was due to the higher number of hydroxyl groups from cellulose counterparts. Also, incorporating coupling agents were suggested to improve adhesion between the cellulose and the PLA matrix [16]. In this work, PLA/cellulose biocomposites were prepared using melt-compounding, followed by 3D printing process. Extracted kenaf cellulose was used as reinforcement. Moreover, PE-g-MA and PP-g-MA were incorporated as compatibilizers to enhance the mechanical performance of the biocomposites. Triacetin was selected as a plasticizer, which is expected to improve the biocomposites’ flowability and processability. The effects of kenaf cellulose fiber loading, type of suitable compatibilizers, and plasticizer on the mechanical and thermal properties, morphology, and water absorption of the biocomposites were examined to optimize the potential application in the automotive area. Materials and Methods Kenaf Cellulose Extraction Untreated kenaf fibers (Khon Kean, Thailand) were treated using 12%w/v NaOH in a 1:20 fiber to liquor ratio, at 80 ± 2oC for 3 h. The treated kenaf fibers were then washed with water and oven dried. The obtained fibers were then treated and bleached in a 50:50 mixture of glacial CH3COOH and 30%w/v H2O2 in a 1:20 fiber to liquor ratio, at 80 ± 2oC for 3 h to obtain cellulose fibers [17]. The fibers were subsequently washed with water followed by warm ethanol and they were then ground to obtained kenaf cellulose fibers (K). PLA/Kenaf Cellulose Biocomposite Filament Fabrication Polylactic acid (PLA) (IngeoTM Biopolymer 4043D, NatureWorks), with the MFR of 6.0 g/10min (190 °C /2.16 kg) and the density of 1.24 g/cm3 was used as a matrix polymer. Polyethylene graft maleic anhydride (PE-g-MA, Licocene PE MA 4351 Fine Grain S1000, Clariant Plastics & Coating) and polypropylene graft maleic anhydride (PP-g-MA, SCONA® TPPP 8112 FA, BYK Additives & Instruments) were used as compatibilizers. Triacetin (99%, Sigma-Aldrich) was also used as processing aid agent. Virgin PLA granules, extracted kenaf cellulose fiber (K), PE-g-MA, PP-g-MA and triacetin were dried in an oven at 70oC overnight to ensure that there was no moisture content. To fabricate the PLA/kenaf cellulose composite filaments, the PLA and kenaf fibers were dried and mixed with PE-g-MA, PP-g-MA and Triacetin according to the compositions shown in Table 1. The premixed materials were then melt compounded together using the microcompounder (Thermo Scientific HAAKE MiniLab II), at 180oC and the screw speed of 40 rpm. Table 1. Material compositions in PLA/K composites. 8

The 28th Special CU-af Seminar 2020 October 20, 2020 3D Printing of PLA and PLA/Kenaf Cellulose Composites The obtained PLA and its composite filaments were then 3D printed using a commercial 3D printer (Wanhao, Duplicator6). All filaments were printed using the same printing conditions, listed in Table 2. Table 2. Parameters setup for 3D printing of PLA and PLA/kenaf cellulose biocomposites. Characterization Non-isothermal crystallization differential scanning calorimetry The thermal properties of PLAand its biocomposites were investigated using the NETZSCH DSC 204F1 Phoenix, under nitrogen atmosphere. The filament samples were heated from 30°C to 220°C with the temperature ramp of 10 K/min, and kept isothermal for 2 min. The samples were then cooled down to 30°C with the temperature ramp of 10 K/min. Following by the second heating scan, the samples were again heated up to 220°C and kept isothermal for 2 min with the same temperature ramping. Consequently, the degree of crystallization (Xc) of PLA and its biocomposites with kenaf cellulose fiber can be determined based on the following equations [18]: where ∆Hm is the enthalpy of melting and ∆Hcc is the enthalpy of cold crystallization and w the weight fraction of PLA in the sample. ∆Hmo is the enthalpy of melting for 100% crystalline PLA material, which was taken as 93.1 J/g [19]. Thermogravimetric analysis (TGA) Thermal degradation of the composites was analyzed using a NETZSCH TG 209 F3 thermogravimetric analyzer (TGA) under a nitrogen atmosphere. The neat PBS and its composites were heated from 35oC to 700oC at a heating rate of 10oC/min. Their thermal degradation temperatures (Td) were determined. Water absorption test Water absorption tests were carried out for the extruded PLA and its composites filaments, with a diameter of 1.75 mm and were cut into sections of length approximately 40 mm and were dried in an oven at 70°C for overnight. The specimens were weighted and submerged in containers filled with water at room temperature. Three specimens were used for each composite and also for the neat PLA filament. The samples were carefully taken from the water container 9

The 28th Special CU-af Seminar 2020 October 20, 2020 and blotted dry with tissue paper before weighting to the nearest 0.0001g. The water absorption was calculated based on the following equation [20]: where Wt is the weight of the specimen after immersion in water and W0 is the original weight before immersion. Scanning electron microscopy (SEM) Morphology of the tensile tested specimens of the neat PLA and its composites were examined using a JEOL JSM-IT500HR scanning electron microscope (SEM) at an accelerating voltage of 5 kV. The specimens were sputter coated with gold before the examination. Tensile testing The dog bone-shape specimens (Type-V) were printed in accordance with ASTM D638. The tensile tests were carried on under a load cell of 5 kN and a crosshead speed of 5 mm/min. The tensile strength, Young’s modulus and elongation at break of the printed samples were evaluated as averages of at least five replicates. Results and Discussion Kenaf Cellulose Fibers The kenaf cellulose fibers were extracted and examined by SEM as shown in Figure 1. The minimum, maximum and average widths of the extracted kenaf cellulose fibers were 4.5 µm, 14.6 µm and 8.9 ± 2.1 µm, respectively. The minimum, maximum and average lengths were 31.8 µm, 304.2 µm and 101.7 ± 57.6 µm, respectively. Thus, the aspect ratios of the extracted kenaf cellulose fibers were 11.5. Figure 1. SEM micrograph of extracted cellulose fibers at x1000 magnification. 10

