sensors Article

Development of a Microforce Sensor and Its Array Platform for Robotic Cell Microinjection Force Measurement Yu Xie *, Yunlei Zhou, Yuzi Lin, Lingyun Wang and Wenming Xi Department of Mechanical and Electrical Engineering, Xiamen University, Xiamen 361005, China; [email protected] (Y.Z.); [email protected] (Y.L.); [email protected] (L.W.); [email protected] (W.X.) * Correspondence: [email protected]; Tel.: +86-151-6006-1679 Academic Editor: I-Ming Hsing Received: 14 February 2016; Accepted: 17 March 2016; Published: 6 April 2016

Abstract: Robot-assisted cell microinjection, which is precise and can enable a high throughput, is attracting interest from researchers. Conventional probe-type cell microforce sensors have some real-time injection force measurement limitations, which prevent their integration in a cell microinjection robot. In this paper, a novel supported-beam based cell micro-force sensor with a piezoelectric polyvinylidine fluoride film used as the sensing element is described, which was designed to solve the real-time force-sensing problem during a robotic microinjection manipulation, and theoretical mechanical and electrical models of the sensor function are derived. Furthermore, an array based cell-holding device with a trapezoidal microstructure is micro-fabricated, which serves to improve the force sensing speed and cell manipulation rates. Tests confirmed that the sensor showed good repeatability and a linearity of 1.82%. Finally, robot-assisted zebrafish embryo microinjection experiments were conducted. These results demonstrated the effectiveness of the sensor working with the robotic cell manipulation system. Moreover, the sensing structure, theoretical model, and fabrication method established in this study are not scale dependent. Smaller cells, e.g., mouse oocytes, could also be manipulated with this approach. Keywords: cell-holding device; cellular force sensor; force measurement; microinjection; micromanipulation; PVDF film

1. Introduction With the fast development of cell biology and related biotechnology, robot-assisted cell manipulation, which is precise and can enable a high throughput, is attracting interest from researchers [1–4]. Robotic cell manipulation, e.g., cell probing, grasping, or microinjection, requires an end effector to establish soft physical contact with the cell. A cell force sensor is, therefore, a crucial element, as it allows the robot to better understand and control the mechanical interaction between cell and manipulator. Many force sensors have been developed for single cell force measurement. These include atomic force microscopes, optical tweezers and magnetic tweezers, which provide small force measurement at or below the nanonewton range. For larger cellular force measurement (in the micronewton range), a set of mechanical sensors has been developed using microelectromechanical systems (MEMS) fabrication techniques. A probe with a flexible beam is used as the force load of the cell; the force between the probe and the cell results in deformation of the beam, as shown in Figure 1. A force-displacement sensing mechanism, e.g., a piezoelectric [2–6], strain gauge [7–9], or capacitive transducer [10] is employed and microfabricated as the beam. Lu et al. [7] developed a piezoresistive micro-force sensor to monitor zebrafish embryo injection forces. A micropipette was bonded to the free end of a sensing beam, while Sensors 2016, 16, 483; doi:10.3390/s16040483

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the whole sensor was mounted on a movable stage. When the micropipette contacted the cell, the to the freeand end the of a change sensing beam, while the whole sensor was mounted on a movable stage. beambonded was deformed, in resistance was measured using a Wheatstone bridge circuit. When the micropipette contacted the cell, the beam was deformed, and the change in resistance was Rajagopalan et al. [9] used a flexible beam attached to a rigid probe to study the mechanical response measured Drosophila using a Wheatstone circuit. Rajagopalan et al. [9] used asensors flexible beam to a of embryonic axons. bridge However, conventional micro-force shareattached some common rigid probe to study the mechanical response of embryonic Drosophila axons. However, conventional limitations, which prevent their integration in a cell microinjection robot. In cell microinjection, the micro-force sensors share some common limitations, which prevent their integration in a cell probing needle must be connected to the pressure tube so that a liquid substance can be injected into microinjection robot. In cell microinjection, the probing needle must be connected to the pressure the cell (see Since thecan needle is connected a flexible ofneedle the pressure tube soalso that Figure a liquid1).substance be injected into theto cell (see alsobeam, Figurethe 1). gravity Since the is tube isconnected far greater the injection i.e.,ofofthe thepressure order oftube micro-newtons. In a study by Liuforce, et al. [11], to than a flexible beam, theforce, gravity is far greater than the injection a vision-based method was developed positioning the force-sensing structure i.e., of the force order measurement of micro-newtons. In a study by Liu et al.by [11], a vision-based force measurement at themethod cell side instead of the beam. elastic polydimethylsiloxane pillar array was was developed byneedle positioning theAn force-sensing structure at the cell side(PDMS) instead of the needle Ancell elastic (PDMS) pillarpillars array was placed at the cell side and slender placedbeam. at the sidepolydimethylsiloxane and the slender forces of these were measured during cell the microinjection. forces of these pillars were measured during cell microinjection. The method overcomes the tube The method overcomes the tube gravity problem associated with the conventional cell force sensor gravity problem associated with the conventional cell force sensor and can be used in a robot-assisted and can be used in a robot-assisted cell microinjection system, but compared with MEMS-based force cell microinjection system, but compared with MEMS-based force sensing, it is difficult to precisely sensing, it is difficult to precisely calculate the contribution of the pillar deflection by a visual tracking calculate the contribution of the pillar deflection by a visual tracking algorithm [12]. In this paper, we algorithm [12]. In this paper, we present a new design of a MEMS-based micro-force sensor capable of present a new design of a MEMS-based micro-force sensor capable of providing real-time cell force providing real-time force feedback in cell microinjection manipulation. A novel beam is feedback in cell cell microinjection manipulation. A novel supported beam is designed forsupported the cell holder designed for the cell holder side. The piezoelectric material polyvinylidine fluoride (PVDF) is chosen side. The piezoelectric material polyvinylidine fluoride (PVDF) is chosen as the sensing element. as theCompared sensing element. Compared with vision-based elastic deformation sensing [11], PVDF-based with vision-based elastic deformation sensing [11], PVDF-based force measuring offers a rapid measurement force more measuring offers a moreresponse. rapid measurement response.

