Self-Focused AlScN Film Ultrasound Transducer for Individual Cell

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Self-focused AlScN film ultrasound transducer for individual cell manipulation Benpeng Zhu, Chunlong Fei, Chen Wang, Yuhang Zhu, Xiaofei Yang, Hairong Zheng, Qifa Zhou, and K Kirk Shung ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.6b00713 • Publication Date (Web): 29 Dec 2016 Downloaded from http://pubs.acs.org on January 3, 2017

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Self-focused AlScN film ultrasound transducer for individual cell manipulation Benpeng Zhu1,2,*, Chunlong Fei3, Chen Wang4, Yuhang Zhu1, Xiaofei Yang 1, Hairong Zheng 4 Qifa Zhou2 , K. Kirk Shung2 1

School of Optical and Electronic Information, Huazhong University of Science and Technology, Wuhan 430074, China 2 Department of Biomedical Engineering and NIH Transducer Resource Center, University of Southern California, Los Angeles, California 90089-1111, USA 3 School of Microelectronics, Xidian University, Xi’an 710071, China 4 Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China

Abstract Precise cell positioning is indispensable in the fields of biophysics and cellular biology. Acoustic micro-beam produced by highly-focused ultrasound transducer has recently been investigated for a particle or cell manipulation. By virtue of relatively good piezoelectric property, Sc doped AlN film was introduced for highly-focused ultrasound transducer application. Using sputtering approach, a self-focused AlScN film based device has been designed, fabricated and characterized at a frequency ~230MHz. It had a narrow lateral beam width (~8.2µm). The AlScN ultrasound transducer was not only shown to be capable of remote controlling a single 10µm polystyrene microsphere in distilled water, but also demonstrated to possess the capability to contactless manipulate individual 10 µm epodermoid carcinoma cell in two-dimension within a range of hundreds of micrometers in phosphate buffered saline. Most importantly, the cell manipulation was realized in continuous mode and no switch-on and off operation was needed. These results suggest that self-focused AlScN film ultrasound transducer is a promising candidate for biomedical and molecular biology applications. Keywords：self-focused AlScN film; Ultrahigh frequency; ultrasound transducer; Cell manipulation；Acoustic radiation force Chunlong Fei is the co-first author of this paper *Corresponding author: Dr. Benpeng Zhu, [email protected]

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In the field of precision medicine, detailed cellular studies are indispensable. Commonly, cells are analyzed en masse, yielding results on the average signals from thousands of cells. Due to the high cellular heterogeneity, the accurate characterization of cellular behavior may only be achieved by studying individual cells respond, one by one. Therefore, research on precise cell positioning has become extremely crucial and attracted much attention lately. Dielectrophorestic 1, magnetic 2 and optical

3

forces are used traditionally to remotely move cells. Among them, the

former two have limited flexibility in controlling a single target 4. Meanwhile optical tweezers have been shown to be capable of contactless manipulating individual cells. Its principle is based on conservation of momentum to trap micro-particle with a tightly focused optical beam. However, the energy of a powerful focused laser may cause photodamage to the targeted biological sample 5. Additionally, the optical radiation force (at pico-newton level) is too small to manipulate larger cells. To address these issues, single-beam acoustic tweezers (abbreviated as SBAT) has been proposed as a possible substitute, the feasibility of which was theoretically and experimentally demonstrated several years ago

6-7

. Similar to optical tweezers,

SBAT takes advantage of acoustic radiation force to trap micro-particle with a steeply focused ultrasound beam. In order to realize individual cell mobilization in the Mie regime (cell diameter > wavelength), the ultrasound beamwidth is required to approach cell size. This will only be achieved at a frequency higher than 150MHz, if a cell with the size of 10µm needs to be manipulated. Nevertheless, it is still a challenge to fabricate such a SBAT which necessitates a high frequency ultrasound transducer with a small f-number (f#=focal length / aperture size). To push to such high frequencies, piezoelectric film technology is preferred8-9, because lapping down piezoelectric bulk material to the desired thickness is a tough and time-consuming work. Till now, only 10µm human leukemia cell has been reported to be successfully controlled by a 200MHz ZnO film transducer using a sapphire lens to focus acoustic beam10. Nevertheless, this manipulation couldn’t be realized in continuous mode, and switch-on and-off operation was needed. Given that ZnO film is reactive and unstable in moisture 11, the stability and reliability is potentially a major problem. Furthermore,


