On-Demand Droplet Spotter for Preparing Pico- to Femtoliter

We call our droplet spotter a pulsed-field droplet spotter. .... Side-view images of droplets were obtained using a video microscope consisted of an ...
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Anal. Chem. 2001, 73, 1896-1902

On-Demand Droplet Spotter for Preparing Pico- to Femtoliter Droplets on Surfaces Osamu Yogi,† Tomonori Kawakami,‡ Masayo Yamauchi,† Jing Yong Ye,† and Mitsuru Ishikawa*,†

Joint Research Center for Atom Technology (JRCAT), Angstrom Technology Partnership (ATP), 1-1-4 Higashi, Tsukuba, Ibaraki 305-0046, Japan, and Hamamatsu Photonics K. K. Central Research Laboratory, 5000 Hirakuchi, Hamakita, Shizuoka 434-0041, Japan

A droplet spotter for on-demand generation of pico- to femtoliter droplets was developed to meet the requirement for high-density spotting of chemicals on a surface. Our droplet spotter involves applying a ∼1000-V and ∼10ms pulse voltage to the tip of a capillary tube (o.d. ∼18 µm; i.d. ∼11 µm) supplied with water or a dye solution. The capability of the spotter was demonstrated by preparing a microarray of dye molecules. The microarray was prepared by spotting ∼30-fL droplets of a dye solution on a surface at the density of one spot per 20 × 20 µm2. Generation of droplets smaller than nanoliters1-10,12-15,17,18 is a fundamental technique in modern analytical chemistry. The * Corresponding author. E-mail: [email protected]. † Joint Research Center for Atom Technology. ‡ Hamamatsu Photonics. (1) Russo, R. E.; Withnell, R.; Hieftje, G. M. Appl. Spectrosc. 1981, 35, 531536. (2) Childers, A. G.; Hieftje, G. M. Appl. Spectrosc. 1986, 40, 688-691. (3) Hager, D. B.; Dovichi, N. J. Anal. Chem. 1994, 66, 1593-1594. (4) Hager, D. B.; Dovichi, N. J.; Klassen, J.; Kebarle, P. Anal. Chem. 1994, 66, 3944-3949. (5) French, J. B.; Etkin, B.; Jong, R. Anal. Chem. 1994, 66, 685-691. (6) Lin, H.-B.; Eversole, J. D.; Campillo, A. J. Rev. Sci. Instrum. 1990, 61, 10181023. (7) Whitten, W. B.; Ramsey, J. M.; Arnold, S.; Bronk, B. V. Anal. Chem. 1991, 63, 1027-1031. (8) Ng, K. C.; Whitten, W. B.; Arnold, S.; Ramsey, J. M. Anal. Chem. 1992, 64, 2914-2919. (9) Barnes, M. D.; Ng, K. C.; Whitten, W. B.; Ramsey, J. M. Anal. Chem. 1993, 65, 2360-2365. (10) Barnes, M. D.; Ng, K. C.; McNamara, K. P.; Kung, C.-Y.; Ramsey, J. M.; Hill, S. C. Cytometry 1999, 36, 169-175. (11) Two types of technique are used to generate droplets for ink jet printing. The first is a continuous generation of droplets (Watanabe, K.; Che, F.; Mutoh, M. Proc. ASME Fluids Eng. Div. ASME 1997, 244, 179-184). The second is on-demand generating droplets.16 In this context, we are involved in the first one. (12) Lemmo, A. V.; Fisher, J. T.; Geysen, H. M.; Rose, D. J. Anal. Chem. 1997, 69, 543-551. (13) Hosokawa, K.; Fujii, T.; Endo, I. Anal. Chem. 1999, 71, 4781-4785. (14) Graves, D. J.; Su, H.-J.; Mckenzie, S. E.; Surrey, S.; Fortina, P. Anal. Chem. 1998, 70, 5085-5092. (15) Onnerfjord, P.; Nilsson, J.; Wallman, L.; Laurell, T.; Marko-Varga, G. Anal. Chem. 1998, 70, 4755-4760. (16) Two types of technique are used for ink jet printing in on-demand generation of ink droplets. The first is bubble-jet technique and the second is piezoelectric technique of pulsing ink (Kretschmer, J. 1998 International Conference on Digital Printing Technologies, IS&Ts NIP 14). Generation of 4-pL droplets was reported in 1440-dpi ink jet printing using a piezoelectric technique (http://www.i-love-epson.co.jp/products/hikaku/color.htm). (17) Kebarle, P.; Ho, Y. In Electrospray Ionization Mass Spectrometry; Cole, R. C., Ed.; John Wiley & Sons: New York, 1997; pp 1-64.