Figure Typicalprobe probebased based micro-force Figure 1. 1.Typical micro-forcesensor. sensor.

In addition, in the study of cell mechanics it has been found that the activity of cells, such as cell

In addition, in the study of cell mechanics it has been found that the activity of cells, such as cell growth, division, signal conversion and gene expression, changes the cell’s mechanical properties, growth, signal and genetoexpression, changes the mechanical anddivision, vice versa [5]. The conversion stiffness (ratio of stress strain) of a developing eggcell’s changes at variousproperties, stages and vice versa [5]. The stiffness (ratio of stress to strain) of a developing egg changes stages of the cell cycle. For a zebrafish embryo, for example, the stiffness at the blastula stageatis various 1.66 times of thethat cellatcycle. For a zebrafish embryo, forrobotic example, stiffness at the blastula stage 1.66 the prehatching stage [6]. From the pointthe of view, the manipulated object (i.e.,isthe cell)times time-varying andstage individually variable. Understanding the cell characteristics that atis the prehatching [6]. From the robotic point of view, themechanical manipulated object (i.e.,via thea cell) force sensorand is thus a key step for the controller design in a robot manipulation system. To this end,via a is time-varying individually variable. Understanding the cell mechanical characteristics trapezoidal array based device isdesign specially Combined withsystem. the micro-force force asensor is thus a key stepcell-holding for the controller indesigned. a robot manipulation To this end, sensor, this will improve the measurement speed and allow the researchers better understand the a trapezoidal array based cell-holding device is specially designed. Combined with the micro-force single cell characteristics by comparing the force characteristics of a batch of cells at same robotic sensor, this will improve the measurement speed and allow the researchers better understand the manipulation environment. We demonstrate the suitability of these sensors in a robot-assisted cell single cell characteristics by comparing the force characteristics of a batch of cells at same robotic microinjection application by determining the real-time injection force on zebrafish eggs. manipulation environment. We demonstrate the suitability of these sensors in a robot-assisted cell microinjection application by determining the real-time injection force on zebrafish eggs. 2. Sensor Design 2. Sensor Design 2.1. Sensor Construction In the cell microinjection process, a high-sensitivity sensor is required, as the cell load force is usually of the order of micronewtons or less. Figure 2 illustrates the structure of the proposed

2.1. Sensor Construction

In the cell microinjection process, a high-sensitivity sensor is required, as the cell load force is usually of the order of micronewtons or less. Figure 2 illustrates the structure of the proposed

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microforce sensor, which is composed of three parts: a mechanical transducer, which converts the needle injection forcewhich to deformation PVDF an electrical transducer, which converts microforce sensor, is composedofofthe three parts:beam; a mechanical transducer, which converts the the charge output force of thetoPVDF film to a corresponding andwhich a cell-holding device. needle injection deformation of the PVDF beam; an voltage electricalsignal; transducer, converts the charge output of the PVDF filmforce to a corresponding voltage When signal; the andneedle a cell-holding device. The principle of operation of the sensor is as follows. penetrates theThe egg, the principle operation of the force sensor is asisfollows. When needle penetrates the friction egg, theforces process can beofconsidered quasi-static. The egg suspended in the culture medium, so the process can be considered quasi-static. The egg is suspended in culture medium, so the friction between the cell and the cell-holding device and the fluid dynamic forces can be ignored. forces According between the cell and the cell-holding device and the fluid dynamic forces can be ignored. According to Newton’s law, the needle probing force equals the force applied on the beam. The piezoelectric to Newton’s law, the needle probing force equals the force applied on the beam. The piezoelectric PVDF film is a lightweight and compliant film that directly attaches to the beam without disturbing PVDF film is a lightweight and compliant film that directly attaches to the beam without disturbing the mechanical motion. As a typical piezoelectric material, it has a wide frequency bandwidth from the mechanical motion. As a typical piezoelectric material, it has a wide frequency bandwidth from 0.0010.001 to 10to9 10 Hz9 Hz andand a high voltage Thus,we wedesigned designed a PVDF beam structure that is fixed at a high voltageoutput. output. Thus, a PVDF beam structure that is fixed at one end and and simply supported at the other end. AAU-shape at the thenon-fixed non-fixed end one end simply supported at the other end. U-shapeconstraint constraint is is designed designed at of the beam tobeam prevent unwanted disturbances. end of the to prevent unwanted disturbances.

Figure 2. Figure 2. Sensor Sensordesign. design.

In our design, the cell-holding device is firmly glued to the PVDF beam so that the center of the In our design, the cell-holding is firmly glued to the PVDF beam soforce, thatthe thebeam center of cell lies on the latitudinal center axisdevice of the beam. When it is subjected to an injection the cell lies on the the latitudinal center axis ofatthe it isbending subjected to an injection force, deforms. Since micropipette is acting thebeam. center When of the cell, is the primary mode of the whilethe torsion is zero. Note that theat sensitivity of the sensor is determined by themode beamdeformation, deforms. Since micropipette is acting the center of the cell,output bending is the primary width and length of torsion the beam, stiffness thethe beam and the piezoelectric strain constant of the of deformation, while is the zero. Note of that sensitivity of the sensor output is determined piezoelectric film. The structure of the beam is an important factor to be considered during design. by the width and length of the beam, the stiffness of the beam and the piezoelectric strain constant Compared with a conventional cantilever beam structure, this beam design—fixed at one end and of the piezoelectric film. The structure of the beam is an important factor to be considered during simply supported at the other end—is novel. Considering the mechanical properties of the materials design. Compared with a conventional cantilever beam structure, this beam design—fixed at one and structural principles, the cantilever structure undergoes greater deformation and produces a end higher and simply at this the other end—is novel. the mechanical properties of voltagesupported output than novel beam design, for Considering the same injection force. However, the the materials andnature structural cantileverproduces structure undergoes greater deformation asymmetrical of theprinciples, bending of the the cantilever a larger puncture wound to the cell and produces higher voltage thandesign. this novel beam design, for the same injection force. However, than isa produced by the output novel beam