The 28th Special CU-af Seminar 2020 October 20, 2020 Thermal Properties According to the DSC second heating scan, the glass transition temperatures (Tg), followed by the cold crystallization temperatures (Tcc) and the melting temperatures (Tm) of the PLAand its biocomposites, can be observed on the thermograms shown in Figure 2. The addition of kenaf cellulose fibers and compatibilizers (PE-g-MA and PP-g-MA) seem to have no effect on the Tg, while the addition of Triacetin intensely reduced the Tg for about 10 -12oC as compared to the neat PLA (see Figure 3(a)). For PLA/kenaf cellulose composites, the cold crystallization peak of the comp becomes broader and is shifted to lower temperatures as compared to that of the neat PLA (Figure 3(c)). Also, the Tm of all composites decreased as compared to the neat PLA (see Figure 3(b)). The lower Tcc and Tm can be an indication of faster crystallization induced by kenaf cellulose fibers, which act as nucleating agents for PLA [21]. Also, the increase in the degree of crystallinity can be observed with the kenaf cellulose fibers addition (see Figure 3(d)). Figure 2. DSC thermograms from the second heating scan of neat PLAand its composite filaments. 11

The 28th Special CU-af Seminar 2020 October 20, 2020 Figure 3. Transition temperatures of neat PLA and its composite filaments from the DSC second heating scans: (a) glass transition temperatures (Tg), (b) melting temperatures (Tm), (c) cold-crystallization temperatures (Tcc) and (d) the degree of crystallinity. Thermal Stability The TGA curves of the neat PLA and its composites are shown in Figure 4. Their onset, maximum and endset decomposition temperatures and residues at 700oC are given in Table 3. The onset decomposition temperature of all composites decreased as compared to the neat PLA. This can be attributed to the lower decomposition temperature of kenaf cellulose (272.0oC, not shown in this report). The PLA/K10 had the lowest onset decomposition temperature. The addition of compatibilizer, PE-g-MA and PP-g-MA resulted in the second decomposition region at higher temperature (around 450-490oC), which might be the effects of polyethylene and polypropylene compositions in those compatibilizers. The addition of Triacetin seems to have a dramatic effect on the thermal stability or the decomposition behavior of the composite, as can see from the first stage of decomposition started at around 150-280oC, which is correlated to the boiling point of Triacetin. Figure 4. Thermogravimetric analysis (TGA) curves of the neat PLA and its composites. 12

The 28th Special CU-af Seminar 2020 October 20, 2020 Table 3. Decomposition temperatures (Td) and residues at 700oC of the neat PLA and its composites. Water Absorption Although PLA is hydrophobic, it is still sensitive to moisture, which may result in the carboxylic acid end groups self-catalyzing causing polymer hydrolysis. Therefore, the effect of the percentage of cellulose reinforcement on the biocomposite’s hygroscopicity was observed in both the neat PLA and the PLA/kenaf cellulose biocomposites. Figure 5 exhibits the water absorption of the PLA and all the biocomposites filaments. It is clearly seen that the water absorption significantly increased during the first 2 days (48 h) and then leveled off, gradually reaching a plateau after passed 10 days. The PLA filament had a moisture uptake of 1.0% after 10 days. The moisture uptake increased with the addition of kenaf cellulose. The moisture uptake of the PLA/K10 was about 1.6% after 10 days. However, the addition of compatibilizers and triacetin resulted in an increase in the moisture uptake, which is reached to 3.0% for the biocomposite with PP-g-MA and Triacetin. This may be attributed to the hydrophilicity of the compatibilizers. Figure 5. Water absorption during 0-30 days of neat PLA and its composite filaments. 13

The 28th Special CU-af Seminar 2020 October 20, 2020 Morphology Figure 6 presents the SEM images of the fracture surfaces of the tensile tested specimens. Some voids that appeared in all samples, which is common for the sample prepared using Fused Deposition Modeling (FDM) 3D printing due to the lack of molding pressure during the printing process. The addition of PE-g-MA led to an improvement in the interfacial adhesion between the PLA matrix and kenaf cellulose fibers. With the addition of kenaf cellulose fibers, some voids were propagated in the composite strand. The poor interfacial adhesion between the hydrophobic PLA and hydrophilic kenaf cellulose fibers resulted in a disconnected area between such two components. The compatibilizers, PE-g-MA and PP-g-MA, were incorporated to improve the interfacial adhesion and the compatibility between the kenaf cellulose fiber and the PLA matrix. It is seen that PE-g-MA gave a more promising result compared to PP-g-MA. For the composites with PE-g-MA, the kenaf cellulose fiber was covered by the polymeric layer and some fibrils connected between the kenaf cellulose fiber and PLA matrix were observed (Figures 6(d) and (e)). For the composites with PP-g-MA, some voids with approximately 100 µm in size were observed (Figure 6(f)). However, such voids were moderated by the incorporation of Triacetin (Figure 6(g)). This might be attributed to the plasticizing effect of Triacetin improved the flowability of the PLA to cover the kenaf cellulose fiber and shorten the voids propagation in the composite. The results may be ascribed to the compatibilizers, and plasticizer incorporation could enhance the fiber-matrix interfacial adhesion, which further improves the mechanical performance of the composites. Figure 6. SEM micrographs of fractured surfaces of printed specimens after the tensile test (a) PLA, (b) PLA/K10, (c) PLA/K10/T5, (d) PLA/K10/E3, (e) PLA/K10/E3/T5, (f) PLA/K10/P3 and (g) PLA/K10/P3/T5 composites. 14

