Micro-systems and Nanotechnologies in ELISA and droplet generation applications

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(1)222nd Meeting of ECS -The Electrochemical Society October 7-12, Society, 7 12 2012 2012, Honolulu Honolulu, Hawaii. Micro-systems and Nanotechnologies in ELISA and Droplet Generation A li ti Applications Speaker: p Yu-Cheng g Lin, Distinguished g Professor Fellow, SPIE Fellow, Royal y Society y of Chemistry y Senior Member, IEEE Department off E D Engineering i i Science, S i National Cheng Kung University, E E-mail: il [email protected] li @ il k d t Date: 2012/10/10.

(2) Ed Education ti . 1996. Ph.D. Electrical Engineering, g g,. University of Illinois at Chicago (Chicago, IL, USA) . 1994. M S Electrical Engineering, M.S. Engineering. University of Illinois at Chicago (Chicago, IL, USA) . 1987. M S Mechanical Engineering, M.S. Engineering. National Cheng Kung University (Tainan, Taiwan) . 1985. BS M B.S. Mechanical h i l Engineering, E i i. National Cheng Kung University (Tainan, Taiwan).

(3) HONORS / AWARDS & COMMITTEES/ BOARDS 2012. SPIE, Fellow. 2012. G Gold ld M Medal d l off K K.T. T L Lee T Technology h l and d Lit Literature t Lectureship L t hi. 2012. Associate Editor, IEEE Sensors Journal. 2009 2012 S 2009-2012 Senior i Editor Edit J Journal l off Micro/Nanolithography, Mi /N lith h MEMS, MEMS and d MOEMS (SCI 2010 Impact factor 1.194) 2009. General Chair, IEEE-NANOMED IEEE NANOMED. 2008. Fellow of Royal Society of Chemistry, FRSC. 2008. Distinguished Research AwardAward College of Engineering, NCKU. 2008. Chair of Technical Program Committee, The 4th Asia-Pacific Conference of Transducers and Micro-Nano Technology (APCOT 2008). 2008. IEEE Senior Member.

(4) 4. O laboratory Our l b t research h GDG-1. . . . Microfluidic chip for emulsion generation -Biomaterial: Alginate, chitosan, PLGA, gelatin…etc. -Encapsulation: BSA, nanoparticle, Vitamin C…etc. Blood separation for plasma collection -Cross flow microfluidic chip -Modified fiber membrane Micro-printing chip for cell migration -Gap size effect -Concentration gradient effect ECs. 0 hr. SMCs. ECs. 12 hr. SMCs. 200 m.

(5) 5. Our Ou laboratory abo ato y research esea c. 10 nm AgNPs g. 20 nm AuNPs 20 nm. . Microchip Mi hi for f Immunoassay I and d DNA hybridization h b idi ti -Method: Optical and impedance detection -Target: Protein A, KET, MET, LH…etc. Goal: Sensitivity, specificity -Goal: Ion separation microchip -Pre-treatment P t t t off the th immunoassay i chip hi for f urine i sample l -Decrease the noise for impedance detection -Increase the discrimination and sensitivity PDMS reaction well. ITO electrode. glass slide. .

(6) Micro-systems and Nanotechnologies in ELISA Sensors and Actuators B-Chemical, 117, 451-456, 2006. Microfluidics and Nanofluidics, 6, 85-91, 2009. Mi fl idi andd Nanofluidics, Microfluidics N fl idi 6, 6 93-98, 93 98 2009. 2009 Biosensors and Bioelectronics, 24, 1661-1666, 2009. Sensors & Actuators: B B-Chemical, Chemical, 139, 387 387-393, 393, 2009. Talanta, 83, 55-60, 2010. Analyst, 135, 2717-2722, 2010 Sensors and Actuators B-Chemical, 154, 185-190, 2011. Sensors and Actuators B-Chemical, 161, 1168-1175, 2012. Microfluidics and nanofluidics, nanofluidics 13, 13 319-329, 319 329 2012 2012..

(7) Overview of Immunoassay Methods Radioimmunoassay (RIA) Enzyme-link immunosorbent assay (ELISA) Chromatographic immunoassay Chemiluminescence immunoassay (CIA) Fluorescent immunoassay (FIA) Metalloimmunoassay. Labels I125, H3 (radioactive element) Yallow & Berson (1959) Horseradish peroxidase, Alkaline phosphatase, β-galactosidase (enzyme) Engvall g & Perlman ((1971)) Carbon black, Colloid gold Alkaline p phosphatase p β-galactosidase Fluorescein isothiocyanate (FITC), Quantum dot (QD) Colloidal gold, Colloidal silver, Quantum dot and Magnetic beads. 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion. 7.

(8) Enzyme-Linked Immunosorbent Assay (ELISA)  The. ELISA is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample.  The ELISA has been used as a diagnostic tool in medicine and provide qualitative or quantitative therapeutic information.  Detect and measure Ab-Ag reactions with greater sensitivity i i i (0.1~ (0 1 10 ng/mL) / L) andd higher hi h throughput h h (96 well ll solid-phase plate) than other classical serological methods. 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion. 8.

(9) 9. Traditional ELISA Formats. Direct:. wash. wash antigen-coated. add sample. Sandwich: antibody-coated. add sample wash. antibody-coated. secondary Ab. add sample. secondary Ab. add substrate. wash. dd enzyme-conjugated j d add. secondary Ag. add substrate. wash. add enzyme-conjugated. wash. Competitive:. wash. add enzyme-conjugated. wash. wash. antigen antibody enzyme enzyme-Ab. add substrate. 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion.

(10) 10. Electrical Detection Chip-Chip Chi D Design i &F Fabrication b i i. The PDMS is easy to be stripped from glass slide, which is suitable for impedance detection.. Glass slide Electrode Patterned P tt d well ll. Combination. 1000 1500 1000, 14000. Unit: m. 100. . MEMS technologies were used to fabricate the Au/Cr (2000/200 Å) microelectrodes with the sizes of 100 m wide and different gaps between electrodes.. 11000. . PDMS (2 mm thick). 20~200. 1500. 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion.