the asymmetrical nature of the bending of the cantilever produces a larger puncture wound to the cell Microfabrication of novel Cell-Holding than2.2. is produced by the beamDevice design. The cell-holding device was fabricated in two steps. First, an inverted trapezoidal grooved

2.2. Microfabrication of Cell-Holding stainless steel mold was machinedDevice three times by electrical discharge machining with a cutting speed

of 0.05cell-holding m/s. To ensure the transparency of thein cell-holding device, polishing was carried The device was fabricated two steps. First,electrolytic an inverted trapezoidal grooved out, to reduce the surface roughness of the mold. The mold was then placed in the center of a glass stainless steel mold was machined three times by electrical discharge machining with a cutting speed Petri dish that had been cleaned with acetone. The second step was the preparation of the PDMS cellof 0.05 m/s. To ensure the transparency of the cell-holding device, electrolytic polishing was carried holding device. Sylgard 184 pre-polymer and curing agent were fully mixed in a ratio of 10:1 by out, weight, to reduce the surface roughness of the mold. The mold was then placed in the center of a glass and the mixture placed in a desiccator to degas for 20 min by allowing bubbles to escape. PetriThis dishmixture that had with acetone. Thedegassed second in step was theheating preparation the°CPDMS wasbeen then cleaned poured into a Petri dish and a vacuum furnace of at 70 cell-holding device. Sylgard 184 pre-polymer and curing agent were fully mixed in a ratio of 10:1 by for 12 h. Using a sharp scalpel to cut around the pattern evenly and gently, the PDMS cell-holding weight, andwas theremoved mixturefrom placed a desiccator to degas for3 20 minthe by fabrication allowing bubbles to escape. This device thein stainless steel mold. Figure shows of the cell-holding device using stainless steel mold.dish and degassed in a vacuum heating furnace at 70 ˝ C for 12 h. mixture was thenthe poured into a Petri

Using a sharp scalpel to cut around the pattern evenly and gently, the PDMS cell-holding device was removed from the stainless steel mold. Figure 3 shows the fabrication of the cell-holding device using the stainless steel mold.

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Figure 3.3.Fabrication Fabrication of PDMS cell-holding device with stainless steel mold. Figure3. Fabricationof ofPDMS PDMScell-holding cell-holdingdevice devicewith withstainless stainlesssteel steelmold. mold. Figure

2.3. Sensor Mechanical Model 2.3.Sensor SensorMechanical MechanicalModel Model 2.3. Since the PVDF force-sensing structure isisnewly newly designed for cell injection force measurement, Sincethe thePVDF PVDFforce-sensing force-sensingstructure structureis newlydesigned designedfor forcell cellinjection injectionforce forcemeasurement, measurement, Since we will analyze the force-mechanical model of the novel beam structure. The left end of the sensor in we will analyze the force-mechanical model of the novel beam structure. The left end of thesensor sensorin in we will analyze the force-mechanical model of the novel beam structure. The left end of the Figure 4 is the clamped end; the right end is the simply supported end. Figure44isisthe theclamped clampedend; end;the theright rightend endisisthe thesimply simplysupported supportedend. end. Figure

Figure Figure 4.4.Simplified (a) force at simple support; (b) force at clamped end. F, applied Figure4. Simplifiedsensor sensorstructure structure(a) (a)force forceat atsimple simplesupport; support;(b) (b)force forceat atclamped clampedend. end.F, F,applied applied force; F for zero movement at A; a, distance between forces; l, length of beam. force; F A , force at A forces; l, length of beam. A force; FA, force at A for zero movement at A; a, distance between forces; l, length of beam.

For clear illustration, the beam of Figure isisredrawn redrawn in Figure 4a. A beam of length and width Forclear clearillustration, illustration,the thebeam beamof ofFigure Figure222is redrawnin inFigure Figure4a. 4a.A Abeam beamof oflength lengthllland andwidth width For isisconsidered. considered. beams [13], the force AAcan be considered.According tomaterial material mechanics theory ofofhyperstatic hyperstatic beams [13], the force can be bbbis Accordingto materialmechanics mechanicstheory theoryof hyperstatic beams [13], the force A can replaced by the force F A with the condition that the movement at point A is zero. Now the beam replaced byby the zero. Now Now the thebeam beam be replaced theforce forceFAFAwith withthe thecondition conditionthat thatthe themovement movement at at point point A is zero. structure isissimplified simplified as cantilever structure, as shown in Figure 4b. IfIfthe the cantilever movement at structureis simplifiedas asaaacantilever cantileverstructure, structure,as asshown shownin inFigure Figure4b. 4b.If thecantilever cantilevermovement movementat at structure point A is W AA,,then: point A is W then: point A is WA , then: W “ WF `W “0 (1) W (1) WAAA W WFF W WFFFAAA  00 (1) where WF and WFA are the cantilever displacement at point A under the force F and FA , respectively. where WFFand and W WFFAAare arethe thecantilever cantileverdisplacement displacementat atpoint pointAAunder under the theforce forceFFand andFFAA,,respectively. respectively. where Then weWget: Then 2 Thenwe weget: get: a p3l ´ aq FA “ F 3 aa22((2l 33llaa)) FFAA  FF and the bending moment along the z-axis is: 22ll33 $ and Fza2is: p3l ´ aq andthe thebending bendingmoment momentalong alongthe the’ z-axis is: &z-axis ,0 ă z ă a 3 2l Mpzq “ (2) 3 3 2 ’ % Fa ´ Fzp2l ` a ´ 3la q , a ă z ă l 2l 3