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current methodology of generating droplets can be divided into three types based on the generation technique: vibrating orifice, extrusion, and electrospray. In the vibrating orifice type, the orifice of a capillary supplied with a liquid stream is vibrated, thus causing the stream to split into droplets. The performance of this technique is represented by one-by-one generation of droplets smaller than 50 pL, typically 1-20 pL, at a fast operation rate (10-100 kHz). The smallest volume of the droplets generated is basically limited by the flow rate of liquid and by the frequency of the vibrating orifice that determines the operation rate. The higher the frequency, the smaller the volume of droplets. This technique is widely used for preparing gas-phase ions in atomic absorption spectroscopy,1 in atomic emission spectroscopy,2and in mass spectrometry3-5 or for preparing uniform liquid droplets in spectroscopic and optical research,6 in single-molecule detection,7-10 and in the ink jet printing technology.11 However, this technique is inconvenient for regularly spotting droplets on a surface because its operation rate is too fast. The faster the operation rate, the harder regularly positioning droplets on a surface. In the extrusion type of droplet generation, liquid in a container is pushed, or extruded, from an orifice, thus generating droplets. The performance of this technique is represented by one-by-one and ondemand generation of droplets smaller than 1 µL, typically 30500 pL, at a moderately fast operation rate (lower than 500 Hz).12-15 The volume of the droplets generated is basically limited by the size of the orifice. This technique is widely known as the key technology of ink jet printing16 and has recently been applied to combinatorial chemistry for drag screening,12 micro total analysis system (µ-TAS),13 preparation of DNA chips,14 and sample preparation in matrix-assisted laser description/ionization timeof-flight (MALDI-TOF) mass spectrometry.15 In the electrospray type of droplet generation, a direct current (dc) voltage is applied to the tip of a capillary supplied with a liquid stream, thus causing the stream to split into droplets. The performance of this technique is represented by generation of a spray of fine (submicrometers in diameter) droplets. Thus, this technique is widely used for preparing gas-phase ions in ionization mass spectrometry17 and for laser plasma X-ray generation.18 In the extrusion type of droplet generation, the moderately fast operation rate is convenient for regularly spotting droplets on a surface when this technique is used as an on-demand system. (18) Mountford, L. C.; Smith, R. A.; Hutchinson, M. H. R. Rev. Sci. Instrum. 1998, 69, 3780-3788. 10.1021/ac0012039 CCC: $20.00

© 2001 American Chemical Society Published on Web 03/16/2001

Figure 1. Block diagram of the pulsed-field droplet spotter. Key components of the droplet spotter are magnified in the inset.

However, this technique is inadequate to help in the further development of combinatorial chemistry, µ-TAS, and DNA chip technologies because of the large droplet volume, 10-100-pL range.12-15 Indeed, droplets smaller than 10 pL generated by ondemand spotting will be required in the development of microchemical technologies. To meet such a requirement, we developed an on-demand droplet spotter for preparing pico- to femtoliter droplets on a surface. This droplet spotter will be useful not only for combinatorial chemistry, µ-TAS, and DNA chip applications requiring high-density arrays of chemicals but also for developing single-molecule chemical analysis. Our droplet spotter is similar to the electrospray technique in that a dc voltage is applied to the tip of a capillary supplied with liquid. However, our technique differs in that it is an on-demand system using pulsed voltage for spotting one by one extremely small droplets, down to femtoliters in volume, on a surface. We call our droplet spotter a pulsed-field droplet spotter. In this paper, we direct our attention to making droplets as small as possible and spotting them regularly on a surface, not spotting droplets as fast as possible. The construction of the pulsed-field droplet spotter is described in the Experimental Section. Dependence of the volume of droplets on pulse width and voltage is described in Results and Discussion to find the optimum pulsed voltage for making droplets as small as possible. Fabrication of 30-fL droplets of a rhodamine B (rhB) solution on a surface at intervals of 20 µm is described in Results and Discussion to demonstrate high-density spotting chemicals on a surface using the optimum pulsed voltage. To the best of our knowledge, a two-dimensional array of femtoliter droplets at intervals of 20 µm is the highest density of on-demand droplet spotting on a surface. EXPERIMENTAL SECTION Overview of Spotting Droplets on a Surface. Here we outline the operating principle of the pulsed-field droplet spotter to find what kinds of phenomena are involved in the droplet