(11) 11. Fabrication Processes-Chipp Designg and Fabrication. Mask. Cr (1) Clean slide. (2) Evaporate Cr. Au (3) Evaporate Au. Resist (4) Spin-coat resist. UV. (5) Exposure. PDMS (6) Develop. (7) Etch Au. (8) Etch Cr. (9) Remove resist. (10) Cleaning and PDMS. 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion.

(12) 12. Electrode Gap Tests -Chip Design and Fabrication. 500 m. 20 m. 1.5 mm. The. gap size of electrode is 20 m.. The Th. l length h off electrode l d is i 1.5 1 5 mm.. Advantages of PDMS:  No leakage  High biocompatibility  Easy to fabrication  No toxicity to sample  Easy to wash bio-samples. 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion.

(13) Preparation of IgG-ANPs Conjugates. 13. –Material & Method. .  . . The surfaces of AgNPs g (pH 8.1)) (p were covered with citrate anions (negative charge). I G (pI~7.5) IgG ( I 7 5) suspended e ded in i PBS should bear a net positive charge. Isoelectric ppoint (p (pI)) is the ppH at which a molecule or surface carries no net electrical charge. IgG AgNPs conjugates by the IgG-AgNPs electrostatic force, hydrophobic force and van der waals.. L. M. Weiner et al, Nat. Biotechnol., 23, 556, 2005.. Ag.

(14) 14. Preparation p of IgG-AuNPs g Conjugates j g 5 nm ANPs IgG Au. binding 20 nm ANPs. Au. Au. Au Protein A.  The. proof of IgG IgG-ANPs ANPs conjugates diagram s. 518 nm 525 nm. UV UV-Vis Vis absorbance spectra comparison of ANPs and IgG-ANPs conjugates . s.  TEM image of IgG IgG-ANPs ANPs (5 nm)  binding with protein A-ANPs (20. 1. Introduction 2. Experimental 3. Results & Discussion 4. Conclusion. nm).

(15) 15. Preparation of IgG-AgNPs Conjugates –Material Material & Method. 10 nm AgNPs. The evidence of IgG-AgNPs conjugates diagram. 20 nm AuNPs 20 nm. . Protein A-20 nm Au is from Sigma.. . IgG-AgNPs (10 nm) are from experiments.. . IgG is specific to Protein A.. . TEM image of IgG-AgNPs (10 nm)) bindingg with pprotein AAuNPs (20 nm). 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion.

(16) 16. Experimental Methods – Modified the microelectrode chip surface Si. Si. Surface modification by the O2 plasma machine. Si. O. O. O. Si. Si. Si. Soaked in the 2% APTESToluene solution in 1 hr. R. Glass slide. NH2. N. (CH2)3 C2H5O. R. Si. C2H5O. O. O. O. Si. Si. Si. Add 20 µL BSA-MET conjugate solution -H2O. (CH2)3. CHO. (CH2)3 Soaked in the 10% GAPBS solution in 1 hr. N. R. CH. CH. Si. O. O. O. Si. Si. Si. ((CH2)3. (CH2)3. C2H5O. C2H5O. R. CH (BSA-MET). C2H5O. C2H5 O. Si. O. O. O. Si. Si. Si. 1. O2 plasma machine 2 APTES 2. APTES-Toluene Toluene 3. Ga-PBS. -OH NH2 -NH -CHO. 1. Introduction 2. Experimental 3. Results & Discussions 4. Conclusion.

(17) 17. Model ode Immunoassay u o ss y & Formats o s Glass slide Au. Au. Au. Au. Au. AuNPs antibody. Modify the surface of glass slide by SAM method. Add 20 µL BSA-MET conjugate solution. MET Mix the 30 µL sample solution (MET) and 30 µL anti-MET antibody-colloidal gold conjugates MET. Au. Au. Au. Au. Au. BSA Add 30 µL mixed solution. The unconjugated anti-MET antibody-colloidal gold conjugates were bound with the BSA-MET. Au. Au. (I (Impedance d ddetection t ti by b LCR meter) t ).   . Immunoassay format: Competitive model and sandwich model Target: MET, Protein A, Luteinizing hormone Detection machine: LCR meter ((Agilent g E4980A)) 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion.

(18) 18. Results & Discussion Protein A detection.

(19) 19. Sensitivityy v.s. Gap p Size and Electrode Length.    . 1.0E+08 1.0E+07 1.0E+06 protein A 10 μg/mL protein A 1 μg/mL protein A 0.1 μg/mL GUD. 1.0E+05 1.0E+04 1.0E+03 0. 3. 6. 9 12 time (min). 15. 18. 1.0E+06 protein A 10 μg/mL protein A 1 μg/mL protein A 0.1 μg/mL GUD. 1.0E+03 0. 3. 6. 9. 1.0E+06 protein A 10 μg/mL protein A 1 μg/mL protein A 0.1 μg/mL GUD. 1.0E+05 1.0E+04 0. 12 15 18 time (min). 3. 6. 9 12 15 time (min). 24. 27. 21. 24. 24. 27. 1.0E+08 1.0E+07 1.0E+06 protein A 10 μg/mL protein A 1 μg/mL protein A 0.1 μg/mL GUD. 1.0E+05 1.0E+04 1.0E+03. 21. 18. 200μm 1mm. 1.0E+09. 1.0E+07. 1.0E+04. 1.0E+07. 21. 1.0E+08. 1.0E+05. 1.0E+08. 1.0E+03. 100μm 1mm. 1.0E+09. 50μm 1mm. 1.0E+09 impedance (oh hm). impedance (oh hm). Gap size: 20~200 m Electrode El t d length: l th 1 mm Protein A: 10~10-1 g/mL IgG-Au: 10-9 M GUD: 1 mg/mL Silver enhancement solution changed every 3 min. impedaance (ohm).  . 20μm 1mm. 1.0E+09. impedaance (ohm). Au Au Au Au. 0. 3. 6. 9. 12 15 18 time (min) ti ( i ). 21. 1. Introduction 2. Experiment 3. Results & Discussion 4. Conclusion.

