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This technique is capable of patterning cells and microparticles regardless of shape, size, charge or polarity. Its power intensity, approximately 5 105 times lower than that of optical tweezers, compares favorably with those of other active patterning methods. Flow cytometry studies have revealed it to be non-invasive. The aforementioned advantages, along with this technique’s simple design and ability to be miniaturized, render the ‘‘acoustic tweezers’’ technique a promising tool for various applications in biology, chemistry, engineering, and materials science.Introduction The ability to arrange cells and microparticles into desired patterns is critical for numerous biological studies and applica- tions such as microarrays,1,2 tissue engineering,3,4 and regenera- tive medicine.5,6 Researchers have developed a variety of patterning techniques such as microcontact printing,7–9 optical tweezers,10,11 optoelectronic tweezers,12,13 magnetic tweezers,14–16 electro-/dielectro-phoresis,17–21 evanescent waves/plasmonics,22,23 hydrodynamic flows,24–32 and bulk acoustic wave-based acous- tophoresis.33–37 The invention of optical tweezers10,11 and developments in optofluidics38–41 have spurned a new platform for manipulating and patterning micro/nanoscale objects with unprecedented precision. Chiou et al. demonstrated optoelectronic tweezers that featured not only the high precision of optical tweezers, but also achieved high throughput and low power consumption, with a power intensity approximately 1 105 times less than that of optical tweezers.12 Most recently, Yang et al. demonstrated trapping and transporting dielectric nanoparticles as small as 75 nm using sub-wavelength slot waveguides.42 Despite the impressive performance of such optics-based patterning and manipulating techniques, they require bulky, complicated optical setups, which are difficult to miniaturize. Techniques based on magnetic/electrical fields (such as magnetic tweezers, electro- phoresis, and dielectrophoresis)14–21 could serve as alternative solutions. These techniques are amenable to device miniaturi- zation, but they have limited versatility. For example, magnetic tweezers require targets to be pre-labeled with magnetic mate- rials. The recently developed bulk acoustic wave (BAW)-basedaDepartment of Engineering Science and Mechanics, The Pennsylvania State University, University Park, PA, 16802, USA. E-mail: junhuang@ psu.edu; Fax: +1 814-865-9974; Tel: +81 14-863-4209 bDepartment of Bioengineering, The Pennsylvania State University, University Park, Pennsylvania, 16802, USA † Electronic supplementary information (ESI) available: Including device fabrication, simulation of standing surface acoustic waves, flow cytometry measurements, qualitative force analysis and ID and 2D patterning of fluorescent microparticles. See DOI: 10.1039/b910595f 2890 | Lab Chip, 2009, 9, 2890–2895acoustophoresis have shown promise in manipulating macro/ micro particles regardless of their optical or electrical proper- ties.33–37 However, it is challenging to implement these BAW- based techniques with the existing fast-prototyping methods, such as soft lithography, that are widely used in microfluidics. Due to the limitations of the existing techniques, researchers are still searching for cell-patterning techniques that simultaneously meet specifications for miniaturization, versatility, throughput, speed, and power consumption. A surface acoustic wave (SAW) is a sound wave that propa- gates along the surface of an elastic material.43 When SAW propagates, most of its energy is confined within one to two wavelengths normal to the surface of the substrate.43 This energy- confining characteristic makes SAW an energy-efficient tool for manipulating particles and biomaterials. Furthermore, SAW- based techniques are free of contamination, only introducing low-power mechanical vibrations to the suspension. Recently, researchers have demonstrated SAW-based mixing,44 pumping,45 concentration,46 and particle focusing.47 In this paper, we reveal an ‘‘acoustic tweezers’’ technique, in which microparticles and cells can be effectively patterned using standing surface acoustic waves (SSAW). This technique is versatile, non-invasive, and amenable to miniaturization, and its power consumption and speed compare favorably to those of existing active cell- patterning techniques. Working mechanism Fig. 1 illustrates the working principle of the ‘‘acoustic tweezers.’’ The device consists of a polydimethylsiloxane (PDMS) micro- fluidic channel and a pair of interdigital transducers (IDTs) deposited on a piezoelectric substrate in a parallel (Fig. 1a) or orthogonal (Fig. 1b) arrangement. A solution of microparticles or cells is infused into the microchannel by a pressure-driven flow. Once the distribution of particles or cells stabilizes in the channel, a radio frequency (RF) signal is applied to both IDTs to generate two series of identical SAWs propagating either in the opposite (Fig. 1a) or orthogonal (Fig. 1b) direction. The inter- ference of these two series of SAWs forms a SSAW, as well asThis journal is ª The Royal Society of Chemistry 2009 Fig. 3 SSAW-based cell patterning. (a) Patterning of bRBC. The wavelength of the applied SAW was 100 mm. (b) Patterning of E. coli cells pretreated with Dragon Green fluorescent dyes. The wavelength of the applied SAW was 300 mm (see Supplementary Video 2, ESI†). I IV shows the dynamic process of E. coli cells aggregate at a pressure node. (c–f), Flow cytometric histograms (DiBAC4(3) fluorescent light intensity (FI) vs. forward scattering (FS)) for different cell types. (c) E. coli cells cultured for 12 h (Positive Control 1); (d) E. coli cells that passed through a microchannel without applying SSAW (Positive Control 2); (e) E. coli cells that experienced the SSAW-based patterning process in a microchannel (SSAW Sample); and (f) E. coli cells heated at 70 C