In Situ Quantification of Living Cell Adhesion Forces: Single Cell Force

Feb 26, 2014 - Institute of Intelligent Systems and Robotics, University of Pierre and Marie Curie, 4 Place Jussieu, 75005 Paris, France ... *E-mail: ...
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In Situ Quantification of Living Cell Adhesion Forces: Single Cell Force Spectroscopy with a Nanotweezer Hui Xie,*,† Munan Yin,‡ Weibin Rong,† and Lining Sun† †

The State Key Laboratory of Robotics and Systems, Harbin Institute of Technology, 2 Yikuang, C1 HIT Science Park, 150080 Harbin, China ‡ Institute of Intelligent Systems and Robotics, University of Pierre and Marie Curie, 4 Place Jussieu, 75005 Paris, France ABSTRACT: A novel method is presented for in situ quantification of living cell adhesion forces using a homemade nanorobotic system provided with two independently actuated probes that form a dual-probe nanotweezer capable of pickand-place manipulation of a single living cell in an aqueous environment. Compared with single-cell force spectroscopy (SCFS) based on traditional atomic force microscopy (AFM), cell immobilization via chemical trapping is unnecessary and the test cell can be efficiently released using the nanotweezer to significantly enhance production of the SCFS. Benefiting from the accurate force sensing capability of AFM, the nanotweezer allows reliable force measurement ranging from picoNewtons to microNewtons and is sufficiently sensitive to characterize short- and long-term adhesion of cell−cell and cell−substrate adhesions. Capabilities of the nanotweezer have been validated through experimental qualification of cell−substrate and cell−cell adhesion events of C2C12 cells (mouse myoblast adherent) with different contact times.

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tweezers,9,10 optical tweezers,11,12 and femtosecond laser impulses.13 The maximum vertical force of hundreds of picoNewtons that can be performed with magnetic and optical tweezers restricts measurement of cell−substrate adhesion from hundreds of nanoNewtons to microNewtons. Alternatively, micropipets14,15 and nanoforks16 have been used to detach cells, but their applications are limited because of the difficulty in analyzing the adhesion process in detail. By contrast, atomic force microscopy (AFM) has been proven to be important in characterizing living biological samples because of its high spatial resolution and ability to work in different conditions (e.g., in aqueous environments). AFM-based SCFS uses a single cell attached to an AFM cantilever and probed on the substrate17−20 or another single cell21−24 for cell−cell and cell−substrate adhesion force measurement, respectively. Detaching forces of cell adhesion ranging from 10 to 106 pN can be recorded by this method.8 Functionalized AFM probes have been used to alternatively probe a single cell immobilized on the substrate.25−27 The mechanism through which surface chemistry or topography affects cell adhesion can be elucidated. AFM-based SCFS still has some limitations despite its versatility and strength. One of them is the poor outcome because the current protocols for attaching a living cell or biomaterials to an AFM cantilever are labor-intensive and

INTRODUCTION Cell adhesion is mainly responsible for tissue cohesion and dynamic regulation of adhesion and de-adhesion processes, which enable tissue remodeling and cell migration. These interactions are fundamental to physiological and physiopathological processes.1 Numerous methods have been developed to qualitatively and quantitatively analyze cell adhesion. Microscopy-based methods have been utilized to qualitatively analyze cell adhesion. For instance, fluorescence resonance energy transfer has been used to determine the composition for dynamic protein−protein interactions.2 Semiqualitative methods have been used to determine cell adhesion in biomaterial research. The washing method is a commonly used method,3,4 in which nonattached or weakly attached cells on the substrate are washed away by flow and the percentage of cells still attached on the substrate can be determined. To elaborately control the detaching process, better-controlled hydrodynamic shear forces,5,6 or centrifugal force,7 have been used to analyze cell adhesion. The cells are laterally sheared off by forces parallel to the substrate. One major drawback of these techniques is the dependence of shear forces on cell geometry, size, and cell−substrate attachment. Thus, shear forces are not uniformly distributed along the cell surface or vary from different cells. To analyze cell adhesion quantitatively and in detail, methods have been developed to directly measure cell−cell and cell− substrate adhesion forces by single-cell force spectroscopy (SCFS).8 Quantitative data have been obtained using magnetic © 2014 American Chemical Society