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the attenuation of the ultrasound beam in lens degrades the tweezers’ performance. Additionally, the sapphire costs too much and the lens fabrication process is complicated. Aluminium nitride (AlN), with the same wurtzite structure as ZnO, is another important non-ferroelectric piezoelectric material. It does possess better chemical and thermal stabilization than ZnO11. A further advantage of AlN is its much higher longitudinal wave velocity11, which benefits AlN for high frequency application. To date, while research on AlN film has been primarily carried out for surface acoustic wave (SAW), film bulk acoustic resonating (FBAR) and micromechanical devices12-14, it has gradually achieved recognition for high frequency ultrasound transducer fabrication15-16. A drawback of AlN film is the weak piezoelectric behavior, which might limit tweezers’ performance. This obstacle can be overcome by partially substituting Sc for Al, the possibility of which has been proved by recent researches17-18. To eliminate the influence of lens, self-focusing technology

19-20

may

be a good choice. In this regard, piezoelectric AlScN film is deposited on the pre-focused substrate and the desired ultrasound mirco beam can be formed. In this paper, the results in an effort to investigate whether the self-focused AlScN film transducer can produce enough force to trap and manipulate a single cell are reported.

Results and Discussion


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Figure 1 Self-focused AlScN film ultrasound transducer characterization : (A) Pre-focused Al substrate; (B) Film deposition technology; (C) AlScN /Al Spherical structure; (D) SEM cross-sectional morphology of AlScN /Al; (E) X-ray diffraction pattern and energy dispersive spectroscopy of AlScN film; (F) Photograph of self-focused AlScN film ultrasound transducer The characterization of self-focused AlScN film ultrasound transducer is illustrated in Figure 1. Aluminum (Al) was selected as the substrate. There are several reasons: it is relatively soft and easy to machine to form the desired focused structure; it is conductive and can be act as the bottom electrode; and its acoustic impedance is 19MRayls which is the lowest value among the metals considered for backing material

19

. A photograph of Al backing substrate is shown in Fig. 1(A). RF

magnetron sputtering (SPF-430H, ANELVA, Japan) was used to deposit the AlScN at 1µm/h with the following settings: N2+Ar (2:1) gas at 3mTorr, rf power 400W, distance between substrate and target 40mm, substrate unheated. Fig. 1(C) depicts the cross-sectional morphology of the AlScN/Al spherical structure. It can clearly be observed that the Arc angle is 60o, indicating that the f-number of the self-focused AlScN film based device is approximately 1. As presented in Fig. 1(D), the sputtered AlScN film was around 10 µm thick. Fig. 1(E) shows the X-ray diffraction (XRD)


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pattern this obtained film. It is obvious that this AlScN film has a typical wurtzite structure with (0002)-preferred orientation. According to the result of energy dispersive spectroscopy (EDS), as shown in the inset, the atom ratio of Al/Sc can be determined to be 0.82:0.18. Using piezoresponse force microscopy (PFM), the effective piezoelectric constant d33 of this AlScN film’s was measured to be 15 pm/V. It is worthy to be noticed that this value is even better than that of ZnO film (~12pC/N)11. The AlScN+Al rod was encapsulated in a brass case using an unload epoxy and a Sub-miniature Version A (SMA) conductor was attached to the Al rod with a small amount of silver epoxy. Then, a thin layer of Cr/Au (100/150nm) was sputtered over the exposed AlScN film and brass case to complete device interconnect and rf shielding. Finally, a matching layer of Parylene C (Specialty Coating Systems, Indianapolis, IN) was vapor deposited over the electroplated AlScN layer as well as the housing. The photograph of the self-focused AlScN film ultrasound transducer is described in Fig. 1(F).

Figure 2 (A) Time-domain pulse/echo response and frequency spectrum of AlScN ultrasound transducer; (B) Lateral beam profile of AlScN ultrasound transducer The performance of AlScN ultrasound transducer was evaluated in the deionized water. In the pulse echo test, an X-cut quartz plate was selected as the target and a pulser receiver (5900PR, Panametrics Inc., Waltham, MA) was used to activate the transducer and receive the echo, whose waveform was recorded using a 1-GHz oscilloscope (LC534, LeCroy Corp., Chestnut Ridge, NY). The measured pulse-echo waveform and frequency spectrum are shown in Fig. 2(A). It can be found that the central frequency of the transducer is 230 MHz and the bandwidth at -6 dB is


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measured to be 69.6%. Since neither calibrated hydrophones nor alternative standardized methods are available for measuring absolute pressure levels at the frequency greater than 60 MHz, the lateral beam profile, instead, was measured by scanning a tungsten wire target with the diameter of 6µm. Wire scanning was conducted using a three-axis positioning system (Inchworm, Burleigh Inc., Fishers, NY) with an in-house computer-controlled exposimetry system at a resolution of about 0.5 µm. The pulse intensity integral (PII) was calculated from the received echo signals reflected back from the wire which was located at the focus point of the ultrasound beam. PII is defined as the time integral of the intensity of a received echo taken over the time where the acoustic pressure is non-zero 21. As shown in Fig.2(B), the beam width is determined to equal to 8.2 µm by detecting its PII value which is reduced by 6 dB from the maximum. This measured result is close to the predicted width of 6.7 µm (f-number×wavelength) 22.

