微流實驗室

The hourglass-shaped aperture was implemented by conventional laser drilling technique. A series of short pulses of CO2 laser were applied to drill on borosilicate cover glass with typical thickness of 150um. The total laser energy was designed to just penetrate the thin glass.

(A) Micro-opening is fabricated to replace the conventional micropipette. The aperture serves for both cell trapping and signal recording.
(B) An additional concentric channel design facilitates precise cell positioning.
(C) Hourglass-shaped aperture provides more contact area to enhance the seal quality.
(D) Lateral trapping approach offers optical and electrical investigation simultaneously.

The side (edge) view of the 150 um glass slide Fig. (A) shows the laser path as predominately Gaussian and penetrated the majority of the glass thickness. At the tip of the Gaussian profile, the hourglass feature is clearly seen; it bottlenecks at the aperture followed by an expanding funnel structure towards the other surface. The diameter of the funnel is approximately 30 um on the glass surface and 20 um deep (from the beam exiting surface to the aperture). Micrographs from the “funnel side” are shown in Figs. (B) (isometric view) and (C) (top view). The aperture is shown to be smooth, circular, and debris-free. The first two characteristics are most likely the benefits of the reflow process; the melted glass aided by local surface tension resulted in the desirable features. Debris-free aperture is due to series of short pulses and just sufficient energy for penetration. In contrast, a single long pulse would generate considerable thermal stress which would cause the brittle glass to crack around the aperture.

Simulation aids to illuminate the physics leading to hourglass formation. Figure above presents the simulated reflow process at different computational time steps. The simulation was commenced just after the laser penetrated through the glass. The gray portion indicated melted glass. As can be seen, the glass reflowed gradually inward and upward
towar laser entering surface to form the hourglass profile. t*=60 time step, the hourglass feature was clearly evident verifying that the simulation captures the dominant physics of th glass reflow process.

(a) Current response of the open resistance of a 1 um aperture was obtained by a 5 mV, 35 ms pulse. The calculated resistance was 1.1 MOhm. (b) PC-12 cells were dropped on the aperture and then gentle suction was applied. Within 1 min, 1.9 GOhm maximum seal resistance was achieved. Smooth, circular, debris-free, and freshly activated surface of the hourglass aperture enable good seal quality.

Illustration of two-stage laser pulses command to fabricate apertures with diameter of 1-10μm. 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 hourglass-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.

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.

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.

Measured ion-channel current via fabricated 1~3μm aperture as a planar patch-clamp chip. (A) Whole cell current recording of endogenous channels in HEK 293T cell without leak subtraction. Strong ion channel activation appeared at positive membrane potential. (B) The current-voltage relation obtained from the whole cell current traces.