spotter, such as deformation of a meniscus induced by a highvoltage pulse (∼1000 V and ∼10 ms), as described in Results and Discussion. Our method of spotting droplets consists of four steps. First, a pulse voltage is applied between the tip of a capillary supplied with a liquid sample and the substrate surface where droplets are to be prepared. Second, due to the applied voltage, the meniscus of the liquid deforms into a cone pointed at the surface, and then a jet of the liquid is ejected from the apex of this cone. The jet then strikes the surface, thus forming a droplet. Third, while the pulse voltage is off, the jet disappears and the cone returns to the initial meniscus of the solution. Fourth, the substrate is moved to the next position where another droplet is to be prepared. Overview of the Pulsed-Field Droplet Spotter. Figure 1 shows a block diagram of essential components of the pulsedfield droplet spotter: a glass capillary supplied with a liquid, mechanical stages for moving the capillary and a substrate, and a video microscope for side-view observation of droplet formation in real time. The stage for the capillary was used in the initial positioning of the capillary. A power supply for generating a highvoltage (∼1000 V) pulse is connected to the capillary, with a tungsten wire as the anode and the substrate holder as the cathode. This selection of the polarity is effective for avoiding the droplet formation being disturbed by corona discharge.19 Two computers were used in the pulsed-field droplet spotter: one for synchronizing the generation of a pulsed voltage (∼2 V) that drives the high-voltage power supply, for acquisition of video images and for operation of the mechanical stages; one for acquiring video images through the video microscope and for processing the acquired video images. The core section of the pulsed-field droplet spotter (Figure 2A) consists of a capillary, capillary holder, mechanical stages, concave mirror for backilluminating the tip of the capillary, and video microscope. The key components (Figure 2B) are the capillary and the capillary (19) Cloupeau, M.; Prunet-Foch, B. J. Aerosol Sci. 1994, 25, 1021-1036.

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Figure 2. (A) Overview of the components of the pulsed-field droplet spotter mounted on the vibration-isolation table. (B) Closeup view of the capillary and capillary holder, which are the key components of the droplet spotter. A coin is placed at the left-hand side of the substrate holder for awareness of the dimensions of the key components.

holder. In our experiments, this core section was set on a vibration-isolation table (VH3648, Newport, CA), and the entire spotter was housed in a class-100 clean-air booth (Air Tech, Japan, Tokyo, Japan), where the ambient temperature was 22 °C and the relative humidity was ∼35%. Detailed Description of the Pulsed-Field Droplet Spotter. Positioning a Capillary and Holding a Substrate. First, the video microscope was mounted on the vibration-isolation table using a laboratory-use jack. Then, the tip of the capillary was positioned at the focal point of the microscope using a manually driven X-Y stage. A substrate was then placed under the tip of the capillary using a motor-driven X-Y-Z stage. The substrate was set on a vacuum chuck as a substrate holder. The holder was mounted on the X-Y-Z stage through a 10-mm-thick insulating ceramic spacer. The vacuum chuck was also used as ground for the cathode. The substrates (20 × 20 mm2 in area and 0.5 mm thick) were p-type (111) silicon, which has low resistance (0.375-0.625 Ω/cm2).20 The silicon surface was coated with a ∼70-nm-thick poly(vinyl alcohol) (PVA) film to prevent fluorescence of dye molecules from being quenched by the silicon itself. The details 1898