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xbd 31l 2 (4 n  n 3  n 2  2) E PVDF Q F  where a is the distance from F to the supported end of the beam. 4 I beam Ebeam

(9)

The bending moment causes tension at the bottom and compression at the top, as illustrated in Figure 5. The small element a distance x fromthe therelationship neutral axisbetween of the bent is:Q and Since d31,strain EPVDF, in Eb,an, x, l and b areatknown constants, thebeam charge

F can be expressed as: εz “

pR ` xqθ ´ Rθ x “ Q Rθ  k1F R

(3) (10)

where radius of the curvature of the neutral axis, and X and θ are defined in Figure 5. where R k1 is is the a constant.

Figure 5. 5. Beam Figure Beam stretching. stretching.

2.4. Sensor Electrical Model The moment is expressed as: ż A PVDF film has a high direct current output impedance and appears as a charge source. Thus, Mpzq “ xσz dxdy (4) a charge amplifier was added to convert the charge output from the PVDF film to a voltage signal. s Following the charge amplifier, low-pass amplifiers were added to remove the power frequency where σz is the in theand z-direction. interference in stress the circuit amplify the weak voltage signal. In addition to the low-pass filters, According to Hook’s law and Equation (2), theelectromagnetic stress in the z-direction is σznoise. “ Eb ε zThe , where Eb is shielding techniques were considered to prevent interference electrical the Young modulus of the beam. According to Equations (2)–(4), we have: circuit is shown in Figure 6. R“´

Eb Ib Mpzq

(5)

where Ib is the inertial moment of the cross-sectional area. We then obtain a new expression for the strain: xMpzq εz “ (6) Eb Ib The force leading to the deformation of the material is mainly determined by the beam since the stiffness of the beam is much greater than that of the PVDF film. The strain of the PVDF film is dominated by the strain of the beam. Then the PVDF stress is σz´PVDF “ EPVDF ε z , where EPVDF is the Young modulus of the PVDF film. Based on these equations, the stress of the PVDF film along the Figure 6. Electrical circuit of the sensor. z-direction is given by: $ The circuit operates by passing an finput current FE pvd xza2 p3l ´ aq that charges the feedback capacitor C1. Because ’ ’ & 0ăzăa of the virtual ground condition of the operational 2Ebeam Ibeam l 3 amplifier, the impedance of the parallel feedback σz´PVDF “ (7) ’ E pvd f x Fzp2l 3 ` a3 ´ 3la2 q resistor R2 and capacitor C1 is:’ % rFa ´ s aăzăl Ebeam Ibeam 2l 3

R2 (11) In our sensor construction, the PVDF beam1isstretching j R2C1 along its length, so the charge Q is mainly Z

affected by the piezoelectric strain constant d31 . The electrical charge developed is: By using Laplace transformation, the output voltage of charge amplifier V1 subjected to the input current dQ/dt is obtained as:

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Q Sensors 2016, 16, 483

xbd 31l 2 (4 n  n 3  n 2  2) E PVDF F 4 I beam Ebeam

(9) 6 of 13

Since d31, EPVDF, Eb, n, x, l and b are known constants, the relationship between the charge Q and F can be expressed as: Q“

r s

Q  k1F

d31 σz´PVDF dA

(10)

where k1 is a constant. rb rl E pvd f xd31 rb ra FE pvd f xzd31 a2 p3l ´ aq Fzp2l 3 ` a3 ´ 3la2 q dzdy ` sdzdy “ rFa ´ 3 Ebeam I 2Ebeam Il 2l 3 0 a 0 0 xE pvd f bd31 p4Fal 4 ´ Fa3 l 2 ` Fa2 l 3 ´ 2Fl 5 q “ 4Ebeam Il 3

(8)

Let a = nl (0 < n < l); then: Q“

xbd31 l 2 p4n ´ n3 ` n2 ´ 2qEPVDF F 4Ibeam Ebeam

(9)

Since d31 , EPVDF , Eb , n, x, l and b are known constants, the relationship between the charge Q and F can be expressed as: Q “ k1 F (10) where k1 is a constant.

Figure 5. Beam stretching.

2.4. Sensor Sensor Electrical Electrical Model Model 2.4. A PVDF PVDF film film has has aa high high direct direct current current output output impedance impedance and and appears appears as as aa charge charge source. source. Thus, Thus, A charge amplifier amplifier was was added added to to convert convert the the charge charge output output from from the the PVDF PVDF film film to to aa voltage voltage signal. signal. aa charge Following the charge amplifier, low-pass amplifiers were added to remove the power frequency Following the charge amplifier, low-pass amplifiers were added to remove the power frequency interference in in the the circuit circuit and and amplify amplify the the weak weak voltage signal. In In addition addition to to the the low-pass low-pass filters, filters, interference voltage signal. shielding techniques techniques were were considered considered to The electrical shielding to prevent prevent electromagnetic electromagnetic interference interference noise. noise. The electrical circuit is shown in Figure 6. circuit is shown in Figure 6.

Figure 6. Electrical circuit of the sensor. Figure 6. Electrical circuit of the sensor.

