Bachelor thesis THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY DUONG VAN HUY TOPIC TITTLE: DEVELOPMENT OF 3D-PRINTED MICRODIALYSIS PROBE FOR DETERMINING GLUCOSE CONCENTRATION IN-VITRO BACHELOR THESIS Study Mode : Full-time Major : Environmental Science and Management Faculty : International Training and Development Center Batch : 2010-2015 Thai Nguyen, 22/01/2015 n Bachelor thesis DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University of Agriculture and Forestry Degree Program Bachelor of Environmental Science and Management Student name Huy Van Duong Student ID DTN 1053110103 Development of 3D-printed Microdialysis probe for Thesis Title determining glucose concentration In-vitro 1) Prof. Yuh-Chang Sun, National TsingHua Supervisors University, Taiwan 2) Assoc. Dam Xuan Van Abstract: To validate the possibility of using 3D-printed microdialysis probe in-Vitro experiment, in this study we described a methodology of printing 3D-printed probe by Miicraft 3D printer and assay for testing its efficiency in comparison with the commercial probe. Perfusate, of testing process, was 0.9% NaCl (Sodium Chloride) pumped into the inlet of the probe at different flow rate (0.5µL, and 2µL), flows past the active area of the dialysis membrane, and flow out the outlet of the probe.
Perfusate, in order of passing by the dialysis membrane, a concentration of glucose is established across the membrane. It facilitates the diffusion of compounds of interest from the extracellular space through the membrane and also the perfusion stream for analysis. After all amount of glucose, set previously about 0.3 mL, pumped into membrane, we collected the sampled glucose from outlet to then analyze and determine the glucose concentration by using fluorescence microplate reader. To come up with final result, the relative recovery was formally calculated by given formula.
The desirable efficiency of 3D-printed microdialysis was expected around 70% to 80% compared that of commercial probe. There were, however, some restrictions as well as limitations found during the conduction such as inaccurate dimension printing due to Huy Van Duong – K42 AEP ii n Bachelor thesis the size of device was ineligible etc. As a result, the identical efficiency of 3D-printed probe was unable to reach the announced targets wherein relative recovery of 3D printed found approximately at 50% to those of introduced device. In detail, the RRs were found at flow rate of 0.2% respectively in comparison to 42.5% of the introduced probe.
Although the result was under the expectation, these data have proven that 3D-printed probe possibility could be touched in the near future, opens a wide application of that device to various fields of study. Key words microdialysis, in-Vitro, 3D-printed microdialysis, relative recovery. Number of page 35 Date of 22/01/2015 submission Huy Van Duong – K42 AEP iii n Bachelor thesis ACKNOWLEDGEMENT This thesis has been greatly conducted from the support as well as assistance of many people whom I would sincerely like to give deep thanks and acknowledgements here. Firstly, I would like to send a great acknowledgement to Thai Nguyen University of Agriculture and Forestry (TUAF) and Advance Education Program (AEP) for arranging me an appreciated internship to National Tsing Hua University, Taiwan.
Surely, without their helpfulness my study would not come be true. Secondly, I am deeply grateful to my supervisor, Prof. Yuh-Chang Sun at Department of Biomedical Engineering and Environmental Science, National Tsing Hua University (NTHU), Taiwan for accepting, and authorizing me to conduct such a valuable and potential research during these 3 months. Without his support, this achievement of my life would not have come to such successes; also, I would like to say thanks to Assoc.
Prof Dam Xuan Van for his enthusiasm in guiding and correcting my report writing. Both of them deserve a special recognition for their always highly competent remarks and suggestions and particular praise for their openness and their calm and friendly manner that permitted him to convey everything most graciously. Thirdly, I would gratefully like to thank Cheng-Kuan Su, an enthusiastic guider, he was the one who has had a very positive influence on me and my orientation from the beginning by suggesting and assisting me this interesting topic during implementation of the study. Thanks to his amiable disposition and motivational strength, I could gain new research experiences and noticeably involve in a variety of Huy Van Duong – K42 AEP iv n Bachelor thesis fantastic work by practicing in several new scientific instruments and chemically professional devices.
He continued to inspire along the way as well as his generous hospitality by providing me with a comfortable place to conduct this research. Fourthly, I also want to thank my lab-mates, friends in NTHU, all of your presences would help my little heart experience the second home with unforgettable memories and events. Ultimately, from my little heart, I would like to express my deep gratitude and motivation to my parents for their continued moral and financial support and my all friends for their encouragement throughout my studies, the former being of much greater importance. The broad education that I was able to enjoy while growing up has proven invaluable.
Thai Nguyen, 22/01/2015 Author Duong Van Huy Huy Van Duong – K42 AEP v n Bachelor thesis TABLE OF CONTENT LIST OF FIGURES.1 LIST OF ABBRIVIATIONS.2 LIST OF ABBRIVIATIONS. Rationale of study. Aims of study. Scope of the study:.
In-Vitro Experiment. The 3D printing technology. Concept of 3D printing. How it works.
History of microdialysis. The Microdialysis membrane. Researchsituation of microdialysis devices. Infinite® 200 PRO – TECAN (96 well plate reader).
Procedure of 3D-printed validation. 23 Huy Van Duong – K42 AEP VI n Bachelor thesis 3. Procedures of microdialysis validation. Technical information of 3D-printed microdialysis probe.
Fluorescence of glucose. DISCUSSION AND CONCLUSION. 33 Huy Van Duong – K42 AEP VII n Bachelor thesis LIST OF FIGURES Figure 3.MiiCraft 3D printer sets a new standard in terms of price and performance within the 3D printing sector and offers the market community, education institution. Also, with a minimum layer thickness of 50 micron, this machine produces stunning, high-resolution part fraction.Infinite® 200 Pro – TECAN (sometimes called 96 well plate reader) used to detect dyed signals from chemical reactions or others assessment.
