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Drop-on-Demand Patterning of Bacterial Cells Using Pulsed Jet Electrospraying Kyoungtae Kim,†,‡,§ Byung Uk Lee,*,‡ Gi Byung Hwang,‡ Jun Hyun Lee,‡ and Sangsoo Kim† Aerosol and Particle Technology Laboratory, Department of Mechanical Engineering, Korea Advanced Institute of Science and Technology, Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea, Aerosol and Bioengineering Laboratory, Department of Mechanical Engineering, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul, 143-701, Republic of Korea, and Department of Mechanical Engineering, University of Minnesota, 111 Church Street, S.E., Minneapolis, Minnesota 55455 In this study, the drop-on-demand patterning of bacterial cells on a raw silicon wafer was newly conducted with an electrospray pulsed jet. We produced various sized line patterns and spot patterns of bacterial cells on the silicon wafer under varying experimental conditions of frequency, flow rate, and translational speed of the electrospray system in one and two-dimensional ways. Especially, the electrospray’s pulsed jet of bacterial solution produced regular patterns of around 10 µm diameter spots, each of which contained a single bacterial cell. By enabling drop-on-demand patterning and manipulation of single bacterial cells on a raw wafer in the gas phase, this new technique broadens the scope of biological experiments beyond liquid phase treatments to solid and gas-phase treatments. Immobilization of biological materials including microorganisms onto solid surfaces has become important for the development of biosensors, drug discovery, and diagnostics.1-3 Microcontact printing, photolithography, and dip-pen nanolithography are considered good methods for patterning biomaterials on substrate.1,4,5 These methods generally require complicated surface modification for binding of specific materials on the surface;1 and when the pattern must be varied, these methods require completely new modified substrates. To our knowledge, a microcontact printing method approached patterning of bacteria with cellular resolution;4 however, none of current methods have produced patterns of single bacteria cells with varying sizes. The optimal method of patterning involves picking up target biomaterials from a liquid suspension and depositing them at * To whom correspondence should be addressed. E-mail: leebu@ konkuk.ac.kr. Tel: 82-2-450-4091. Fax: 82-2-447-5886. † Korea Advanced Institute of Science and Technology. ‡ Konkuk University. § University of Minnesota. (1) Park, T. J.; Lee, S. Y.; Lee, S. J.; Park, J. P.; Yang, K. S.; Lee, K.; Ko, S.; Park, J. B.; Kim, T.; Kim, S. K.; Shin, Y. B.; Chung, B. H.; Ku, S.; Kim, D. H.; Choi, I. S. Anal. Chem. 2006, 78, 7197–7205. (2) Park, J. P.; Lee, S. J.; Park, T. J.; Lee, K.; Choi, I. S.; Lee, S. Y.; Kim, M.; Chung, B. H. Biotecnol. Bioprocess Eng. 2004, 9, 137–142. (3) Liu, V. A.; Jastromb, W. E.; Bhatia, S. N. J. Biomed. Mater. Res., Part A 2002, 60, 126–134. (4) Xu, L.; Robert, L.; Quyang, Q.; Taddei, F.; Chen, Y.; Lindner, A. B.; Baigl, D. Nano Letter. 2007, 7, 2068–2072. (5) Mrksich, M.; Dike, L. E.; Tien, J.; Ingber, D. E.; Whitesides, G. M. Exp. Cell. Res. 1997, 235, 305–313. 10.1021/ac9027966 2010 American Chemical Society Published on Web 02/09/2010
specific sites on a surface without any chemical treatments. In this study, we conducted the optimal patterning of bacterial cells by using an electrospray pulsed jet. An electrospray can generate picoliter and femtoliter droplets of a liquid substance in the gas phase by using an intense electric field between the nozzle and the deposition plate.6 The droplets have a monodisperse size distribution and a high electric charge; furthermore, the charged droplets can be controlled easily by adjustment of the electric field. The droplets of spray or residue that remain after the evaporation of the liquid have been used in various fields, particularly in catalytic combustion,7 the generation of medical powder,8 the generation of metal oxide particles,9 and film coating processes.10 Recently, an electrospray jet was found to be able to produce patterns of ink droplets with a resolution of a few micrometers.11,12 The application of electrospray jets has been broadening