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of fluorescence quenching by silicon were previously described.21 The distance between the capillary tip and a substrate surface (usually 20-30 µm) was easily evaluated as half of the distance between the video image of the capillary tip and its mirror image, as depicted in Figure 6A, without determining the borderline between the video image and its mirror image. Video Microscope for Observation of Droplet Formation. Sideview images of droplets were obtained using a video microscope consisted of an objective lens with a long-working distance, a microscope tube containing a fiber-optic wire, a digital CCD camera system including a camera controller, and a computer containing a video frame grabber composed of a video interface and frame memories. Light for illuminating the tip of the capillary was guided to the objective lens from a halogen lamp through a fiber-optic wire attached to the microscope tube. To facilitate the (20) The use of a conducting silicon surface is essential to the success in the pulsed-field droplet spotter. When using a slide glass as a substrate, we failed in generating droplets. However, the use of a thin insulating film on a silicon surface has no effect on the performance of the droplet spotter. (21) Ishikawa, M.; Yogi, O.; Ye, J. Y.; Yasuda, T.; Maruyama, Y. Anal. Chem. 1998, 70, 5198-5208.

visualization of the capillary tip by the video microscope, the light passing across the capillary tip was reflected off an aluminum concave mirror (50 mm in diameter; 60 mm of the focal length) mounted on an X-Y-Z-θ stage, to again illuminate the capillary tip. Top-view fluorescence images of microarrays of dye molecules (see Fabrication of Microarrays of Dye Molecules in Results and Discussion) were obtained using another video microcopy system containing a conventional upright microscope (figure not shown). Furthermore, to obtain insight into the mechanism of droplet generation, we observed the meniscus of water at the capillary tip and a PVA-coated silicon surface using the video microscope shown in Figure 2A. Capillary as an Electrode. A capillary was fabricated by thermally extending a glass tube (o.d. 1 mm × i.d. 0.6 mm) using a glass tube puller. The outer diameter of the capillary tip thus obtained was 5-40 µm, typically ∼17 µm, and the inner diameter was always ∼60% of the outer diameter . The capillary was then mounted on the capillary holder. This holder is originally an electrode holder for the patch-clamp technique, which is used for measuring cell membrane capacitance in vivo. This capillary holder looks like a three-way faucet (Figure 2B). One faucet was connected to the capillary, another to a tungsten wire (0.1 mm in diameter) as an anode, and the third to a microinjector, which is commonly used for cell biology research, for withdrawing a liquid sample from a vial through the capillary before starting droplet formation. The tungsten wire anode was immersed in a liquid sample (Milli-Q water or a rhB Milli-Q water solution) supplied with the capillary (see the inset of Figure 1) and was connected to the power supply through the second faucet of the capillary holder. The capillary holder was attached to the manually driven X-Y stage through an insulating ceramic spacer. The power supply has three terminals: one for input (input terminal), one for output use (output terminal), and one for monitoring the output voltage (monitor terminal). A voltage Vin from the first computer is supplied to the input terminal and is linearly amplified by a factor of 500, thus generating a high-voltage Vout from the output terminal. Because Vin is variable from 0 to 10 V, we can obtain a range for Vout of 0 to 5 kV. Also, the output current of this power supply is variable from 0 to 3 mA. In our experiments, we used the maximum rating of Imax ) 3 mA for every Vout. A positive pulse voltage Vout was supplied to the capillary electrode to generate a droplet of a liquid sample. To improve temporal response of Vout, a resistor was inserted between the capillary electrode and the substrate holder, as illustrated in the inset of Figure 1. The temporal width ∆t of Vin is variable from several microseconds to several seconds. The waveform of Vout was not directly observed through the output terminal; instead, the waveform of Vmon was observed through the monitor terminal using a 500-MHz digital oscilloscope (TDS 754C, Tektronix). Because the waveform of Vmon is equivalent to that of Vout except for the reduced voltage by a factor of 500, here we refer to Vmon as Vout after multiplying Vmon by a factor of 500. Synchronization of Generating a Droplet, Operating the Mechanical Stages, and Imaging the Droplet. Generation of Vin, operation of the mechanical stages, and acquisition of video images were synchronized with each other using analog, general-purpose interface bus (GP-IB), and transistor-transistor-logic (TTL) signals from three interface boards contained in the first computer;

