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“Single Cell Force Spectroscopy” for Probing Steerable Cell-Material Interactions Hongrui Zhang, Gordon, G. Wallace, Michael J. Higgins* ARC Centre of Excellence, Intelligent Polymer Research Institute, University of Wollongong, Australia

Backgrounds

Methods: Experimental Setup 200

Results 200 180 160

0.2µm

19pN Count Count

140 120 100 80 60 40 20

0 0 20 40 60 80 100 120 140 160 180 200

0 0 1 2 3 4 5 6 7 8

Plateau Force(pN)

Plateau length(µm)

(A) 60

(B) 100

525pN 50

5E-16J 80

40

60 Count

30

Count

40

20

Figure 1. Cell-material interactions are important in biomedical engineering, tissue engineering and bionics, and pivotal for understanding the integration of biomaterials with living cells and tissues (Figure 1, top left). Previous studies in our group have demonstrated the ability to control extracellular matrix protein and cell adhesion on conducting polymers via electrical stimulation. However, the underlying mechanisms of controlling the cellular response are still not known, particularly as a function of electrical stimulation. Elucidating these mechanisms becomes even more of challenge when we consider the complexities of the cellular-material interface. To address this, we are currently undertaking research using Biological-AFM (Bio-AFM), combined with electrochemicalAFM, to study the adhesive interactions of living cells with conducting polymers (Figure 1, right). The aim is to resolve, and temporally control, molecular adhesion at the cellularmaterial interface with a view to designing smart materials and interfaces (e.g. Figure 1, bottom - electrically switchable conducting polymers) for cell-based applications. (Figure 1 remake from Leal-Egaña, Díaz-Cuenca et al. 2013)

Figure 3. Schematic representation setup of the experimental setup to enable single cell force spectroscopy on conducting polymers within an electrochemical cell under AFM. The AFM scanning stage sits above an inverted optical microscope (Nikon) and the electrochemical fluid cell comprises three-electrodes with the conducting polymer substrate as the working electrode. The system can be temperature, humidity and gas controlled, and force measurements conducted in cell media using the live cell probes.

10

20

0 0 500 1000 1500 2000 2500 3000 3500

0 0.00E+000

2.00E-015

4.00E-015

6.00E-015

8.00E-015

1.00E-014

Maximum Force(pN)

Energy(J)

(C) (D) Figure 7. Analysis of parameters, including maximum adhesion force, total adhesion energy, membrane tether (plateau) force and membrane tether length (total of 10 cells with 50 force curves for each cell). The histograms show a mean distribution of 19pN for the membrane tether force (A), 200 nm for the plateau length (B), 5e-16 joules for the adhesion energy (C) and, interestingly, a quantized distribution for the maximum adhesion of single cell with a primary distribution of 425 pN. These values correspond to the very early and initial stages of cell-substrate adhesion (i.e. cell-surface contact time of 1 sec).

Electrical stimulation results 0.003 0.002 0.001 0.000 -0.001 -0.002 -0.003 -0.004 -1.0 -0.8 -0.6

Methods: Principle of SCFS -2.62V

Biotin-Bovine serum albumin

Streptavidin

Biotin-Concanavalin-A

Current(A)

-5.14V -0.4 -0.2 0.0 0.2 0.4 0.6

Voltage(V)

Figure 4.: A representative force curve for the interaction between L929 cell and PPy-DBSA film obtained using single cell force spectroscopy (approach/extension curve (red) and retracting(blue) to and from the substrate. The cell is moved towards the surface and no force is detected in the first part of extension. When the cell is pressed on the surface, the force increases until a pre-set value is reached (we are able to quantify the stiffness and modulus of a cell from this part). After a given contact time, the cell is retracted from the surface and the bonds that have been formed, including jumps (peaks) and membrane tethers (plateaus) break sequentially and record a force. The maximum adhesion force (star) is the force needed to separate the majority of the cell-surface contacts. For the whole interaction, the energy required to completely separate the cell from the surface is given as the area under the retraction curve (blue dashed region). Figure 8. A cyclic voltammogram for the PPy-DBSA film in CO2 independent medium without serum and within the electrochemical cell under the AFM is shown at the top of the figure. To investigate the cell adhesion as a function electrical stimulation, we applied an oxidation (constant) potential of +300mV and reduction (constant) potential of -800mV. Force curves of single cell adhesion were first taken on non-stimulated films followed by reduced and then oxidized films. Representative force curves for L929 cell adhesion on the

Results

non-stimulated PPy-DBSA (blue curves), reduced PPy-DBSA (red curves) and oxidized PPy-DBSA substrate (green curves). We observed that adhesion increased on the reduced films followed by a significant decrease upon oxidation. This switching of the adhesion is due to rearrangement of the DBSA at the polymer surface, as shown in Figure 1.

Future work Acknowledgement Figure 2. We are developing and applying a Bio-AFM technique termed Single cell force spectroscopy experiment (SCFS). Figure 2 shows a schematic demonstrating the key approaches of SCFS experiment. Firstly, a tipless AFM cantilever is functionalized with Biotin-Bovine serum albumin(Biotin-BSA), followed by Streptavidin and then BiotinConcanavalin-A (Figure 2 A-C). Secondly, a single cell is picked up and attached to the end of the functionalized cantilever (Figure 2 D-F). The live single cell AFM probes are then brought into contact with a surface and the adhesion forces are measured by conducting AFM force curves. Figure 2A-E shows a schematic representation of a force curve during cell detachment from a surface. The cell-surface adhesion can be separated into different phases. A The cell is in contact with the substrate B,C during cell detachment, the established interactions(specific and non-specific) bonds rupture and the contact zone shrinks. D When the cell body is further separated from the substrate, membrane tethers (nanotubes) are formed and link the cell and substrate until E. the cell is fully detaches from the substrate. (Figure 2 remake from Friedrichs, Legate et al. 2013) Figure 5. Picture (A) shows picking up a cell at the end of cantilever in the electrochemical fluid cell consisting of CO2 independent medium. A cell is picked immediately after settling on the surface and prior to establishing long-term adhesion. (B) Shows other cells spreading on the conducting polymer film (working electrode) after their injection and a longer-period in the electrochemical fluid cell. (A) (B)    Quantifying single cell adhesion as a function of oxidized and reduced conducting polymers. Elucidate and quantify the specific molecular interactions (e.g. single integrin binding)

invol ved in single cell adhesion on conducting polymers. Investigate the reversible switching of integrin binding on conducting polymers via

electrical control. Undertake SCFS measurements on pseudo 3-D structures such as wetspun and electrospun fibres. . This work has been supported by the Australian Research Council under the Australian Research Fellowship and DP110104359 (Michael Higgins). We also greatly acknowledge the ARC Centre of Excellence for Electromaterials Science (ACES) and Australian National Fabrication Facility (ANFF) for providing instrumentation.


Hongrui zhang  
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