Development of 3d printed microdialysis probe for determining glucose concentration in vitro

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 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) Supervisors Prof Yuh-Chang Sun, National TsingHua University, Taiwan 2) Assoc Prof 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, 1µL, 1.5µL, 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 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 0.5µL, 1µL, 1.5µL and 2.0µL were around 23.2%, 15.4%, 13.9% and 11.2% respectively in comparison to 42.2%, 35.3% 26.7% and 21.5% 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 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 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 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 Bachelor thesis TABLE OF CONTENT LIST OF FIGURES LIST OF ABBRIVIATIONS LIST OF ABBRIVIATIONS PART I INTRODUCTION .3 1.1 Rationale of study .3 1.2 Aims of study .4 1.3 Research Questions .4 1.4 Scope of the study: PART II LITERATURE REVIEW 2.1 In-Vitro Experiment 2.2 The 3D printing technology 2.2.1 Concept of 3D printing .6 2.2.2 How it works 2.2.3 Applicability 2.2.4 Future promising 2.3 Microdialysis .9 2.3.1 History of microdialysis 10 2.3.2 The microdialysisexperiment 11 2.3.3 The Microdialysis membrane 13 2.3.4 Applicability 13 2.3.5 Researchsituation of microdialysis devices 18 PART III METHODS 21 3.1 Reagents 21 3.2 Equipments 21 3.2.1 MiiCraft 3D printer 21 3.2.2 Infinite® 200 PRO – TECAN (96 well plate reader) 22 3.3 Procedure of 3D-printed validation 23 3.3.1 Chemical prepared 23 Huy Van Duong – K42 AEP VI Bachelor thesis 3.3.2 Procedures of microdialysis validation 23 PART IV RESULTS 25 4.1 Technical information of 3D-printed microdialysis probe 25 4.2 Fluorescence of glucose 26 4.3 Relative recovery 29 PART V DISCUSSION AND CONCLUSION 31 5.1 Discussion 31 5.2 Conclusion 31 REFERENCES 33 Huy Van Duong – K42 AEP VII Bachelor thesis LIST OF FIGURES Figure 3.1.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 21 Figure3.2.Infinite® 200 Pro – TECAN (sometimes called 96 well plate reader) used to detect dyed signals from chemical reactions or others assessment 22 Figure 3.3 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 24 Figure 4.1 3D sketch of microdialysis in direction views on Solidwork display 25 Figure 4.2 3D-printed microdialysis a) and commercial MD probe b) with membrane 26 Figure 4.3 The fluorescence signal of different glucose concentration of 3D-printed and introduced probes was detected at flow rate 0.5µL.min-1 (a) and 1.0µL.min-1 (b) 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 0.9%NaCl solution 27 Figure 4.4 The fluorescence index of glucose concentration of different devices was at different flow rate, 1.5µL.min-1(a) and 2.0µL.min-1(b) 28 Figure 4.5 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 Bachelor thesis LIST OF ABBRIVIATIONS AUR Amplex® UltraRed CAD Computer Aided Design CEO Chief Executive Officer CSF Cerebrospinal Fluid Barrier EF Extraction Fraction HPLC High –performance liquid chromatography HRP Horseradish peroxidase RR Relative Recovery SPE Solid Phase Extraction Huy Van Duong – K42 AEP Bachelor thesis PART I INTRODUCTION 1.1 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 wellknown 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 Bachelor thesis PART III METHODS 3.1 Reagents Stock solutions of glucose 0.1mM obtained by diluting 15µL Glucose in 1485µL Sodium chloride 0.9% in concentration (NaCl 0.9%), glucose Oxidize was concentrated at 30 units Similarly, catalyst horseradish peroxidase (HRP) and dye AmplexUltraRed (AUR) also diluted to prepare their concentration as 0.64 unit and 60µM respectively from their initial concentration 3.2 Equipments 3.2.1 MiiCraft 3D printer The MiiCraft 3D printer has been specifically developed as a well made robust unit to overcome the limitation of low-cost, deposition-based 3D printers It offers a new paradigm at the entry-level by virtue of the factbased on the Stereolithography technology using Pico DLP projectors-providing high resolution parts MiiCraft 3D printer considers as a fraction of the cost of comparable machines Figure 3.1 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 Huy Van Duong - K42 AEP 21 Bachelor thesis In this case, we used this printer to print the sample 3D microdialysis sketched by Solidwork software About its dimension which is listed above, we tried to partially mimic the commercial device’s dimension to obtain closely the efficiency which is known as relative recovery of commercial probes’ Additionally, a semi-membrane is attached between the tip of inner shaft and the outer shaft 