A HIGH-THROUGHPUT DRUG-SCREENING SYSTEM TARGETING ABC … · Figure 2: Process flow of a device...
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A HIGH-THROUGHPUT DRUG-SCREENING SYSTEM TARGETING ABC-TRANSPORTERS: AN APPLICATION OF A MICROFLUIDIC GRADIENT GENERATOR Y. Abe 1,3 , H. Sasaki 1 , R. Yamamoto 1,3 , T. Osaki 1 , R. Kawano 1 , K. Kamiya 1 , N. Miki 1,3 , and S. Takeuchi 1,2 1 Kanagawa Academy of Science and Technology, 2 Institute of Industrial Science, The University of Tokyo, 3 Keio University, Japan ABSTRACT We describe a high-throughput methodology to analyze the function of ATP binding cassette transporter (ABC- transporter) by using a microfluidic device that generates a concentration gradient of substrates. Taking advantage of the basic procedure that we established for functional analysis of ABC-transporters, we here show an advanced system utilizing multi-channel microfluidic device. We applied a microfluidic gradient generator, which makes various concentrations of substrates in a single device. After incubation of ABC-transporters at 5 different substrate concentrations in a gradient gen- erator, we succeeded in detecting the difference of substrate transport revealed by the different fluorescence intensity from ABC-transporter vesicles. KEYWORDS: ABC-transporter, Microfluidics, PDMS, Concentration gradient INTRODUCTION ATP-binding cassette (ABC) transporters are mem- bers of a protein superfamily involved in various dis- eases, such as tumor resistance, cystic fibrosis and can- cer [1]. The proteins are expressed in the luminal membrane of the small intestine, in the blood–brain barrier, and in the apical membranes of excretory cells, such as hepatocytes and kidney proximal tubule epithe- lia, and transport a wide variety of substrates including metabolic products and drugs utilizing the energy of ATP hydrolysis. Dysfunction of ABC-transporters in- fluences the disposition and/or side effects of drugs in human body. Because in vitro studies of drug interac- tions with ABC-transporters are required in drug devel- opment [2], the high-throughput systems to conduct the analyses are highly demanded. The drug discovery at its early stage primarily con- sists of experiments that can prove the interaction of a compound (future drug candidate) and a specific recep- tor (target). Nowadays, high-throughput screenings (HTS) are the principal tool for pharmaceutical compa- nies to investigate the properties of new chemical enti- ties. Traditionally, multiple-well plates are used to per- form screenings. Further miniaturization, however, is important to reduce sample consumption and reaction time and, hence, costs of the process. Several possible miniaturized formats have been suggested in order to achieve drug or library formation and drug testing. One of the promising HTS platforms is the miniaturized reactors using microfluidics technology [3]. Microflu- idics technology can provide continuous and semi- continuous reactors that promise faster operation and throughput and improved automation prospects. For drug discovery, concentration gradients generated in a microfluidic channel have been used for testing multiple drug does simultaneously or for lead optimization [4]. Figure 1: Conceptual illustrations of a concentration- gradient generator and the influx of fluorescent substrates to an ABC-transporter vesicle tethered to a glass. 978-0-9798064-4-5/μTAS 2011/$20©11CBMS-0001 885 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 2-6, 2011, Seattle, Washington, USA
Transcript of A HIGH-THROUGHPUT DRUG-SCREENING SYSTEM TARGETING ABC … · Figure 2: Process flow of a device...