Figure 3. Synchronization of generating a droplet and imaging the droplet. Five pulses were involved in the synchronization: Vprep, Vcam, Vexp, Vin, and Vout.

a multifunctional I/O board generates an analog signal of Vin, a GP-IB board generates GP-IB signals, and a counter-and-timer board generates a TTL signal. The video camera is triggered by a TTL signal Vcam. Both Vin and Vcam are variably triggered using a master TTL clock Vpre in the counter-and-timer board. Image acquisition, which is triggered using a TTL signal Vexp from the camera controller, is delayed 35 ( 1 ms after receiving Vcam. Note that this delay time accompanied by the large fluctuation ((1 ms) is not controllable and is intrinsic to the video camera. An exposure time for the image acquisition was determined by a temporal width ∆t of Vexp, which is variable from 100 µs to 60 s. We selected 500 µs as sufficient for visualizing the capillary tip using a halogen lamp. Figure 3 shows synchronization of Vpre, Vcam, Vexp, Vin, and Vout. Note that the start of timing was defined as the time at the beginning point of Vin. Generation of a Pulsed Voltage for Fabricating Droplets. From preliminary experiments (data not shown), we found that the voltage suitable for the pulsed-field droplet spotter was 1000 ( 200 V. To understand what waveforms of Vout we observe, the waveform of Vout on Vin using Vin ) 2.0 V and ∆t ) 7.5 ms was observed with and without a resistor inserted between the capillary electrode and the substrate holder (see the inset of Figure 1) and then the dependence of the waveform of Vout on Vin using Vin ) 2.0 V and ∆t ) 1.5, 4.5, 7.5, 10.5, and 13.5 ms was examined. For easy discrimination of the observed waveforms, the values of ∆t Analytical Chemistry, Vol. 73, No. 8, April 15, 2001

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Figure 5. Representative waveforms of Vout using (A) ∆t ) 1.5 ms and Vin ) 2.0 V, (B) ∆t ) 1.5 ms and Vin ) 2.95 V, (C) ∆t ) 4.5 ms and Vin ) 2.0 V, (D) ∆t ) 7.5 ms and Vin ) 2.0 V, (E) ∆t ) 10.5 ms and Vin ) 2.0 V, and (F) ∆t ) 13.5 ms and Vin ) 2.0 V. Note that temporal rise of all waveforms was delayed by 2 ms from the start of timing. The start of timing was set to the leading edge of Vin (see Figure 3). In (A), the peak voltage of Vout was reduced to 620 V. To make Vout 1000 V we set Vin ) 2.95 V, as shown in (B). The amplification factor 500 of the power supply was reduced in (A) and (B).

Figure 4. Waveform of (A) Vin ) 2.0 V and ∆t ) 7.5 ms, (B) Vout without a resistor when Vin ) 2.0 V and ∆t ) 7.5 ms, and (C) Vout with a 1-MΩ resistor when Vin ) 2.0 V and ∆t ) 7.5 ms. Note that the temporal rise of the waveform in (B) and in (C) started 2 ms after that in (A), although identification of the 2-ms delay is difficult from these figures. See Figure 5 for clear evidence of the 2-ms delay.

were selected. Moreover, the Vout was changed at intervals of 25 V between 800 and 1300 V, and ∆t was 13.5, 10.5, 7.5, and 1.5 ms to find optimum pulsed voltage for making droplets as small as possible. RESULTS AND DISCUSSION Characterization of Pulsed-Voltage Waveforms. Figure 4A shows a waveform of Vin ) 2.0 V when ∆t ) 7.5 ms, and Figure 4B shows a waveform of Vout when Vin ) 2.0 V and ∆t ) 7.5 ms without inserting a resistor between the capillary electrode and the substrate holder. The trailing edge of Vout continued more than 2 s. This waveform is too long for our purpose of generating droplets smaller than 10 pL. Figure 4C shows a waveform of Vout after inserting a 1-MΩ resistor. This waveform provides an improved temporal response of Vout while keeping the peak voltage of 1000 V, which was expected from Vin ) 2.0 V and the 500 gain of the power supply. When a resistor smaller than 1 MΩ was used, such as 300 and 200 kΩ, we obtained Vout ) 1000 V and a small (within 10%) reduction of the full width at half-maximum (fwhm) of Vout. However, when a 100-kΩ resistor was used, we obtained a reduced voltage of Vout ) 300 V from Vin ) 2.0 V. This reduction 1900 Analytical Chemistry, Vol. 73, No. 8, April 15, 2001