Received: January 5, 2014 Revised: February 25, 2014 Published: February 26, 2014 2952

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Figure 1. Schematic image of cell adhesion force quantification using the dual-probe nanotweezer. (a) System configuration of the dual-probe nanotweezer. When the cell is grasped by the nanotweezer, the adhesion force measurement begins by moving the NP I downward and upward on the Z axis while the nanotweezer is immobilized. The detaching force is synthesized from bending forces on both probes individually detected by their own optical levers. This scheme is also suitable for cell−substrate adhesion force measurement. (b) Optical microscope image of the nanotweezer while grasping a cell. Scanning electron microscope image (inset) shows the configuration of the nanotweezer constructed from two AFM probes with protruding tips. The scale bar is 20 μm. (c) Adhesion force computation model, where F is the detaching force and kn is the normal spring constant stiffness of the nanotweezer. δn, δc, and δdet denote the normal deflection, cell deformation, and detaching distance of the nanotweezer, respectively.

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require specific expertise,19 thus limiting the measurements to a few cells per day.27 These procedures must be repeated to detach the cell from the cantilever with specific chemicals for new testing or replace the functionalized cantilever because the surface becomes restructured and contaminated with debris from the cell.28 When the contact time exceeds 20 min, the cell−substrate adhesion force measurement becomes difficult because the existing thermal drifts among the cell, cantilever, and substrate are hardly determined; cell−substrate adhesion may become stronger than the adhesion of the cell to the AFM cantilever.19 Therefore, faster, more accurate, and more skillful approaches and manipulation systems for cell adhesion measurement are necessary. In this study, we propose a novel AFM-based nanorobotic system for quantification of living cell adhesion forces. This system comprises two independently actuated and sensed probes that construct a dual-probe nanotweezer capable of pick-and-place manipulation of micro- and nanoscale objects with real-time force sensing.29 The nanotweezer can be used for in situ quantification of cell−cell and cell−substrate adhesion forces. Compared with the conventional AFM-based SCFS, in this method, the cell need not be immobilized on the nanotweezer using chemical glue, thereby eliminating the influence of chemical binding to cell morphology and activity, as well as the thermal drift of the system for long contact times. Furthermore, the target cells can be removed from the nanotweezer with ease. These advantages enable the attainment of high-throughput adhesion force measurements for multiple cells. To validate the capability of the nanotweezer, we performed SCFS experiments to analyze the adhesion of C2C12 cells (mouse myoblast adherent) via cell−substrate and cell−cell adhesion measurements; we also demonstrated the applicability of this method for various cell types with a wide range of force applications.

MATERIALS AND METHODS Nanotweezer Setup. Figure 1a shows the configuration of the AFM-based nanorobotic system for cell adhesion force measurement, which is equipped with a nanotweezer constructed from two opposed probes (probes I and II) with protruding tips. Each probe is independently actuated and sensed. Probe I is supported by micropositioning stage I (MP I), and the applied forces are detected by optical lever I, comprising laser I and position-sensitive detector I (PSD I, a four-quadrant photodiode). Probe II, which is sensed by optical lever II comprising laser II and PSD II, is mounted on nanopositioning stage II (NP II, travel range: 10 × 10 × 10 μm) that is supported by MP II. The sample platform is placed on NP I (travel range: 75 × 75 × 50 μm) that is supported by MP III. An optical microscope (equipped with a 20× lens) is used to position the laser spots on the probes and coarsely locate probes above target cells for adhesion force measurements. A multithread planning and control system is developed for AFM image scanning and dual-probe coordination control during manipulation. This system enables programming of complex tasks of cell−substrate and cell−cell adhesion quantifications, which is placed in a sealed mini-environment, wherein the CO2 concentration and temperature (37 °C) of the system are controllable. The nanorobotic system was originally developed for three-dimensional nanomanipulation and nanoassembly. More detailed specifications can be obtained from our previous paper.29 Figure 1b shows an optical microscope image (top view) of the nanotweezer while grasping a cell. An inset of a scanning electron microscope image (side view) shows the ‘V’ configuration of the nanotweezer formed by two AFM probe tips tilted from the probe beam with an angle θ ≈ 63°. This angle enables a stronger hold, resulting from the combined effect of the clamping, friction forces, and tip−cell adhesion forces during cell detachment.30 Figure 1c shows the computational model of adhesion force measurement. The detaching force F = knδn loaded on the

