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Detection of Molecular Interactions with Modified Ferrocene Self-Assembled Monolayers Man Yi Ho,† Peng Li,† Pedro Estrela,‡,† Sarah Goodchild,§ and Piero Migliorato*,† UniVersity of Cambridge, Engineering Department, Electrical Engineering DiVision, 9 J.J. Thomson AVenue, Cambridge CB3 0FA, U.K., and Defence Science and Technology Laboratory, Porton Down, Salisbury SP4 0JQ, U.K. ReceiVed: May 19, 2010; ReVised Manuscript ReceiVed: July 4, 2010
Ferrocene-terminated self-assembled monolayers (Fc-SAMs) are one of the most studied molecular aggregates on metal electrodes. They are easy to fabricate and provide a stable and reproducible system to investigate the effect of the microenvironment on the electron transfer parameters. We propose a novel application for Fc-SAMs, the detection of molecular interactions, based on the modification of the SAM with target-specific receptors. Mixed SAMs were fabricated by coimmobilization on Au electrodes of thiolated alkane chains with three different head groups: hydroxy terminating head group, ferrocene head group, and a functional head group such as biotin. Upon binding, the intrinsic electric charge of the target (e.g., streptavidin) modifies the electrostatic potential at the plane of electron transfer, causing a shift in the formal potential E°′. The SAMs were characterized by AC voltammetry. The detection mechanism is confirmed by measurements of formal potential as a function of electrolyte pH. 1. Introduction Ferrocene-terminated self-assembled monolayers (Fc-SAMs) on metal electrodes have been a topic of investigation for many years.1–23 As they provide a robust reproducible system, FcSAMs have been extensively used to study mechanisms affecting electron transfer.1–3 Smith and White4 proposed a model highlighting the interplay between electrochemical double layer (DL) potential, SAM capacitance, and distance of the redox centers from the electrolyte in controlling the formal potential and voltammetric peak current. Improved models were later developed by Fawcett and co-workers5,6 to take into account the discreteness of redox molecule charges, the nature of the SAM-electrolyte interface, and ion association effects. Experimental studies on the role of the DL,7,8 microenvironment,9 and ion pairing10–12 have also been published in past decades. We propose here a novel application of Fc-SAMs: the detection of molecular interactions, based on the incorporation of targetspecific receptors in the SAM. To demonstrate the principle, we used mixed Fc-SAMs with charged head groups. Three different thiol-terminated chains were coimmobilized on a Au electrode: (1) a chain with ferrocene (Fc) head groups; (2) a hydroxy alkane chain; (3) an alkane chain with a charged head group, namely, -NH3+ or -COO-. The hydroxyl alkane thiol passivates the Au and is essential to create a well-organized SAM. When the redox molecules are incorporated in the charged SAM, we observe a directional shift of the formal potential (E°′) in AC voltammetry upon chemical neutralization of the charged head groups. Neutralization of -NH3+ produces a negative shift in E°′, while a positive shift is observed upon neutralization of -COO- groups. Since many biomolecular interactions result in a change of charge and a variety of receptors can in principle be attached to alkane chains, this approach can provide a generic * Corresponding author. E-mail:
[email protected]. Phone: +44-1223748302. Fax: +44-1223-748348. † University of Cambridge. ‡ Current address: University of Bath, Department of Electronic & Electrical Engineering, Bath BA2 7AY, U.K. § DSTL, Porton Down.
label-free biodetection platform. We have demonstrated the approach by successfully detecting the biotin-streptavidin interaction. For this system, we find that the measured formal potential shifts vs pH show good agreement with the theoretical streptavidin titration curve. Our results can be explained on the basis of the model proposed by Smith and White.4 2. Methods 2.1. Apparatus. Electrochemical measurements were performed in a three-electrode cell consisting of a Hg/Hg2SO4 reference electrode, a Pt wire counter electrode, and a bulk polycrystalline Au electrode with a diameter of 2 mm. An AUTOLAB PGSTAT302/FRA2 electrochemical analysis system (Eco Chemie, The Netherlands) was used. 2.2. Chemicals. All materials were used as received. Deionized water was used in all solutions. Streptavidin, sodium monobasic phosphate, sodium dibasic phosphate, phosphoric acid, n-hydroxysulfosuccinimide (sulfo-NHS), 3-(maleimide)propionic acid NHS ester, 6-hydroxy-1-hexane thiol (HHT), 11hydroxy-1-undecane thiol (HUT), 11-mercaptoundecanoic acid (carboxylic acid thiols), DMSO, and EDC were purchased from Sigma-Aldrich. 6-Ferrocenyl-1-hexane thiol (FcHT) and 11ferrocenyl-1-undecane thiol (FcUT) were purchased from NBS Biological. 11-Amino-1-undecane thiol hydrochloride (amine thiols) and biotin-terminated tri(ethylene glycol) undecanethiol (biotin-PEG-UT) were purchased from Assemblon. Ovalbumin (OVA)-specific scFv antibody (MW 27000) was provided by the Defence Science and Technology Laboratory (DSTL). Decon, a cleaning agent, is purchased from Veltek Associates. 2.3. Electrode Cleaning and Modification. Bulk polycrystalline Au electrodes with a radius of 1 mm (CH Instrumetns, U.S.) were first sonicated in 3% Decon for 5 min. After rinsing with deionized water, the electrode was polished with 0.03 µm alumina for 5 min. Further sonication was carried out for another 5 min in deionized water. The electrode was then polished with blank polishing paper. Finally, it was rinsed under deionized water and sonicated again in deionized water for a further 5 min. After the mechanical polishing steps, electrochemical