(20) 20. Sensitivityy v.s. Gap p Size and Electrode Length.    . 1.0E+08 1.0E+07 1.0E+06. protein A 10 μg/mL protein A 1 μg/mL protein A 0.1 μg/mL GUD. 1.0E+05 1.0E+04 1.0E+03. 50μm 1.5mm. 1.0E+09 impedance (ohm). impedance (oh hm). Gap size: 20~200 m Electrode length: 1.5 mm Protein A: 10~10-1 g/mL IgG-Au: 10-9 M GUD: 1 mg/mL Silver enhancement solution l i changed h d every 3 min. 1.0E+08 1.0E+07 1.0E+06 protein A 10 μg/mL protein A 1 μg/mL protein A 0.1 μg/mL GUD. 1.0E+05 1.0E+04 1.0E+03. 0. 3. 6. 9 12 time (min). 15. 18. 21. 0. 100μm 1.5mm. 1.0E+09 impedan nce (ohm).  . 20μm 1.5mm. 1.0E+09. 1.0E+08 1.0E+07 1.0E+06 protein A 10 μg/mL protein A 1 μg/mL protein A 0.1 μg/mL GUD. 1.0E+05 1.0E+04 1.0E+03 0. 3. 6. 9. 12 15 18 time (min). 3. 6. 9 12 15 time (min). 24. 27. 21. 24. 24. 27. 1.0E+08 1.0E+07 1.0E+06 protein A 10 μg/mL protein A 1 μg/mL protein A 0.1 μg/mL GUD. 1.0E+05 1.0E+04 1.0E+03. 21. 18. 200μm 1.5mm. 1.0E+09 impedan nce (ohm). Au Au Au Au. 0. 3. 6. 9. 12 15 18 time (min). 21. 1. Introduction 2. Experiment 3. Results & Discussion 4. Conclusion.

(21) 21. Sensitivityy v.s Gap p Size and Electrode Length The relationship between electrode length, gap size and reaction time Length of probe. Gap p size of electrode 20 m. 50 m. 100 m. 200 m. 1 5 mm 1.5. 15 min i. 18 min i. 21 min i. 21 min i. 1.0 mm. 15 min. 18 min. 21 min. 21 min. The relationship between electrode length and impedance Gap size of Electrode electrode length 20 m. Concentration of protein A (g/mL). Control. 10. 1. 0.1. GUD. 1.5 mm. 17.2 K. 73.1 K. 120 K. 1480 K. 1.0 mm. 210 K. 500 K. 830 K. 1500 K. 1. Introduction 2. Experiment 3. Results & Discussion 4. Conclusion.

(22) 22. Silver-Precipitation Immunoassay -AuNPs El t d Gap Electrode G (20 m)). Conditions  Voltage: . . Au 200 nm. Cr 20 nm. Cr 20 nm. 0.5 0 5 V (< silver ion reduction potential). Electrode gaps: 20 m.  Avoid. Au 200 nm. Glass slide. environmental light in detection. Negative controls GUD which is non-specific to IgG in replacement of Protein A 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion.

(23) 23. Impedance Immunoassay.  . Au Au Au. 1.0E+09. Gap size: 20 m Electrode El d length: l h 1.5 1 5 mm. . IgG: 100 g/mL. . Protein A: 10~10-1 g/mL 10-9. . IgG-Au conjugates:. . Control: GUD 1mg/mL. M. im mpedancee (ohm). Au. 1.0E+07 1.0E+05 1.0E+03 1.0E+01 0. Reaction eact o  The Th impedance i d off protein t i A time. 3. 6. 9 12 time (min). 155. Concentration of p protein A (g (g/mL)). 188. 21. Control. 10. 1. 0.1. GUD. 15 min. 28.5 k. 1.1 k. 233 k. 200000 k. 18 min. 0.5 k. 0.2 k. 1.1 k. 100000 k. 0.1 g/mL > 1 g/mL. The concentration effect is not exist.. protein A 10 μg/mL protein A 1 μg/mL protein A 00.1 1 μg/mL GUD. 1. Introduction 2. Experiment 3. Results & Discussion 4. Conclusion.

(24) 24. Impedance Immunoassay Au. Au Au Au.  . Gap size: 20 m Electrode El d length: l h 1.5 1 5 mm.  IgG:. 10 g/mL.  Protein A:. 10~10-1 g/mL 10-9.  IgG-Au. conjugates:.  Control:. GUD 1mg/mL. impedancee (ohm). 1 0E+09 1.0E+09 1.0E+07 1.0E+05 1.0E+03 1.0E+01 0. M. Reaction  The impedance of protein A time. 3. 6. 9 12 time ((min)). 15. Concentration of protein A (g/mL). 18. 21. Control. 10. 1. 0.1. GUD. 15 min. 670 K. 110000 K. 610 K. 200000 K. 18 min i. 0 9 K 0.9. 40 K. 1 7 K 1.7. 200000 K. 1 g / m L > 1 0 g/mL. The concentration effect is not exist.. protein A 10 μg/mL protein A 1 μg/mL protein A 0.1 μg/mL GUD. 1. Introduction 2. Experiment 3. Results & Discussion 4. Conclusion.