The circuit operates by passing an input current that charges the feedback capacitor C1. Because circuit operates by passing anoperational input current that charges the feedback C1 .feedback Because of theThe virtual ground condition of the amplifier, the impedance of capacitor the parallel of the virtual ground condition of the operational amplifier, the impedance of the parallel feedback resistor R2 and capacitor C1 is: resistor R2 and capacitor C1 is: R R22 Z“ (11)  1 ` jωR (11) 1  j R2 C C1 2

1

By using Laplace transformation, the output voltage of charge amplifier V 1 subjected to the input By using Laplace transformation, the output voltage of charge amplifier V1 subjected to the input current dQ/dt is obtained as: current dQ/dt is obtained as: R2 s V1 “ Q (12) 1 ` R2 C1 s The charge amplifier network works as a high-pass filter where the cut-off frequency is: fc “

1 “ 0.16Hz 2πR2 C1

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It is worth noting that the input force is a limited-width signal in the time domain for force measurement. This means that the spectrum of the input signal is continuous and that some of the signal frequencies must be lower than the cut-off frequency. A digital compensator (1 + s)/s is thereby added to Equation (11) to let the sensor output V 1 be proportional to charge Q. Then we have V 1 = k2 Q, where k2 = R2 . After there, a low-pass amplifier is then designed with an adjustable amplifier: Vout “ p1 `

R5 qV R4 1

and a cut-off frequency: f2 “

1 “ 40Hz 2πR3 C2

to eliminate the power frequency interference. Combined with the charge amplifier, the transfer function of the complete electrical circuit becomes: Vout “ k3 Q

(13)

where k3 is the electrical circuit gain. Substituting Equation (9) into Equation (12), the linear theoretical model for the PVDF force sensor is obtained as V out = k1 k3 F, where the proportional parameter k1 k3 can be obtained in calibration experiments. 2.5. Analysis of Force Sensor Configuration In the sections above, we have obtained an analytical model of the injection force and the sensor voltage output of the PVDF beam structure. Because the V-shaped PDMS cell-holding device is attached to the beam, the beam bending caused by the force not only depends on the properties of the beam, but is also affected by the PDMS. Further analysis of the effect of the groove is obtained using finite element software ANSYS. The PVDF force beam structure models with and without PDMS were established, respectively, using finite element analysis. The suitable boundary conditions were defined using the software. The material is assumed to be isotropic and the analysis is of the linear type. The modulus of elasticity of the PDMS is 1.8 MPa, the density is 0.95 g/cm3 , and the Poisson ratio is 0.48. The piezoelectric beam packaging material is polyethylene; its elastic modulus is 2200 MPa, its density is 1.01 g/cm3 , and its Poisson ratio is 0.38. The material parameters are affected by the synthesis process and are not exact values. This does not affect the analysis of the influence of the groove on the bending of the beam, as they are generally sufficiently accurate under an external force of 0.2 N applied perpendicular to the same position of the beam (the center of the middle groove). Figure 7 shows the strain distribution of the PVDF beam structure with and without the PDMS cell-holding device. It can be seen that the forms of the bending is similar. The maximum deformation of the beam without the groove is 0.915 mm while that with the groove is 0.746 mm. This is mainly because the elastic modulus of the PDMS material is very small and far less than the modulus of elasticity of the packaging material. The packaging material has a large elastic modulus and is more rigid; the bending of the beam is mainly influenced by the more rigid material. 3. Calibration Experiments Prior to force-sensing application experiments, force calibration was performed to quantify the relationship between the applied force and sensor output. The calibration set-up is shown in Figure 8. When a needle moves towards the PVDF beam, the force exerted on the film equals that on the electrical scale. The needle was manipulated by a linear motor (M-403.4DG, PI, Karlsruhe, Germany) at a fixed speed, to ensure repeatability and consistency. A high-precision microforce sensor (Nano17, ATI, Apex, NC, USA) was attached to the needle to measure the needle force. An oscilloscope

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(DS1102E, Rigol, Beijing, China) was used to display the voltage signal of the electrical circuit. The PVDF film (SDT1-028K, MEAS., Hampton, VA, USA) has a length of 30 mm, a width of 13 mm and a thickness of 28 µm. The electrodes of the PVDF film were sealed with seal glue (HMG-628X, HOMEEN, Shengzheng, since the sensor is used in aqueous solution. Sensors 2016, 16,China), 483 8 of 13 Figure 7. Strain distribution of the beam structure with (left); and without (right) cell-holding device.

3. Calibration Experiments Prior to force-sensing application experiments, force calibration was performed to quantify the relationship between the applied force and sensor output. The calibration set-up is shown in Figure 8. When a needle moves towards the PVDF beam, the force exerted on the film equals that on the electrical scale. The needle was manipulated by a linear motor (M-403.4DG, PI, Karlsruhe, Germany) at a fixed speed, to ensure repeatability and consistency. A high-precision microforce sensor (Nano17, ATI, Apex, NC, USA) was attached to the needle to measure the needle force. An oscilloscope (DS1102E, Rigol, Beijing, China) was used to display the voltage signal of the electrical circuit. The PVDF film (SDT1-028K, MEAS., Hampton, VA, USA) has a length of 30 mm, a width of 13 mm and a thickness of 28 µm. The electrodes of the PVDF film were sealed with seal glue Figure 7. 7. Strain distribution of of the the beam beam structure structure with with (left); (left);and andwithout without (right)cell-holding cell-holdingdevice. device. Figure Strain distribution (HMG-628X, HOMEEN, Shengzheng, China), since the sensor is used in(right) aqueous solution. 3. Calibration Experiments Prior to force-sensing application experiments, force calibration was performed to quantify the relationship between the applied force and sensor output. The calibration set-up is shown in Figure 8. When a needle moves towards the PVDF beam, the force exerted on the film equals that on the electrical scale. The needle was manipulated by a linear motor (M-403.4DG, PI, Karlsruhe, Germany) at a fixed speed, to ensure repeatability and consistency. A high-precision microforce sensor (Nano17, ATI, Apex, NC, USA) was attached to the needle to measure the needle force. An oscilloscope (DS1102E, Rigol, Beijing, China) was used to display the voltage signal of the electrical circuit. The PVDF film (SDT1-028K, MEAS., Hampton, VA, USA) has a length of 30 mm, a width of 13 mm and a thickness of 28 µm. The electrodes of the PVDF film were sealed with seal glue (HMG-628X, HOMEEN, Shengzheng, China), since the sensor is used in aqueous solution.