3.2.2 Infinite® 200 PRO – TECAN (96 well plate reader) The Infinite 200 PRO accordingly knows as a user-friendly and affordable multimode reader It designed to serve the needs of today’s applications The 96 well plate readers, based on the highly successful infinite 200 series of microplate reader, can provide a full domain of detective techniques and methods Currently, such equipment popularizes as an essential machines for micro-analytical studies Figure3.2.Infinite® 200 Pro – TECAN (sometimes called 96 well plate reader) used to detect dyed signals from chemical reactions or others assessment For the determination of glucose fluorescence or final concentration of collected samples of glucose through flowing into commercial and 3D-printed microdialysis, a fluorescence microplate reader, infinite® 200- TECAN with 96-well Huy Van Duong - K42 AEP 22 Bachelor thesis microplates have been introduced wherein the Amplex®Red reagent, in combination with Horseradish peroxidase (HRP), was used to detect H2O2 (catalog no A22188)released from reaction of glucose and glucose oxidizes The dye AUR functions as a supporter of fluorescence detections because it strengthens signals of hydro peroxide H2O2 by dyeing them 3.3 Procedure of 3D-printed validation 3.3.1 Chemical prepared To validate the possibility of 3D-printed microdialysis probe we needed to inadvance prepare a numbers of chemical essential for intended experiment as well as detection as listed following: • 10 to 15 ml of perfusion 0.9% NaCl • 2ml of Glucose 0.1 mM • Glucose oxidize 10mM • Catalyst and dye (AUR and HRP) 3.3.2 Procedures of microdialysis validation The testing assay would be done through the perfusionNaCl 0.9% was fully filled ml injector, then, pumped into membrane through inlet tubing with identified flow rates In this assay, we decided to setmicrodialysis probe suffered different flow rate which are 0.5µL, 1µL, 1.5µL and2µL in tern Later on, the pumped fluid past the active area of the dialysis membrane, and then flows the outlet of probe Note that, the microdialysis membrane must be soaked in 1500 mL glucose which was created by diluting 15µL glucose 10mM into 1485µLNaCl 0.9% The amount of perfusion suggested to stably maintaining at 0.3mL During process, as 0.9% NaCl passes by the dialysis membrane, a concentration gradient was established across the membrane Huy Van Duong - K42 AEP 23 Bachelor thesis causing higher pressure inside membrane compared extracellular space, so that, the NaCl molecules tended to escape membrane Inversely, when NaCl molecules escaped membrane causing lower pressure which leads glucose molecular passes backward membrane from extracellular space Only compound or molecules, however, below the molecular0weight cutoff of the membrane can diffuse through membrane pores (Watson et al., 2006) After finishing the flows, sample of glucose collected in a clean eppendoft serving for later analysis by microplate reader instrument Figure 3.3 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 Note that, validating both efficiency of commercial and 3D-printed microdialysis device, so we can similarly repeat this process for commercial probe for subsequent comparisons In addition, this validated experiment should be implemented in silence and room temperature conditions, avoiding collision and outside factor impact to the flow rates of perfusion, to obtain the most creditable results as possible Huy Van Duong - K42 AEP 24 Bachelor thesis PART IV RESULTS 4.1 Technical information of 3D-printed microdialysis probe The CMA 20 Elite Microdialysis Probe, in this study, was chosen as a qualified and comparable device whose parameters were mimicked for the technical dimension of 3D-printed microdialysis device So the technical dimension exactly computed during sketching on Solidwork In this case, we sketched differently the shape and formation of 3D-printed compared to CMA 20 Elite probe Figure 4.1 3D sketch of microdialysis in direction views on Solidwork display • Technical information of 3D-printed microdialysis device - Membrane material: Polyarylethersulfone - Membrane molecular cut off: 20 000 Daltons - Membrane length: 4mm - Length inlet and outlet tubes: 200 mm - Total Probe Length: 24 mm - Outer diameter of probe shaft: 1.20 mm Huy Van Duong - K42 AEP 25 Bachelor thesis - Outer diameter of probe membrane: 0.5 mm - Inner diameter of probe shaft: 0.85 mm a) b) Figure 4.2 3D-printed microdialysis a) and commercial MD probe b) with membrane However, these technical parameters of 3D-printed device differed partially from introduced information In detail, the outer diameter of probe shaft was noticeably enlarged to fit the membrane