(25) 25. Impedance Immunoassay.  . Au Au Au. Gap size: 20 m Electrode El d length: l h 1.5 1 5 mm. . IgG: g 10 g g/mL. . Protein A: 10-1~10-4 g/mL. 1.0E+09 impedancce (ohm). Au. 1.0E+07 1 0E+05 1.0E+05 1.0E+03 1.0E+01 0. IgG-Au conjugates: 10-9 M  Control: GUD 1mg/mL . . Detection limit:. Reaction 10-3 g/mL. Concentration effect is exist. protein A 0.1 μg/mL protein A 0.01 μg/mL protein A 0.001 μg/mL protein A 0.0001 μg/mL GUD. 3. 6. 9 12 15 time (min). 18. Concentration of protein A (g/mL). 21. 24. Control. time. 01 0.1. 0 01 0.01. 0 001 0.001. 0 0001 0.0001. GUD. 18 min. 2.7 K. 6.6 K. 600 K. 200000 K. 200000 K. 21 min. 0.1 K. 0.7 K. 3.5 K. 780 K. 700 K. 1. Introduction 2. Experiment 3. Results & Discussion 4. Conclusion.

(26) 26. Results & Discussion MET detection.

(27) 27. Contact Angle Detection – ITO glass slide Angle = 74.33 degrees. Angle = 15.89 degrees. 74. 16. (a) (a) Control A l = 40 Angle 40.11 11 ddegrees. (b) (b) O2 plasma Angle = 32.63 degrees. 40. ( )T l (c) ( ) APTES-Toluene (c) APTES. 37. (d)Ga-PBS (d). 1. Introduction 2. Experimental 3. Results & Discussions 4. Conclusion.

(28) 28. Immunofluoresence method – ITO glass slide Control.  . Experiment. Target : Goat anti-Rabbit IgG, F(ab’)2, X-Adsorbed (DTAF) After SAM method was used, used DTAF is bound on the chip. 1. Introduction 2. Experimental 3. Results & Discussions 4. Conclusion.

(29) 29. Impedance Detection Dilution of MET-GC – Concentration of MET-GC 1. Mean (kohm) 0.9. 1X. 2X. 10X. 20X. 30X. 40X. 80X. 100X. 120X. NC. 3.57. 13.8. 99.6. 171. 241. 413. OD532=0.93. 714. 1714. 47525. ±6 2 ±38.4 ±6.2 ±38 4. ±14. 60000. A u. A u. 50000. Impedance (kohm). MET 40000 30000. MET. ±32 9 ±103.1 ±32.9 ±103 1 ±8.5 ±8 5 ±13.9 ±13 9 ±1912.4 ±1912 4 ±11614.9 ±11614 9. 0.7 A 0.6 u. A u. Parameter. A u. 0.5. MET-GC. 0.4. MET BSA MET-BSA MET. 0.3Add. mixed solution. 0.2. Concentration of the samples. 3500. 0 5  g/mL 0.5 / L 250 ng/mL. 2500. Dil t d MET-GC Diluted MET GC stock, t k 2X, 2X 10X, 10X 20X 20X, 30X 30X, 40X 40X, 80X 80X, 100X 100X, and d 120X 2000. Stock MET-GC 13 min. Reaction time 1500. 0.1 20000. F Frequency. 0 400. 10000. 4000. 3000. Impedance (kohm). 70000. Absorban A nce (AU). 0.8 STDEV (kohm) ±5 6 ±5.6. 696. 450. 1000. 100 H Hz. 500 500. BSA. 550. 600. Wavelength (nm). 0. 0. NC. 120X. 100X. 80X. 40X. 30X. 20X. 10X. Concentration of MET-GC. 2X. 1X. 120X. 100X. 80X. 40X. 30X. 20X. 10X. 2X. Concentration of MET-GC. 1. Introduction 2. Experimental 3. Results & Discussions 4. Conclusion. 1X.

(30) 30. Impedance Detection. 60 300. – MET-GC diluted 10X. 50 250. A u. nce (kohm m) IIImpedan mpedanc ce (kohm m). MET. A u. A u. A u. A u. A u. MET-GC. 40 200. Mix the 30 µL sample solution (MET) and 30 µL 30 150 antibody-AuNPs (MET-GC). A u 20. 100. A u. A u. A u. Parameter. Concentration of the samples. MET-BSA MET BSA. 0.5  g/mL. MET. NC, 0.1, 1, 10, 50, 100, 150, 200, 250, and 300 ng/mL. Diluted MET-GC. 10X. Reaction time. 13 min. Frequency. 100 Hz. Add 30 µL mixed solution. 1050. MET. 00 0. BSA. 0. 50. 0.1 100. 150. 1. 200. 10 250. 300 50. 350. Concentration of MET in urine (ng/mL). 1. Introduction 2. Experimental 3. Results & Discussions 4. Conclusion.

(31) 31. Impedance Detection. 60 900. MET. A u. A u. Im mpedancce m) IImpedan ce(kohm (kohm m). A u. A u. – MET-GC diluted 30X. 800 50 700A. A u. u. A u. MET-GC. 40 600. 500Mix the 30 µL sample solution (MET) and 30 µL 30 antibody-AuNPs (MET-GC) 400 A u. A u. 300 20. 200 10 MET 100. A u. Add 30 µL mixed solution. Parameter. Concentration of the samples. MET-BSA. 0.5  g/mL. MET. NC, 0.1, 1, 10, 50, 100, 150, 200, 250, and 300 ng/mL. Diluted MET-GC. 30X. Reaction i time i. 13 min i. Frequency. 100 Hz. 00 0. BSA. 0. 50. 100 0.1. 150. 1. 200. 250 10. 300 50. 350. Concentration Concentration of of MET MET in in urine urine (ng/mL) (ng/mL). 1. Introduction 2. Experimental 3. Results & Discussions 4. Conclusion.

