Direct Observation of Reversible Biomolecule Switching Controlled By Electrical Stimulus

Control and reversibility of biomolecular interactions at engineered interfaces presents opportunities to develop highly effi cient substrates and devices for a wide range of biomedical applications. [ 1‐4 ] A major challenge nowadays in the fi eld of stimuli-responsive interfaces is to acquire a molecular understanding of the changes occurring at the biointerface upon external stimulation. Herein, we used in situ Sum-FrequencyGeneration (SFG) spectroscopy to study changes in molecular orientations in electrically switchable biofunctionalized selfassembled monolayers (SAMs). The bioactivity of a mixed SAM on gold consisting of a biotin-terminated positively charged oligopeptide (biotin-KKKKC) and a tri(ethylene glycol)-terminated thiol is shown to be related to a switch between upward exposure and random orientation of the biotin group in response to positive and negative applied potentials, respectively. The fi ndings reported here support the mechanism by which charged biomolecules control biomolecular interactions, for example, protein binding affi nities, and lay the foundation for future studies aiming to explore molecular conformational changes in response to electrical stimuli. Dynamic surfaces are particularly attractive for biomedical applications and are playing an increasingly important part in the development of highly sensitive biosensors, [ 5‐7 ] novel


SAM Preparation
The gold substrates were cleaned by exposure to UV light for 1 h and immediately rinsed with Ultra High Pure (UHP) H 2 3 to prevent the formation of hydrogen bonds between the NH 2 functional groups of the bound thiolate peptide on Au surface and that of free thiol peptide in the bulk solution. [1]

Contact Angle
Contact angles were determined using a home-built contact angle apparatus, equipped with a charged coupled device (CCD) KP-M1E/K camera (Hitachi) that was attached to a personal computer for video capture. The dynamic contact angles were recorded as a micro-syringe was used to quasi-statically add liquid to or remove liquid from the drop. The drop was shown as a live video image on the PC screen and the acquisition rate was 4 frames per second. FTA Video Analysis software v1.96 (First Ten Angstroms) was used for the analysis of the contact angle of a droplet of UHP H 2 O at the three-phase intersection. The averages and standard errors of contact angles were determined from five different measurements made for each type of SAM.

Ellipsometry
The thickness of the deposited monolayers was determined by spectroscopic ellipsometry. A Jobin-Yvon UVISEL ellipsometer with a xenon light source was used for the measurements.
The angle of incidence was fixed at 70°. A wavelength range of 280-820 nm was used. The DeltaPsi software was employed to determine the thickness values and the calculations were based on a three-phase ambient/SAM/Au model, in which the SAM was assumed to be isotropic and assigned a refractive index of 1.50. The thickness reported is the average and standard error of six measurements taken on each SAM.

X-ray photoelectron spectroscopy (XPS)
XPS spectra were obtained on the Scienta ESCA300 instrument based at the Council for the Central Laboratory of the Research Councils (CCLRC) in The National Centre for Electron Spectroscopy and Surface Analysis (NCESS) facility at Daresbury, UK. XPS experiments were carried out using a monochromatic Al K α X-ray source (1486.7 eV) and a take-off angle of 15°. High-resolution scans of N (1s) and S (2p) were recorded using a pass energy of 150 eV at a step size of 0.05 eV. Fitting of XPS peaks was performed using the Avantage V 2.2 processing software. Sensitivity factors used in this study were: N (1s), 1.73; S (2p), 2.08; Au (4f 7/2), 9.58; Au (4f 5/2), 7.54.

