FABRICATION OF HOURGLASS-SHAPED MICROAPERTURE VIA TWO-STAGE LASER

PULSES AND ITS APPLICATION Chang-Yu Chen1, Ting-Yuan Tu1, De-Shien Jong2, and Andrew M. Wo1

1 Institute of Applied Mechanics and 2 Department of Animal Science and Tech-nology, National Taiwan University, Taipei, TAIWAN

ABSTRACT

This work presents the fabrication of hourglass-shaped aperture by applying two-stage CO2 laser pulses on glass substrate to control the size of the aperture to 1-10μm, and applies the fabricated aperture to measure ion-channel activities and to trap single cells in array configuration. First stage laser pulses with longer duration was utilized to generate apertures larger than 10μm, and a series of shorter duration laser pulses was added for maintaining the glass reflow to reduce its diameter. This easy fabrication procedure can be operated without complicated micro-fabrication process for applications of single-cell analysis. KEYWORDS: Single-cell analysis, laser drilling, microaperture, patch-clamp INTRODUCTION

Single-cell analysis has known to constrain cells in a certain confined area or fixed location for studying their morphological or physiological behaviors under en-vironmental stimuli. Recently varies approaches have been presented to locate single cells in place. Rettig et al. showed large-scale single cells trapped by seeding of cells in PDMS microwells [1]. Besides, micro-openings were created in varies ways to trap a single cell for measuring ion-channel responses [2]. However, most of these techniques for creating microapertures are time-consumed, or need to be proceeded through complicated micro-fabrication processes. In our previous research [3], a fast and easy method was developed to fabricate an hourglass-shaped microaperture by CO2 laser drilling on glass material. The size of the apertures typically ranged between 5-15μm, and smallest size could be at-tained to 1-3μm under strictly control of laser machine. In order to create apertures with more stable and controllable size, a new laser drilling strategy was proposed. The size of the aperture was much stable and predictable by this method. The fabri-cated aperture could be used in planar patch-clamp application or single-cell trap-ping in microarray configuration.

THEORY

A two-stage laser pulses command illustration is demonstrated in Fig. 1A. The first stage is composed of a number of pulses with longer duration than the second stage pulses. These laser pulses drill through the glass substrate and form an hour-glass-shape aperture with diameter typically larger than 10μm. The second stage comprised a series of shorter duration pulses is designed to maintain the reflow of melted glass back to the core of the aperture to reduce its diameter to 1-10μm. Fig-

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

Figure 2. SEM image of fabricated apertures with different number of second stage pulses. The aperture size gradually narrowed down by implementing different amount of the second stage laser pulses, from 0 to 10 in 2 interval.

Figure 3. Fabricated aperture diameter due to different number of second stage laser pulses. Results indicated the size of the aperture was progressively reduced by increasing the number of the second stage laser pulses.

ure 1B and 1C shows side-view micro-graph and isometric-view SEM image of the hourglass-shaped aperture respec-tively. EXPERIMENTAL ASPECT

A 25W CO2 laser (Synrad 48-2) was driven by LabVIEW program via data acquisition card (NI-6251) to execute the drilling process. Borosilicate cover glass (Matsunami Glass) with typical thick-ness of 150μm was used as substrate. The size of the fabricated aperture was measured under 500× microscope.

HEK 293T cells were grown in DMEM supplemented with 10% FBS at 5% CO2 incubator. Electrophysiological recording was performed with conven-tional Axon 200B amplifier in whole-cell configuration. Cells were stained with calcein AM 15 min before trapping on microaperture array, and the fluorescent image was taken by Olympus DP70 CCD on IX71 microscope.

RESULTS AND DISCUSSION

SEM image of fabricated apertures with different number of second stage pulses is showed in Fig. 2. The apertures correspond to different number of pulses between 0 to 10 in 2 interval revealed gradually reduced aperture diameters. The smallest size of the aperture in this demonstration was around 3μm. Figure 3 shows the relation-ship between the number of second stage laser pulses verses aperture diameter. Re-sults show the more the second stage pulses, the smaller the aperture diameter. This result verifies the concept of the two-stage laser pulses approach. The error bars rep-resent the standard deviations which located in the acceptable range.

Figure 1. (A) Illustration of two-stage laser pulses command to fabricate apertures with diameter of 1-10μm. (B) Side-view micrograph of the hourglass-shaped aperture. (C) Isometric SEM image of the aperture captured from the laser exiting surface.

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

Figure 4. Measured ion-channel current via fabri-cated 1~3μm aperture as a planar patch-clamp chip. (A) Whole cell current recording of endoge-nous channels in HEK 293T cell without leak sub-traction. Strong ion channel activation appeared at positive membrane potential. (B) The current-voltage relation obtained from the whole cell cur-rent traces.

Figure 5. (A) SEM image of microaperture ar-ray fabricated by two-stage laser drilling tech-nique. The size of the apertures was uniformly less than 8μm. (B) Fluorescent image of single-cell trapping on microaperture array. Individual HEK 293T cells with calcein AM stain were trapped on the aperture by applying negative suction.

Planar patch-clamp application with fabricated aperture in the range of 1-3μm was presented. Whole cell current recording of endogenous channels in HEK 293T cell without leak subtraction is shown in Fig. 4A. Voltage steps were elicited be-tween -100 and +80 mV in 20 mV interval from holding potential of -80 mV. Strong ion channel activation appeared at positive membrane potential. Seal resistance at whole cell configuration was around 1 GΩ. Figure 4B indicates the current-voltage relation obtained from the whole cell current traces.

A 3x3 mirco-aperture array fabricated via the two-stage laser drilling technique was described. Figure 5A shows the SEM image of microarray with uniform size of the apertures smaller than 8μm. HEK 293T cells stained with calcein AM were trapped as a microaperture array by application of suction, as shown in Fig. 5B. CONCLUSIONS

The two-stage laser pulses approach proves feasible for fabricating 1-10μm aper-tures. We can acquire a decreasing aperture size by increasing the number of second stage laser pulses. The fabricated aperture can be used in single-cell analysis such as planar patch-clamp application or microarray for single-cell trapping. REFERENCES [1] J. R. Rettig and A. Folch, "Large-scale single-cell trapping and imaging us-

ing microwell arrays," Analytical Chemistry, vol. 77, pp. 5628-5634, 2005. [2] J. Dunlop, M. Bowlby, R. Peri, D. Vasilyev, and R. Arias, "High-

throughput electrophysiology: an emerging paradigm for ion-channel screening and physiology," Nature Reviews Drug Discovery, vol. 7, pp. 358-368, 2008.

[3] C. Y. Chen, K. T. Liu, D. S. Jong, and A. M. Wo, "Hourglass-shaped aper-ture for cellular electrophysiological study," Applied Physics Letters, vol. 91, 123901, 2007.

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