insertion and eased for printing process due to the internal surface of probe’s shaft was not as smooth as causing hard to insert membrane Also, the outer diameter of introduced probe shaft was 0.77 mm while 3Dprinted device’s has set at 1.20 mm because of reducing the weakness of 3D-printed plastic shaft As a result; the inner diameter of shaft was greater than at 0.85 mm to fit the membrane insertion The inlet tubing used for 3D-printed microdialysis device was peek tub (NAT) with dimension of 0.025x0.20x5’ whereas the outlet tubing was the same as introduced devices’ 4.2 Fluorescence of glucose As mentioned in previous reports, the relative recovery of microdialysis device is not affected by the concentration of dialysate under the investigation(Torto et al., 2000).The complexity of the matrix does not disturb significantly the extraction Huy Van Duong - K42 AEP 26 Bachelor thesis fraction So in this circumstance, we established the concentration of dialysate was 0.1mM glucose diluted in 0.9% NaCl and then, the collected samples was analyzed by fluorescence microplate reader a) b) Figure 4.3 The fluorescence signal of different glucose concentration of 3D-printed and introduced probes was detected at flow rate 0.5µL.min-1 (a) and 1.0µL.min-1 (b) 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 0.9%NaCl solution In detail, figure demonstrates the fluorescence index of glucose concentration in sample solution at flow rate 0.5 and 1.0µL.min-1, higher fluorescence means greater concentration of glucose Generally, the signal of glucose 0.1mM, at flow rate 0.5µL.min-1 (figure 1a),was the highest value which varied roundly 20,000 and tended downward while those of introduced probe increased from 6131 to 9678 approximately, and a half less than was seen in the fluorescence of detected sample of 3D-printed device over the investigative time at where it went up to 5799 units Similarly, we considered these data in 1µL.min-1 (figure 1b) wherein the fluorescence Huy Van Duong - K42 AEP 27 Bachelor thesis of 0.1mM glucose was still unchanged; those figures in 3D-printed and commercial devices were remarkably increased from1387 to 3716 and from 2400 to 8600 Explanation for those data is that once the peroxidase appeared (product of glucose and glucose oxidize), the Amplex® Red reagent (AUR) reacts with H2O2 in a 1:1 ratio to produce the red-fluorescent oxidation product which will be recorded by microplate reader under the excitation and emission wavelength of 535 and 590 respectively Figure 4.4 The fluorescence index of glucose concentration of different devices was at different flow rate, 1.5µL.min-1(a) and 2.0µL.min-1(b) The fluorescence signal of dialysate, in fact, declines upon flow rate of perfusion rise In figure 2a, therefor, the fluorescence at flow rate 1.5µL.min-1was greater than those in figure 2b which was obtained by detecting glucose concentration in flow rate 2µL.min-1 In detail, the fluorescence of glucose of 3D-printed device was found between 1400 and 3500 while those values of commercial probe recorded nearly twice higher than, it started at about 2400 then increased till 6900 units In the same way, the fluorescence of glucose of 3D probe was gone up from 900 to 2500 units and increasing from 1400 to 5000 units was illustrated in the fluorescence index of glucose of standard probe in flow rate 2.0µL.min-1 of perfusion Huy Van Duong - K42 AEP 28 Bachelor thesis 4.3 Relative recovery The microdialysis generally knows as applicable to solutes that not form complexes in solutions This is because the relative recovery only reflects the freely diffusing analytes that can pass through the permi-selective microdialysis membrane Theoretically, the relative recovery (RR) is a proportion of the analytes in the sample solution, compared those that are detected after sampling with a microdialysis device as shown in below equation The feasibility of applying microdialysis sampling is routinely identified by the RR obtained for the analytes of interest Greater RR is always expectable in every case of experiments; even it does not require very sensitive analyte detection as compared to lower RR Since the relative recovery increases its efficiencies by reducing the time of analysis, higher relative recovery rates are essential once lowering flow rates In addition, different perfusion and sample concentration will result different relative recovery of device The results discussed here is the comparison of 3D-printed to commercial microdialysis device.From above-mentioned fluorescence of glucose concentration of 3D-printed and commercial probe, we were able to compute the relationship between relative recoveries (RR) and