Characterisation of the biotin-KKKKC, TEGT and biotin-KKKKC:TEGT SAMs
The formation of pure biotin-KKKKC, pure TEGT and mixed biotin-KKKKC:TEGT SAMs was studied by means of contact angle and ellipsometry (Table S1). As expected, the water advancing and receding contact angles for the pure TEGT SAM revealed a hydrophilic monolayer. The pure biotin-KKKKC SAM formed a less hydrophilic surface, with biotin-KKKKC:TEGT SAM exhibiting contact angles in between those observed for pure monolayers of either components. This intermediates contact angles values obtained support the formation of the mixed SAM. Ellipsometry analysis of the three surfaces showed the formation of monolayers after 12 h immersion time with thickness values close to the theoretical measurements (obtained from Chem 3D software). In particular, the thickness values observed for the biotin-KKKKC:TEGT SAMs were closer to those found for the TEGT SAMs revealing an higher concentration of TEGT compared to biotin-KKKKC on the surface. The mixed biotin-KKKKC:TEGT SAM has been previously [1] characterised by X-ray photoelectron spectroscopy (XPS) and an average ratio on the surface of 1:16 ± 4 was observed. Furthermore, Fourier transform infrared reflection-absorption spectroscopy (FT-IRRAS) analysis of the modified gold surfaces were performed and the characteristic peaks for each of the three SAMs are highlighted in Figure S1. All the SAM surfaces presented peaks in the CH region between 2800-3000 cm -1 . In particular, three peaks for the biotin-KKKKC SAM and the biotin-KKKKC:TEGT SAM and two peaks for the TEGT SAM were observed. In addition, the biotin-KKKKC SAM displayed a band at ~1675 cm -1 which was readily assigned to the amide band of the peptide groups. [2][3] This strong band in the amide region pertaining to the ureido bicycle of the biotin molecule end-group. [5] The presence of the additional small and sharp band at ~3020 cm -1 could be associated to a Fermi resonanceenhanced overtone coming from the amide II entities [6] as well as the NH 3 + stretching of the oligolylisine backbone of biotin-KKKKC. However, available literature report these stretching at higher frequencies (~3300 cm -1 ) [7], [8][9] . The fact that this contribution is appearing as a dip on the nonresonant signal is indicative of an orientation of the transition dipole moment (TDM) away from the surface. Within the mixed SAM, features in these spectral regions are less pronounced and of more difficult interpretation. However, a small peak at ~3220 cm -1 could still be observed.
The differences between the SFG spectra of biotin-KKKKC and biotin-KKKKC-TEGT could be mainly imputed to the lower surface density for the latter surface and the higher rate of disorder of the molecules which can arrange in a greater variety of conformations. As a result, the sum of TDM from this more isotropic system is lower in comparison to an ordered system such as the biotin-KKKKC SAM in air. Therefore, as the strength of SFG signals depends on both density and order, the intensity is less pronounced in the case of the mixed SAM.
Furthermore, this SAM might not be uniformly mixed throughout the sample area and domains of either biotin-KKKKC or TEGT may be present on the surface.

Electrochemical Sum Frequency Generation (SFG)
The

Control studies performed with biotin-KKKKC SAM
The SFG experiment described above was performed on the biotin-KKKKC SAM in order to prove that switching was only possible due to the space between the peptide moieties provided in the biotin-KKKKC:TEGT SAM. Figure S3a shows the overlapping of the biotin-KKKKC SFG spectra recorded at positive and negative potential. Both the SFG spectra and the SPR results show that there are no significant changes in the film between positive and negative potential applied. Isotropic arrangement largely remains for the biotin-KKKKC SAM due to steric hindrance, therefore no detectable reorientations for the biotin occurs as opposed to the biotin-KKKKC:TEGT film, in which the peptides have sufficiently space to reconfigure towards a random orientation. In particular, the SFG spectra at positive and negative potential are almost completely overlapping but the peak at 3220 cm -1 is not present suggesting a higher disorder rate of the surfactant molecules compared to the same surface analysed in air ( Figure S2). Figure S3b shows the Relative Response Units (RRU) found by electrochemical SPR analysis for biotin-KKKKC SAMs. The difference between the sensorgrams obtained at the two potentials is less than 100 RRU and the affinity of the biotin group for the Neutravidin is still high in both cases, indicating that the interaction biotin-NeutrAvidin is independent from the alignment of the biotin groups. Figure S4