representation of of the the calibration calibration set-up. set-up. Figure 8. Schematic representation

The relationship relationshipbetween between needle movement voltage is examined. first examined. The needle was The needle movement andand voltage is first The needle was moved moved forward through different distances at a speed of 2.5 mm/s for five times. The five distances forward through different distances at a speed of 2.5 mm/s for five times. The five distances are aremm, 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, respectively. corresponding output voltages 0.1 0.2 mm, 0.3 mm, 0.4 mm, and and 0.5 mm, respectively. The The corresponding output voltages are are shown in Figure 9, which shows that the sensor output has high repeatability and linearity. Within shown in Figure 9, which shows that the sensor output has high repeatability and linearity. Within the the 0–0.5 mm measurement range, the linear sensitivity is 4.5 V/mm. The linearity is 1.82% full scale. 0–0.5 mm measurement range, the linear sensitivity is 4.5 V/mm. The linearity is 1.82% full scale. It is noticed that an offset exists in Figure 9. It may be from the capacitors since the PVDF, cable and charge amplifier contain ones. We observed the voltage output of the circuit at point 1 and V 1 simultaneously without any force acts on the PVDF film in Figure 10. It can be seen that the offset comes from the PVDF and cable capacitors are almost undetectable while the offset at V 1 is steady. This shows the offset may come from the non-ideal behaviour of the capacitor of C1 . Figure 8. Schematic representation of the calibration set-up.

The relationship between needle movement and voltage is first examined. The needle was moved forward through different distances at a speed of 2.5 mm/s for five times. The five distances are 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, and 0.5 mm, respectively. The corresponding output voltages are shown in Figure 9, which shows that the sensor output has high repeatability and linearity. Within the 0–0.5 mm measurement range, the linear sensitivity is 4.5 V/mm. The linearity is 1.82% full scale.

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Figure 9. Displacement–voltage response of the sensor. Figure 9. Displacement–voltage response of the sensor.

It is noticed that an offset exists in Figure 9. It may be from the capacitors since the PVDF, cable It is noticed that an offset exists in Figure 9. It may be from the capacitors since the PVDF, cable and charge amplifier contain ones. We observed the voltage output of the circuit at point 1 and V1 and charge amplifier contain ones. We observed the voltage output of the circuit at point 1 and V1 simultaneously without any force acts on the PVDF film in Figure 10. It can be seen that the offset simultaneously without any force acts on the PVDF film in Figure 10. It can be seen that the offset of the sensor. Figure Displacement–voltage sensor. comes from the PVDF and cable9. capacitors are almostresponse undetectable while the offset at V1 is steady. comes from the PVDF and cable capacitors are almost undetectable while the offset at V1 is steady. This shows the offset may come from the non-ideal behaviour of the capacitor of C1. This It shows the offset may come frominthe non-ideal behaviour thecapacitors capacitor since of C1. the PVDF, cable is noticed that an offset exists Figure 9. It may be fromofthe and charge amplifier contain ones. We observed the voltage output of the circuit at point 1 and V1 simultaneously without any force acts on the PVDF film in Figure 10. It can be seen that the offset comes from the PVDF and cable capacitors are almost undetectable while the offset at V1 is steady. This shows the offset may come from the non-ideal behaviour of the capacitor of C1.

Figure 10. 10. Voltage Voltage output output at atpoint point11(left); (left);and andpoint pointVV1 (right). (right). Figure Figure 10. Voltage output at point 1 (left); and point V11 (right).

Figure 11 shows the relationship between force input and sensor output while the cell injection Figure Figure 11 11 shows shows the the relationship relationship between between force force input input and and sensor sensor output output while while the the cell cell injection injection was switched between cell-holding grooves from the simply supported end to the fixed end of the was was switched switched between between cell-holding cell-holding grooves from the the simply simply supported supported end to the fixed fixed end end of of the the PVDF beam. The slope of the force–voltage curve changes as the cell groove changes. PVDF beam. The slope of the force–voltage curve changes as the cell groove changes. PVDF beam. Figure 10. Voltage output at point 1 (left); and point V1 (right).

Figure 11 shows the relationship between force input and sensor output while the cell injection was switched between cell-holding grooves from the simply supported end to the fixed end of the PVDF beam. The slope of the force–voltage curve changes as the cell groove changes.

(a) (a)

(b) (b)

Figure 11. Calibration results of the microforce sensor towards different cell holding grooves Figure 11. 11. Calibration results of microforce sensor towards towards different different cell cell holding grooves grooves Figure Calibration of the the microforce sensor (a) position of the force's results application point; (b) relationship between force input andholding sensor output. (a) position of the force's application point; (b) relationship between force input and sensor output. (a) position of the force's application point; (b) relationship between force input and sensor output.

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Figure 11. Calibration results of the microforce sensor towards different cell holding grooves (a) position of the force's application point; (b) relationship between force input and sensor output.

