Báo cáo toàn văn Kỷ yếu hội nghị khoa học lần IX Trường Đại học Khoa học Tự nhiên, ĐHQG-HCM II-P-1.39 DETERMINATION OF THE CONCENTRATION OF FLUORESCENT POLYMERS USING THE LASER – µTAS Tran Hong Nhan1, Tran Quang Nguyen1,2, Nguyen Nang Dinh3, Tran Quang Trung1 Department of Solid State Physics, Faculty of Physics, University of Science, VNU-HCM University Information Technology, VNU-HCM University of Engineering and Technology, VNU Hanoi Corresponding emails: tranhongnhan223@gmail.com, trungvlcr@yahoo.com.sg ABSTRACT Microfluidic analysis systems, also known as Micro total analysis systems (µTAS) or “Lab-on aChip” are a rapidly developing field There are many kinds of µTAS due to the purpose of the experiment we needed In this report, we use the Laser - Micro total analysis system (Laser-µTAS) to determine the concentration of the fluorescent polymer such as AlQ conducting polymer and R6G fluorescent polymer The modulation signal with frequency 5Hz was applied to µTAS system for modulating the excitation light and referencing to Lock-in Amplifier This way resulted in the limit of detection (LOD) of the Laser – µTAS for AlQ3 down to 10nM and R6G down to 1nM based on the calculation of the received “Lock-in amplifier SR510” data Keywords: µTAS, Microfluidic chip INTRODUCTION Microfluidic analysis systems, also known as Micro total analysis systems (µTAS) or “Lab-on a-Chip” are a rapidly developing field A lab-on-a-chip (LOC) is a device that integrates one or several laboratory functions on a single chip of only millimeters to a few square centimeters in size LOCs deal with the handling of extremely small fluid volumes down to less than picoliters Lab-on-a-chip devices are a subset of MEMS devices and often indicated by "Micro Total Analysis Systems" (µTAS) as well Fluorescence is the most commonly used form of detection in microscale biological and chemical analysis, primarily due to high sensitivity, and is becoming increasingly important as many assays are being miniaturized into credit card size microfluidic lab-on-a-chip (LOC) systems A number of investigators have attempted to integrate fluorescence detection with microfluidic chips Edel et al [1] have recently demonstrated the use of a polyfluorene based pLED (polymer LED) as light source, avalanche photodiode as detector with limit of detection (LOD) of 1mM for fluorescein dye Yao et al [2] fabricated a microfluidic device with an integrated OLED excitation source and a PMT as detector for on-chip fluorescence detection using a pinhole and interference filter for masking the excitation light In this report, we use the homemade Micro Total Analysis System (µTAS) and the homemade microfluidic chip to determine the the concentration of fluorescent polymers such as AlQ3 and R6G EXPERIMENT The Laser - Micro total analysis system (Laser-µTAS): a The Laser-µTAS structure The Laser – Micro Total Analysis System (Laser-µTAS) had four parts including of the excitation source, the excitation light filter, the solution tank and the optical detector The excitation source was violet laser diode (𝑆𝐴𝑁𝑌𝑂, 𝜆 = 405𝑛𝑚) and the Pulse Generator at frequency 5Hz was used to control the excitation light in pulse Two cross-polarized glasses (Edmundoptics) which the transmission of crossed polarizers was 0.15% used for the excitation light filter Quartz cuvette or microfluidic chip was used for solution tank placed in the middle of two cross-polarized glasses and finally the Silicon photodiode was used for optical detector The whole system was placed in black box to prevent the noise from the outside light For signal treatment, we used the “Lock-in amplifier SR510” connecting simultaneous the signal of detector and reference pulse from pulse generator For getting data, the “lock-in amplifier” connected to computer via RS232 port