Figure 3 (A) The configuration used in the trapping experiments; (B) and (C) Simulated acoustic pressure field distributions at x–z plane at a frequency of 230MHz; (D) plot of transverse radiation force against the horizontal position of the center


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within a lateral distance (the red dashed line shows Fx = 0). The configuration used in the trapping experiments is described in Fig. 3(A). The highly-focused transducer was set in a chamber filled with distilled water or phosphate buffer saline (PBS); the chamber had an acoustically transparent mylar film at the bottom of the opening. The transducer was mounted on a motorized three-axis positioner (SGSP 26-50, Sigma KOKI Co., Japan), and was controlled using customized LabVIEW programs. A CMOS camera (ORCA-Flash 2.8, Hamamatsu, Japan), in combination with an inverted microscope (IX-71, Olympus, Japan), was placed under the chamber, to record the motion of the trapped sample. In order to achieve desirable peak-to-peak voltage amplitude, duty factor and pulse repetition frequency, the transducer was driven in a sinusoidal burst mode by a function generator (AFG3251, Tektronix, Anaheim, CA) and then amplified by a 50-dB power amplifier (525LA, ENI, Rochester, MN). In order to ensure that the targeted sample was located on the focal plane, a pulse-echo test was performed before the acoustic trapping experiment. Prior to singular cell manipulation, micro particle immobilization was conducted to check the AlScN ultrasound transducer’s capability. Polystyrene microspheres with diameter of 10 µm (Microbead NIST traceable particle size standard, Polyscience, Inc., Warrington, PA) were selected. The density, longitudinal wave velocity, and transverse wave velocity of these particles were 1050 kg/m3, 2340 m/s, and 1100 m/s, respectively. During the experiment, such microspheres were suspended above the mylar membrane in distilled water. Due to no experimental method available to evaluate the trapping force of transducers at frequencies higher than 200 MHz, the finite-element analysis and solver software package Comsol Multiphysics was used as an alternative. The simulations results of acoustic pressure field distributions at x–z plane are shown in Fig. 3 (B) and (C), in agreement with the experimental results. The acoustic radiation force in a single focused Gaussian beam can be evaluated by integrating the time-averaged radiation stress tensor ST the object 23:


over a surface enclosing

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F =

∫s

t ST

⋅ dA ,

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(1)

where the integral is carried over the whole surface of the particle and the differential area dA points to its outer normal. ST is the time-averaged Brillouin radiation stress tensor and its expression is given below:  ρ uu * p2 ST =  0 −  4 4 ρ 0 c02 

 ρ uu * , I − 0  2 

(2)

with ρ0 (1000 kg/m3) and c0 (1540 m/s) being the static mass density and sound velocity of the surrounding fluid, respectively, and the asterisk denoting the usual complex conjugation. Here, I is a unit tensor. p = −iωρ0ψ and u = −∇ψ are the first-order velocity and pressure fields, respectively, and they are related via the velocity potential ψ , with angular frequency ω. In this system, the resultant of force on polystyrene particle in z direction is always zero, so only transverse radiation force needs to be considered. Ideally, the device meets the requirement of acoustic impedance matching, so high frequency ultrasound attenuation along the propagation path is the main factor affecting acoustic pressure. According to Ref24, the ultrasound attenuation coefficient (α) is 2.2×10-4dB/MHz2/mm. Obviously, as for this transducer, the focus distance is approximately 0.7mm and the operational frequency is 230MHz. Therefore, the ultrasound attenuation can be calculated to be 8.1dB, suggesting that the acoustic pressure is just 15.5% of the theoretic value. With this in mind, the maximum value of Fx is estimated to be 10nN. A plot of the transverse radiation force (Fx) against the lateral distance away from focal beam center is shown in Fig. 3 (D). It is worthy to be noticed that Fx points to the focal beam center and the focal point (x=0) is a force equilibrium state for the polystyrene particle. This result indicates that if a small lateral distance is produced between the particle center and the focal beam center by the movement of the transducer, a transverse radiation force will appear and push the particle back to the focal beam center.


