本研究使用電穿孔技術將螢光染劑輸送至微藻內，藉此觀察在電場中細胞所含油脂的染色情形。微藻細胞可產生豐富的油脂並可藉由轉脂化反應將其轉為生質柴油以取代石油。傳統檢測藻體內油量的方法須經過一連串複雜且耗時的處理步驟。自從1987年螢光感測分子—尼羅紅—被發現可用於定量細胞內部脂質的含量，此螢光檢測法隨即被廣泛地運用於生物體內油脂檢測中。然而，雖然使用尼羅紅染劑可輕易地觀察細胞內油脂，但由於不同的藻種(如：不同厚度及組成的細胞膜和細胞壁)具有不同的螢光強度、染色條件和螢光激發週期，導致難以使用螢光法來準確地定量油脂的含量。
電穿孔技術已被廣泛地應用於將大型分子輸送至細胞內。本研究使用交流轉換電源產生交流電場，藉由輸出不同振幅和頻率破壞微藻細胞膜，加快尼羅紅染劑運輸至細胞內與脂質體染色的速率。實驗中分別將小球藻、柵藻及螺旋藻固定在表面並施加100~2000 V⁄cm (10 MHz)的電場強度，在2000 V⁄cm下，柵藻及螺旋藻的螢光強度相較於對照組(0 V⁄cm)，在10分鐘內可增加300%及120%。因此，藉由電穿孔技術加速染色過程，避免不同藻種和染色時間所造成的誤差是可實行的。另外，不同電場強度影響螢光染色的機制亦被詳細地討論。除了尼羅紅之外，本研究也嘗試使用LipidTOX來觀察微藻的螢光強度變化，發現LipidTOX在電場中可與脂質較穩定的結合，所誘發的螢光強度並不會像尼羅紅隨著時間快速減少。在本文最後利用不同微流道設計產生不同的螢光染劑濃度梯度，以利於在單一晶片中整合螢光標定、細胞穿孔、偵測和樣品篩選。

This study aims to use electroporation to facilitate the transport of the fluorogenic dye, Nile red, into microalgae for labeling the cellular neutral lipids. Microalgae produce profuse neutral lipids that can be transformed to biofuels by esterification. The traditional methods for quantifying the lipid amounts inside microalgae usually require a series of processes, which are complicated and time consuming. A fluorogenic probe, Nile red, was used to detect the lipid bodies inside cells since 1987. This optical method has been utilized widely. However, even if the neutral lipids can be observed easily, it was hard to accurately quantify the lipid abundance since the fluorescence intensity varied with algae strains (i.e. different thickness and compositions of cell membrane and wall) and staining conditions and durations.
Electroporation is widely applied to transport large molecules into cells. In our study, an alternating current power source was used to produce alternating electric fields with different intensities and frequencies to compromise the microalgae cell membrane and transport Nile red into cells. The fluorescence intensity of microalgae(Chlorella sp., Scenedesmus and Spirulina sp.) fixed on the surface was observed under electric fields ranging from 100 V⁄cm to 2000 V⁄cm. The fluorescence intensity of Scenedesmus and Spirulina increased to 300% and 120% compared with the control (0 V⁄cm) in 2000 V⁄cm (10 MHz) in 10 min. Therefore, it was feasible to use electroporation to accelerate the labeling process and prevent biased results due to the differences in microalgae strains and staining durations. The effects of different electric field strengths and fluorogenic dyes (Nile red, LipidTOX) on the fluorescence labeling process of microalgae were also discussed. LipidTOX-lipid complex was more stable inside electric field compared with Nile red-lipid complex; therefore, the rapid decrease of fluorescence intensity was not observed when LipidTOX was used. We have also produced controllable gradients of fluorescein concentration using microchannels with different geometries. The information provided in this study is valuable for integrating the labeling, electroporation, detection and sorting into one device.

[1] P. Greenspan, E. P. Mayer, and S. D. Fowler, "Nile red - a selective fluorescent stain for intracellular lipid droplets," Journal of Cell Biology, vol. 100, pp. 965-973, 1985.