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Figure 2. Schematic of the protocol for single-cell adhesion force measurement and the optical images taken during measurement with the nanotweezer. (a) The procedure for the cell−substrate adhesion force measurement. (I) Both tips are aligned to the left and right sides of the cell. (II) Probe I is in contact with first with the cell and then with probe II. (III and IV) The cell is grasped and picked up by moving NP I on the Z axis with a rate of 0.5 μm s−1 and (V) detached from the substrate. (b) The procedure for the cell−cell adhesion force measurement. (I) The detached cell (in step V of Figure 2a) is transferred over the target cell and is (II) brought in contact with the target cell with a contact force and a setting value between 0.5 and 1.5 nN. Both probes can retrace a suitable distance to reduce the clamping force for long contact times. (III and IV) The cell is picked up from the target cell and is completely detached in step (V). (C) Optical images captured during the adhesion force measurement. (I) Before grasping. (II) Probe I is in contact with the cell and (III) then with probe II. (IV) The cell is detached from the substrate and (V) transferred to the target cell. (VI) Both cells are brought in contact for cell−cell adhesion force measurement. Scale bars represent 20 μm.

Petri dish (Corning, no. 3294) containing normal DMEM/F12 medium and 10% fetal bovine serum. The Petri dish was immediately mounted in a fluid cell (with a temperature control unit at 37 ± 0.2 °C) that was further mounted on the sample platform of the nanorobotic system placed in a minienvironment control (5% CO2). Nanotweezer Preparation. The cantilevers (ATEC− CONTAu, Nanosensors) were cleansed in pure alcohol for 1 h and thoroughly rinsed with Milli-Q water. The cantilevers were then placed on a glass slide and inserted into a hightemperature vacuum sterilizer. The cantilever holders were cleansed in pure alcohol for 1 h and thoroughly rinsed with Milli-Q water. The cantilevers were placed on the holders, which were put into a high-temperature vacuum sterilizer. Prior to the experiment, the spring constants of both cantilevers were calibrated using the method proposed by Sader et al,31 with values of 0.19 and 0.18 N/m. The respective normal sensitivities of the two probes in cell-culture medium were calibrated as 4.51 and 4.53 V/μm, which yielded force conversional factors of 42.1 and 39.7 nN/V on probes I and II. With consideration of the voltage noise of about 0.94 mVp−p, force accuracies were 39.6 and 37.3 pN on probes I and II, which were both better than 10 pN measured using an electrical system with higher signal-to-noise ratio and soft probes. To reduce the thermal drift of the system, starting the nanorobotic system, the mini-environment controller, and the temperature controller of the fluid at least 1 h prior to the sample preparation was necessary. Upon stabilizing the thermal drift, a Petri dish containing prepared cells was fixed on the sample platform of the nanorobotic system. The two AFM headers were immediately mounted on this system. By driving MP I and MP II, probes I and II were located at the center of

nanotweezer is synthesized from bending forces on each probe individually detected by the optical lever. kn is the sum of the stiffness of both probes, δn is the deflection of the nanotweezer, and δdet is the detaching distance, which is the sum of cell deformation (δc) and nanotweezer deflection (δn). The adhesion force of the cell grasped by the nanotweezer is given by the bending forces applied on the probes during cell detachment, that is, the pickup force applied on the cell that balances the adhesion forces from the substrate or another target cell. To detect the pickup force, determining the normal sensitivities and voltage outputs of the PSDs of both probes is necessary. The bending force Fn−I on probe I can be estimated by the voltage output ΔVn−I from PSD I as follows: Fn − I = βI × ΔVn − I

(1)

where βI is the normal force sensitivity of PSD I. The same treatment applies to the pickup force Fn−II on probe II. Thus, the pickup force applied on the cell, which is the cell adhesion force Fdet, can be synthesized from the bending forces on both probes via Fdet = Fn − I + Fn − II = βIΔVn − I + βIIΔVn − II