was due to the limited rating (Imax ) 3 mA) of current in the power supply. Thus, in our all experiments for generating droplets, we used a 1-MΩ resistor to minimize the current flowing through the resistor and to ensure that Vout is consistent with a temporal response sufficient for generating droplets of pico- to femtoliters. Figure 5 shows representative waveforms of Vout when Vin ) 2.0 V; ∆t ) 1.5, 4.5, 7.5, 10.5, and 13.5 ms. Note that the beginning point of Vout delayed by 2 ms. Our observations can be divided into two classes. In the first class, the observed waveforms of Vout were convolutions of each waveform of Vin with the intrinsic response waveform prescribed by the impedance of the 1-MΩ resistor, the capillary electrode, and the substrate plus the substrate holder. Every Vout decreased exponentially with a time constant τd ) 10 ms. The waveforms A and B in Figure 5 represent the intrinsic response waveform because the temporal width Vin ) 1.5 ms was shorter than τd ) 10 ms. Furthermore, the observed waveforms A and B of Vout were identical to each other even when Vin was changed while keeping ∆t constant (data not shown except for ∆t ) 1.5 ms). In the second class, the peak voltage Vout was always 1000 V, as expected from Vin ) 2.0 V and the 500 gain of the power supply if we used ∆t longer than 4.5 ms. However, the peak voltage Vout became smaller than 1000 V if we used ∆t shorter than 4.5 ms. The peak voltage was 620 V when ∆t ) 1.5 ms and Vin ) 2.0 V. This reduced voltage may be due to the disappearance of Vin before Vout reached 1000 V. We thus set Vin ) 2.95 V (∆t ) 1.5 ms) to keep the peak voltage at 1000 V. These two classes of observations provide two criteria for our selection of ∆t of Vin. First, ∆t shorter than 4.5 ms should be selected if a rapid temporal rise and decay of Vout are required. In fact, the effect of ∆t on Vout was negligible when ∆t ) 1.5 ms was used. Second, if ∆t is shorter than 4.5 ms, Vin should be increased to obtain an expected voltage of Vout. Visualization of Droplet Formation. Here, we generated a large (˜3 pL) water droplet to make visible a change in the meniscus of the capillary tip, especially forming a jet of water. Under the conditions for generating a droplet smaller than 1 pL, a jet of water was invisible. Before the Vout was applied (Figure 6A), the meniscus of the capillary tip was undisturbed. Five

Figure 7. (A) Dependence of the volume of droplets on the peak voltage of Vout for each ∆t: (0) 13.5 (9) 10.5, (O) 7.5, and (b) 1.5 ms. Each point represents the average of 50 droplets. Error bars were estimated from the standard deviation of the 50 observations. Specifications of the objective lens used were ×20, NA 0.42, and WD 20 mm. The outer diameter of the capillary tip was 18.0 µm. Distance between the tip and the surface was 25 µm. Video exposure time was 500 µs, and resolution of the video image was 0.485 × 0.485 µm2/pixel. (B) Representative histogram of the volume of the droplets generated at ∆t ) 7.5 ms and Vout ) 1075 V. Average volume was 2.62 pL with a standard deviation of 0.25 pL.