[2] K. L. Yeh, J. S. Chang, and W. M. Chen, "Effect of light supply and carbon source on cell growth and cellular composition of a newly isolated microalga Chlorella vulgaris esp-31," Engineering in Life Sciences, vol. 10, pp. 201-208, Jun 2010.
[3] C. Q. Lan, Y. Q. Li, M. Horsman, B. Wang, and N. Wu, "Effects of nitrogen sources on cell growth and lipid accumulation of green alga neochloris oleoabundans," Applied Microbiology and Biotechnology, vol. 81, pp. 629-636, Dec 2008.
[4] Y. N. Liang, N. Sarkany, and Y. Cui, "Biomass and lipid productivities of Chlorella vulgaris under autotrophic, heterotrophic and mixotrophic growth conditions," Biotechnology Letters, vol. 31, pp. 1043-1049, Jul 2009.
[5] A. Widjaja, C. C. Chien, and Y. H. Ju, "Study of increasing lipid production from fresh water microalgae chlorella vulgaris," Journal of the Taiwan Institute of Chemical Engineers, vol. 40, pp. 13-20, Jan 2009.
[6] A. P. R. F. Canela, P. T. V. Rosa, M. O. M. Marques, and M. A. A. Meireles, "Supercritical fluid extraction of fatty acids and carotenoids from the microalgae Spirulina maxima," Industrial & Engineering Chemistry Research, vol. 41, pp. 3012-3018, Jun 12 2002.
[7] Q. Y. Wu, C. F. Gao, W. Xiong, Y. L. Zhang, and W. Q. Yuan, "Rapid quantitation of lipid in microalgae by time-domain nuclear magnetic resonance," Journal of Microbiological Methods, vol. 75, pp. 437-440, Dec 2008.
[8] Q. Hu, W. Chen, C. W. Zhang, L. R. Song, and M. Sommerfeld, "A high throughput nile red method for quantitative measurement of neutral lipids in microalgae," Journal of Microbiological Methods, vol. 77, pp. 41-47, Apr 2009.
[9] S. D. Fowler and P. Greenspan, "Application of nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue-sections - comparison with oil red o," Journal of Histochemistry & Cytochemistry, vol. 33, pp. 833-836, 1985.
[10] P. Greenspan and S. D. Fowler, "Spectrofluorometric studies of the lipid probe, nile red," Journal of Lipid Research, vol. 26, pp. 781-789, 1985.
[11] B. Raleigh, D. Elsey, D. Jameson, and M. J. Cooney, "Fluorescent measurement of microalgal neutral lipids," Journal of Microbiological Methods, vol. 68, pp. 639-642, Mar 2007.
[12] G. Diaz, M. Melis, B. Batetta, F. Angius, and A. M. Falchi, "Hydrophobic characterization of intracellular lipids in situ by nile red red/yellow emission ratio," Micron, vol. 39, pp. 819-824, Oct 2008.
[13] J. F. Deye, T. A. Berger, and A. G. Anderson, "Nile red as a solvatochromic dye for measuring solvent strength in normal liquids and mixtures of normal liquids with supercritical and near critical fluids," Analytical Chemistry, vol. 62, pp. 615-622, 1990.
[14] M. M. G. Krishna, "Excited-state kinetics of the hydrophobic probe nile red in membranes and micelles," Journal of Physical Chemistry A, vol. 103, pp. 3589-3595, May 13 1999.
[15] M. S. Cooper, W. R. Hardin, T. W. Petersen, and R. A. Cattolico, "Visualizing "green oil" in live algal cells," Journal of Bioscience and Bioengineering, vol. 109, pp. 198-201, 2010.
[16] W. T. Wu, C. H. Su, C. C. Fu, Y. C. Chang, G. R. Nair, J. L. Ye, and I. M. Chu, "Simultaneous estimation of chlorophyll a and lipid contents in microalgae by three-color analysis," Biotechnology and Bioengineering, vol. 99, pp. 1034-1039, Mar 1 2008.