The homemade program was built up from Labview software to control the system operation and calculated the concentration of fluorescent dye in solution The setup of the Laser-µTAS was shown on Figure ISBN: 978-604-82-1375-6 282 Báo cáo toàn văn Kỷ yếu hội nghị khoa học lần IX Trường Đại học Khoa học Tự nhiên, ĐHQG-HCM Fig.1 The setup of Laser – Micro Total Analysis System (Laser-µTAS) b The Laser-µTAS operation A square pulse with the frequency 5Hz created by the Pulse Generator was fed into the violet laser diode The excitation light in pulse went through the first cross-polarizer glass Because the laser light was the crosspolarized light, the plane polarization of first cross-polarizer glass was adjusted to parallel position with the plane polarization of laser light for getting the highest intensity of incident excitation light to sample Then the plane polarization of second cross-polarized glass was adjusted to perpendicular position with the plane polarization of the first cross-polarized glass for blocking the excitation light After that, the AlQ solution was pumped into quartz cuvette or microfluidic chip The AlQ fluorescent dye was stimulated by the excitation light and emitted the yellow characterized light (λ = 520 nm) Because of the emission light was unpolarized, it went easily through the polarization plane of second cross-polarized glass The second cross-polarized glass blocked the excitation light because its polarization plane is perpendicular with the polarization plane of the second cross-polarized glass As a result, the fluorescence light came to the photodiode without the excitation light The “lock-in amplifier” will eliminate the noise when it compares the signal from photodiode and reference signal from pulse generator and sends data to computer via RS232 interface The “Lock-in amplifier SR510” with minimum scale down to nV is convenient for determining the small signal The received data from “lock-in amplifier” will be analyzed by homemade program for determining the concentration of fluorescent dye in solution Fabrication of microfluidic chip: Microfluidic chip was fabricated by using soft-lithography method [3] The microfluidic mold was created by using photolithography processes The rectangle mold had cm width and cm height The micro-channel width was 200 µm and its height was 20µm Microfluidic chip had two input ports, one output port, and the circle tank The diameter of the input/output port was mm and the diameter of circle tank was mm Polydimethylsiloxane (PDMS) (Sylgard 184, Dow Corning), was mixed at a 10:1 (m/m) ratio of base to curing agent, degassed and then poured over the microfluidic mold to achieve 4mm thickness The PDMS was cured at 80°C for 2.5 hours on a leveled hotplate, cut and peeled from the mold with a sharp razor blade or scalpel, and input/output ports were carefully pierced using a mm (OD) punch PDMS mold and 4cmx10cm glass microscope slide were cleaned in a 1:5 ratio (v/v) of Hydrochloric acid and water for about 15 minutes and then dried using nitrogen gas to remove all moisture from it The PDMS mold was bonded to the glass slide using microwave oxygen plasma [4] Preparation AlQ3 solutions and R6G solutions: The Tris(8-hydroxyquinolinato) aluminium (AlQ3) fluorescent dye was prepared in ethanol with different concentrations: 1000 µM, 100 µM, 10 µM, µM, 100 nM and 10 nM We did the same progress with the R6G fluorescent polymer which have the concentrations ranged from 1000 µM to nM Figure 3a showed the fluorescence of AlQ3 concentration varying from 1000 µM to µM and Figure 3b showed the fluorescence of R6G concentration ranged from 1000 µM to µM ISBN: 978-604-82-1375-6 283 Báo cáo toàn văn Kỷ yếu hội nghị khoa học lần IX Trường Đại học Khoa học Tự nhiên, ĐHQG-HCM (a) (b) Fig.3 (a) The fluorescence of AlQ3 