(2)

where βII and ΔVn−II are the normal force sensitivity and the normal voltage output of PSD II. Cell Cultivation and Sample Preparation. C2C12 cells (mouse myoblast adherent) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, pH 7.5) supplemented with 1.0 g/L glucose, L-glutamine, sodium pyruvate, 5958 mg/L HEPES, 100 units/mL penicillin, and 100 units/mL streptomycin. The cell line was maintained in 5% CO2 atmosphere at 37 ± 0.1 °C. Prior to the experiments, an appropriate number of cells was transferred into a Ø35 mm 2954

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recommended. Otherwise, the cell can be completely released at step IV in Figure 4 for longer contact times. Holding the Cell for Cell−cell Adhesion Force Measurement. Figure 2a-V shows that the cell completely detached from the substrate was still held by the nanotweezer for cell− cell adhesion force measurement and was located above a target cell by moving MP III on the x−y plane (Figure 2b-I). Attachment of the Grasped Cell to the Target Cell. Figure 2b-II shows that after at least 10 min of cell recovery, the grasped cell vertically approached the target cell attached on the substrate with real-time force monitoring by driving NP-I. The contact force should be between 0.5 and 1.5 nN, and the approach rate should be less than 0.5 μm s−1. Note: if the contact time is longer than 30 s, opening the nanotweezer to revert the cell to an original form of phantom lines (Figure 2bII) is necessary to effectively reduce the effects on the building of the cell−cell adhesion from the clamping force or the thermal drift of the system. Detachment of the Grasped Cell from the Target Cell. Figure 2b-III and 2b-IV shows that the grasped cell is vertically retraced from the target cell after a suitable contact time while driving NP I, in which the retraction rate is less than 0.5 μm s−1. F−D data on both probes are recorded for cell−cell adhesion force computation. Adjusting the contact time, multiple cell− cell adhesion force measurements from step (6) for the same target cell can be restarted, from step (5) for different target cells, or from step (1) for different grasped cells. Figure 2c shows images captured during the adhesion force measurement. Figure 2c-I shows that the nanotweezer is located at the testing cell that is brought in contact with probe I in Figure 2c-II and clamped by the nanotweezer in Figure 2c-III when probe II is in contact with the cell. Once the cell is completely detached from the substrate (Figure 2c-IV), the former is transferred to the target cell for cell−cell adhesion measurement (Figure 2c-V). The grasped cell is brought in contact with the target cell (Figure 2c-VI). Compared with time-consuming cell immobilization performed using traditional SCFS, cell grasping can be efficiently completed with the nanotweezer within 1.2 min with from steps (1) to (3), thereby significantly reducing the cycle time for each cell adhesion measurement. Serial quantification of cell−substrate adhesion could also be reduced to less than 3 to 4 min, which is mainly determined by the detaching speed and distance. Thus, more than 100 serial quantifications of cell− substrate adhesion could be obtained in a work day. Although cell−cell adhesion measurement was less efficient because the nanotweezer should be held up during the contact time, the nanotweezer can simply and efficiently perform the cell−cell adhesion measurement of different cells. Clamping Detection during Cell Grasping. Clamping detection and control yield successful cell grasping and protect the cell from damage. To detect the interaction between the cell and the tip during cell grasping, bending force control on the probe begins when the probes approach and further clamp the cell. Figure 3 shows an example of the contact detection on the cell with probe I. The force curve starts from a noncontact state between the probe and the cell, and the probe tip is in contact with the cell at 0.4 μm. As the probe tip moves further toward the cell, this tip is bent downward to about 10.5 nm, leading to negative forces. In this part, the bending force decreases gradually to −2 nN with a displacement of about 200 nm. Further movement leads to cell deformation that causes a slow decrease of the bending force to the minimum value of