flow rate as shown in figure below Huy Van Duong - K42 AEP 29 Bachelor thesis Figure 4.5 Comparison and dependence of relative recovery (RR) on different flow rate of perfusing solution for a commercially available and 3D-printed microdialysis The possibility validation of 3D-printed microdialysis was in parallel implemented by the same assay, times and instruments to available probe, so that we could objectively evaluate the opportunity of 3D-printed device The trend in Fig 4, obviously, shows a decrease in RR with an increase in flow rate of perfusion for two devices investigated wherein the 3D-prrited device’s relative recovery was nearly50% lower than those figures of compared probe In detail, the relative recovery (RR) of commercial device, presented in figure 4, at 0.5µL.min-1 of flow rate, results at around 42%, those values decline to 34%, 28% and 21% approxi corresponding to perfusion rate increased at 1µL, 1.5µL and 2.0µL.min-1 respectively For 3D-printed device’s efficiency, its RR reached about 20%, 15.5%, 13% and 11% equivalent to flow rate went down Huy Van Duong - K42 AEP 30 Bachelor thesis PART V DISCUSSION AND CONCLUSION 5.1 Discussion The assessment of capability of 3D-printed microdialysiswhich is hopefully able to substitute, in future,commercial microdialysis has shown a positive prospects about 3Dprinting devices even when its working efficiency has not reached desired standards yet In this case, the relative recovery of 3D-printed probe has been clearly demonstrated; it has workedeven its relative recovery (RR) was noticeably lower than the introduced device’s after validation process The reasons that cause lower RR of 3D-printed probe addressed as membrane cutoff, inlet tubing’s dimension and materials Predictably, the RR of 3D-printed microdialysis device would be closer to those of introduced probe if using the same parts and components or as long as improving its extraction fraction through careful calibrations and resolve those difficulties we have met in this progress This investigation, however, hasmarked anaccessible development of 3Dprinted microdialysis in the near future; opened a new bright future for manufacturing scientific equipment to reduce costs and expenses in scientific researches In particularly, in environmental study, the perfusion liquid may use for the separation, modification of the outer microenvironment in term of predicting solid and liquid sample’s pollution and metal mobility during natural and accidental occurrences 5.2 Conclusion The relative recovery (RR) of 3D-printed microdialysis wasquiet lower than the expectation, approximately 50% compared to commercial ones, meaning that it might not be able to apply to scientific researches and replace commercial ones In details, the extraction fraction of 3D-printed microdialysis device was 0.23, 0.15, 0.14 and 0.11 corresponding with flow rates of 0.5µL, 1.0µL, 1.5µL and 2.0µL respectively Further, the Huy Van Duong - K42 AEP 31 Bachelor thesis ununiform of inner dimension of probe shaft and inlet tubing’s dimension and materials as well cause lower extraction fraction of 3D-printed probe.However, this result has not shown that the capability of 3D printermicrodialysis device was impossible because this study targeted objectively the primary evaluation of 3D-printed microdialysis tailoring with a short time conducted, so in substantial studies, the calibration of 3D-printed microdialysis should carefully implement in order to increase the relative recovery of the device Huy Van Duong - K42 AEP 32 Bachelor thesis REFERENCES 3D Printing Group (2014) How does 3D printing works Retrieved from: http://3dprinting.com/what-is-3d-printing/#howitworks(accessed on Jan 6th2015) 3D Printing group (2014) What is 3D printing.Retrieved from: http://3dprinting.com/what-is-3d-printing/ (accessed on 14/12/2014) Benveniste, H and Huttemeier, P.C (1990) Microdialysis – Theory and Application.Progress in Neurobiology, 3(5), pp 195-215 Bungay, 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365-373 Ungerstedt, U., and Rostami, E (2004) Microdialysis in Nuerointensive care Current Pharmaceutical Design, 10(18), pp.2145-2152 Watson, C.J., Venton, B.J., and Kennedy, R.T (2006) In Vivo Measurement of Neurotransmitters by Microdialysis Sampling Analytical chemistry, 78(5), pp 1391-1399 Huy Van Duong - K42 AEP 35 ... 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... 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?... because of reducing the weakness of 3D- printed plastic shaft As a result; the inner diameter of shaft was greater than at 0.85 mm to fit the membrane insertion The inlet tubing used for 3D- printed microdialysis