for 30 min (Negative Control).reached the pressure nodes, the acoustic pressures, and thus acoustic forces applied to the cells, were nearly zero; cells were steadily patterned in the ‘‘wells’’ defined by the pressure gradients around the pressure nodes. At the same time, the pressure oscillations inside the patterned cells (at pressure nodes) are also nearly zero, minimizing the heat generation. These characteris- tics suggest that the ‘‘acoustic tweezers’’ technique would be non- invasive to cells. In order to confirm the non-invasive nature of our technique, we studied the integrity of the cell membranes before and after the patterning process. We used a membrane-potential-sensitive dye, DiBAC4(3) (bis-(1,3-dibarbituric acid)-trimethine oxanol), as an indicator for cell viability. DiBAC4(3) is an anionic lipo- philic fluorescent dye which is found mainly in the outer cytoplasmic membranes of intact cells.47 It enters the cell body and accumulates in the cytoplasm when the cell membrane is damaged. Thus, the amount of dye accumulation, which is indicated by the average fluorescent intensity (FI), can be used to quantify the degree of membrane disruption caused by our technique. We performed flow cytometry experiments to quan- titatively analyze the viability of cells, in which FI and the forward scattering (FS) signals represented the cell viability and size distribution, respectively.53 As shown in Fig. 3c–e, the E. coli cells after the SSAW patterning process (average FI ¼ 0.536; Fig. 3e) exhibited distribution peaks almost identical to those of the cells prior to the pattering process (positive controls of average FI ¼ 0.530–0.536; Fig. 3c and 3d). On the other hand, the cells incubated at 70 C for 30 min (negative control; Fig. 3f)This journal is ª The Royal Society of Chemistry 2009exhibited a much higher FI (2.2), implying that the cell membranes were severely damaged. These results (as detailed in Supplementary Table 1, ESI†) confirm that our SSAW-based ‘‘acoustic tweezers’’ technique is non-invasive.Quantitative force analysis The behavior of cells or other objects in a SSAW field can be predicted via theoretical force analyses. The primary forces involved in the SSAW patterning process are as follows: (1) acoustic radiation forces;35,44–47,49 (2) viscous forces; (3) buoyant forces; and (4) gravity. Among these forces, buoyant forces are typically balanced by gravitational forces as they are of similar magnitudes and in opposite directions. Fig. 4a shows the dependence of these forces on particle size. It reveals that when the diameters of the particles are >1 mm, acoustic forces domi- nate under the applied power intensity. Fig. 4b shows that when the SAW wavelength is smaller than 100 mm, the acoustic forces acting upon the targets (polystyrene beads with diameter of 1.9 mm, bRBC, and E. coli cells) are significantly stronger than the viscous forces. In this study, all the particles were trapped at the pressure node, where the acoustic force was minimal and particles were immobilized due to the force gradient around the pressure node. Fig. 4c shows the acoustic force distribution within a half wavelength region centered at a pressure node. The results show that the acoustic forces change sinusoidally and point to the pressure node, forming a trapping well (inset of Fig. 4c). To release a particle from the trapping well, one mustLab Chip, 2009, 9, 2890–2895 | 2893 Fig. 4 Theoretical analyses. (a) Size dependence of acoustic force (AF), viscous force (VF), buoyant force, and gravitational force. (b), Dependence of AF and VF on the working wavelength for different objects. (c) The distribution of trapping force around a pressure node (PN). The forces on beads and E. coli are magnified 10 times for clear visualization. The inset indicates the force vectors and equal force contours around the PN. The SAW wavelength was 100 mm. (d) Calculated and experimental patterning time. The SAW intensity was 2000 W m2, and the particles speed was 1 mm s1 in VF calculation.overcome the maximum acoustic force in the field (25 pN for bRBC). By increasing the operation power or reducing the SAW working wavelength, one can further increase the stiffness of the trapping well built by the acoustic forces gradient around the pressure nodes or antinodes, making it possible to manipu- late and pattern nanoscale particles.35,42,44–47,49 In addition, our experimental and calculated results (Fig. 4d) show that the process for patterning polystyrene beads and bRBC takes less than 3 s, a speed faster than those of the previously reported studies.12,17,18,25 Detailed description of the theoretical model used in Fig. 4 can be found in the ESI.† In conclusion, we have demonstrated a SSAW-based ‘‘acoustic tweezers’’ technique that enables one to actively pattern cells and microparticles. This technique does not require pre-treatments on the substrates or cells/microparticles. It is applicable to virtually any type of cell/microparticle regardless of size, shape, or electrical/magnetic/optical properties. We verified this versa- tility by patterning polystyrene beads, bRBC, and E. coli cells. The required power intensity of acoustic tweezers (2000 W m2) is 5 105 times lower than that of optical tweezers (109 W m2) and compares favorably to those of other patterning methods.12,23 Such a low power intensity also contributes to the technique’s non-invasive nature, as confirmed by our cell viability studies. With its advantages in versatility, miniaturiza- tion, power consumption, speed, and technical simplicity, our ‘‘acoustic tweezers’’ technique is a powerful tool for applications2894 | Lab Chip, 2009, 9, 2890–2895such as tissue engineering, microarray, cell studies, and drug screening and discovery. Acknowledgements We thank Dr Bernhard R. Tittmann, Dr Kenji Uchino, Subash Jayaraman and Seyit Ural for help with equipments, and Yanjun Liu and Thomas R. 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