concentration varying from 1000µM to 1µM in UV-light (b) The fluorescence of R6G concentration ranged from 1000 µM to µM in UV-light The measuring method: After preparing the flourescent solutions, we built up the standard curve of dye concentration via collected data from Laser-µTAS for determining the signal to background ratio Both conducting polymer AlQ3 and flourescent polymer R6G are solubed in Ethanol Firstly, Ethanol was pumped into microfluidic chip and then the second polarized-glass was fine adjusted so that the signal received from “lock-in amplifier” was smallest These data, which we got was called the background data After that, we removed the quartz cuvette or microfluidic chip containing the ethanol solution out from Laser-µTAS tank and pumped 1000 µM AlQ3 solution into microfluidic chip Those data, which we got was called the signal data of 1000 µM AlQ3 solution Steps by steps, we got the background data of ethanol solutions and the signal data of AlQ dye solution with different concentrations varying from 1000 µM to 10 nM We did the same process with the fluorescent polymer R6G concentrations ranged from 1000 µM to nM Figure shows the fluorescent of R6G polymer in the microchannel of the microfluidic chip under UV-light Fig.4 The fluorescent of R6G polymer in the microchannel of the microfluidic chip under UV-light RESULTS AND DISCUSSION The background data is the collected signal from photodiode (voltage) when quartz cuvette or microfluidic chip contains ethanol solution This signal could be the combination of dark current of photodiode and noise signal of system due to the excitation light leaking though polarizers and excitation light leaking through due to device assembly and alignment The signal data is sum of background signal and photo-luminescent signal of fluorescent dye when quartz cuvette or microfluidic chip contains AlQ3 concentration varying from 1000 µM to 10 nM The time measurement is 30 seconds and the sampling rate is 50 points per second from “lock-in amplifier” Thus, the background data and signal data have the same 1500 data points After that, we calculate the average value of background data and the signal data with different concentration of dye solution Figure 5a shows the background data and signal data of AlQ3 concentration varying from 1000 µM to 100 nM and the inset picture shows the distinction of the signal of AlQ concentration varying from 10 µM to 100 nM The same process was done with the fluorescent polymer R6G Figure 5b shows the background data and signal data of R6G concentration varying from 10 µM to 10 nM and the inset picture shows the distinction of the signal of R6G concentration varying from µM to 10 nM ISBN: 978-604-82-1375-6 284 Báo cáo toàn văn Kỷ yếu hội nghị khoa học lần IX Trường Đại học Khoa học Tự nhiên, ĐHQG-HCM (a) (b) Fig.5 (a) The background data and signal data of AlQ3 concentration varying from 1000µ to 100nM (b) The background data and signal data of R6G concentration ranged from 10 µM to 10 nM The ratio of average signal data and the average background data is defined S/B When the ratio S/B=1, the average signal data is as same as the average background data It is called the limit of detection of Laser-µTAS Table shows the calculation of signal and background for 100µM AlQ3 solution with “lock-in amplifier” with time average of readings for 30 seconds Table shows the calculation of S/B ratio for all different concentration of AlQ3 solutions As the result of the S/B ratio of 10 nM AlQ solution is equal to one, the limit of detection of Laser-µTAS is 10 nM AlQ3 Table Calculation of signal and background for 100 µM AlQ3 solution with “lock-in amplifier” with time average of readings for 30 seconds We continued to calculate the signal data and the background data of the R6G fluorescent polymer