The 28th Special CU-af Seminar 2020 October 20, 2020 Tensile Properties Figure 7 shows the tensile properties of the PLA and its composites. As expected, the addition of the kenaf cellulose fibers shows adverse effects on the mechanical properties. Due to the hydrophilic character of the cellulose, the poor interfacial adhesion and the poor dispersion of the kenaf cellulose fibers in the PLA matrix can be promoted. The tensile strengths of all composites were slightly decreased as compared to the neat PLA. This might be due to the phase separation structure and voids appeared in the composites. However, Young’s moduli of the composites were improved with the PE-g-MA addition. This might be attributed to the improvement of interfacial adhesion as well as compatibility between the kenaf cellulose fibers and the PLA matrix. Moreover, the elongation at break was significantly improved with the addition of Triacetin. This suggests that all the PLA/kenaf cellulose biocomposites developed in this study could be used in the same field as the neat PLA. Also, all the composites could be processed and 3D printed using the same condition used for the neat PLA. Figure 7. Tensile properties of 3D printed neat PLA and its composites. Conclusion This study arrived at a methodology of fabricating good-quality PLA/kenaf cellulose biocomposite filaments for Fused Deposition Modelling (FDM) 3D printing. The critical parameters, water absorption, thermal, morphological, and mechanical properties were examined. The PLA/ kenaf cellulose fiber biocomposite filaments were successfully fabricated, and subsequently, 3D printed using the conventional FDM 3D printer. The addition of compatibilizers (PE-g-MA and PP-g-MA), and plasticizer (Triacetin) improved the compatibility as well as the interfacial adhesion between the kenaf cellulose fibers and the PLA matrix, which further enhanced the composites’ mechanical performance. The results suggested that the addition of PE-g-MA, together with Triacetin, was the best promising method to achieve good-quality filament for the FDM 3D printing process. These biocomposite materials offer the opportunity for customized and rapid prototyping of degradable biocomposite or green composite products, which will be further useful in automotive applications. 15

The 28th Special CU-af Seminar 2020 October 20, 2020 References 1) Bajpai, P. K., Singh, I. and Madaan, J., J. Thermoplast. Compos. Mater., 2014(1): 52-81. 2) Anuar, H., Zuraida, A., Kovacs, J. G. and Tabi, T., J. Thermoplast.Compos. Mater., 2012(25) : 153-164. 3) Balakrishnam, H., Hassan, A., Imran, M. and Wahit, M. U., Polym.Plast. Technol. Eng., 2012(51): 175-192. 4) Bouzouita,A., Notta-Cuvier, D., Raquez, J.-M., Lauro, F. and Dubois, P. (2017). Poly(lactic acid) -based materials for automotive applications. In Advances in Polymer Science. (pp. 1-43). Berlin, Heidelberg: Springer. 5) Lim, L.-T., Auras, R. and Rubino, M., Prog. Polym. Sci., 2008(33): 820-852. 6) Rasal, R. M., Janorkar, A. V. and Hirt, D. E., Prog. Polym. Sci.,2010(35):, 338-356. 7) Razak, N. I. A., Ibrahim, N. A., Zainuddin, N., Rayung, M. and Saad,W. Z., Molecules, 2014(19): 2957-2968. 8) Xiao, L., Wang, B., Yang, G and Gauthier, M. (2012). Poly (lactic acid)-based biomaterials: Synthesis, modification and applications. In D. N. Ghista (Ed.), Biomedical Science, Engineering and Technology (pp. 247-282). InTech. 9) Anuar, H., Zuraida, A., Kovacs, J. G. and Tabi, T., J. Thermoplast.Compos. Mater., 2012(25) : 153-164. 10) Yussuf, A. A., Massoumi, I. and Hassan, A., J. Polym. Environ.,2010(18): 422-429. 11) Yu, T., Li., Y. and Ren, J., T. Nonferr. Metal. Soc., 2009(19): s651-s655. 12) Graupner, N., Herrmann, A. S. and Müssig, J., Compos. Part A-Appl.S., 2009(40): 810-821. 13) Serizawa, S., Inoue, K. and Iji, M., J. Appl. Polym. Sci., 2006(100): 618-624. 14) Ibrahim, N. A., Yunus, W. M. Z. W., Othman, M., Abdan, K. and Hadithon, K. A., J. Reinf. Plast. Comp., 2010(29): 1099-1109. 15) Taib, R. M., Hassan, H. M. and Mohd Ishak, Z. A., Polym. Plast.Technol. Eng., 2014(53) : 199-206. 16) Penjumras, P., Rahman, R. A., Talib, R. A. and Abdan, K., Int. J. Adv, Sci. Eng. Inf. Techno., 2015(5): 343-349. 17) Rautiainen, R. and Alen, R., Cellulose, 2009(16): 349-355. 18) Boruvka, M., Behalek, L., Lenfeld, P., Ngaowthong, C. and Pechociakova, M., Mater. Techno., 2019(34): 143-156. 19) Hamad, K., Kaseem, M., Yang, H.W., Deri, F. and Ko, Y.G., Express Polym. Lett. 2015(9) : 435-455. 20) Murphy, C.A. and Collins, M.N., Polym. Compos., 2018(39) : 1311-1320. 21) Frone, A.N., Berlioz, S., Chailan, J.-F. and Panaitescu, D.M., Carbohydr. Polym., 2013(91) : 377-384. 16

