m22f sequentially addressable two-dimensional microwell array for high

SEQUENTIALLY ADDRESSABLE TWODIMENSIONAL MICROWELL ARRAY FOR HIGHTHROUGHPUT SINGLE CELL-BASED ASSAY

Young-Ji Kim1, Hyung-Kew Lee2, Jaehoon Chung1, Il-Joo Cho3 and Euisik Yoon1,3 1

Dept. of Electrical and Computer Engineering, University of Minnesota, USA 2 Seagate Technology, Bloomington, MN, USA 3 Dept. of EECS, University of Michigan, Ann Arbor, USA

ABSTRACT This paper proposes a new microfluidic array which can sequentially load different types of reagents into a two-dimensional microwell array for high-throughput single cell assay. Proposed array is composed of more than 200 chambers and each sub-array can be selected by microfluidic logics for row and column selection. By applying pneumatic pressure and injecting a different set of reagents, we could load different reagents into the two-dimensional array. In order to verify the sequential addressing scheme, we fabricated and tested a 3x3 device for 9 different reagent loading demonstrated by various colors in food dye. KEYWORDS: Single cell assay, Microwell array, Sequentially addressable INTRODUCTION Previously, several microfluidic platforms have been reported for cell assay; however, most of them could only load cells in an array of capturing sites in microchamber and inject reagents only one-dimensional direction. None of them could either isolate cells from others or inject different reagents to the 2-D arrays in parallel for high-throughput assays [1-4]. We propose a two-dimensional microfluidic microwell array which can sequentially select a block of microwells for injection of multiple reagents and isolate microwells by pneumatic operation using row and column selection logics as shown in Fig. 1. WORKING PRINCIPLE We used a single-cell isolation scheme for loading cells in each microwell, which was reported in our previous paper [5]. Fig. 2 shows the schematic view of operation and experimental procedures for loading single cells and isolating them from one another. By using two-way pneumatic actuation of membranes, single cells are trapped at the capture sites as shown in Fig. 2 (a, b) and loaded into each microwell by lifting the membrane as shown in Fig. 2 (c, d). After that, positive pressure is applied to the membrane for isolating microwells (Fig. 2 (e)). To verify the isolation, we injected ink into the microchannel and confirmed the complete isolation (Fig. 2 (f)). The isolation can be maintained at a flow rate of 100µl/min at an applied pressure of 5psi. Fig. 3 shows the photographs of myoblast cells cultured for 3 days in the fabricated microwell array.

Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences October 12 - 16, 2008, San Diego, California, USA 978-0-9798064-1-4/µTAS2008/$20©2008CBMS

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Figure 1: Schematic comparison of the conventional 1-D reagent addressing scheme with the proposed 2-D reagent addressing by microfluidic logics for row and column selection

Figure 3: Myoblasts (muscle stem cells) cultured in the fabricated microchamber array for three days.

Figure 2: Conceptual diagrams and corresponding experimental results for singlecell capture and isolation: (a, b) trapping single-cells at each capture sites; (c, d) loading single-cells to microwells; and (e, f) isolating single cells in microwells.

EXPERIMENTAL RESULTS Using this single-cell isolation scheme, we expanded an array size up to 3x3 to incorporate more than 240 microwells in a single platform and demonstrated its functionality. Fig. 4 shows the basic operation principle for injecting different reagents in each section. Initially, all channels and microwells are filled with the buffer media and all the chambers are closed for isolation by applying positive pressure. Then, the first set of reagents (three reagents in this 3x3 array) are injected into the first row in the microwell array through the drug injection logic by lifting up the microwells in the first row by selectively applying negative pressure as shown in Fig. 4 (a).

Figure 4: Conceptual diagram of sequential loading of multiple reagents into each section of microwell array: (a) 3 different reagents are injected through drug injection logic to the first row which is open; (b) and (c) show the sequential reagent injection to microwell arrays in the second and third rows, respectively; and (d) all chambers are closed for cell assay

Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences October 12 - 16, 2008, San Diego, California, USA

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Then, the microwells in the first row are closed by applying positive pressure to the control channel. After that, the microwells in the second and third rows are sequentially filled with the predetermined sets of reagents in the same way as shown in Fig. 4 (b-c). After loading reagents into the entire array, all the microwells are closed for a parallel assay of different reagents as shown in Fig. 4 (d). In order to verify the chip operation, we injected nine different food dyes of different colors to the fabricated 3x3 microwell array and Fig. 5 demonstrates its successful operation.

Figure 5: Photograph of the prototype device for sequentially addressable 2-D microwell arrays filled with 9 different colors of food dye.

CONCLUSIONS In summary, we have proposed and successfully demonstrated the prototype of sequentially addressable two-dimensional 3x3 microwell arrays for single cell assay. By incorporating the embedded fluidic logic, the proposed two-dimensional addressing scheme can be easily expanded into a much larger array and be used for various high-throughput bio-assays. ACKNOWLEDGEMENTS This work has been partially supported by the Intelligent Microsystems Program (IMP) of the ‘21C Frontier R&D program’ from KIST. REFERENCES [1] P. Hung, P. Lee, and L. P. Lee, “A Continuous Perfusion Microfluidic Cell Culture Array for High Throughput Cell-based Assays,” Biotechno. Bioeng., Vol. 89, pp. 1-8, (2005). [2] K.-S. Yun and E. Yoon, “Micro/Nanofluidic Device for Single-Cell-Based Assay,” Biomedical Microdevices, 7:1, pp. 35-40, (2005). [3] Z. Wang, M.-C. Kim, M. Marquez and T. Thorsen “High-density microfluidic arrays for cell cytotoxicity analysis,” Lab Chip, 7, pp. 740-745, (2007). [4] K. R. King, et. al., “A high-throughput microfluidic real-time gene expression living cell array,” Lab Chip, 7, pp. 77-85, (2007). [5] Y.-J. Kim, J. Chung, H.-K Lee and E. Yoon, “Microfluidic Array Chip For Single-Cell Isolation Using Two-Way Pneumatic Actuation,” IEEE MEMS Conference, Technical Proceeding, pp. 14-17, (2008).

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