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sensitivities of of the the three three positions positions are are0.0439 0.0439V/N, V/N, 0.0399 0.0399 V/N V/N and and 0.0354 0.0354 V/N V/N The force sensitivities respectively. The The linearity linearity is is 1.82% 1.82% full full scale. scale. As As shown shown in in Equation Equation (9), the proportional relationship respectively. the injection injection force FF varies varies along along the the beam. beam. These results show that the holds as the acting point of the sensor is consistent with the theoretical analysis and has a good linear response. The slight offset in the experimental curves might be due to the inverse piezoelectric effects of the PVDF film, which has model derivation. derivation. not been considered in the model 4. CellInjection InjectionExperiment Experimenton onZebrafish Zebrafish Embryo Embryo 4. Cell 4.1. Experiment Materials Materials and 4.1. Experiment and Setup Setup To To verify verify the the effectiveness effectiveness of of the the proposed proposed microforce microforce sensor, sensor, the the PVDF PVDF sensor sensor was was adopted adopted in in the real-time injection force measurement of a robot-assisted microinjection system for manipulating the real-time injection force measurement of a robot-assisted microinjection system for manipulating zebrafish The zebrafish zebrafish is is one one of of the zebrafish embryos. embryos. The the commonly commonly used used animal animal models models of of developmental developmental genetics and embryonic development. Microinjection of zebrafish embryos is a standard genetics and embryonic development. Microinjection of zebrafish embryos is a standard procedure procedure used to analyze the effects of introduced materials. The zebrafish embryos used in our cell used to analyze the effects of introduced materials. The zebrafish embryos used in our cell microinjection force measurement experiments were collected in accordance with standard microinjection force measurement experiments were collected in accordance with standard embryo embryo preparation The zebrafish zebrafish embryo embryo is is about about 800–1200 800–1200 µm µm in in diameter, diameter, with preparation procedures procedures [14]. [14]. The with the the cytoplasm and nucleus at the animal pole sitting upon a large mass of yolk. The egg coat is called cytoplasm and nucleus at the animal pole sitting upon a large mass of yolk. The egg coat is called the the chorion, chorion, and and is is pierced pierced by by aa needle needle when when injecting injecting genes genes into into the the embryo. embryo. The robot-assisted microinjection system consists of the sensor, an inverted microscope The robot-assisted microinjection system consists of the sensor, an inverted microscope (AE31, (AE31, Motic, Xiamen, China) with a CCD camera (UI-1540SE-M-GL, IDS, Obersulm, Germany), Motic, Xiamen, China) with a CCD camera (UI-1540SE-M-GL, IDS, Obersulm, Germany), and a threeand a three-degrees-of-freedom (MP-285, Sutter, Novato, CA,controlling USA) for controlling degrees-of-freedom micro-robot micro-robot (MP-285, Sutter, Novato, CA, USA) for the needle,the as needle, as shown in Figure 12. The PVDF beam creates an extrusion when the needle is injected shown in Figure 12. The PVDF beam creates an extrusion when the needle is injected into the embryo. into the embryo. The output the PVDF signaltoisa then transformed to aelectronic voltage output The output of the PVDF chargeof signal is then charge transformed voltage output by the circuit by the electronic circuit of Figure 6. The injection needles were fabricated by a micropipette puller of Figure 6. The injection needles were fabricated by a micropipette puller (P2000, Sutter, Novato, (P2000, Sutter, Novato, CA, USA). The different diameters of needle tip were obtained by adjusting the CA, USA). The different diameters of needle tip were obtained by adjusting the laser heating time. laser heating time. Tip diameters of 20 µm, 40 µm, and 60 µm were selected. Tip diameters of 20 µm, 40 µm, and 60 µm were selected.

Figure 12. 12.Experimental Experimentalset-up set-up zebrafish embryo injection force measurement. three Figure forfor zebrafish embryo injection force measurement. 3-DOF, 3-DOF, three degrees degrees of freedom. of freedom.

4.2. Experiment and Results Results 4.2. Experiment Method Method and We performed performed robot-assisted robot-assisted microinjection microinjection on on 18 18 embryos. embryos. The The embryos embryos were were randomly randomly and and We averagely divided divided into averagely into three three groups. groups. For For different different group group the the diameter diameter of of the the inject inject needle needle is is different. different. The first, three zebrafish embryos were placed in the trapezoidal PDMS groove, as shown in Figure 13. It can be seen that the transparency of the PDMS is satisfactory for use as the cell-holding device in a robot-assisted cell manipulation system. Compared with the horizontal bottom and top

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The first, three zebrafish embryos were placed in the trapezoidal PDMS groove, as shown in Figure 13. It can be seen that the transparency of the PDMS is satisfactory for use as the cell-holding Sensors 2016, 16, 483 11 of 13 device in a16, robot-assisted cell manipulation system. Compared with the horizontal bottom and top Sensors 2016, 483 11 of 13 of the trapezoidal groove, the image of the groove slope is darker. This results from the surface of the trapezoidal groove, the image of the groove slope is darker. This results from the surface of the trapezoidal groove, the of the groove slope is to darker. Thissloping resultssides fromsmooth the surface roughness of sloping side of image the groove; groove; it is is more difficult difficult to make the the sloping sides smooth than roughness of the the sloping side of the it more make than roughness of the sloping of the groove; it is more difficult to make the sloping sides smooth than the horizontal sections in the process. the horizontal sections in side the fabrication fabrication process. the horizontal sections in the fabrication process.

Figure Figure 13. 13. Zebrafish Zebrafish embryos embryos in in cell-holding cell-holding device. device. Figure 13. Zebrafish embryos in cell-holding device.