Figure 6. Time course of droplet generation on a surface: (A) before applying a pulse voltage, (B) after applying a pulse voltage of 5.0 ms, (C) 6.0 ms, (D) 8.5 ms, (E) 11.5 ms, and (F) 15.5 ms. Specifications of the objective lens used were ×20, NA 0.42, and WD 20 mm. The outer diameter of the capillary tip was 18.0 µm. Distance between the tip and the surface was 25 µm. Here, ∆t ) 7.5 ms and Vout ) 1075 V. Video exposure time was 500 µs, and resolution of the video image was 0.485 µm/pixel. (G) Schematic showing the volume of a droplet defined by rotating the arc of a circle around the z-axis. The volume Vd of the droplet in (F) was 2.8 ( 0.2 pL.

milliseconds after Vin was applied (Figure 6B),22 the meniscus was deformed into a cone pointed at the surface. During this 5 ms, Vout was in the leading edge (Figure 5). At 6.0 ms (Figure 6C), the applied electric force exceeded the surface tension of water, causing a jet of water to be ejected from the apex of the cone and to strike the PVA-coated silicon surface, thus creating a droplet. At 8.5 ms (Figure 6D), the supply of water through the jet continued, thereby increasing the volume of the droplet. At 11.5 ms (Figure 6E), because the surface tension of water exceeded the applied electric force, the supply of water stopped. In this time regime (11.5 ms), the pulse voltage decreased to the trailing part of Vout (Figure 5). Furthermore, although the jet disappeared at 11.5 ms, the meniscus and the droplet were still deformed due to a residual electric force. At 15.5 ms (Figure 6F), the cone returned (22) Note the point of the start of timing (Figures 3 and 5) and the large fluctuation (1 ms in the appearance of Vexp.

to the initial meniscus (Figure 6A). The droplet in Figure 6F shows no deformation due to an electric force because of the reduced Vout in this time regime. Formation of a cone and jet at the meniscus of the capillary tip is a widely known phenomenon in electrohydrodynamics. Cloupeau and Prunet-Fock described the formation of a cone and jet.19 Our observation of the cone and jet in Figure 6A-D is essentially identical to the observation by Cloupeau and PrunetFock. Volume of a Droplet as a Function of the Parameters of a Pulse Voltage. To determine the criteria needed to generate a droplet of pico- to femtoliters, we examined the dependence of the volume of a droplet on Vout and the temporal width ∆t of Vin. The droplet volume was evaluated using eq A1 in Supporting Information. Figure 7A shows the dependence of the droplet volume on Vout for each ∆t. Each data point was an average value of 50 droplets. Figure 7B is a representative histogram of the droplet volume for ∆t ) 7.5 ms and Vout ) 1075 V. The average volume was 2.62 pL, with a standard deviation of 0.25 pL. Our observations can be divided into two classes. In the first class, the droplet volume saturated with increasing Vout for each ∆t value. In the second class, as ∆t increases, the minimum Vout necessary to generate a droplet decreases. A tentative explanation of the first class of the observations is as follows. The electric force between the capillary tip and the substrate surface is saturated by the accumulation of a positive charge on the surface. This positive charge is concurrently transferred with the liquid from the capillary. The positive charge remains on the surface during the application of Vout, because the surface is covered with a polymer thin film as an insulator. Note Analytical Chemistry, Vol. 73, No. 8, April 15, 2001