[17] H. B. Yu, G. Y. Zhou, C. F. Siong, S. H. Wang, and F. W. Lee, "Novel polydimethylsiloxane (pdms) based microchannel fabrication method for lab-on-a-chip application," Sensors and Actuators B-Chemical, vol. 137, pp. 754-761, Apr 2 2009.
[18] A. Folch, A. Ayon, O. Hurtado, M. A. Schmidt, and M. Toner, "Molding of deep polydimethylsiloxane microstructures for microfluidics and biological applications," Journal of Biomechanical Engineering-Transactions of the Asme, vol. 121, pp. 28-34, Feb 1999.
[19] D. C. Duffy, J. C. McDonald, O. J. A. Schueller, and G. M. Whitesides, "Rapid prototyping of microfluidic systems in poly(dimethylsiloxane)," Analytical Chemistry, vol. 70, pp. 4974-4984, Dec 1 1998.
[20] R. Zaouk, B. Y. Park, and M. J. Madou, "Fabrication of pdms microfluidics using su-8 molds," in Methods in molecular biology, vol. 321, pp. 17-21, 2006
[21] S. W. Smye, C. Chen, M. P. Robinson, and J. A. Evans, "Membrane electroporation theories: A review," Medical & Biological Engineering & Computing, vol. 44, pp. 5-14, Mar 2006.
[22] J. C. Weaver, "Electroporation of biological membranes from multicellular to nano scales," Ieee Transactions on Dielectrics and Electrical Insulation, vol. 10, pp. 754-768, Oct 2003.
[23] J. C. Weaver and Y. A. Chizmadzhev, "Theory of electroporation: A review," Bioelectrochemistry and Bioenergetics, vol. 41, pp. 135-160, Dec 1996.
[24] A. J. H. Sale and W. A. Hamilton, "Effects of high electric fields on microorganisms .I. Killing of bacteria and yeasts," Biochimica Et Biophysica Acta, vol. 148, pp. 781-&, 1967.
[25] S. H. White, "Electrical breakdown of bimolecular lipid-membranes as an electromechanical instability," Biophysical Journal, vol. 14, pp. 155-158, 1974.
[26] K. Kinosita, Jr. and T. Y. Tsong, "Formation and resealing of pores of controlled sizes in human erythrocyte membrane," Nature, vol. 268, pp. 438-41, Aug 4 1977.
[27] S. W. Hui, "Effects of pulse length and strength on electroporation efficiency," Methods Mol Biol, vol. 48, pp. 29-40, 1995.
[28] T. Kotnik, L. M. Mir, K. Flisar, M. Puc, and D. Miklavcic, "Cell membrane electropermeabilization by symmetrical bipolar rectangular pulses. Part i. Increased efficiency of permeabilization," Bioelectrochemistry, vol. 54, pp. 83-90, Aug 2001.
[29] M. Tarek, "Membrane electroporation: A molecular dynamics simulation," Biophysical Journal, vol. 88, pp. 4045-53, Jun 2005.
[30] J. C. Weaver, "Electroporation - a general phenomenon for manipulating cells and tissues," Journal of Cellular Biochemistry, vol. 51, pp. 426-435, Apr 1993.
[31] T. Kotnik, F. Bobanovic, and D. Miklavcic, "Sensitivity of transmembrane voltage induced by applied electric fields - a theoretical analysis," Bioelectrochemistry and Bioenergetics, vol. 43, pp. 285-291, Aug 1997.
[32] H. S. Radomska and L. A. Eckhardt, "Mammalian cell fusion in an electroporation device," Journal of Immunological Methods, vol. 188, pp. 209-217, Dec 27 1995.
[33] H. R. Azencott, G. F. Peter, and M. R. Prausnitz, "Influence of the cell wall on intracellular delivery to algal cells by electroporation and sonication," Ultrasound in Medicine and Biology, vol. 33, pp. 1805-1817, Nov 2007.
[34] L. Alibaud, P. Cosson, and M. Benghezal, "Dictyostelium discoideum transformation by oscillating electric field electroporation," Biotechniques, vol. 35, pp. 78-+, Jul 2003.