The microdialysis’s dialyzing process is illustrated in the above image. Perfusion contained in injector and then pumped into probe through inlet, the perfusion after passed active area of membrane will escape into another beaker by outlet tubing. 3D sketch of microdialysis in 4 direction views on Solidwork display. 3D-printed microdialysis a) and commercial MD probe b) with membrane.
The fluorescence signal of different glucose concentration of 3D-printed and introduced probes was detected at flow rate 0. Blank item means no glucose presence; 3D-printed device’s glucose was the glucose concentration after dialyzing by 3D-printed microdialysis, similarly, commercial probe’s glucose presented the amount of glucose which underwent dialyzing by commercial probe, and Glucose 0.1mM prepared by diluting glucose10mM into 0. The fluorescence index of glucose concentration of different devices was at different flow rate, 1. Comparison and dependence of relative recovery (RR) on different flow rate of perfusing solution for a commercially available and 3D-printed microdialysis.30 Huy Van Duong – K42 AEP 1 n Bachelor thesis LIST OF ABBRIVIATIONS 1.
AUR Amplex® UltraRed 2. CAD Computer Aided Design 3. CEO Chief Executive Officer 4. CSF Cerebrospinal Fluid Barrier 5.
EF Extraction Fraction 6. HPLC High –performance liquid chromatography 7. HRP Horseradish peroxidase 8. RR Relative Recovery 9.
SPE Solid Phase Extraction Huy Van Duong – K42 AEP 2 n Bachelor thesis PART I. Rationale of study Microdialysis device recently has a huge potential in automatic sampling and sampling clean-up technique for environmental study wherein it is demonstrated with selected examples classified following to the comprehensive state of the sample matrix (Míroand Frenzel, 2005). Once microdialyzer, as a passive sampler, is implanted into solid materials opening new paths in soil science, scientists can eliminate or purify aqueous and solid selected samples. More recently, microdialysis sampling has been shown to be a powerful technique for various study areas such as very popular in pharmacokinetic, neurochemistry, biotechnology and environmental studies.
As a result, there are hundreds of scientific papers published with the content correlating to microdialysis application study such as emerging trends in in vivo neurochemical monitoring by microdialysis (Kennedy, 2013) a high-throughput microdialysis-parallel solid phase extraction-inductively coupled plasma mass spectrometry hyphenated system for continuous monitoring of extracellular metal ions in living rat brain (Cheng et al., 2013) and so on. In another side, the tendency of 3D-printed lab research instruments is well- known as a strategy for cutting the cost of scientific research by using 3D printers and micro-controllers (Pearce,2013). With a promise of various designs can be 3D-printed, which means everyone can have an exact replica for the cost of materials. Such equipment has been proven in the open-source scientific design community at where the academic world is on “the verge of a new era where low-cost scientific equipment” puts increasingly sophisticated tools into the hands of not only our top universities and governmental labs but also every school and public as well (Elsevier group, 2014).
Huy Van Duong - K42 AEP 3 n Bachelor thesis 3D-printed microdialysis possibility, however, is still a myth for researchers on its workability for in-vitro experiment so as to its sensitivity and ineligible dimension that cause difficulties for 3D-printers. With its various applications, the 3D printers are extensively popularizing at where consumers, middle-school students have 3D-printed stock cars for physics lessons, scientist have 3D-printed tissues, cell and others scientific instruments for human organs, studies and scientific researches. An affirmation is that, according to CEO of 3D system, AviReichental,“The 3D printing is one of those technologies that literally touches everything we do” (Torto et al. Aims of study From those above-mentioned ideas, in this study, we have come up study with detailed purposes as following: • Validate the relative recovery (RR) of 3D-printed microdialysis probe in comparison commercial devices.
• Reduce the cost and investment in microdialysis related studies. • Strengthen printability of 3D printers to micro objects. • Encourage further applicability of microdialysis device to various fields, especially in environmental studies. Research Questions i) What arethe Relative Recovery (RR) of 3D-printed microdialysis probe in comparison those of commercial probefor determining glucose concentration? ii) How is 3-printed microdialysis device’s replicability? Huy Van Duong - K42 AEP 4 n Bachelor thesis 1.
Scope of the study: Accordingly, the main purpose of study is to combine efficiently hi-tech applications into scientific study at where we firstly focused on creating or producing scientific instruments, at least simple stuffs, in order to lower cost and expense in scientific researches to accelerate the possibilities and inspirations of study. As a result, the possibility assessment of 3D-printedmicrodialysis probe is firstly focused on lab-scaled experimentswith glucose determinations for experimental validation. However, after this process, the 3D-printed microdialysis would be considered applicable into further studies in various researches, at larger scale, such as in vivo and in vitro experiments and an efficient separator of metals ions or nanoparticles in order to eliminate undesirable molecules in matrix solutions. Limitation of designing 3D-printed MD device: Unfortunately, there have some limitations, which we addressed during the implementation, restricted the 3D-printed probe need to be solved to improve its possibility, that is, the printing accuracy is not concisely standardized as sketched parameters of probe, sometimes the inner dimensions is smaller than decided ones, so that the fitness of membrane and inner cannula was not qualified.
Furthermore, due to the limited capacity of our lab at which we have lacked some necessary materials and appropriated details such as inlet and outlet tubes were not uniformed, different dimensions as introduced devices’ leading the extraction fraction was affected. So, these suggested problems are recommended to solve for improving the capability of 3D-printed probe to make it is substitutable to formal one. Huy Van Duong - K42 AEP 5 n Bachelor thesis PART II. In-Vitro Experiment In-Vitro research generally refers to as the manipulation of organs, tissues, cells and biomolecules in controlled, artificial environment.