(32) 32. Impedance Detection. 70 3000. – MET-GC diluted 100X. 60 2500 A u. MET. A u. I Impedan ce (kohm m). A u. A u. 50 2000. A u. A u. A u. MET-GC. Mix the 30 µL sample solution (MET) and 30 µL 1500 antibody-AuNPs (MET-GC). 40. Parameter. Concentration of the samples. MET-BSA. 0.5  g/mL. MET. NC, 0.1, 1, 10, 50, 100, 150, 200, 250, and 300 ng/mL. Diluted MET-GC MET GC. 100X. Reaction time. 13 min. Frequency. 100 Hz. 30. A u. A u. 1000 20. 500 MET10. A u. Add 30 µL mixed solution. 0 0 0. BSA. 0. 50. 100 0.1. 150. 1. 200. 10 250. 300 50. 350. Concentration Concentration of of MET in in urine (ng/mL). 1. Introduction 2. Experimental 3. Results & Discussions 4. Conclusion.

(33) 33. Impedance Detection – MET-GC diluted 120X A u. 70000. A u. A u. A u. A u. 3500. MET-GC 3000. 60000. IImpedance (k kohm). 40000 30000. IImpedance (k kohm). Mix the 30 µL sample solution (MET) and 30 µL antibody-AuNPs (MET-GC). 50000. MET. 2500. Parameter. Concentration of the samples p. MET-BSA. 0.5  g/mL. 2000 1500. MET. NC, 0.1, 1, 10, 50, 100, 150, 200, 250及 300 ng/mL. 20000. Diluted 1000 MET-GC MET GC. 120X. 10000. Reaction time 500. 13 min. Frequency. 100 Hz. A u. A u. A u. A u. A u. Add 30 µL mixed solution MET. 0 0. 50. 0 100. 150. 200. 250. 300. Concentration of MET in urine (ng/mL). 350. 0. 0.1. 1. 10. Concentration of MET in urine (ng/mL). BSA. 1. Introduction 2. Experimental 3. Results & Discussions 4. Conclusion. 50.

(34) 34. The Effect of MET-GC Dilution MET GC dilution MET-GC 10X. 30X. 100X. detection limit. 50 ng/mL. 10 ng/mL. 1 ng/mL. detection slope. 267.85. 920.32. 2494.1. R square. 0.99. 0.78. 0.73. 1. Introduction 2. Experimental 3. Results & Discussions 4. Conclusion.

(35) Micro-systems and Nanotechnologies in Droplet Generation Applications Lab on a Chip, 6, 954-957, 2006. Journal of Micromechanics and Microengineering, 17, 14281434 2007. 1434, 2007 Frontiers in Bioscience, 12, 3061-3067, 2007. Sensors and Actuators B: Chemical, Chemical 124, 124 510-516, 510-516 2007. 2007 Microfluidics and Nanofluidics, 6, 277-283, 2009. y 151, 231-236, 2009. Sensors & Actuators: A. Physical, Microfluidics and Nanofluidics, 8, 115-121, 2010. Microfluidics and Nanofluidics, 11, 245-253, 2011. Mi fl idi andd Nanofluidics, Microfluidics N fl idi 12, 12 475-484, 475 484 2012. 2012.

(36) 36. Conventional dose  Periodic P i di administration d i i i  High concentration could. cause side effects or damage  Low concentration could be ineffective . Controlled-release  Stable effective. Adverse side effects Min. toxic conc. Therapeutic range Min. effective conc. No therapeutic effects. 1. Drug conccentration. . Drug concentratiion. Conventional Dose vs. Controlled-Release. 2. 3. Frequencies of dosing. Injection Oral intake Controlled-release Adverse side effects Min. toxic conc. Therapeutic range Min. effective conc. No therapeutic effects. concentration for an Time extended period of time 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion.

(37) 37. Controlled-Release System . What is controlled-release system  Delivers D li an agentt att controlled t ll d rate t for f extended t d d time ti  Localizing drug action by specific placement. . G l off controlled-release Goal ll d l  To achieve a stable blood level of the drug for a. period i d off time i  Uniform microcapsule size and narrow distribution. 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion.

(38) Chip Design -Channel Layer Channel Layer Containing a cross-junction channel where the sheath flow effect occurring.. 200 μm Scale bar 200 m. Scale bar 200 m. 1. Introduction 2. Experimental 3. Results & Discussion 4. Conclusion.

(39) (b). (a). W/O E Emulsion li G Generation ti (d). (c). Fixed oil flow rate.  1400. Micro 1200. oil flow rate 0.5 mL/min oil flow rate 0.3 (e)mL/min oil flow rate 0.1 mL/min channel. w/o emulsions.  1400. Oilwater flow rate 0.10 mL/min. (f). Sheath flow effect 1200. 1000. water flow rate 0.05 mL/min water flow rate 0.01 mL/min. 1000 sizee ( m). sizee ( m). Fixed water flow rate. 800 600. 800. (scale bar 200 m) Water Flow rate (mL/min). 600 No.. Flow direction. 400 200 0 0.005. 0.01 0.03 0.05 water flow rate (mL/min). 0.1. 400. a b 200 c d0 e f. 01 0.1. water oil 0.030 200 m 0.010 0.100 0.005 0.050 03 0.3 0 5 0.300 0.8 0.5 08 0 010 0.010 oil flow rate (mL/min) 0.010 0.5. OIl O. 1. Introduction 2. Experimental 3. Results & Discussion 4. Conclusion.

(40) 40. Gradient-microfluidic Droplet Generator.

(41) 41. Gradient-microfluidic Droplet p Generator - CFD simulation: Conditions setting Property . Problem type:  Flow  Grid deformation. . Model option:  Transient solution  Auto time step. . Boundary condition:  Fluid 1, Q1 = 0.0033 m/s  Fluid 2, Q2 = 0.0033 m/s. Fluid 1 997. 997 kg/cm3. 0.001. 0.001 kg/m-s. Density Viscosity. Fluid 2 Unit. Inlet 1 X Y. Inlet. Inlet 2. Mixer. Outlet. Outlet Reservoir. Schematic diagram of chip Schematic diagram of channel 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion  .