Table shows the calculation of S/B ratio for all different concentration of R6G solutions Since the S/B ratio of nM R6G solution is equal to one, the limit of detection of Laser-µTAS is nM R6G Due to the higher quantum yield[5][6], the limit of detection of R6G is higher than that ones of AlQ3 Base on the S/B ratio from Table and Table 3, we plot the S/B ratio of AlQ and the S/B ratio of R6G Figure 6a demonstrated the S/B ratio of AlQ3 concentrations varying from 1000 µM to 10 nM and the inset picture illustrated the others ranged from 100 µM to 10 nM Figure 6b demonstrated the S/B ratio of R6G concentrations varying from 1000 µM to nM and the inset picture illustrated the others ranged from 10 µM to nM Table The S/B ratio of AlQ3 concentrations varying from 1000 µM to 10 nM ISBN: 978-604-82-1375-6 285 Báo cáo toàn văn Kỷ yếu hội nghị khoa học lần IX Trường Đại học Khoa học Tự nhiên, ĐHQG-HCM Table The S/B ratio of R6G concentrations ranged from 1000 µM to nM (a) (b) Fig.6 a The S/B ratio of different AlQ3 concentrations and the inset picture illustrated the others ranged from 100 µM to 10 nM b The S/B ratio of different R6G concentrations and the inset picture illustrated the others ranged from 10 µM to nM CONCLUSION By using the Laser-µTAS, we determined the concentration of conducting polymer AlQ down to 10 nM and the concentration of fluorescent polymer R6G down to nM For getting the lower LOD, the detector with higher response coefficient is necessary and applying this system to determine the antibody by using nanoparticles will be done in next step Acknowledgments: This research is funded by Vietnam National University – Ho Chi Minh City(VNUHCM) under grant number B2012-18-12TĐ XÁC ĐỊNH NỒNG ĐỘ CỦA POLYMER PHÁT QUANG BẰNG HỆ ĐO LASER-ΜTAS Trần Hồng Nhân(1), Trần Quang Nguyên(1,2), Nguyễn Năng Định(3), Trần Quang Trung(1) (1) Khoa Vật lý - Vật lý Kỹ thuật, Trường ĐH KHTN, ĐHQG-HCM (2) Trường ĐH Công nghệ Thông tin, ĐHQG-HCM (3) Trường ĐH Công nghệ, ĐHQG-Hà Nội TÓM TẮT Hệ thống phân tích vi lưu, hay gọi µTAS “Lab-on-chip”, lĩnh vực quan tâm nghiên cứu nhiều Tùy vào mục đích thí nghiệm mà hệ µTAS có kiểu thiết kế khác Trong báo này, sử dụng hệ Laser - Micro total analysis system (Laser-µTAS) nhằm xác định nồng độ polymer phát quang polymer dẫn AlQ3 polymer phát quang R6G Tín hiệu điều biến với tần số 5Hz đưa vào hệ µTAS nhằm điểu biến ánh sáng kích thích dùng làm xung tham chiếu cho khuếch đại Lock-in Dựa liệu thu từ khuếch đại Lock-in, xác định giới hạn hệ đo Laser – µTAS với AlQ3 10nM với R6G nM ISBN: 978-604-82-1375-6 286 Báo cáo toàn văn Kỷ yếu hội nghị khoa học lần IX Trường Đại học Khoa học Tự nhiên, ĐHQG-HCM REFERENCES [1] Edel J B., Beard N P., Hofmann O., deMello J C., Bradley D D C., Mello A J - Thin-film polymer light emitting diodes as integrated excitation sources for microscale capillary electrophoresis, Lab Chip (2004) 136-140 [2] Yao B., Luo G., Wang L., Gao Y., Lei G., Ren K., Chen L., Wang Y., Hu Y., and Qiu Y - A microfluidic device using a green organic light emitting diode as an integrated excitation source, Lab Chip (2005) 1041-1047 [3] Kim P., Kwon K W., Park M C., Lee S H., Kim S M., Suh K Y - Soft Lithography for Microfluidics, A Review, Biochip Journal (2008) 1-11 [4] Hui A Y N., Wang G., Lin B C., and Chan W T - Microwave plasma treatment of polymer surface for irreversible sealing of microfluidic devices, Lab Chip (2005) 1173–1177 [5] V V N Ravi Kishore, K L Narasimhan and N Periasamy, - On the radiative lifetime, quantum yield and fluorescence decay of Alq in thin films, Phys Chem Chem Phys ,5, (2003), 1386–1391 [6] M Fischer, J Georges, - Fluorescence quantum yield of rhodamine 6G in ethanol as a function of concentration using thermal lens spectrometry, Chemical Physics Letters , 260 (1996) 115-118 ISBN: 978-604-82-1375-6 287