pH Response of Carboxy-Terminated Colorimetric Polydiacetylene Vesicles Simon J. Kew and Elizabeth A. H. Hall* Institute of Biotechnology, University of Cambridge, Tennis Court Road, CB2 1QT, United Kingdom Carboxy-terminated polydiacetylene vesicles are known to undergo dramatic color transitions in response to exposure to external stimuli such as pH, temperature, and receptor-ligand binding. FTIR spectroscopy was used to identify the breakdown in the interfacial hydrogen-bonding interactions of the carboxylic acid headgroups of polym- erized 10,12-tricosadiynoic acid (TRCDA) vesicles in aqueous solution during pH chromic transition. The headgroup structure was monitored as the chromic tran- sition takes place and the dissociation dependence of the pK a was determined. Due to the attenuated acidity of the interfacially confined carboxy groups, which exhibit pK a values in the range 9.5-9.9, it was found that the deprotonation-triggered blue-red chromic transition oc- curred in the pH range 9.0-10.1 and that the mechanism of the transition required interaction with the surface carboxyl group, which is of importance in the design of a biochromic mechanism using PDA assemblies. Transmis- sion electron microscopy and FTIR spectroscopy revealed that the surface ionization and the pH-induced chromoge- nic transition was also accompanied by a dramatic vesicle- planar morphological transition alongside subtle changes to the alkyl chain conformation and packing. A two-step mechanism was implicated as causing the chromic transi- tion that first involves surface deprotonation and then specific cation binding, which can aid the design of sensitive surface-ligand chemistry for new PDA struc- tures. The conjugated ene-yne backbone of polymerized diacetylene (PDA) lipids, first reported by Wegner in 1969, 1 has two spectro- scopically distinct phases absorbing at ca. 640 and 540 nm, termed the blue and red phases, respectively. Polydiacetylene membranes have been produced that undergo striking chromogenic transitions in response to external stimuli, typically converting the polymer from the blue phase to the red phase. This has enabled PDA membranes to be used as sensitive probes of environmental perturbations including pH changes, 2 thermal changes, 3 and mechanical stresses. 4 In 1993, Charych et al. reported the colorimetric detection of influenza virus using Langmuir-Blodgett thin films of sialic acid functionalized PDA. 5 This so-called biochromic color transition has also been exploited to detect a wide range of interfacial recognition phenomena reporting ligand- receptor binding processes, 6 so that it appears to have some potential as a bioanalytical platform for colorimetric biosensors based on organized Langmuir-Blodgett monolayers 7 and bilayer vesicles. 8 Of these, vesicle assemblies of 10,12-tricosadiynoic acid (TRCDA) and phospholipids have become the focus of extensive recent investigations by Jelinek and Kolusheva, 9 who have demonstrated the capability to specifically detect interfacial recognition processes ranging from antibody-epitope 10 recogni- tion to ion-ionophore 11 binding. Despite the correlation between a recognition event and biochromic color transition, the role (if any) of the TRCDA carboxy functional group in enabling the chromogenic response mechanism is not well-described. Notwithstanding the nature of the ligand-receptor binding process occurring at the interface, an ionisable headgroup also appears to be required for the biochromic response. This may suggest that the recognition event is not the process that causes the biochromic response directly, but that it influences particular properties of surface structure, leading to a perturbation in the PDA assembly causing a chro- mogenic transition. Recent studies by Cheng et al. 12,13 have established that pH responsiveness in aqueous assemblies of amino acid-terminated amphipathic polydiacetylene is qualitatively dependent on the ionization of the hydrophilic headgroup. The chromatic change upon adjusting pH has been attributed to the repulsive Coulombic interactions developed during surface ionization, which force adjacent chains apart and trigger a conformational change. It is also known that interfacial hydrogen bonding has an effect on the ability of PDA membranes to convert between the blue and red forms, and it has been suggested that the chromatic transition * Corresponding author. E-mail: lisa.hall@biotech.cam.ac.uk. (1) Wegner, G. Z. Naturforsch 1969, 24B, 824. (2) Song, J.; Cheng, Q.; Kopta, S.; Stevens, R. C. J. Am. Chem. Soc. 2001, 123, 3205-3213. (3) Chance, R. R.; Baughman, R. H.; Mu ¨ller, H.; Eckhardt, C. J. J. Chem. Phys. 1977, 67, 3616-3618. (4) Carpick, R. W.; Sasaki, D. Y.; Burns, A. R. Langmuir 2000, 16, 1270-1278. (5) Charych, D. H.; Nagy, J. O.; Spevak, W.; Bednarski, M. D. Science 1993, 261, 585-588. (6) Charych, D. H.; Cheng, Q.; Reichert, A.; Kuziemko, G.; Stroh, M.; Nagy, J. O.; Spevak, W.; Stevens, R. C. Chem. Biol. 1996, 3, 113-120. (7) Geiger, E.; Hug, P.; Keller, B. A. Macromol. Chem. Phys. 2002, 203, 2422- 2431. (8) Okada, S.; Peng, S.; Spevak, W.; Charych, D. H. Acc. Chem. Res. 1998, 31, 229-239. (9) Jelinek, R.; Kolusheva, S. Biotechnol. Adv. 2001, 109-118. (10) Kolusheva, S.; Kafri, R.; Katz, M.; Jelinek, R. J. Am. Chem. Soc. 2001, 123, 417-422. (11) Kolusheva, S.; Shahal, T.; Jelinek, R. J. Am. Chem. Soc. 2000, 122, 776- 780. (12) Cheng, Q.; Peng, T.; Stevens, R. C. J. Am. Chem. Soc. 1999, 121, 6767- 6768. (13) Cheng, Q.; Yamamoto, M.; Stevens, R. C. Langmuir 2000, 16, 5333-5342. Anal. Chem. 2006, 78, 2231-2238 10.1021/ac0517794 CCC: $33.50 © 2006 American Chemical Society Analytical Chemistry, Vol. 78, No. 7, April 1, 2006 2231 Published on Web 03/08/2006