Engineering a biospecific communication pathway between cells and electrodes

Collier and Mrksich. 10.1073/pnas.0504349103.

Supporting Information

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Supporting Figure 5
Supporting Figure 6
Supporting Figure 7
Supporting Figure 8
Supporting Figure 9
Supporting Text

Supporting Figure 5

Fig. 5. Cyclic voltammetry of hydroquinone-terminated self-assembled monolayers (SAMs) of various hydroquinone densities. The electrolyte was PBS. These surfaces demonstrate redox peaks from 160 to175 V and from –75 to –95 V, corresponding to those produced by Chinese hamster ovary cells expressing the cutinase construct (CHO-CUT) action upon 4-hydroxyphenyl valerate monolayers.

Supporting Figure 6

Fig. 6. Cyclic voltammetry of a 30% 4-hydroxyphenyl valerate monolayer before and after a 10-min reaction with 100 nM soluble cutinase in PBS at room temperature.

Supporting Figure 7

Fig. 7. Fluorescence-phase overlays of Chinese hamster ovary (CHO) cells transiently transfected with the cutinase construct containing both an intact b1 integrin tail and intact cutinase domain (a), a mutated cutinase domain (b), and the final construct with a mutated b1 integrin domain (c), compared with untransfected CHO cells (d). The antibody is anti-HA-FITC (catalog no. A488- 101L, Covance). As expected with cationic lipid-based transient transfection, only a subset of CHO cells express the cutinase construct (green). CHO cells transfected with constructs containing an unmutated integrin segment displayed highly rounded morphology (a and b), whereas CHO cells transfected with the b1 integrin mutant (c) spread comparably to untransfected cells (d). (c) Cells were propagated in selection medium and sorted two times via FACS to produce stable transfects, shown in Fig. 3a, reproduced for comparison. These cells appear to spread with wild-type behavior (untransfected cells seen in Fig. 3b).

Supporting Figure 8

Fig. 8. Synthesis scheme of 4-hydroxy-(3 mercaptopropyl) phenyl valerate.

Supporting Figure 9

Fig. 9. ¹H NMR spectra of 4-hydroxy-(3-mercaptopropyl)phenyl valerate.

Supporting Text

Here we present cyclic voltammagrams of monolayers containing 10–30% surface-bound hydroquinone, the redox-active species produced by cutinase action on 4-hydroxyphenyl valerate. These data demonstrate the presence of redox peaks consistent with those observed in the cell studies (Fig. 5). Hydroquinone monolayers were synthesized by the same chemistry as for cell studies, except that in the final step 3-mercaptopropyl hydroquinone was coupled onto the maleimide S-adenosylmethionine instead of the cutinase substrate 4-hydroxy-(3-mercaptopropyl)phenyl valerate. Cyclic voltammetry of these constitutively redox-active hydroquinone surfaces at the same scan rate as used for the cell studies (100 mV·s^–1) produces reduction peaks from 158-177 V and oxidation peaks from –75 to –95 V (Fig. 5). These peaks correspond to the redox peaks generated by CHO-CUT cell action on the 4-hydroxyphenyl valerate surfaces presented in the manuscript (165 V and –105 V). We also demonstrate the evolution of these same redox peaks when 4-hydroxyphenyl valerate monolayers are incubated in the presence of soluble cutinase (Fig. 6). Importantly, we conducted these control experiments in PBS, thus minimizing the potential for artifacts generated by complex cell culture medium constituents. The redox peak positioning for hydroquinone surfaces and soluble cutinase-generated hydroquinone surfaces in these control experiments indicate that the peaks observed in our cell experiments are attributable to hydroquinone production.