Optical fiber sensor based on surface plasmon resonance for sensitive and selective detection of Atrazine herbicides Assoc. Prof. Dr. Pakorn Varanusupakul



The 28th Special CU-af Seminar 2020 October 20, 2020 Optical fiber sensor based on surface plasmon resonance for sensitive and selective detection of Atrazine herbicides Assoc. Prof. Dr. Pakorn Varanusupakul Abstract A screening device based on surface plasmon resonance (SPR) spectroscopy was developed on an optical fiber and made to be selective to atrazine by molecularly imprinted polymer. The SPR probe was fabricated by deposition of gold nanoparticles on the unclad surface of an optical fiber using self-assembly method. The gold surface was further modified for selective sensing of atrazine with the molecularly imprinted polymer (MIP) by in-situ polymerization via thermal/UV polymerization method or atom-transfer radical polymerization (ATRP) method. The SPR signals were observed by the dip of the resonance wavelength. The advantages and diadvantages of polymerization methods were discussed. Despite the SPR probe assembled with MIP was successfully fabricated, the probe did not provide satisfactory sensitivity as expected. The detection limit was in ppm level. The working range for quantitative analysis was 0-30 ppm. The % recoveries of spiked atrazine standard in real water samples were in the range of 88-109%. Department of Chemistry Faculty of Science, Chulalongkorn University Bangkok, Thailand 21

The 28th Special CU-af Seminar 2020 October 20, 2020 Introduction and Objectives Surface plasmon resonance (SPR) is a spectroscopic feature of a conductive metal nanoparticle that provides an optical technique for highly sensitive analytical sensor. SPR takes place at the interface of different refractive index mediums between light guidance (prism)/nano-metal/dielectric (sensing medium) interfaces under the total internal reflection condition. The SPR occurs when the evanescent waves generated at the light guidance /nano-metal interface excite the electron density oscillation at the nano-metal/ dielectric (sensing medium) interface as illustrated in Figure 1 [1]. The SPR spectrum is recognized by the dip of the resonance wavelength. This phenomenon is very sensitive to any change such as the adsorption of molecules to the surface that causes the shift of the resonance wavelength. Figure 1 Schematic diagram of surface plasmon resonance (Kretschmann’s configuration) Generally, SPR probe sensor is based on Kretschmann’s configuration (Figure 1), where the light guidance is a prism configuration. In 2008, Greg and et al. developed general SPR probe based on Kretschmann’s configuration, using gold nanorod as metal surface to study bimolecular binding activity without any label [2]. Recently, SPR probe using optical fiber has been developed instead of prism based on the same principle [3] as illustrated in Figure 2. The advantages of optical fiber SPR probe are small and portable. SPR probe has been developed for detection of analyte by modification of the dielectric interface with antigen or antibody in biosensor works [4]. The change in dielectric surface led to the shift of resonance wavelength, which was directly corresponding to the amount of the analyte. In 2011, Jeong and et al. [5] developed the optical fiber SPR sensor for study of antigen-antibody reaction of interferon-gamma. The optical fiber SPR was fabricated by assembling gold nanoparticles on the end-face of an optical fiber. The density of the gold nanoparticle was optimized by varying dipping time of the optical fiber in the gold nanoparticle colloid solution. The interferon-gamma antibody was immobilized on the surface of the gold nanoparticle. The resonance wavelength shifted when the concentration of antigen increased, according to the activity of antibody/antigen 22

The 28th Special CU-af Seminar 2020 October 20, 2020 Figure 2 Schematic diagram of surface plasmon resonance (Optical fiber’s configuration) The conventional SPR technique has been limited to biosensor works. Recently, SPR probe has been developed for environmental analysis. In 2016, Gupta et al. [6] used molecularly imprinted polymer (MIP) instead of antigen and antibody in SPR works. The optical fiber SPR probe was modified by assembled with MIP to analyze profenofos in water sample. The probe was fabricated by coating silver nanoparticle on the part of the optical fiber. The uniformity of silver nanoparticle was controlled by thermal evaporation method. Then the MIP was assembled on the silver film by using thermal polymerization method. The shift of the resonance wavelength was observed when the concentration of profenofos in the sample changed. The probe showed high sensitivity for detection of profenofos. Molecularly imprinted polymer or MIP is the synthesized material resembling to lock and key model providing binding sites that have specific size and shape with the template analyte. MIP has been widely used for selective extraction of desire analytes. The advantages of MIP are easy to synthesize, low cost and tunable with desire types of analytes [7]. There are many pathways to synthesize MIP. The conventional polymerization has been done by heat and UV light, using metacrylic acid as monomer. This pathway is very easy and fast to synthesize, however, attaching or growing the MIP on the surface of the nanometal with controlled thickness is challenging. In this research, fabrication of an optical fiber SPR probe with surface modification by MIP is studied and developed for selective sensing of herbicide in environmental samples. Atrazine is selected as a model of this study because it is possibly carcinogenic to human and its effect to endocrine system and reproductive system in human and animals. Atrazine has been widely used in Thailand especially in corn filed at the northern part of Thailand. Besides, there have been reports of Atrazine contamination in soils and water likely due to its transportation from cultivated areas by runoff or leaching. Our developed optical fiber SPR probe would provide alternative screening tool for atrazine to conventional methods, which requires specific reagents such as enzyme linked immunosorbent assay (ELISA) or expensive instruments such as gas or liquid chromatographic methods. Experimental Fabrication of optical fiber SPR probe with gold nanoparticle by self-assembly method An optical fiber (600 μm diameter core multimode fiber, solarizatin resistant with acrylic coating, Thorlabs, Singapore) was first cut into 12 cm. About 1 cm in the middle of the optical fiber was unclad, washed and sonicated in a surfactant for 10 min followed by DI water for 10 min prior to use. Gold nanoparticle was coated on the unclad optical fiber using self-assembly method as summarized in Figure 3. The optical fiber was immersed into gold (III) chloride solution 23