the optical microscope view. The apexes of both tips were precisely aligned to form a nanotweezer with an opening distance of 2 to 4 μm wider than the cell diameter. The laser spots were focused on the backside of the corresponding probe beams, and the positions of PSDs were adjusted to set the signals around zero where PSDs held the most sensitive responses. For the entire experiment, the CO2 concentration within the mini-environment and the temperature of the fluid cell were stabilized at 5% and 37 °C, respectively. Protocol of the Adhesion Force Measurement. Figure 2 shows a schematic of the protocol for single-cell adhesion force measurement using the proposed nanotweezer. Cellsubstrate adhesion force measurement was performed (Figure 2a). When the cell was completely picked up from the substrate, the detached cell was released from the nanotweezer to serially detach the next cell or used as a testing cell for cell− cell adhesion experiments (Figure 2b). Figure 2c shows six optical microscope images (under a 20× objective) obtained during the experiment. The following processes describe the cell adhesion measurement performed with the nanotweezer. System Initialization. Each axis of the nano- and micropositioning stages was set in appropriate positions that provide pick-and-place manipulation with sufficient motion range on each axis. MP III was driven to move a favorable cell into the center of the optical microscope view. Nanotweezer Location at the Testing Cell. Figure 2 (panels a-I) shows that by driving MP III, the nanotweezer was placed over the favorable cell under the optical microscope, aligning tips I and II on the left and right sides of the cell, respectively. MP I and MP II were moved on the z axis, until both tips were close to the substrate with a distance less than 10 μm. The force servo control was started to move NP I and NP II alternatively to make both tips in contact with the substrate. To avoid grasping the cell−substrate interactive interface, the substrate was moved down to create a gap of 2 to 3 μm (1/4 to 1/3 of the cell diameter) from the nanotweezer by driving the NP I on the z axis. Grasping the Cell with the Nanotweezer. Figure 2 (panel aII) shows that tip I initially approached the cell by moving NP I on the x axis. Similarly, tip II approached the cell by moving NP II on the x axis. Once both tips were in contact with the cell, a nanotweezer was configured to grasp the cell with a clamping force for cell−substrate adhesion force measurement. The clamping force should be well-controlled to achieve successful grasping and protect the cell from damage during this process. Detachment of the Cell from the Substrate. Following the detection of reliable grasping on the cell with force monitoring, the cell−substrate adhesion force measuring process was started by moving the NP I downward on the z axis (Figure 2, panels a-III and a-IV). For the computation of the cell−substrate adhesion force, force−distance (F−D) data on both probes were recorded during the approach and retraction processes. Critical step: the cell release operation (retraction process) is presented in detail in the next section. Once the cell is released from the nanotweezer, the cell−substrate adhesion force measurement can be restarted from step (1) for serial adhesion measurement of different cells. The measurement is restarted from step (3) if multiple tests on the same cell are required. In this case, the release operation will be different from the required contact time. To ensure that the time needed for release (from steps II to IV in Figure 4) and grasping is less than the contact time, cell release operation being processed to step II in Figure 4 for contact times less than several minutes is 2955

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Figure 3. Clamping detection by normal force sensing on probe I. The force curve shows the bending behavior of the cantilever during approach, contact, and retraction.

−4.8 nN at a total distance of 2.5 μm, wherein the cell is trapped with a clamping force of about 43 nN through mechanical analysis.30 During retraction, the tip slowly reverts to the original shape. Before the tip pulls off from the cell, the adhesion force between the tip and the cell induces an increase of the bending force to 1.5 nN. After the probe-cell adhesion breakage, the cell no sooner recovers when the bending force sharply reaches zero at 0.6 μm, with a deformation of 0.2 μm. The force response described (Figure 3) is sufficient to detect not only the contact between the tip and the cell but also the clamping state. Building a computational model for contact between the soft cell and the nanotweezer to estimate an exact clamping force that is sufficient to overcome cell adhesion is complicated compared with solid−solid contact between the nanotweezer and micro/nano-objects. Compared with the maximum grasping force of several microNewtons occurring at the solid−solid contact interface,30 the deformed soft cell can be more reliably held and picked up by the nanotweezer. This assumption has been validated by experiments that indicate successful cell detachment when the cell is trapped by the nanotweezer with an empirical normal bending force of subnanoNewtons to several nanoNewtons on each probe for cell−cell adhesion and dozens of nanoNewtons for the case of the cell−substrate adhesion. Cell Release. To analyze the adhesion force of different cells with the traditional SCFS, an operation was performed to bind a single cell to a clean and freshly mounted cantilever. However, along with the release of the tested cell, this process is time-consuming. The cell attached on the cantilever is removed with chemicals or is directly changed to a clean cantilever for new testing. In this study, cell sticking often occurs in the nanotweezer after adhesion force measurement. Fortunately, the cell is not strongly stuck to the nanotweezer because of the small contact area between the cell and the unfunctionalized probe tip, thereby successfully releasing the cell on the substrate by a simple scheme (Figure 4). (1) Figure 4a-I shows the retraction process by moving up NP I on the z axis to place the cell in contact with the substrate, with a contact force of several nanoNewtons. F−D data on both probes were recorded during the retraction process. (2) To reduce the effects of the cell−substrate adhesion on the setup, the clamping force is immediately reduced by opening the nanotweezer until the cell recovers its original circular shape. A contact time ranging from 10 to 30 s is normally set to obtain sufficient cell−substrate adhesion force for cell release in the succeeding steps. (3) By simultaneously moving NP I and NP