Second, the needle was moved to the 3 o’clock edge of the embryo by the manipulator. Second, the was moved moved to to the the 33 o’clock o’clock edge of of the the embryo embryo by by the the manipulator. manipulator. Second, thea needle needle was Figure 14 shows single embryo and a needle before the edge robot-assisted microinjection (2× objective, Figure 14 aa single embryo and before the robot-assisted microinjection (2ˆ objective, Figureadapter). 14 shows showsThe single embryo and aa needle needle before robot-assisted microinjection (2× objective, 0.65× needle microinjection speed was the fixed as 2 mm/s and the move distance was 0.65ˆ adapter). The needle microinjection speed was fixed as 2 mm/s and the move distance 0.65× adapter). needle microinjection was fixed as402 and mm/s move distance was was 800 µm. For eachThe group the diameter of the speed inject needle is 20, 80 and µm, the respectively. 800 800 µm. µm. For For each each group group the the diameter diameter of of the theinject injectneedle needleisis20, 20,40 40and and80 80µm, µm,respectively. respectively.

Figure 14. Zebrafish embryo before needle injection. Figure 14. 14. Zebrafish Zebrafish embryo embryo before before needle needle injection. injection. Figure

The typical microinjection force trajectories for different needle tip diameters are shown in The typical microinjection force trajectories for different needle tipforce diameters arethe shown in Figure 15,typical where microinjection the vertical axisforce denotes the corresponding between needle The trajectories for differentpenetration needle tip diameters are shown in Figure 15, where the vertical axis denotes the corresponding penetration force between the needle tip and15, embryo. can be seen that the injection force nonlinearly increases the needle Figure where It the vertical axis denotes the corresponding penetration forceasbetween the advances. needle tip tip and embryo. It can bewere seen associated that the injection force nonlinearly increases as of thethe needle advances. The with the stiffness andincreases inner pressure embryo. and nonlinear embryo. Itproperties can be seen that the injection force nonlinearly as the needle advances. The The nonlinear properties were associated with the stiffness and inner pressure of the embryo. The force was a were maximum whenwith the the embryo chorion was penetrated, can be used as a nonlinear properties associated stiffness and inner pressure of which the embryo. The force was a to maximum when penetration the embryo chorion was The penetrated, which can be used as a signal for the robot acknowledge of the cell. penetration force force) The force was a maximum when the embryo chorion was penetrated, which can (peak be used as a signal for the robot to acknowledge penetration of the cell. The penetration force (peak force) increases as the needle diameter increases. The average punctuation force of are 7.3 mN, 12.1 mN signal for the robot to acknowledge penetration of the cell. The penetration force (peak force) increases increases as the needle diameter increases. punctuation force offorce are 7.3 mN, 12.1 mN and 17.9 mN corresponding to the 20,average 40 andThe 80 average µm diameter. the decreased while as the needle diameter increases. The punctuation forceAfter of arethat, 7.3 mN, 12.1 mN and 17.9 mN andchorion 17.9 mNpressure corresponding to the 20, 40 and 80 µmthe diameter.trajectory After that, the force decreased while the released. during thewhile released corresponding to thewas 20, 40 and 80 The µm vibration diameter. of After force that, the force decreased the process chorion the chorion was of released. Thebeam. vibration of the force trajectory during the released process is a result of pressure the vibration the PVDF pressure was released. The vibration of the force trajectory during the released process is a result of is a result of the vibration of the PVDF beam. the vibration of the PVDF beam.

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The force was a maximum when the embryo chorion was penetrated, which can be used as a signal for the robot to acknowledge penetration of the cell. The penetration force (peak force) increases as the needle diameter increases. The average punctuation force of are 7.3 mN, 12.1 mN and 17.9 mN corresponding to the 20, 40 and 80 µm diameter. After that, the force decreased while the chorion was released. The vibration of the force trajectory during the released process Sensors 2016, 16,pressure 483 12 of 13 is a result of the vibration of the PVDF beam.

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(c) Figure 15. 15. Injection Figure Injection force force trajectories trajectories for for different different needle needle diameters. diameters. (a) (a) Force Force curve curve of of20 20µm µm diameter; diameter; (b) Force Force curve curve of of 20 20 µm µmdiameter; diameter;(c) (c)Force Forcecurve curveofof20 20µm µmdiameter. diameter. (b)

5. Conclusions 5. Conclusions To measure measure the the real-time real-time cell cell injection injection force force trajectory trajectory in in real real cell cell microinjection microinjection experiments, experiments, aa To novel force-sensing beam structure was designed. Theoretical analysis showed that the force force was was novel force-sensing beam structure was designed. Theoretical analysis showed that the proportional to to the the charge charge output output of of the the piezoelectric piezoelectric sensor. sensor. A A mechanical mechanical model model of of the the sensor sensor was was proportional verified experimentally. The traditional mold machining method was used to make a cell-holding verified experimentally. The traditional mold machining method was used to make a cell-holding device. This can be be applied applied to to smaller smaller scales scales [15]. [15]. Finally, the effectiveness effectiveness of of the the designed designed device. This approach approach can Finally, the sensor when when working working with with aa micro-robotic micro-robotic cell cell manipulation manipulation system system was wasverified verifiedexperimentally. experimentally. sensor Acknowledgments: This National Natural Natural Science Science Foundation Foundation of of China China Acknowledgments: This work work was was supported supported by by the the National (No. 61403322) 61403322)and and the Research for the Doctoral Program of Higher Education (No. partpart from from the Research Fund for Fund the Doctoral Program of Higher Education (20130121120013). (20130121120013). Author Contributions: Yu Xie and Yunlei Zhou designed the sensors, conducted the experiments; Yuzi Lin performed thesimulation;Yu Wenming and Lingyun Wang help fabrication cell holding device. Author Contributions: Xie and Xi Yunlei Zhou designed theto sensors, conducted the experiments; Yuzi Lin performed Wenming Xi and Wang help to fabrication cell holding device. Conflicts ofthesimulation; Interest: The authors declare noLingyun conflict of interest.

Conflicts of Interest: The authors declare no conflict of interest.

References

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