The 28th Special CU-af Seminar 2020 October 20, 2020 (Sigma-Aldrich, Germany) mixed with 500 mM of sodium formate (Carlo-Erba, France) at 1:5 ratios [8]. The effect of concentration of gold (III) chloride and immersion time on SPR characteristic of the optical fiber were investigated and optimized. The concentrations of gold (III) chloride were varied from 5-20 mM and the immersion times were varied from 5-80 min. Figure 3 Fabrication of optical fiber SPR probe with gold nanoparticle by self-assembly method Assembly of MIP on the optical fiber SPR probe via thermal/UV polymerization The MIP was synthesized using methacrylic acid (Merck, Germany) as a monomer, 2,2- Azobis(2 methylpropionitrile) (Sigma-Aldrich, Germany) as initiator, ethylene glycol dimethylacrylate (EGDMA) (Sigma-Aldrich, Germany) as a cross linker and atrazine as a template. As summarized in Figure 4, a 30 mg of atrazine, 2 mL of monomer, 1 mL of crosslinker were mixed under nitrogen gas for 5 min, then a 0.5 mL of initiator was added into the mixture and stired under nitrogen gas for 2 min. The mixture was heated on water bath (about 70 °C) for 10 min. The viscous liquid of pre-polymer was formed. The pre-polymer was dropped on the gold layer on the optical fiber and radiated under UV lamp for 1 hr to form the MIP. The probe was washed by ethanol and the template was removed by washing with the basic solution (pH 8). The non-imprinted polymer (NIP) was assembled on the optical fiber sensor in the same way without addition of atrazine template. Figure 4 Assembly of MIP on the optical fiber SPR probe via thermal/ UV polymerization 24

The 28th Special CU-af Seminar 2020 October 20, 2020 Assembly of MIP on the optical fiber SPR probe via modified atom-transfer radical polymerization The MIP was in situ assembled on the gold film coated optical fiber. The gold coated optical fiber probe was treated with 4-bromo thiophenol (Sigma-Aldrich, Germany) by dipping in the solution to create thiol-gold interaction leaving bromine part as initiator for the polymerization process.The pre-polymer was prepared by mixing methacrylic acid as a monomer, ethylene glycol dimethylacrylate (EGDMA) as a cross linker, Copper (II) chloride (Sigma-Aldrich, Germany) as a catalyst, N,N,N2,N2-Tetramethylethylenediamine (Sigma-Aldrich, Germany) as a ligand and atrazine as template in a small vial. As summarized in Figure 5, a 30 mg of atrazine, 315 μL of monomer, 2 mL of cross-linker were mixed under nitrogen gas for 5 min, then a 5 mL of acetonitrile was added as a solvent. The mixture was stirred for 5 min then 0.7 g Copper (II) chloride was added and stirred until the solid was dissolved. A green solution was obtained. Then a 20 μL of ligand was added. The treated optical fiber was immersed in the prepolymer solution and heated (80 °C) in the oven for 1 hr to form the MIP. The probe was washed by ethanol and the template was removed by washing with the basic solution (pH 8). The NIP was assembled on the optical fiber sensor in the same way without addition of atrazine template. Figure 5 Assembly of MIP on the optical fiber SPR probe via modified atom-transfer radical polymerization Set up of optical fiber SPR probe for measurement The optical fiber SPR probe assembled with MIP was set up for measurement by connecting one end to the UV-Vis light source (DH2000, Ocean optics, Inc., USA) and the other end to the spectrophotometer (USB4000, Ocean optics, Inc., USA) by using temporary connector Selection Guide (Thor lab, Singpore) as the connector as illustrated in Figure 6. The optical fiber SPR probe was immersed in a sample solution covered by a black box to prevent ambient light for a certain of time prior to SPR signal acquisition. 25

The 28th Special CU-af Seminar 2020 October 20, 2020 Figure 6 Set up of optical fiber SPR probe for measurement Results and Discussion Fabrication of optical fiber SPR probe with gold nanoparticle by self-assembly method In deposition of gold nanoparticle on the unclad optical fiber by self-assembly method, the particle size of gold nanoparticle would affect the SPR signal [9]. The concentration of gold and immersion time were studied. The resulting SPR signals were summarized in Figure 7. Figure 7 Spectrum profiles obtained from optical fibers deposited with gold nanoparticles. 26