Figure 4. A schematic of cell release to the substrate. (a) Protocol of the cell release. Step I: the nanotweezer is constantly holding the cell and NP I is moved upward on the z axis to place the cell in contact with the substrate. Step II: to reduce the effects of cell−substrate adhesion on the setup, the clamping force is immediately reduced by separating both probes until the cell recovers its original circular shape. Step III: following a contact time of 10 to 30 s, both probes are simultaneously separated from the cell until the cell is thoroughly released in step IV. (b) Optical microscope images (20×) I−IV capture using the steps performed in (a).

II on the x axis, both probes retrace from the cell with a low rate of 0.1 μm s−1. Figure 4a-III shows the gradual detachment of the probes from the cell and the successful detachment of the probes from the cell (Figure 4a-IV). Figure 4b-I−IV show the optical images related to the release scheme. In the experiments, almost all cells can be successfully released with a contact time of 10 to 30 s if the cell is active and the probes are not severely contaminated with cell debris. However, the probes should be changed after about 20 testing procedures for the next batch of adhesion force measurements to reduce effects from the contaminated probes on the cell activities and to increase the force accuracy of testing.

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EXPERIMENTAL RESULTS

Cell-Substrate Adhesion Force Measurement. When the setup is ready for adhesion force measurement, the nanotweezer is initially used to quantify the cell−substrate adhesion forces in the first set of experiments. Using the optical microscope and force feedback, the favorable cell is clamped by the nanotweezer. The sample platform is moved downward to detach the cell from the substrate by moving the NP I on the z axis while recording the nanotweezer bending force required to detach the cell. Once the cell is completely detached from the substrate, the cell is transferred to another target cell attached to the substrate for cell−cell adhesion force measurement (Figure 2b), or the cell is released from the nanotweezer to the substrate (Figure 4), such that next cell can be tested. Instead of chemically immobilizing the cells to the AFM cantilever as in conventional SCFS, we used robotic grasping to immobilize the cell to the nanotweezer. The cell is not attached on the nanotweezer during the cell−substrate adhesion setup. This method can be used to quantify time-dependent cell adhesion forces with contact times of several hours, which is impractical with conventional SCFS. The latter is prone to failure at these times because the cell−substrate adhesion forces often surpass those used for the fixation of the cells to the cantilever and the thermal 2956

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Figure 5. Cell-substrate adhesion of C2C12 cells to the bottom of the Petri dish. (a) Representative example of F−D curves obtained with a contact time of 20 min. Data show a maximum adhesion force of 47.5 nN that is synthesized (green) from forces measured on probes I (blue) and II (red). (b) Time-dependent maximum adhesion forces with contact times ranging from 5 to 125 min. The analysis in (b) involved the recording of at least 10 F−D curves per time frame. The data is represented as mean and standard error (±).