with the use of various sprayed substances, especially the biological materials used in chemical analysis. The electrospray has been widely used in electrospray ionization as part of the mass spectrometry of biological materials.13 Electrospray jet systems can produce protein nanoparticles,14 biologically active DNA, and biologically active protein substances.15-17 Our research team recently discovered that the electrospray system could produce not only active biological materials but also viable bacteria cells.18 (6) Hayati, I.; Bailey, A. I.; Tadros, TH. F. Nature 1986, 319, 41–43. (7) Kyritsis, D.; Roychoudhury, S.; McEnally, C.; Pfefferle, L.; Gomez, A. Exp. Therm. Fluid Sci. 2004, 28, 763–770. (8) Xie, J.; Lim, L. K.; Phua, Y.; Hua, J.; Wang, C. J. Colloid Interface Sci. 2006, 302, 103–112. (9) Nakaso, K.; Han, B.; Ahn, K. H.; Choi, M.; Okuyama, K. J. Aerosol Sci. 2003, 34, 869–881. (10) Jaworek, A. J. Mater. Sci. 2007, 42, 266–297. (11) Paine, M. D.; Alexander, M. S.; Smith, K. L.; Wang, M.; Stark, J. P. W. J. Aerosol Sci. 2007, 38, 315–324. (12) Park, J.; Hardy, M.; Kang, S. J.; Barton, K.; Adair, K.; Mukhopadhyay, D. K.; Lee, C. Y.; Strano, M. S.; Alleyne, A. G.; Georgiadis, J. G.; Ferreira, P. M.; Rogers, J. A. Nat. Mater. 2007, 6, 782–789. (13) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64–71. (14) Gomez, A.; Bingham, D.; Juan, L. d.; Tang, K. J. Aerosol Sci. 1998, 29, 561–574. (15) Morozov, V. N.; Morozova, T. Y. Anal. Chem. 1999, 71, 3110–3117. (16) Moerman, R.; Frank, J.; Marijnissen, J. C. M.; Schalkhammer, T. G. M.; Dedem, G. W. K. V. Anal. Chem. 2001, 73, 2183–2189. (17) Bhatnagar, P. Appl. Phys. Lett. 2007, 91, 014102. (18) Kim, K.; Kim, W.; Yun, S. H.; Lee, J. H.; Kim, S. S.; Lee, B. U. J. Aerosol Sci. 2008, 39, 365–372.
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Figure 1. Schematic diagram of electrospray patterning system for bacterial particles.
Coincidently, a recent study focused on the patterning of bacteria via electrohydrodynamic forces.19 However, because the line patterns of the study have a width of 160 µm to several millimeters and the dot patterns have irregular sizes, that study is significantly different from our cellular resolution scale experiments. Our approach is novel. We apply the electrospray method of generating highly charged viable bacterial cells to the production of various sized one-dimensional and two-dimensional patterns of bacterial cells on a raw (untreated) silicon wafer with a cellular resolution; the patterns include a specific pattern in which each spot contains one bacterium. MATERIAL AND METHODS Figure 1 shows a schematic diagram of the experimental setup. The experimental system consists of a capillary, a ground plate, a translation system of the ground plate, a liquid feeding part, a high-voltage power supply system (HV), and a visualization system. The silica capillary (PicoTip emitter, New Objective, USA) has a diameter of 30 µm with a sharp tip. The ground plate is a stainless steel plate with a diameter of 12 cm, to which a silicon wafer is attached. The wafer on the ground plate was installed 100 µm below the capillary tip. The motorized stage translation apparatus translates the ground plate in two-dimensional ways at an adjustable constant speed. For the liquid feeding part, we use a 25 µL syringe (1702TLL, Hamilton, USA) and a syringe pump (Model 220, KD Scientific, USA). The syringe and the capillary are connected through a microflow fitting set (P-662, 1572, F-242, U-322, Upchurch, USA). We applied sinusoidal high-voltage electricity of several kilovolts to the capillary by means of an AC high-voltage power supply (AC +15 kV, Korea Switching, Korea) and a function generator (FG-7002C, EZ Digital, Korea). We applied high-voltage electricity to the spraying liquid by means of a stainless steel ZDV union (U-322, Upchurch, USA) in which the passing spraying liquid was exposed to the surface of the charged union. To observe the characteristics of the applied electric voltage, we used a 1/1000 AC high-voltage reduction probe (P6015A, Tektronix, USA) and an oscilloscope (TDS2014, Tektronix, USA). We visualized the capillary tip and the pulsed jet by (19) Kim, J. H.; Lee, D. Y.; Hwang, J.; Jung, H. I. Microfluid. Nanofluid. 2009, 7, 829–839.
