IROS2019国际学术会议论文集 0313

1 Abstract The capability to precisely rotate the cells and other microscale objects is invaluable in biomedicine bioengineering and biophysics We propose a novel on chip three dimension 3D cell rotation method using whirling flows generated by oscillating asymmetrical microstructures In an acoustic field excited by the vibration of a piezoelectric transducer two different modes of microvortices are generated around our custom designed microstructures that are utilized to precisely achieve in plane and out of plane rotational manipulation of microparticles and cells The rotation mechanism is studied and verified using numerical simulations We also investigate the effect of various parameters on the acoustically induced flows such as the frequency the driving voltage and the distance from the microstructure tip to the oocyte center thus indicating the rotational speed can be effectively tuned on demand for single cell studies Finally by observing the maturation stages of M2 after excluding the first polar body of operated oocytes the proposed method is proved noninvasive Comparing with the conventional works our acoustofluidic cell rotation approach is simple to fabricate and easy to operate thereby allowing rotations irrespective of the physical properties of the specimen under investigation I INTRODUCTION With the development of MEMS technology 1 2 Cellular studies have attracted great attention in the field of modern bioscience 3 because the cell is the basic structural functional and biological unit of all known living organisms Precise rotational manipulation of microbeads and cells is an essential capacity in biotechnology that impacts various disciplines including cell observation 4 5 cell analysis 6 and cell microsurgery 7 For example since the characteristics of zona pellucida ZP and spindle of embryo cell such as the retardance measured by polarization microscope shows great influences on the genetic studies it is necessary to properly orient the target cell to a specific angle via 3D cell rotation for observation Furthermore controllable 3D cell rotation is also required for the enucleation and microinjection in the cloning process and transgenic animal research 8 For instance after the removal of nuclei using Reseach supported by the Natural Science Foundation of Beijing Grant No 17L20128 and Beihang University Zhuobai project ZG216S1751 and ZG226S188D Bin Song is with Beihang University School of Mechanical Engineering School of Mechanical Engineering School of Mechanical Engineering School of Mechanical Engineering each microstructure is designed to be of a constant height of 80 m and a tip angle of 20 which is utilized to achieve OPR Fig 1d Fig 1e illustrates that when an acoustic wave excited by a piezoelectric transducer is propagated into the microchannel different types of local steady microvortices are induced in the vicinity of corresponding microstructures because of viscous dissipation inside the microchannel Furthermore the response to time harmonic forcing is generally not harmonic due to the dissipative property of the fluid The fluid s response to harmonic forcing can be viewed as a combination of a time harmonic response generally referred to as acoustic response and the remainder referred to as acoustic streaming 18 19 Hence the latter provides a unique method of utilizing the dominant viscous nature of microfluidic flows Overall as the demand of cell on chip tools for manipulating microparticles and cells increases our method will become well suited for biological research II METHODS AND MATERIALS A Fabrication of the microfluidic chip Fig 2a shows the detailed fabrication process of the microfluidic chip At first the chip mold Fig 2b was fabricated via a high quality 3D printing machine using a photosensitive resin NanoArch P140 BMFPrecision Shenzhen China The fabrication of the microstructures was implemented with a two step successive PDMS molding technology A Sylgard 184 silicone elastomer base was mixed with a Sylgard 184 silicone elastomer curing agent at a ratio of 10 1 weight weight and was used for mold casting In the first curing process the temperature was require at 60 C for 3 h to prevent the deformation of the 3D printing mold After the first molding Fig 2c the surface of negative mold structures was treated with C 4 to allow the execution of the second molding in an easier manner followed by casting Subsequently the final molding microstructure and a 40 mm 50 mm 0 15 mm width length thickness glass slide were treated with oxygen plasma YZD08 2C SAOT Beijing China for 20 s and bonded at 90 C for 10 min Finally a piezoelectric transducer 81 7BB 27 4L0 Murata Electronics Kyoto Japan was attached to the glass slide next to the PDMS channel by using epoxy 84101 Permatex Hartford CT USA 679 Fig 2 Microfluidic chip fabrication a fabrication process of the microfluidic chip b SEM image of the 3D printing microstructure mold and c the negative PDMS microstructure mold B System setup Fig 3 shows a concept design of the manipulation system platform including the observation and driving systems A differential interference contrast DIC microscope CX41 Olympus Co Ltd Tokyo Japan equipped with a charge coupled device CCD camera GS3 U3 23S6C C Point Grey Co Ltd Richmond BC Canada was attached