The 28th Special CU-af Seminar 2020 October 20, 2020 The spectrum profiles obtained from optical fibers deposited with gold nanoparticles using gold chloride concentration of 10 mM showed SPR characteristic where the resonance wavelenght was observed at 570.0 nm. When immersion time was increased, the intensity of resonance wavelength increased but at immersion time above 60 min, the intensity of resonance wavelength decreased. The SEM images of the optical fibers using gold chloride concentration of 10 mM at different immersion time were shown in Figure 8. The particle sizes of the gold nanoparticles were bigger and denser with increased immersion time. At immersion time of 80 min (Figure 8D), the gold particle layer started to get dense because of the compilation of particles resulting in decrease in SPR signal. Therefore, the gold chloride concentration of 10 mM at 60 min was chosen to prepare the optical fiber for SPR sensing probe. Figure 8 SEM images of the optical fiber surfaces using gold chloride concentration of 10 mM at different immersion time; (A) 35 min, (B) 50 min (C) 60 min and (D) 80 min Assembly of MIP on the optical fiber SPR probe via thermal/UV polymerization The MIP was first synthesized in batch and tested for atrazine adsorption sucessfully compared to the NIP. It was found that there was difficulty to in situ synthesize the MIP on the gold surface of the optical fiber SPR probe. Therefore, to assemble the MIP on the gold surface of the optical fiber SPR probe, the prepolymer was prepared and dropped on the gold surface, then irradiated under UV-light with wavelength of 350-400 nm for 1 hr. The white solid of MIP was formed on the optical fiber SPR probe. The optical fiber SPR probe modified with MIP and NIP were tested for the SPR signals and sensing performance of atrazine. The results showed broad and shallow SPR spectrum. Besides, it was hard to see the shift of the SPR wavelength when tested for sensing atrazine. It was probably due to rapid polymerization process resulting to poor and inconsistant formation of MIP on the gold surface of the SPR probe. 27

The 28th Special CU-af Seminar 2020 October 20, 2020 Assembly of MIP on the optical fiber SPR probe via modified atom-transfer radical polymerization Fisrt, the MIP was sucessfully synthesized via atom-transfer radical polymerization in batch using methyl-bromo isobutyrate as initiator. Nevertheless, to assemble the MIP on the surface of gold, the thiol-gold interaction was required. 4-bromo thiophenol was used as initiator because it contains thiol group to interact with gold and bromine group to initiate polymerization. The optical fiber SPR was treated with 6 mM of 4-bromo thiophenol prior to soaking in the pre-polymer solution. The optical fiber SPR probes were tested for SPR signal for each stage of fabrication as shown in Figure 9. The SPR signal appeared at 570.0 nm. When the optical fiber SPR assembled with MIP was tested for sensing of atrazine, the SPR wavelength was shifted to 574.3 nm since the adsrption of atrazine could alter dielectric nature of the sensing medium. The reproducibility was tested by weighing the probes before and after assembling the MIP. The average amount of MIP deposited on the probes was 0.0102 g with %Relative standard deviation of 6%. Figure 9 Spectrum of the optical fiber SPR probes at each stage of fabrication and when sensing atrazine. Quantitation analysis of atrazine SPR probe with MIP prepared by ATRP technique was set up for the spectrometric determination of atrazine. Since the SPR wavelength would shift according to the adsorption of atrazine, the degree of shifted SPR wavelength would correspond to the amount of atrazine. The optical fiber SPR probe assembled with MIP was immersed in atrazine standard solution in various concentrations from 5-30 ppm. The probe was left in the solution for 1 hr for fully adsoprtionofatrazine.Theprobewasdriedinroomtemperaturepriortospectrumacquisition.The SPR signal of each concentration was shown in Figure 10 and the delta SPR wavelength at various concentrations of atrazine were summarized in Table 1. 28

The 28th Special CU-af Seminar 2020 October 20, 2020 Figure 10 SPR spectrum obtained from each concentration of atrazine standard Table 1 SPR wavelength and delta delta SPR wavelength of each concentration of atrazine obtained from the optical fiber SPR probe assembled with MIP The calibration curve was established as displayed in Figure 11, the linear relationship was observed in the range of 0-30 ppm with the coefficient of determination (R2) of 0.984. The limit of detection calculated by 3 x standard deviation of regression/slope was 4.6 ppm. The detection limit was not as low as expected compared to the previous report [10], where the optical fiber SPR probe was fabricated by assembled MIP over coated silver film. The dielectric properties of the metals would influence the characteristic of the SPR spectrum, where silver usually gives sharper dip in reflected light intensity than gold [11]. Another critical factor might be attributed to the thickness of the assembled MIP, which in our work was not properly controlled. Because the evanescent wave penetrates only a short distance from the interface, if the layer of sensing medium is too thick, the sensitivity would be subsided [11]. 29

The 28th Special CU-af Seminar 2020 October 20, 2020 Figure 11 Calibration curve for determination of atrazine by optical fiber SPR probe assembled with MIP The optical fiber SPR probe assembled with MIP was applied for determination of atrazine in water samples collected from Nan province, Thailand. The recovery of atrazine was studied by spiking various amount of atrazine into the real water samples. The results were summarized in Table 2. Despite our developed SPR probe was not sensitive enough to detect trace amount of atrazine in the water samples, the %recoveries were in the range of 88-109%, which were in an acceptable range without significant effect from the matrices. Table 2 Optical fiber SPR probe assembled with MIP for determination of atrazine in water samples Conclusion The optical fiber surface plasmon resonance probe assembled with molecularly imprinted polymer was successfully fabricated for sensing atrazine in water samples. The unclad optical fiber was first coated with gold nanoparticle by self-assembly method and assembled with molecularly imprinted polymer by modified atom-transfer radical polymerization method. The ATRP method was slow polymerization process giving consistant amount of MIP. Despite the sensing of atrazine can be observed by the shift of SPR wavelength, the detection limit (in ppm level) was not satisfactory for trace analysis. The sensitivity could be attributed to the thickness or the distance of the sensing layer to the surface of gold layer. 30