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means of a CCD camera (Marlin F-145C2, Allied Vision Tech., Germany), a zoom lens (70XL, OPTEM, Korea), and a light source (LS-100W, Light Solution, Korea). We used an optical microscope (ECULIPSE ME600, Nikon, Japan) connected to a CCD camera (INFINITY1, Lumenera Co., Canada) to observe the produced pattern. We used bacteria suspensions (a mixture of 8 g of a nutrient broth media (beef extract 0.3%, peptone 0.5%, Difco) with 1 L of deionized filtered water; electrical conductivity values of 1.39 × 10 -3 S/cm) with a range of 3.0-9.0 × 107 CFU/mL concentration of the test microorganism Staphylococcus epidermidis (KCTC 1917). Figures S1 and S2 (Supporting Information) also illustrate one example of the experimental setup. The experimental procedure is as follows. A syringe pump supplies a liquid bacterial suspension in a syringe to the capillary of the electrospray at flow rates ranging from 0.1 to 20 µL/h (where 1 µL ) 10-9 m3). A high-voltage power supply system (HV) with a function generator supplies electricity to the electrospray system. The supplied electricity, which has a maximum voltage range of 0.7-1.2 kV and a central point voltage range of 0.35-0.6 kV, has a sinusoidal waveform with a frequency ranging from 4 to 20 Hz. The supplied liquid flow and the sinusoidal high electric field form the pulsed jet of the electrospray. When the jet is formed, the ground plate of the electrospray is translated horizontally at a fixed speed ranging from 0.4 to 2 mm/s; a pulsed jet from the tip of the capillary then makes contact with the clean silicon wafer on the ground plate regularly. We observed the capillary tip and the pulsed jet by means of the visualization system. In addition, we monitored the waveform, peak voltage, and frequency of the applied electricity by means of a 1/1000 AC high voltage reduction probe and an oscilloscope. One electrospray jet is formed for each pulse of the supplied electric voltage, and this jet forms regular patterns of spots of bacterial suspension when it touches the silicon wafer. RESULTS AND DISCUSSION First, we generated line patterns of electrosprayed bacterial cells. The line patterns can be formed by the pulsed jet electrospraying under conditions of relatively high flow rates, high frequency, and low translation speed in comparison with a dot patterning. Figure 2a shows one of the line patterns, which has a width of 190 µm of pile-up dots. The applied electricity was a 1.2 kV sinusoidal waveform with a frequency of 10 Hz. The flow rate of the supplied suspension was 20 µL/h. The translational speed of the silicon wafer was 0.8 mm/s. As a previous study reported the use of an electrospray to generate viable bacterial cells,18 we checked the viability of patterned cells by incubating a wafer that contained patterns at 37 °C for 14 h and then observing the line patterns. Figure 2b shows one of the incubated line patterns. The number of bacterial cells in the pattern was greatly increased by incubation; therefore, we can confirm that viable bacteria cells were patterned on this untreated silicon wafer. Figure 3 shows a two-dimensional V shape pattern of spots. The applied sinusoidal wave electricity had a maximum voltage range of 0.8-1.2 kV with a frequency of 10 Hz. The flow rate of the supplied suspension was 1.0 µL/h. The translational speeds of the silicon wafer ranged from 0.4-0.6 mm/s. We artificially changed the sizes of the spots, which range from 8 to 20 µm, to show the capability of generating various sized spots in one pattern
Figure 2. Line patterns of electrosprayed bacterial cells. (a) Sprayed pattern containing S. epidermidis just after the deposition process. (b) Pattern that was incubated at 37 °C for 14 h after deposition.