(42) 42. Gradient-microfluidic Droplet Generator - chip design 0.1. 60. 30. . Mask designed by AutoCAD® 2010 software  The dimension was 60 × 30 × 6 mm. Mixer area (the chaotic flow). . 80o. 01 0.1. 0.2. Unit: mm. Flow-focusing device 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion .

(43) 43. Fabrication of the microfluidic chip - Fabricate THB-151N mold. Photolithography process PR. ~~ ~~ ~~ ~~ ~~ ~~ ~~ ~~ ~~. . (1) Mask design (2) Clean wafer (SiO2) (3) Photoresist (THB-151N) (4) Soft bake, 120℃, 10 min. Mask UV. ~~ ~~ ~~ ~~ ~~ ~ ~~ ~ ~~ ~~. (5) Exposure. (6) Developing (8) THB-151N mold. (7) ( ) Hard bake,, 130℃, ℃, 10 min. 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion.

(44) 44. Fabrication of the microfluidic chip p - Replica molding and Bonding method. . . Replica molding process. Bonding method. S PDMS. (1) Pour PDMS. PMMA mold & THB-151N mold. ~~ ~~ ~~ ~~ ~~ ~~ ~~ ~~ ~~ (2) Bake, 70℃, 45 min. . O2 plasma machine: 20 mTorr, 70 Watt, 30 s C Si. (3) PDMS S cchip p. C O. Si. C O. Si. CH3. CH3. CH3 O2 plasma. CH3. CH3. CH3. Si C. O. Si C. O. C Si. C O. OH. Si. C. C. O. OH O. Si C. OH O. C. Si OH. OH. OH. Si. Si. C. Si C. Si Bonding. C O. O Si C. Si. C O. Si. O. Si. O O. Si C. O. C. 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion.

(45) 45. Experimental Methods – Gradient-microfluidic Droplet Generator Reservoir Sample phase 1. 1. Flow-focusing. A. Oil phase 2. X B. Sample phase 2. Y. Oil phase 3. C. Teflon tube. Oil phase 4. Na-alginate droplet. Micro-mixer Flow-focusing device. Oil phase. Oil solution. (a). 2 Na-alginate + CaCl2 → Ca-alginate + 2 NaCl GDG-1. GDG-3. GDG-2. GDG-4. Ca-alginate microparticle. (b).    . Ca+2 buffer. (c). Optimal width of X and Y 70% of the trypan blue solution and D.I. water 5% BSA and 1% BSA mixed with the 0.5% alginate g solution BSA release: the absorption at 280 nm. 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion.

(46) Results of Simulation I. 46. I. -Different Different width of bifurcate channels 100 m. Inlet 2 L/min. II. 80 m. Inlet 2 L/min. II. I 100 m. 100 m. Inlet. III. 100 m. I 100 m Outlet. 100 m. III. II. I. Inlet 2 L/min. X. II. I. III. The velocity of the microchannels (m/s) Outlet I. X (µm) 100. 76 m 0.0029 II. Inlet 2 L/min. 80 100 m 78 76. 100 m. 0.0032 100 m 0.0032 0 III 0.0033 0033. 100 m. Outlet II 0.0039. Outlet III. 0.0033. II 0.0029 0.0032. 0.0032. 0.0032. 0 0031 0.0031. 0 0033 0.0033. Inlet 2 L/min. 100 m. 78 m. 100 m. III 1. Introduction 2. Experiments 3. Results & DiscussionIII 4. Conclusion.

(47) 47. Results of Simulation I. I. -Different Different width of bifurcate channels 78 m Inlet 2 L/min. 74 m. II Inlet 2 L/min. 78 m. I. 100 m. II. 100 m. IV 78 m. 72 mThe II. 74 73 72. 100  m. 100 m. IV I. III. 78 m. Outlet I Y (µm) Inlet 2 L/min 100 m 78 m 78 0.0023. Outlet. II. I. Inlet 2 L/min. III. 100 m. Y. Inlet. 78 m. I. III 78 µm. II. IV. velocity of the microchannels (m/s). III Outlet II 0.0025. 73 m. Outlet III. II Outlet IV. 78 m 0.0025. 0.0023. Inlet 2 L/min. 0.0024 III 100 m 0.0024. 0.0025. 0.0025. 0.0024. 0.0024 100 m . 0.0024 III 0.0024. 0.0025. 0.0024. 0.0024. 0.0025. 100 m. IV. 100 m. IV 1. Introduction 2. Experiments 3. Results & Discussion 4.IVConclusion.

(48) Results of Concentrations Distribution 2 L/min. I ：70% II ：43% III：21% IV：0%. 2.5 L/min. I ：70% II ：46% III：20% IV 0% IV：0%. 3 L/min. I ：70% II ：43% 43% III：22% IV：0%. 2 μL/min 190. 2.5 μL/min. Gray value G. 3 μL/min 170. 150. 130 Ⅰ. 70 m. Ⅱ. Ⅲ. Channel number. 630 m. Ⅳ. 48. 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion.

(49) 49. W/O Emulsion Generation -Uniform size with different concentrations I. Channel I. II. Channel II. Channel III. Channel IV. II. 2/4. III. III 2/6 QW/Q QO (L/minn). IV. I. IV. Sample/Oil flow rate: 2 /10 2/8. Sample/Oil flow rate: 2.5/10. I 2/10 2/12. II III IV Sample/Oil flow rate: 3 /10. 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion.

(50) 50. Alginate Microcapsules Generation -Uniform size with different BSA concentrations I. Channel h l III. Channel h lI. II. Channel h l III. II. 2/4. Sample flow rate 2 μL/min Sample flow rate 2.5 μL/min Sample flow rate 3 μL/min Linear(Water flow rate 2 μL/min) Linear(Water ( flow rate 2.5 μ μL/min)) Linear(Water flow rate 3 μL/min). 120. IV. QSD //QO (L/mi Droplet size in) (μm). III. III. 2/6. 100. IV. 80 2/8 flow rate: 2 /6 Sample/Oil. Channel h l IV. Sample/Oil flow rate: 2.5/6 2. y = -5.5487x + 122.49, R = 0.9891. 60. 2/10. I. y = -5 -5.276x 276x + 117.53, 117 53 R2 = 00.9795 9795 y = -5.2987x + 112.12, R2 = 0.9863. II. 40 3. 2/12. III. 5. 7. 9. 11. 13. Oil flow rate (μL/min). IV Sample/Oil flow rate: 3/6. 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion.