The 28th Special CU-af Seminar 2020 October 20, 2020 The thickness of MIP should be properly controlled. Nevertheless, the optical fiber SPR probe assembled with MIP was attempted for quantitative analysis of atrazine in water samples. The %recoveries of spiked atrazine in real water samples suggested that the probe can be directly used in the real samples with no significant matrix effect. References 1) Daghestani, H. N. and Day, B. W., Sensors (Basel), 2010, 10(11): 9630-46. 2) Nusz, G. J.,Marinakos, S. M.,Curry, A. C.,Dahlin, A.,Höök, F.,Wax,A. and Chilkoti, A., Analytical Chemistry, 2008, 80(4): 984-989. 3) Gupta, B. D. and Verma, R. K., Journal of Sensors, 2009, 2009(979761. 4) Liu, Y.,Liu, Q.,Chen, S.,Cheng, F.,Wang, H. and Peng, W., Sci Rep, 2015, 5(12864. 5) Jeong, W. I.,Park, O.,Suh, Y. G.,Byun, J. S.,Park, S. Y.,Choi, E.,Kim,J. K.,Ko, H.,Wang, H.,Miller, A. M. and Gao, B., Hepatology, 2011, 53(4): 1342-51. 6) Gupta, B. D.,Shrivastav, A. M. and Usha, S. P., Sensors (Basel), 2016, 16(9). 7) Zhou, T.,Ding, L.,Che, G.,Jiang, W. and Sang, L., TrAC Trends in Analytical Chemistry, 2019, 114(11-28. 8) Ahmed, S. R.,Kim, J.,Tran, V. T.,Suzuki, T.,Neethirajan, S.,Lee, J. and Park, E. Y., Sci Rep, 2017, 7(44495. 9) Yeh, Y. C.,Creran, B. and Rotello, V. M., Nanoscale, 2012, 4(6): 1871-80. 10) Agrawal, H.,Shrivastav, A. M. and Gupta, B. D., Sensors and Actuators B: Chemical, 2016, 227(204-211. 11) Markey, F., Principles of Surface Plasmon Resonance. In Real-Time Analysis of Biomolecular Interactions, Nagata, K.; Handa, H., Eds. Springer: Tokyo, 2000. 31



Evaluation of in vitro biocompatibility and efficiency of novel bioactive calcium silicate glass-Thai silk fibroin hybrid scaffolds for bone tissue engineering Dr. Peerapat Thongnuek



The 28th Special CU-af Seminar 2020 October 20, 2020 Evaluation of in vitro biocompatibility and efficiency of novel bioactive calcium silicate glass-Thai silk fibroin hybrid scaffolds for bone tissue engineering Dr. Peerapat Thongnuek Abstract Reinforcement of bioactive calcium silicate glass (BCSG) with Thai silk fibroin (TSF) polymers is challenging, as TSF is prone to gel and thus undergoes phase-separation from the solution. Our study using (3-glycidyloxypropyl)trimethoxysilane (GPTMS) as a crosslinker discovered the ratio between BCSG and TSF that allowed both BCSG and TSF to homogeneously form into porous scaffolds. Scanning electron microscopy showed the existence of desirable interconnected pores in our hybrid materials. Fourier transform infrared spectroscopy confirmed the crosslinks of BCSG and TSF by GPTMS. Analysis of the dissolubility in water and in vitro biodegradation revealed the significantly enhanced stability of the crosslinked hybrid scaffolds. The resulting hybrid scaffolds presents the admixture of soft and elastic TSF fibres with stronger but brittle glass, mimicking bone organic and inorganic composition to simultaneously replace and regenerate bone tissue in fractures. Biomaterials Engineering for Medical and Health Research Unit, Biomedical Engineering Program, Faculty of Engineering, Chulalongkorn University Bangkok, Thailand 35

The 28th Special CU-af Seminar 2020 October 20, 2020 Introduction and Objectives As the world population is becoming aged, an increase in the demand for bone-graft materials is predicted as bone has been one of the most frequently grafted organs. Bone grafts can be derived from a second anatomic location of the same patient (autografts) or from another patient (allografts). Despite being a gold standard for bone grafting, autografts may lead to donor site morbidity, and allografts raise concerns over disease transmission.1 These limitations provide for finding alternative methods. Bone tissue engineering has widely been researched as a promising alternative to the autografts and the allografts. In tissue engineering, scaffolds are important because they support fractures in tissue, provide 3D framework for reconstructing functional tissues, release bioactive compounds for regeneration, and attract regenerative cells such as stem cells. Therefore, bone scaffolds are designed to have mechanical performance comparable to that of the native bone. The scaffolds are created to have 3D structure with suitable porosity and interconnected pores to achieve more specific surface area, and support the transport of important molecules. They are also designed to be biodegradable such that bioactive molecules can be released during degradation. Most importantly, they must be biocompatible.2 Silk fibroin (SF) from cocoons of silkworms Bombyx mori is a biocompatible and biodegradable polymeric protein.3 It has been used for making biomaterials because of impressive mechanical properties, biocompatibility, biodegradability and low immunogenicity.4 SF can be used for various tissue engineering applications such as bone, cartilage and skin. However, there are still a number of problems regarding the use of SF in bone tissue engineering. The most important problem is the lack of bioactivity, as the regenerated tissue does not bond very well to the surface of SF. Bioactive calcium silicate glass (BCSG) has been developed as a material for bone tissue engineering.5 The advantages of BCSG over other materials are its excellent bioactivity and degradability. BCSG was shown to bond with living bone through the formation of an apatite interface layer.6 Furthermore, properties of BCSG can be fine-tuned through compositional and structural variation.7 However, its disadvantages are the brittleness and unstability.7 To overcome the mentioned problems, a crosslinking technique has been applied to covalently bond organic polymers and the silicate network of BCSG. In 2001, Ren and his colleague were the first to use 3-glycidoxypropyltrimethoxysilane (GPTMS) (Fig. 1) to crosslink gelatin chains.8 Later, Mahony et al. produced silica-gelatin hybrid scaffolds with GPTMS acting as a crosslinking agent between the silica network and the gelatin.9 It was established that the epoxy ring on the GPTMS reacts with an amine group on a gelatin chain, whilst the methoxysilane on another end of the GPTMS get hydrolyzed becoming silanol groups. Some of which then undergo polycondensation to integrate into the silicate network.9 Figure 1. Molecular structure of GPTMS. 36