Figure 3. Two-dimensional V shape pattern of spots which contain bacterial particles.
by means of controlling a voltage. The size of the spot is determined by several variables such as a suspension flow rate, a frequency, and a voltage. A strong electric field tends to pull a liquid suspension out of a capillary and the tendency temporarily increases the flow rate and the size of the spot. However, this
Figure 4. (a) One-dimensional pattern of electrosprayed spots containing bacteria with an average diameter of 12 µm. (b) Onedimensional patterns of electrosprayed spots, which contain single bacteria per pattern. The microscopic picture was taken after drying liquid solutions of droplets.
variation of the size of spots due to the control of voltage is restricted by the overall suspension flow rate. In this patterning process, we suddenly increased the voltage from 0.8 to 1.2 kV and the spot size was temporarily increased up to 20 µm then slowly decreased. As shown in Figure 3, it can be observed that small spots contain a few bacterial cells and large spots contain dozens of bacterial cells. Figure 4a shows a one-dimensional pattern of spots with a regular diameter of 12 µm. The average interval between spots is 76 µm. The applied electricity was a 0.8 kV sinusoidal waveform with a frequency of 10 Hz. The flow rate of the supplied suspension was 0.1 µL/h. The translational speed of the silicon wafer was 0.8 mm/s. We also took a micrograph of the patterned spots after they had been dried for 12 h to evaporate the droplets of liquid; the drying enabled us to investigate the bacteria in the spots more clearly. Figure 4b shows that the surrounding liquid in the spots was evaporated and that only a single bacterial cell remained in each spot. Staphylococcus epidermidis has a diameter of 1-2 µm, and it usually appears as a spherical coccus under a microscope. We are therefore sure that the small dots in the spots are Staphylococcus epidermidis. Also, this can be observed in a twoAnalytical Chemistry, Vol. 82, No. 5, March 1, 2010
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Figure 5. (a) Rectangular two-dimensional pattern of bacterial suspension spots. The average diameter of the one spot is 7 µm. (b) Magnified picture of a two-dimensional pattern with a scale bar of 10 µm.
dimensional pattern, as shown in Figure 5b. The dark marks surrounding the bacteria show the original size of the patterned dots. In other patterns of this operating condition, we observed that most spots contain a single bacterial cell and that some spots contain no bacterial cell or only a few bacterial cells (Figures S13-S16 of the Supporting Information). Figure 5a shows a two-dimensional pattern of small spots. We translated the ground plate in a rectangular form, the pulsed electrospray jet produced rectangular patterns of small spots. The average diameter of dots is around 7 µm, and the average interval between spots is 20 µm. To produce the pattern of Figure 5a, we adjusted the frequency and voltage of the applied electricity at 20
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Hz and 0.7 kV, respectively. The translational speed of the wafer below the tip was 0.4 mm/s, and the flow rate of a liquid bacterial suspension was 1.0 µL/h. Figure 5b shows that a single bacterial cell was patterned in one spot of two-dimensional patterns. Other two-dimensional patterns are shown in Figures S17-S20 (Supporting Information). Operating conditions of a low flow rate and high frequency produced smaller patterns, and the translational speed of the ground surface and frequency determined the intervals between the patterned spots. The concentration of the bacteria in suspension and the size of the patterns determine the number of bacteria in each patterned spot. By adjusting these operating conditions, we could produce various sized patterns, many of which are provided in the Supporting Information (Figures S3-S20). We used Staphylococcus epidermidis as the test bacterium. If other microorganisms are used, the viability of the patterned bacteria can vary for each species. The effects of different experimental conditions on the detailed ratio of the viability of patterned bacteria should be the subject of future electrospray patterning research. Figures 2-5 and Supporting Information confirm that we can generate bacterial patterns on a clean wafer by using the electrospray system for several seconds without any chemical treatment, any pressure exposure, and any heat treatment and that we can control the size and shape of the patterns by adjusting the experimental conditions. In this experiment, we succeeded in producing specific patterns of spots; each spot had a diameter of around 10 µm and contained a single bacterial cell. Our technique enables a single bacterial cell to be manipulated in the solid and gas phase; an example of such manipulation is a biochemical test on individual cells as a single-cell bioreactor in a gas environment. The high electric charge of deposited cells has the potential to enable efficient treatment of cells in the gas phase: for example, the blowing of some airborne charged compounds over electrosprayed charged cells on a wafer can induce the efficient attachment of the compounds to the cells. This capability can lead to new methods of cell treatment in the solid and gas phase. ACKNOWLEDGMENT This work was supported by Konkuk University. SUPPORTING INFORMATION AVAILABLE Figures S1-S20 and Table S1. This material is available free of charge via the Internet at http://pubs.acs.org.
Received for review July 21, 2009. Accepted January 25, 2010. AC9027966