Figure 6. Cell−cell adhesion of C2C12 cells. (a) Representative example of the F−D curves obtained with a contact time of 2 min. Data show a maximum adhesion force of 2.23 nN synthesized (green) from forces measured on probes I (blue) and II (red). (b) Time-dependent maximum adhesion forces with contact times ranging from 1 to 16 min. The analysis in (b) involved the recording of at least 5 F−D curves per time frame. Data is represented as the mean and standard error (±). drift of the system imposes difficulty in holding the cantilever at the correct position. The nanotweezer is demonstrated to successfully detach dozens of cells from the glass substrate (bottom of the Petri dish), with contact times ranging from 5 to 125 min. Figure 5a shows a representative example of the F−D curves obtained with a contact time of 20 min between the cell and the substrate. The green curve represents the force response during cell− substrate adhesion force measurement, that is, the synthesized forces measured on probes I (blue curve) and II (red curve). Response differences on the probes are mainly due to the cell’s large asymmetrical deformation deduced by strong adhesion that might be nonuniformly distributed at the cell−substrate interface (the adhesion force is not through the center of the nanotweezer), which causes nonsymmetrical force loading on both probes during the detaching process. The curve starts from the contact state between the nanotweezer and the cell. When a load is applied to the cell−substrate adhesion by moving NP I downward to pick up the cell at a constant rate of 500 nm s−1, both cantilevers are bent from the strength of the cell− substrate adhesion, so that the nanotweezer exerts a detaching force on this adhesion. The receptor remains anchored in the cell cortex and unbinds as the force increases when the position reaches 2.26 μm during pickup, and the cell jumps from the substrate with a maximum adhesion force of 47.5 nN. As NP I is further moved down, the force magnitude

constantly and slightly decreases with the discontinuous jumps (part I). In this part, receptor anchoring is lost and membrane tethers are pulled out of the cell at a detachment distance of 17 μm, denoted as the long-distance tethers. Once the cell is completely detached from the substrate (part II), the nanotweezer retracts to the starting position and the cell is released using a previously discussed strategy (part III). Following the retraction at 2.2 μm, further retraction leads to a continuous increase until the nanotweezer is back at the starting point (part IV). The force on the nanotweezer increases because of the extending cell deformation during the pickup manipulation, which places the cell in contact with the substrate before retraction of the nanotweezer to the starting point. The maximum downward force exerted on the nanotweezer is defined as the detachment force (Fdet = 47.5 nN). Detachment tasks are repeated with the protocol described above for different incubation times. A total of 64 cells were measured serially using the same probes, obtaining reliable force data. Incubation time of the tested cells ranged from 5 to 125 min, indicating that long incubation time on the substrate results in higher adhesion force (Figure 5b). The maximum average adhesion force is 228 ± 38 nN with an incubation time of around 2 h. The adhesion will increase with longer incubation time; the cell simultaneously spreads more widely on the substrate and will eventually be stabilized after several hours of contact, which was demonstrated by previous works.32 We tested adhesion of cells incubated within 125 min because the nanotweezers 2957

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formed by the selected contact mode cantilevers are stiff enough to pick up the cells, and the corresponding force response is in the linear range. Thus, stiffer cantilevers (e.g., ATEC-FMAu, Nanosensors, stiffness: 2.8 N/m) should be used to build the nanotweezer for cells incubated with longer contact time. The adhesion will reach from hundreds of nanoNewtons to several microNewtons. Cell−Cell Adhesion Force Measurement. Cell−cell adhesion force measurement was also performed with our developed nanotweezer. Figure 6a shows a representative example of the F−D curves of the cell−cell adhesion measurement obtained with a contact time of 2 min. Once the grasped cell is optically located over the target cell with a vertical distance sufficient to cover the detaching distance, this cell approaches the target cell by moving NP I upward at a constant rate of 500 nm s−1. When the position reaches 1.8 μm, two cells are brought into contact with a snap-in response followed by slight fluctuations before the cells are compressed with a preload of 1 nN. Following the given contact time of 2 min, a load to the cell−cell adhesion is applied by moving NP I downward to pick up the cell at a constant rate of 500 nm s−1. Both cantilevers are bent from the strength of cell−cell adhesion, so that the nanotweezer exerts a detaching force on the grasped cell. During retraction, the grasping force on the nanotweezer continuously increases before the position reaches 5 μm. The grasped cell jumps from the target cell with a maximum adhesion force of 2.23 nN. As NP I is further moved down, the force magnitude constantly decreases initially with discontinuous jumps, followed by the unbinding of membrane tethers before the grasped cell is thoroughly detached from the target cell at about 12 μm. The maximum force exerted on the nanotweezer is defined as the detachment force (Fdet = 2.23 nN). As the nanotweezer retracts to the start position, pick-and-place tasks were repeated with a setting time ranging from 1 to 16 min. A total of 10 cells were measured, thereby obtaining reliable data. The mean Fdet increases from 1.12 ± 0.36 nN to about 12.14 ± 2.08 nN with the increase of the time interval. Figure 6b shows that the required cell separation force increased rapidly within 8 min. The detaching force became gradually stabilized around 12 nN after 10 min of contact. A similar phenomenon was observed in cell adhesion quantification of two S180 cells, such that the adhesion forces stabilized after 1 h contact.14 However, the magnitude of the maximal adhesion force and the stable period measured by each group are different because of the different cells and culture media, validating the capability of the proposed nanotweezer in measuring cell−cell adhesion force demonstrated at the nanoNewton scale.