(51) 51. BSA Microcapsules Release -Measurement of UV-Vis spectrophotometry Channel I. 12 1.2. Channel II. Channel III. Channel IV. Abso orbance (a..u.). 1 08 0.8 0.6 0.4 0.2 0 0. 1. 2. 3. Time (hr). . 100 mg microcapsules in 5 mL PBS. . BSA absorbed wavelength: 280 nm. 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion.

(52) 52. Electro spraying Microfluidic Chip Electro-spraying.

(53) 53. Electro-spraying Microfluidic Chip - chip design. 76. 1.2 10. 1.2. 4 1.7. 20. 0.1. 60o. 3. 26. Unit: mm. Mask designed by AutoCAD® 2010 software  The dimension was 76×26 mm  Gap size was 4 mm . Flow-focusing device The dimension 30 × 30 × 6 mm  The angle was 60 degree  . 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion.

(54) 54. Fabrication of the microfluidic chip p - Fabricate ITO electrodes Photolithography process. S1818. PR (1) Mask design. (2) Clean chip (ITO). (3) Photoresist. ~~ ~~ ~~ ~~ ~~ ~ ~~ ~ ~~ ~~. Mask UV (6) Developing. ~~ ~~ ~~ ~~ ~~ ~~ ~~ ~~ ~~. . (4) Soft bake, 110℃, 1 min. (8) Etching. (7) Hard bake, 130℃, 2 min. (5) Exposure ITO electrodes. 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion.

(55) 55. Experimental Methods – Electro-spraying Microfluidic Chip Oil phase (continuous phase). + 0.1 m mm. _. Emulsion. Silicone tube. PLGA emulsion. Taylor cone Oil phase (continuous phase) 4 mm. _. Sample phase ((dispersed p p phase)). +. ITO electrode. Parallel electrodes and flow-focusing device Dispersed p pphase: D.I. water and 1% PLGA mixed with DMSO solution  Electro-spray phenomenon (Taylor cone)  Dispersed phase should be slightly conducting. Oil DI water PLGA microparticle.  . DMSO out. PLGA. DI Water. 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion.

(56) 56. Oil phase (continuous phase). + 0.1 mm. _. Emulsion. Taylor cone Oil phase (continuous phase) 4 mm. _. Sample phase (dispersed phase). Formation of water emulsions under a electric field. +. ITO electrode. 0V. 2000 V. 4000 V. 6000 V. 80/400 200 m. 0V. 200 m. 2000 V. 40/400 200 m. Qs/Qo. 0V. 200 m. 2000 V. 200 m. 4000 V. 6000 V. 200 m. 4000 V. 200 m. α=40.5o. α=32.2o. 6000 V. 200 m. 6000 V. 50 m. 0/0. 80/400. 50 m. (b). (a). 20/400 200 m. 0V. 200 m. 2000 V. 10/400 200 m. 0V. 200 m. 2000 V. 200 m. 4000 V. 200 m 200 m. 4000 V. 200 m. 6000 V. α=26.1o 200 m. 6000 V. 50 m. 5/400 200 m. α=18.0o. 200 m. 200 m. 200 m.  At a 5% Span p 80 concentration.  When the flow rate ratio was decreased. 40/400 (c). 50 m. 10/400 (d). or the voltage was increased,. the emulsion size decreased.  When the flow rate ratio was decreased from 80/400 to 5/400 at 6000 V, the angle of the Taylor cone was decreased from 32.2o to 18o..

(57) 57. Formation of water emulsions under a electric field.  The. lowest driving voltages used to generate the stable electrop y g condition at Span p 80 concentrations of 3%,, 5%,, and 7% were spraying 4000 V, 4000 V, and 3000 V. 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion.

(58) Formation of PLGA emulsions under a electric l i field fi ld 0V. 2000 V. 3000 V. 200 m. 200 m. 4000 V. 80/400 200 m. 0V. 200 m. 2000 V. 3000 V. 4000 V V 4000. 200 m. 200 m. 200 m. 2000 V. 3000 V. 200 m. 200 m. 200 m. 2000 V. 3000 V. 4000 V. 200 m. 200 m. 200 m. 40/400. Qs/Qo. 200 m. 0V. 4000 V. 20/400 200 m. 0V 10/400 200 m. At a 0% Span 80 concentration, concentration PLGA emulsion size ranged from 40 µm to 70 µm.  At a 3% Span 80 concentration, PLGA emulsion size ranged from 42 µm to 14 µm.  The PLAG jet at the 4000 V under the high flow rate ratio . 58.

(59) Formation of PLGA emulsions under a electric l i field fi ld 3% span 80 concentration i.  When Wh. 59. 5% span 80 concentration t ti. the h Span S 80 concentration i was increased i d to 5%, 5% the h minimum PLGA emulsion was 7 µm  A PLGA jet appeared at a high flow rate ratio when the voltage exceeded 2000 V 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion.