to a control computer to acquire real time and stereoscopic images A microfluidic chip was fixed on a stage The other system consists of a function generator Wave Station 2012 LECROY New York NY USA a high voltage amplifier ATA 2042 Agitek Xi an China and a high resolution syringe pump PHD ULTRA NANOMITE Harvard Cambridge MA USA A sine wave was generated by a function generator and amplified up to a required voltage by a high voltage amplifier Later this output signal was then transmitted to a piezoelectric transducer for acoustic excitation In addition a high resolution syringe pump PHD ULTRA NANOMITE Harvard Cambridge MA USA was connected with the inlet of the microfluidic chip to inject liquid and cells steadily Fig 3 Components of the experimental system C Modeling We denote vectors and scalars by bold and regular fonts respectively Numerical simulations were performed to investigate the fluid response using the continuity equation and the Navier Stokes equation for a linear viscous compressible fluid 20 0 1 1 3 2 2 where is the mass density is the velocity of fluid p is the pressure of fluid and and are bulk and shear dynamic viscosities respectively The relation between and was assumed to be linear 0 2 3 where 0 is the velocity of sound in the fluid at rest We employed Nyborg s perturbation technique where the field of fluid velocity density and pressure were expanded as follows 4a 0 1 2 4b 0 1 2 4c Substituting 3 and 4 into 1 and 2 respectively the following equations referred to as the first and second order equations of acoustic are obtained 1 0 0 5 0 1 4 3 6 and 2 0 1 0 7 0 1 0 2 4 3 8 where x denotes the time average of quantity x over a full oscillation time period The above sets of equations with appropriate boundary conditions allow one to predict the motion of the fluid Furthermore Fig 4 shows the simulation results of the oscillating microstructures which also provide detailed streaming profiles with streamlines and zoomed arrows at the closed microchannel Based on the perturbation approach in plane and out of plane microvortices were generated by setting the oscillation function in the x y and x z planes respectively These closely match with our previous numerical analyses 680 Fig 4 Simulation results of the velocity vector field indicating the a in plane and b out of plane streaming flows The arrows represent the direction of motion unit m s D Concept and methods Here we propose a novel on chip 3D cell rotation method based on local whirling flows generated by custom designed microstructures in the surrounding fluid The specific designs of the in plane rotational and trapping manipulations have been performed in our prior works 17 Furthermore Fig 5 shows the concept design of the out of plane rotation and transportation manipulation of an oocyte at different frequencies because the vibrating pattern became more complex as the frequency change which could result in different orientations of the acoustic streaming field 21 E Cell and microparticle preparation Ovaries were collected from prepubertal gilts at a local slaughterhouse and transported to the laboratory in 0 9 NaCl containing 1 penicillin streptomycin Gibco Life technologies USA Oocytes were aspirated from medium sized follicles with a 20 gauge needle fixed to a 10 ml disposable syringe Oocytes were washed three times in the MEM culture medium Gibco Life technologies USA which contained 10 fetal bovine serum Gibco Life technologies USA and then transferred into a 35 mm polystyrene culture dish containing a 500 L drop of the same media 20 L of the polystyrene beads suspension was centrifuged and then diluted to 200 L using the deionized water DI at room temperature Fig 5 a f Concept of the 3D rotation and transportation of an oocyte by oscillating microstructures using different excited frequencies III EXPERIMENTS AND RESULTS A Evaluation of New Driving Method Fig 6 illustrates the sound pressure level S P L increased upon increasing the voltage Meanwhile the reason why S P L is different is that the vibration amplitude of piezoelectric transducer vary with the driving frequency changes therefore the energy applied on the vibrating microstructures is different B Observed trajectories Fig 7 a d shows the typical bead trajectories observed around the oscillating solid microstructures at diverse frequencies To visualize and characterize the acoustic flows inside the channel 1 m diameter fluorescent polystyrene beads were treated with the experimental system to confirm the fluid field distribution around the two types of solid microstructures Fig 7 a and b depicts that an oscillating solid structure induces two local acoustic flows in its vicinity when a sine wave was applied and the driven oscillation is harmonic with a frequency equal to 4 6 kHz Moreover the size of the microvortices on both sides of the microstructure is different because of asymmetric design Fig 7 c and d shows parallel out of plane microvortices rotating around X axis at a slightly lower frequency 4 2 kHz Fig 6 Relationship between sound pressure level S P L and acoustic frequency distance from the sound source 10 cm Fig 7 Experimentally observed trajectories of the 1 m diameter fluorescent beads in our acoustically vibrating microstructures a the driving voltage is 0 Vp p while b the driving voltage