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that the saturated volume of a droplet for each ∆t converged on the same value, ∼3.4 pL. From this convergence, we infer that the saturation occurs when a certain amount of positive charge Qsat concurrently transferred with the liquid is accumulated on the surface. This Qsat is determined by a capacitor composed of the capillary tip and the surface separated by an air gap of 20-30 µm. This Qsat can be supplied by several pairs of ∆t and Vout, e.g., ∆t ) 13.5 ms and Vout ) ∼920 V in Figure 7A. A tentative explanation of the second class of the observations is as follows. This inverse relationship between ∆t and Vout means that a certain physical quantity proportional to the product of ∆t and Vout plays an important role in the formation of a droplet. Considering the use of pulse voltage, such a physical quantity is the electric charge. Once a positive charge accumulated on the capillary tip exceeds a threshold value, generation of a droplet starts. The positive charge of a threshold value Qth can be supplied by several pairs of ∆t and Vout, such as ∆t ) 1.5 ms and Vout ) ∼1000 V in Figure 7A. Thus, ∆t increases and the minimum Vout needed to produce Qth decreases. From Figure 7A, we found the criteria for selecting the parameters of Vout to obtain a minimal droplet volume. First, the relatively slow increase in the fitting curve for the data points of ∆t ) 1.5 ms and Vout ) 975-1280 V is preferable to generating smaller droplets. Second, the use of ∆t ) 1.5 ms is also preferable for avoiding long exposure of a liquid sample to the electric current.25 In the following droplet spotting experiments, we therefore generated various droplet volumes by controlling Vout and mainly using ∆t ) 1.5 ms. Fabrication of Microarrays of Dye Molecules. To demonstrate the capability of the pulsed-field droplet spotter, we used the spotter to fabricate microarrays of fluorescent dye molecules on a PVA-coated surface. Such fabrication demonstrates possible applications of this spotter, such as preparation of DNA chips. Figure 8A shows a microarray of rhB molecules at intervals of 20 µm. Figure 8B shows a side-view image of a droplet of a rhB aqueous solution before evaporation of water. Using rf,i ) 3 µm and the fitted curve in Figure 10 in Supporting Information, the estimated droplet volume in Figure 8A was ∼30 fL. CONCLUSION We developed a droplet spotter to fabricate pico- to femtoliter water droplets and microarrays of dye molecules on a surface at a density of one fluorescent spot per 20 × 20 µm2. The key to the pulsed-field droplet spotter is the use of a ∼1000-V and ∼10-ms pulse voltage. The fundamental technique used in the pulsed-field droplet spotter is similar to that used in the electrospray technique in which high concentrations of fine droplets are generated. In contrast, our technique generates and fabricates droplets one by one on a surface. The core mechanism in the pulsed-field droplet spotter is the formation of a cone and jet of liquid at the meniscus. (23) We tested several fitting functions, including polynomials, and found that without deformation the side-view profile of a droplet on a surface is well approximated by an arc of a circle. (24) The resolution of the images in Figure 6 is 0.485 µm/pixel. Thus, errors in the measurements of H and R are δH ) δR ) 0.485 µm/pixel. Accordingly, errors in hd and rd are δhd ) δrd ) 0.243 µm/pixel. From eq 1 we obtain δVd ) π(hd2/2 + rd2/2) δh + πhdrd δr. From rd ) 12.4 µm and hd ) 9.7 µm, δVd/Vd ∼ 7% is evaluated. (25) When selecting ∆t ) 13.5 ms and using a rhB solution in water (2.2 × 10-6 M), we found fiberlike solid material deposited on the tip of a capillary after applying a pulse voltage.

1902 Analytical Chemistry, Vol. 73, No. 8, April 15, 2001

Figure 8. (A) Microarray of rhB molecules formed using droplets of a 2.2 × 10-6 M rhB water solution. Average droplet volume was 30 fL. Specifications of the objective lens used were ×100, NA 0.75, and WD 4.7 mm. Resolution of the video image was 0.216 × 0.216 µm2/pixel. (B) Side-view video image of a droplet of a rhB solution in water used for the microarray. Specifications of the objective lens used were ×20, NA 0.42, and WD 20 mm. The outer diameter of the tip of the capillary was 17.0 µm. Distance between the tip and the surface was 20 µm. Here, ∆t ) 1.5 ms and Vout ) 1001 V. Video exposure time was 500 µs, and resolution of the video image was 0.485 × 0.485 µm2/pixel.

This mechanism has already been described in previous work on electrospray.17-19 In our droplet spotter, a cone and jet appeared when an applied voltage was smaller than that required for electrospray. Indeed, if the applied voltage is high enough to generate multiple jets from the cone apex, then the multiple jets will break up into fine droplets in a high concentration.17 The throughput of the pulsed-field droplet spotter is basically limited by the decay time (∼10 ms) of an applied voltage Vout. The power supply we used in the current work has now been improved to obtain a quick temporal response of Vout, and thus, this limitation might be overcome. ACKNOWLEDGMENT This work was performed with a support from New Energy and Industrial Technology Development Organization at the Joint Research Center for Atom Technology. We thank Professor Akira Mizuno and Professor Shinji Katsura, Toyohashi University of Science and Technology, for their advice on fabricating the capillary electrode. SUPPORTING INFORMATION AVAILABLE The methods of evaluating the volume and the evaporation rate of a droplet; components used for assembling the droplet spotter. These materials are available free of charge via the Internet at http://pubs.acs.org.

Received for review October 11, 2000. Accepted January 31, 2001. AC0012039