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Figure 4 Microsphere manipulation using 230MHz AlScN ultrasound transducer. The microsphere moved in different directions: (A)-(B) Down, (C)-(D) Right, (E)-(F) Up, (G)-(H) Left and (I) close to reference microsphere (in red circle). (Video S1) Figure 4 shows the realization of the contactless manipulation of single microsphere. The device was driven by sinusoidal burst with the frequency of 230MHz, a driving voltage of 20Vpp, a duty cycle of 0.2% and a pulse repetition frequency of 1kHz. A series of images were captured at a frame rate of 10frames/s. As seen in the images, the bright circular structure is the projection of the focused AlScN/Al rod and the targeted microsphere is in its center. Another 10µm microsphere labeled in red circle is chosen as a reference point to show the relative location change. It can be observed that a single 10µm microsphere is manipulated with the movement of the AlScN-based device. No matter how close between the two microsphere is, even if they touch each other (shown in Fig.4(I)), no effects has been found acting on the reference microsphere. This result indicates that self-focused AlScN film ultrasound transducer does possess that capability of singular micro particle manipulation in Mie regime.


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Figure 5 Singular epidermold carcinoma cell motion with acoustic micro beam. The cell was trapped (A)-(B) and moved in different directions: (C) Left, (D) Down, (E)-(G) Right and (H) Up (Video S2) A431 cells are a model human cell line (epidermoid carcinoma) used in biomedical research. In our work, such cells (A-431 cell line, ATCC CCL-243) were cultured in Dulbecco’s modified eagle medium (DMEM, GIBCO, Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin – Streptomycin–Neomycin (PSN, GIBCO, Invitrogen) in an incubator supplied with 5% CO2 and set at 37oC and replenished with fresh media every 2-3days. Then, the cells were harvested using TrypLE (Thermofisher, ) and centrifuged and resuspended in DPBS (with Ca2+ and Mg2+). During the experiment, the cultured cells were suspended above the mylar membrane in Phosphate Buffered Saline (PBS). As shown in Fig. 5(A), the epodermoid carcinoma cell has a diameter around 10µm, but it is in an irregular spherical shape. One cell in the center of the bright circular projection is being trapped by the AlScN ultrasound transducer and the other cell labeled by a red triangle is the reference point. The distance between these two cells is less than 50 µm. When the targeted cell is immobilized with the acoustic micro beam, the reference cell is unaffected and remains stationary. Form Fig. 5(B) to Fig. 5(H), it is easily seen that the a single cell can be successively manipulated in the direction of left, down, right and up around reference point. It is worthy to note that previously

10

to move a

leukemia cell, the tweezer needed to be operated in three steps: switched off, moved and switched on. In our work, single cell manipulation is achieved with the


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continuous motion of AlScN ultrasound transducer without the need of switch-on and off. This result is significant for arranging the cells in different patterns and studying cell signaling pathways.

Conclusion Based on sputtering technology, 10µm Al0.82Sc0.18N film with (0002)-preferred orientation was sputtered on the pre-focused Al substrate and the single beam acoustic tweezers have been fabricated. The self-focused AlScN film ultrasound transducer built had a higher operational frequency (~230MHz) and narrower acoustic micro beam (~8.2µm), in comparison to other acoustic micro-beams reported in the literature. In the acoustic trapping experiment, both 10µm polystyrene microsphere and 10 µm epodermoid carcinoma cell were individually contactlessly manipulated. Most importantly, cell manipulation in continuous trapping mode has been realized two-dimensionally within a range of hundreds of micrometers in phosphate buffered saline. These promising results demonstrate that self-focused AlScN film ultrasound transducer is a good candidate for biophysical and molecular biology applications.

Supporting Information Supporting Information Available: The following files are available free of charge. Video S1 showing singular particle contactless manipulation by self-focused AlScN film ultrasound transducer; Video S2 reporting the manipulation of single epodermoid carcinoma cell using self-focused AlScN film ultrasound transducer.

Acknowledgments This work was supported by the Natural Science Foundation of China (Grant no. 61371016, 11574096,11604251), National Key Scientific Instrument and Equipment Development Projects of China under contract 2013YQ160551, The Science and Technology Support Project of Hubei Province (2015BHE012), the Fundamental Research Funds for the Central Universities(2016YXZD038),and it also partially supported from NIH Grant # R01-EB12058 and P41-EB002182. We thank Mr.


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Xiangli Liu and Mr. Baiqian Yan for their assistance in discussion and we also thank the Analytical and Testing Center of Huazhong University of Science & Technology.


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Self-Focused AlScN Film Ultrasound Transducer for Individual Cell

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