(60) Formation of PLGA emulsions under a electric l i field fi ld Span 80 conc.. 0%. 3%. 5%. Water emulsion (μm). PLGA emulsion (μm). Flow rate ratio. Flow rate ratio. Driving voltage (V). 80/400. 40/400. 20/400. 10/400. 5/400. 80/400. 40/400. 20/400. 0. 98 (1). 90 (0.92). 84 (0.86). 80 (0.82). 76 (0.78). 70 (1). 65 (0.93). 61 (0.87) 57 (0.81). 1000. 92 (0.93). 86 (0.88). 69 (0.7). 69 (0.7). 51 (0.52). 63 (0.9). 61(0.87). 57 (0.81). 2000. 80 (0 (0.82) 82). 60 (0 (0.61) 61). 58 (0 (0.59) 59). 38 (0.39) (0 39). 37 (0 (0.38) 38). 62(0 88) 62(0.88). 60 (0 (0.86) 86). 57 (0 (0.81) 81) 55 (0 (0.79) 79). 3000. 68 (0.69). 53 (0.54). 43 (0.44). 37 (0.37). 35 (0.36). 60 (0.86). 59 (0.84). 56 (0.8). 4000. 65 (0.66). 50 (0.51). 41 (0.42). 35(0.35). 33 (0.34). 59 (0.84). 59 (0.84). 53 (0.76) 52 (0.74). 5000. -. -. -. -. -. 58 (0.82). 58 (0.83). 52 (0.74) 44 (0.63). 0. 88 (0.9) (0 9). 75 (0 (0.77) 77). 73 (0 (0.74) 74). 68 (0.69) (0 69). 64 (0 (0.65) 65). 51 (0.73) (0 73). 44 (0 (0.63) 63). 42 (0 (0.6) 6). 1000. 86 (0.88). 71 (0.72). 63 (0.64). 61 (0.62). 55 (0.56). 50 (0.71). 41 (0.59). 38 (0.54) 26 (0.37). 2000. 81 (0.83). 65 (0.66). 54 (0.55). 50 (0.51). 40 (0.41). 48 (0.69). 38 (0.54). 27 (0.38). 3000. 78 (0.8). 60 (0.61). 45 (0.46). 41 (0.42). 28 (0.29). 46 (0.66). 36 (0.51). 24 (0.34) 17 (0.24). 4000. 75 (0 (0.76) 76). 48 (0 (0.49) 49). 40 (0 (0.41) 41). 30 (0.31) (0 31). 2 (0 (0.02) 02). -. -. 18 (0 (0.26) 26) 13 (0 (0.18) 18). 5000. -. 43 (0.44). 4 (0.04). 5 (0.05). 2 (0.02). -. -. -. -. 6000. -. -. -. -. 1 (0.01). -. -. -. -. 0. 80 (0.81). 68 (0.69). 65 (0.66). 62 (0.63). 51 (0.52). 41 (0.58). 30 (0.43). 23 (0.33) 13 (0.18). 1000. 77 (0.79). 64 (0.65). 61 (0.62). 56 (0.57). 42 (0.42). 40 (0.57). 28 (0.4). 20 (0.28). 11 (0.15). 2000. 75(0.77). 62 (0.63). 59 (0.6). 52 (0.54). 30 (0.3). -. -. 16 (0.23). 9 (0.12). 3000. 73(0.74). 58 (0.59). 50 (0.51). 45 (0.46). 23 (0.23). -. -. -. 7 (0.1). 4000. 69(0.7). 52 (0.53). 43 (0.43). 33 (0.34). 2 (0.02). -. -. -. -. 5000. 58(0.59). 33 (0.34). 6 (0.06). 2 (0.02). 2 (0.02). -. -. -. -. 6000. 4(0.04). 2 (0.02). 2 (0.02). 2 (0.02). 1 (0.01). -. -. -. -. 60. 10/400. 56 (0.8) 54 (0.77). 34 (0 (0.49) 49) 21 (0.3). 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion.

(61) 61. Conclusion.

(62) 62. C Conclusion l i . . The results show using AuNPs as the biolabel, electrodes as reaction well, well silver enhancement as magnifying the detection signal could be work successfully in immunoassay. Detection limit: Protein A 0.1 0 1 ng/mL. ng/mL (IgG 10 g/mL). . Using SAM method could enhance the protein bound on the glass chip. . The best detection frequency was 100 Hz and only used 30 L of sample in the detection process (strip use 70 L). . Diluted 100X MET-GC has the best distinction for detection which detection limit was 1 ng/mL (Strip detection limit was 300 ng/mL) 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion.

(63) 63. C l i Conclusion . Simultaneous generation uniform Ca-alginate microcapsules with different BSA concentrations in the gradient-microfluidic droplet generator, through varying the flow rate ratio that the emulsion size could be controlled from 60 m to 105 m.. . An electro-spraying electro spraying microfluidic chip generated a uniform W/O emulsion measuring less than 5 µm with a high driving voltage. voltage The PLGA emulsion size ranged from 7 µm to 70 µm.. 1. Introduction 2. Experiments 3. Results & Discussion 4. Conclusion.

(64) 64. Summary Microfluidic chip. Material. Width and height of the channel. Polymer material. Emulsion size. Cross-junction. PMMA. 200 μm and 1.5 mm. Alginate (AuNPs). 50 μm-2000 μm. Cross-junction. PC. 50 μm and 50 μm. Alginate (BSA). 20 μm-70 μm. T junction T-junction. PMMA. 200 μm and 1.5 1 5 mm. Alginate(AgNPs). 70 μm-300 μm 300 μm. T-junction. PDMS. 200 μm and 1.5 mm. UV-polymer (AuNPs). 40 μm-1000 μm. Gradientmicrofluidic droplet generator. PDMS. 100 μm and 80 μm. Alginate (BSA). 60 μm-105 μm. Adjustablemicrofluidic droplet p ggenerator. PDMS. 100 μm and 80 μm. Chitosan (MNPs). 44 μm-83 μm. Electro-spraying microfluidic chip. PDMS. 100 μm and 80 μm. PLGA. 7 μm-70 μm. Heatable H t bl Microfluidic chip. PDMS. 100 μm and 80 μm. Gelatin (Vitamin C). 45 μm-120 μm.

(65) 65. MEMS llabb members b.

(66) 66. Thanks for Your Attention.

(67)