is 20 Vp p for in plane acoustic flows c the driving voltage is 0 Vp p while d the driving voltage is 40 Vp p for out of plane streaming microvortices 681 Furthermore as shown in Fig 8 the sizes of acoustic flows were different because the vibration amplitude of the piezoelectric transducer varied with changes of the driving frequency When the resonant frequency was close to the resonant frequency of the microstructures the force of acoustic streaming was strong In contrary the force of acoustic streaming was weak and would not have any impact on the motion of cell rotation when the frequency was far from the resonant frequency of the microstructures C In plane and out of plane rotational manipulation Fig 9 shows the in plane rotational manipulation of a swine oocyte At the tip of the oscillating microstructure a strong torque was created at 4 6 kHz and 20 Vp p peak to peak voltage value which was used to achieve in plane cell rotation Accordingly detailed works about in plane rotational manipulation and cell trapping have been investigated in our previous research 17 Moreover this work extends prior experimental studies to characterize the effect of the distance from the microstructure tip to the oocyte center D which could evaluate the size of acoustic streaming force varied with corresponding distance D Fig 8 Acoustic flows excited by diverse frequencies a microstructure and b microstructure Fig 9 In plane rotation of the oocyte at the different distance from microstructure tip to oocyte center D with 4 6 kHz and 20 Vp p a D 100 m and b D 460 m Fig 10 shows the out of plane rotation of the microparticle Fig 10a and oocytes Fig 10b and 10c using oscillating inclined bottom microstructures at a slightly lower frequency 4 3 kHz Compared to the driving voltage used in microstructure to achieve in plane rotational manipulation microstructure needed to be applied relative higher voltage 40 V Here the microparticles and oocytes are positioned on the custom designed slopes of the microstructure and rotated by the torque generated through the out of plane streaming flows To generate the out of plane streaming flows different streaming modes of the microstructures are utilized The out of plane rotational manipulation of different sizes of oocytes shows the out of plane streaming force is enough flexible to manipulate diverse large cells Furthermore Fig 11 shows the transportation manipulation of an oocyte between the patterning microstructures by adjusting frequencies Fig 10 Out of plane rotation of microparticles and oocytes rotational manipulation of a the 100 m diameter fluorescent polystyrene beads b the mouse oocyte and c the swine oocyte at 4 3 kHz and 40 Vp p Fig 11 Transportation manipulation of an oocyte between the patterning microstructures by adjusting frequencies 4 9 kHz 682 Fig 12 Plot of the rotation speed versus the voltage of the 100 m microbead showing that the former can be tuned by the applied peak to peak voltage Fig 13 The process of excluding the first polar body of a swine oocyte after the application of acoustic excitation To identify the relation between the in plane and out of plane rotational speeds for the microbeads with diameters of 100 m and the applied voltage the sinusoidal voltage was changed from 20 Vp p to 80 Vp p which could be as large as approximately 500 revolutions per minute rpm and 370 rpm respectively Fig 12 To verify the viability of swine oocytes influenced by our proposed method the target oocytes were collected from the microchip and were placed in the maturation medium At the end of the culture the swine oocyte maturation stages of M2 excluding the first polar body were determined under microscopic observation Fig 13 which implied that the proposed method caused no significant damage to the cells IV CONCLUSION AND FUTURE WORK In this study a novel acoustic based on chip method has been proposed for 3D rotational manipulation of microparticles and oocytes Compared with prior works our acoustofluidic cell rotation approach has the following advantages i it has been shown to be capable of achieving 3D rotational manipulation based on the use of vibrating microstructures especially in out of plane rotational manipulations ii it demonstrated the feasibility of noninvasive noncontaminated and stable transportation and rotation of microparticles and oocytes at different vibration modes iii it demonstrated the flexibility of rotational speed for various applications based on the choice of the required speeds Finally our future work will be focused on the improvement of the precision and motion stability of 3D cell rotations REFERENCES 1 K B hringer B Donald and N MacDonald Programmable Force Fields for Distributed Manipulation with Applications to MEMS Actuator Arrays and Vibratory Parts Feeders in Proc 1999 IEEE Int Conf Robot Automation ICRA Michigan USA vol 18 no 2 pp 168 200 1999 2 B Donald and J Jennings Sensor interpretation and task directed planning using perceptual equivalence classes in Proc 1999 IEEE Int Conf Robot Automation ICRA Sacramento USA pp 190 197 1991 3 L Feng P Di and F Arai High precision motion of magnetic microrobot with ultrasonic levitation for 3 D rotation of single oocyte Int J Rob Res vol 35 no 12 pp 1445 1458 2016 4 X Liu M 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