A HIGH-THROUGHPUT DRUG-SCREENING SYSTEM TARGETING ABC-TRANSPORTERS: AN APPLICATION OF A MICROFLUIDIC GRADIENT GENERATOR
Y. Abe1,3, H. Sasaki1, R. Yamamoto1,3, T. Osaki1, R. Kawano1, K. Kamiya1, N. Miki1,3, and S. Takeuchi1,2 1Kanagawa Academy of Science and Technology, 2Institute of Industrial Science, The University of Tokyo,
3Keio University, Japan ABSTRACT
We describe a high-throughput methodology to analyze the function of ATP binding cassette transporter (ABC-transporter) by using a microfluidic device that generates a concentration gradient of substrates. Taking advantage of the basic procedure that we established for functional analysis of ABC-transporters, we here show an advanced system utilizing multi-channel microfluidic device. We applied a microfluidic gradient generator, which makes various concentrations of substrates in a single device. After incubation of ABC-transporters at 5 different substrate concentrations in a gradient gen-erator, we succeeded in detecting the difference of substrate transport revealed by the different fluorescence intensity from ABC-transporter vesicles. KEYWORDS: ABC-transporter, Microfluidics, PDMS, Concentration gradient
INTRODUCTION
ATP-binding cassette (ABC) transporters are mem-bers of a protein superfamily involved in various dis-eases, such as tumor resistance, cystic fibrosis and can-cer [1]. The proteins are expressed in the luminal membrane of the small intestine, in the blood–brain barrier, and in the apical membranes of excretory cells, such as hepatocytes and kidney proximal tubule epithe-lia, and transport a wide variety of substrates including metabolic products and drugs utilizing the energy of ATP hydrolysis. Dysfunction of ABC-transporters in-fluences the disposition and/or side effects of drugs in human body. Because in vitro studies of drug interac-tions with ABC-transporters are required in drug devel-opment [2], the high-throughput systems to conduct the analyses are highly demanded.
The drug discovery at its early stage primarily con-sists of experiments that can prove the interaction of a compound (future drug candidate) and a specific recep-tor (target). Nowadays, high-throughput screenings (HTS) are the principal tool for pharmaceutical compa-nies to investigate the properties of new chemical enti-ties. Traditionally, multiple-well plates are used to per-form screenings. Further miniaturization, however, is important to reduce sample consumption and reaction time and, hence, costs of the process. Several possible miniaturized formats have been suggested in order to achieve drug or library formation and drug testing. One of the promising HTS platforms is the miniaturized reactors using microfluidics technology [3]. Microflu-idics technology can provide continuous and semi-continuous reactors that promise faster operation and throughput and improved automation prospects. For drug discovery, concentration gradients generated in a microfluidic channel have been used for testing multiple drug does simultaneously or for lead optimization [4].
Figure 1: Conceptual illustrations of a concentration-gradient generator and the influx of fluorescent substrates to an ABC-transporter vesicle tethered to a glass.
978-0-9798064-4-5/µTAS 2011/$20©11CBMS-0001 885 15th International Conference onMiniaturized Systems for Chemistry and Life Sciences
October 2-6, 2011, Seattle, Washington, USA
In this paper, taking advantage of the basic procedure that we have established for functional analysis of ABC-transporters [5], we apply a microfluidic device [6], which makes various concentration of substrates in a single device, for high-throughput analysis of ABC-transporters function (Figure 1). Generating the concentration gradient of fluorescence sub-strates, we could detect the difference of substrate transport revealed by the different fluorescence intensity from ABC-transporter vesicles.
EXPERIMENTAL
Our device consists of a polydimethylsiloxane (PDMS) microfluidic channel and a glass substrate. The cross-sectional width and height of the channels were both 100 µm. The microfluidic devices were fab-ricated using standard soft lithography. The PDMS chip was then coated with a poly-p-xylylene derivative (parylene N) to prevent the absorption of fluorescent substrates onto the inner surface of the PDMS channels [7]. Following the parylene deposition, the PDMS chip was attached to an aldehyde-coated glass with single-stranded DNA (ssDNA) tethers using O2 plasma treatment. To prevent DNA removal at the plasma-exposure step, we placed masks on the glass. After the device preparation, a cholesterol-modified ssDNA was applied to the channels to form duplexes with the ssD-NA tethered to the glass surface. ABC-transporter ves-icles were then injected into microfluidic channels to be immobilized, and incubated with Rhodamine123 (Rh123) solutions at different concentrations for 1 hour and 37℃. The fluorescence intensities of ABC-transporter vesicles after rinsing with buffer were de-tected by microscopic images (Figure 4). RESULTS AND DISCUSSION
Mask effect to protect glass-surface DNA from O2 plasma
We found that the glass-surface DNA tethers, which are required for vesicle immobilization, were removed when exposed to O2 plasma. To prevent the DNA removal, we placed various masks on the glass in the plasma-exposure step. Figure 3 shows fluorescence images of glass-patterned ssD-NA in the device after exposure to O2 plasma. The line profiles crossing the boundaries of the patterned DNA reveal that the masks made of glass and PDMS can effectively protect the DNA tethers from O2 plasma (Figure 3).