methodologies and software will be developed for future studies to complete an automated and high-throughput measurement of cell adhesion and batch analysis of the F−D data recorded by the nanotweezer-SCFS system. However, the developed nanotweezer still has some limitations that need to be addressed. (1) Compared with the single-probe SCFS, the force resolution of the dual-probe nanotweezer is reduced by half if the same probes are used. In the case of cell−cell adhesion force measurement, the force resolution at the picoNewton level is indispensable to observe any detail during the detaching process, such as characterizing force responses when membrane tethers are pulled out of the mammalian cells. To improve the force sensitivity of the nanotweezer, a laser force measuring system will be properly designed and softer probes with excellent reflectivity will be chosen. (2) The nanotweezer is more suitable for grasping suspension cells or adherent cells having globular shapes rather than cells that are fully spread on the substrate.27 The tips of the nanotweezer will intrude on the cell−substrate interface and peel the cell’s edge if a spread cell is grasped by the nanotweezer. Thus, a specific protocol and patterned substrates16 are required to quantify spread cell adhesion with the proposed nanotweezer. (3) The method is limited by the probe tip length used to form the nanotweezer that ranges from 15 to 20 μm. Should the cell size be much more than the tip length, grasping will be difficult. In this case, tip modification techniques are necessary, such that the length of the probe tip is extended by welding micro- or nanowires on the tip apex33 or cantilevered micropipets with a tip length of several hundreds of micrometers will be used instead of the conventional silicon probe.

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CONCLUSION A nanorobotic system equipped with a dual-probe nanotweezer is developed for SCFS experiments. The nanotweezer has a high positioning accuracy and easily manipulates biological cells and biomolecules. Protocols for in situ quantification of cell− substrate and cell−cell adhesion forces are given. Experiments on time-dependent cell−substrate (Petri dish substrate) and cell−cell adhesion forces of C2C12 cells have been successfully performed using the nanotweezer. Cell immobilization with chemical binding is not required initially, so this method is practical to significantly improve the efficiency of SCFS experiments. Long-term adhesion force measurement is also logical because the proposed nanotweezer is capable of detecting forces ranging from picoNewtons to microNewtons. This method is considerably suitable for studying cell adhesion with different biomaterials,34−36 analyzing cell adhesion with different contact areas,37 and examining temperature dependence of single-cell adhesion.38 This study elucidates single-cell interaction with external matrices and would be beneficial in medical and biological fields.

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DISCUSSION Efficiency is the main concern for the application of SCFS. Chemical fixation of a single cell on the cantilever in traditional SCFS is time-consuming.19,26 Contact times ranging from several seconds to hours are needed for each test to build adhesion between the cell and target surface, limiting the adhesion measurement to a few cells per work day and inducing a labor-intensive acquisition of statistically significant amounts of data. Compared with the traditional SCFS, cell fixation is not needed using the proposed nanotweezer, by which adhesion testing can be performed rapidly and serially cell-by-cell, instead of holding up during the contact time. Probes forming the nanotweezer can be used to grasp and release more than 20 cells with a mount of testing before contamination. Newly cleaned probes can be changed rapidly for the next batch of adhesion force measurements. These measurements can be more than one hundred of the cells daily, which substantially improves the efficiency of SCFS testing compared with the traditional AFM-SCFS. Nevertheless, current adhesion measurement and data analysis are not ideally rapid because of manual and semiautomatic operations. To further improve the efficiency of SCFS testing with the proposed nanotweezer,

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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