[35] H. Wolf, M. P. Rols, E. Boldt, E. Neumann, and J. Teissie, "Control by pulse parameters of electric field-mediated gene-transfer in mammalian-cells," Biophysical Journal, vol. 66, pp. 524-531, Feb 1994.
[36] M. Kanduser, D. Miklavcic, and M. Pavlin, "Mechanisms involved in gene electrotransfer using high- and low-voltage pulses - an in vitro study," Bioelectrochemistry, vol. 74, pp. 265-271, Feb 2009.
[37] H. Y. Wang and C. Lu, "Microfluidic electroporation for delivery of small molecules and genes into cells using a common dc power supply," Biotechnology and Bioengineering, vol. 100, pp. 579-586, Jun 15 2008.
[38] T. Geng, Y. H. Zhan, H. Y. Wang, S. R. Witting, K. G. Cornetta, and C. Lu, "Flow-through electroporation based on constant voltage for large-volume transfection of cells," Journal of Controlled Release, vol. 144, pp. 91-100, May 21 2010.
[39] M. Khine, A. Lau, C. Ionescu-Zanetti, J. Seo, and L. P. Lee, "A single cell electroporation chip," Lab on a Chip, vol. 5, pp. 38-43, 2005.
[40] Y. Huang and B. Rubinsky, "Flow-through micro-electroporation chip for high efficiency single-cell genetic manipulation," Sensors and Actuators a-Physical, vol. 104, pp. 205-212, May 15 2003.
[41] H. Lu, M. A. Schmidt, and K. F. Jensen, "A microfluidic electroporation device for cell lysis," Lab on a Chip, vol. 5, pp. 23-29, 2005.
[42] W. G. Lee, U. Demirci, and A. Khademhosseini, "Microscale electroporation: Challenges and perspectives for clinical applications," Integrative Biology, vol. 1, pp. 242-251, Mar 2009.
[43] Y. C. Lin and M. Y. Huang, "Electroporation microchips for in vitro gene transfection," Journal of Micromechanics and Microengineering, vol. 11, pp. 542-547, Sep 2001.
[44] Y. C. Lin, C. M. Jen, M. Y. Huang, C. Y. Wu, and X. Z. Lin, "Electroporation microchips for continuous gene transfection," Sensors and Actuators B-Chemical, vol. 79, pp. 137-143, Oct 15 2001.
[45] H. Y. Wang and C. Lu, "Electroporation of mammalian cells in a microfluidic channel with geometric variation," Analytical Chemistry, vol. 78, pp. 5158-5164, Jul 15 2006.
[46] V. Hessel, H. Lowe, and F. Schonfeld, "Micromixers - a review on passive and active mixing principles," Chemical Engineering Science, vol. 60, pp. 2479-2501, Apr-May 2005.
[47] C. K. Chen and C. C. Cho, "A combined active/passive scheme for enhancing the mixing efficiency of microfluidic devices," Chemical Engineering Science, vol. 63, pp. 3081-3087, Jun 2008.
[48] V. Mengeaud, J. Josserand, and H. H. Girault, "Mixing processes in a zigzag microchannel: Finite element simulations and optical study," Analytical Chemistry, vol. 74, pp. 4279-4286, Aug 15 2002.
[49] S. Hardt, K. S. Drese, V. Hessel, and F. Schonfeld, "Passive micromixers for applications in the microreactor and mu tas fields," Microfluidics and Nanofluidics, vol. 1, pp. 108-118, May 2005.
[50] W. Jeon and C. B. Shin, "Design and simulation of passive mixing in microfluidic systems with geometric variations," Chemical Engineering Journal, vol. 152, pp. 575-582, Oct 15 2009.
[51] J. Aubin, M. Ferrando, and V. Jiricny, "Current methods for characterising mixing and flow in microchannels," Chemical Engineering Science, vol. 65, pp. 2065-2093, Mar 15 2010.
[52] N. Periasamy and A. S. Verkman, "Analysis of fluorophore diffusion by continuous distributions of diffusion coefficients: Application to photobleaching measurements of multicomponent and anomalous diffusion," Biophysical Journal, vol. 75, pp. 557-567, Jul 1998.