The 28th Special CU-af Seminar 2020 October 20, 2020 In this work, the fabrication of hybrid scaffolds based on TSF and BCSG was investigated. The stability and pore characteristics of the hybrid scaffolds were then examined. Experimental Materials The cocoons of Thai silkworm “Nangnoi Srisaket 1” were obtained from Queen Sirikit Sericulture Center, Nakhonratchasima province, Thailand. Other chemicals used in this study were of analytical grade. Scaffold fabrication Preparation of TSF solution TSF solution was prepared according to the method previously described by Kim et al.10 40 grams of silk cocoon were degummed with 1 L of 0.02 M Na2CO3 and rinsed thoroughly with de-ionised water to remove sericin. The degummed TSF was solubilized in 9.3 M LiBr solution for 4 h at 60 °C. The solution was then dialyzed in de-ionised water for 3 days. Preparation of BCSG solution BCSG solution was prepared by hydrolysis of TEOS in de-ionised water containing 1 N hydrochloric acid (volume ratio DI water : HCl = 3 : 1 and molar ratio DI water : TEOS = 4 : 1) .The same volume of DI water was used for dissolving calcium chloride and the two solutions were mixed together to obtain a sol with 70:30 molar ratio between SiO2 and CaO.6 Scaffold fabrication The ratios of the TSF, BCSG and GPTMS for the fabrication of hybrids are presented in table 1. Table 1. Composition of TSF-BCSG hybrids For synthesis of the hybrid scaffolds, TSF was either functionalized with 10 wt% GPTMS or not. After that, the resulting TSF was added to the BCSG solution, and the mixture was then foamed by vigorous agitation for 20 min. The foamed gels were aged for 3 days at 60 °C in closed beakers. The beakers were then left open to dry for another 24 h at 60 °C. 37

The 28th Special CU-af Seminar 2020 October 20, 2020 Scaffold characterization Chemical structures and crosslinking The structures and chemical bonds were evaluated with an Attenuated Total Reflectance Fourier Transform Infrared Spectrometer (ATR-FTIR). The spectra were collected in the range of 4000-510 cm-1. Morphology and porous structure The morphology and pore structure of TSF-BCSG 0 and TSF-BCSG 10 were charac- terized by scanning electron microscopy (SEM) at an accelerating voltage of 12-15 kV after sputter-coating with gold. The samples were imaged at 100X. The SEM images were analyzed using ImageJ to determine the average pore size. Stability in water The TSF-BCSG scaffolds (10±1 mg) were immersed in 1 ml of de-ionised water for 24 h at 37 °C. The scaffolds were then retrieved from the de-ionised water, and dried over night at 60 °C. The weight loss was used as an indicator for the instability of the scaffolds in water. To determine the weight loss, the following equation was used: Weight loss (%) = W0 - W1 x 100 W0 where W0 is the dry weight before immersion in de-ionised water. W1 is the dry weight after immersion in de-ionised water. In vitro biodegradation test 50 mg of the scaffolds were degraded at 37 ºC in PBS containing 1 unit/ml of protease XIV (Sigma) and 0.01 wt% sodium azide as an antibacterial agent. The enzyme solution was changed every 2 days to ensure continuous enzyme activity. At 1, 3, 5, 7 and 10 days after degradation, the remaining scaffolds were washed with DI water, and freeze-dried. The dried scaffolds were weighed, and the percentage of remaining weight was calculated as follows: Remaining weight (%) = (Wt/W0) x 100 where W0 and Wt represent the weight of scaffolds before and after degradation at different time. Results and Discussion Chemical structures and crosslinking FTIR spectra of GPTMS alone, TSF alone, 10% GPTMS-functionalized TSF and TSF-BCSG 10 confirmed the incorporation of TSF with BCSG, and they also gave evidence for the successful crosslinking by GPTMS (Fig. 2). The spectrum of the pure GPTMS showed a band at 1072 cm-1 assigned to Si-O-C in the methoxysilane end (Fig. 1 and Fig. 2, bottom line). In addition, the three bands at 1254.17, 909.37, and 854.06 cm-1 assigned to bonds in the epoxy ring on another end of the GPTMS molecule were evident. Scaffolds made solely of TSF exhibited amide I (C=O stretching vibrations), amide II (C-N stretching vibrations) and amide III (N-H deformation vibrations) bands at 1623.20, 1514.92 and 1233.79 cm-1, respectively (Fig. 2, 2nd line from bottom). Both GPTMS-functionalized TSF and TSF-BCSG 10 showed the conservation of the amide I, II and III bands (Fig. 2, 3rd line from bottom and top line), confirming 38