Figure 2: Process flow of a device fabrication and the immobilization of ABC-transporter vesicles in the microfluid-ic channels. (a) PDMS fabrication and glass surface modification with single-strand DNA. (b) O2 plasma treatment with a DNA-protecting mask. (c) bonding PDMS to a glass. (d) injection of vesicles and rhodamine substrate into channels. (e) a photograph of a concentration-gradient generator .
Figure 3: Fluorescence images of patterned DNA in the device after exposure to O2 plasma. The DNA on a glass was protected from the O2 plasma by using various masks:(a) without cover, (b) parafilm, (c) glass, (d) PDMS. Graphs in the right column show the line profiles along the red lines drawn in the images.
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The substrate concentration dependence of the transport by ABC-transporters Figure 4 shows fluorescence intensities of ABC-transporter vesicles after incubation at 5 different concentration of
Rh123. After rinsing the substrates, the fluorescence intensity generated from ABC-transporter vesicles increased in propor-tion to the substrate concentration (Figure 4), suggesting that our gradient generating device is useful to detect the substrate concentration dependence of the transport by ABC-transporter. CONCLUSION
In this study, we developed the platform of functional assay of ABC-transporters on one chip by using concentration gradients generator. We believe that our approach would pave the way for the high-throughput analysis of the function of ABC-transporters using micro electro mechanical systems (MEMS) device. ACKNOWLEDGEMENTS
We thank Utae Nose for technical assistance in device preparation. REFERENCES [1] M. Gottesman and S. V. Ambudkar, "ABC Transporters and Human Disease," J. Bioenerg. Biomembr., vol. 33, pp.
453-458, Dec. 2001. [2] K. M. Giacomini et al., "Membrane transporters in drug development," Nat. Rev. Drug Discov., vol. 9, pp. 215-236, Mar.
2010. [3] D. Lombardi and P. S. Dittrich, "Advances in microfluidics for drug discovery," Expert Opin. Drug Discov., vol. 5, pp.
1081-1094, Nov. 2010. [4] J. Pihl, J. Sinclair, E. Sahlin, M. Karlsson, F. Petterson, J. Olofsson and O. Orwar, "Microfluidic gradient-generating
device for pharmacological profiling," Anal. Chem. vol. 77, pp. 3897-3903, Jul. 2005. [5] H. Sasaki, H. Onoe, T. Osaki, R. Kawano and S. Takeuchi, "Transporters on a Chip: A Fluorescence analysis of an
ATP-binding Cassette (ABC)-transporter," Proc. µTAS 2010, pp. 842-844, 2010. [6] S. K. W. Dertinger and G. M. Whitesides, "Generation of Gradients Having Complex Shapes Using Microfluidic Net-
works," Anal. Chem., vol. 73, pp. 1240-1246, Mar. 2001. [7] H. Sasaki, H. Onoe, T. Osaki, R. Kawano and S. Takeuchi, "Parylene-coating in PDMS microfluidic channels prevents
the absorption of fluorescent dyes, " Sens. Actuators B, vol. 150, pp. 478-482, Sep. 2010. CONTACT *Y. Abe, Kanagawa Academy of Science and Technology (KAST), 3-2-1 Sakado, KSP East 303, Takatsu-ku, Kawasaki, Kanagawa 213-0012, Japan, Tel: +81-44-819-2037, Email: [email protected]
Figure 4: Fluorescence intensities of ABC-transporter vesicles after incubation at 5 different concentrations of Rh123. The Rh123 incubation was performed in the presence of ATP for 1 hour. Fluorescence images after incubation at sev-eral concentrations are shown as insets in the graph.
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