10.1021/jp104560e 2010 American Chemical Society Published on Web 07/23/2010
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cleaning with 0.5 M H2SO4 as the electrolyte was employed where the potential was swept from -0.05 V to 1.1 V vs Hg/ Hg2SO4 for 60 cycles. Following this pretreatment, electrodes were immersed in an ethanol solution containing the thiols of interest for 48 h. Three different compositions were used to create: (i) Positively charged redox SAM The coating solution of a positively charged redox SAM contained FcHT, HHT, and the functional strands amine thiol in ethanol. The composition of the surface layer was related to the ratio of the different types of thiol in the coating solution; hence, it was possible to prepare the monolayer with desired ferrocene coverage by adjusting the composition of the coating solution. The ratio used was 1:5:5 (FcHT:HHT:amine thiol) with the concentration of FcHT being 0.2 mM. (ii) Negatively charged redox SAM The coating solution of a negatively charged redox SAM contained FcHT, HHT, and the functional strands carboxylic acid thiol in ethanol. The ratio used was 1:5:5, respectively, with the concentration of FcHT being 0.2 mM. (iii) Biotin functionalized redox SAM The biotin functionalized redox SAM was obtained from a solution containing FcUT, HUT, and biotin-PEG-UT in the ratio 1:5:2.5, with the concentration of FcUT being 0.2 mM. 2.4. Neutralization of Amine Head Groups. The positive charge of the SAM is due to protonation of the amine head groups in pH7 phosphate buffer. To neutralize these head groups, the electrode was immersed in a solution containing 1 mM 3-(maleimide) propionic acid NHS ester in DMSO for two hours. AC voltammetry was used to characterize the SAM before and after neutralization. The potential scan ranged from -0.6 to 0.25 V vs Hg/Hg2SO4, the rms amplitude and frequency of the superimposed AC excitation were 10 mV and 18 Hz. All measurements were done in 5 mM pH7 phosphate buffer. 2.5. Neutralization of Carboxylic Acid Head Groups. The negative charge of this SAM is due to deprotonation of the carboxylic acid head groups at pH 7. Neutral head groups were obtained by immersing the electrodes for 2 h into a solution containing EDC, sulfo-NHS, and ethanolamine at 40, 10, and 20 mM in deionized water, respectively. AC voltammetry was used to investigate the redox properties before and after the neutralization. The settings were the same as in section 2.4. All measurements were done in 5 mM pH 7 phosphate buffer. 2.6. Streptavidin-Biotin Interaction. Electrodes were incubated with streptavidin in 10 mM pH 7 phosphate buffer at a concentration of 20 µM for 1 h. As a control, electrodes were incubated with 10 µg/mL of ovalbumin-specific scFv antibody in PBS for 1 h. Measurements were carried out in two sets of buffers, 13 mM pH 2.5 phosphate buffer or 5 mM pH 7 phosphate buffer. AC voltammetry was used. The potential scan ranged from -0.45 to 0.45 V vs Hg/Hg2SO4; the amplitude and frequency of the superimposed rms signal were 10 mV and 2 Hz. The dependence of the E°′ shift with pH after binding of streptavidin was also investigated. AC voltammetry and phosphate buffers with pH ranging from 7 to 2.5 were used, with the buffer ionic strength being kept constant. 3. Results 3.1. Effect of Neutralizing Positively Charged Head Groups on Formal Potential. Figure 1 shows the AC voltammogram after baseline correction before and after neutralization. E°′ changed by -50 mV, from -47 to -97 mV.
Ho et al.
Figure 1. AC voltammograms at 18 Hz after baseline correction, before and after neutralization of -(CH2)11NH3+; 5 mM pH 7 phosphate buffer was used.
Figure 2. AC voltammograms at 18 Hz after baseline correction, before and after neutralization of -(CH2)11COO-; 5 mM pH 7 phosphate buffer was used.
Figure 3. AC voltammograms measured at 2 Hz after baseline correction upon (a) adding streptavidin and (b) adding OVA antibody as a control. The measurement buffer was 13 mM pH 2.5 phosphate buffer.
3.2. Effect of Neutralizing Negatively Charged Head Groups on Formal Potential. The AC voltammograms before and after neutralization are shown in Figure 2. E°′ shifts by +104 mV, from -286 to -182 mV. 3.3. Biotin-Streptavidin Interaction. Figure 3 shows the AC voltammograms at pH 2.5, for the interaction between streptavidin and the biotin SAM. As shown in Figure 3a, a positive shift in E°′ by 43 mV was observed after incubation with streptavidin. The control experiment with OVA antibody, which is not expected to specifically bind to the SAM, is shown in Figure 3b; a negative shift in E°′ by 6 mV was observed. A reduction in peak current was also observed in both cases. At pH 7, no significant shift in E°′ was observed for either streptavidin or for the control.
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φPET
E°' ) E° + φPET - φS σM + σPET + σ 2RT - φS ) sin h-1 zF 8RTε ε c
[
[
√
o 3 el
σM ) C1 E° + (φref - φS) σPET ) FΓT[zof + zR(1 - f)]
Figure 4. Schematic illustration of irreversibly adsorbed redox SAM: (a) before specific molecular interaction;4 (b) proposed model after specific molecular interaction. φM, φPET, φF, and φS are the absolute potentials at the metal, plane of electron transfer (PET), the end of the film, and the bulk solution. ε1, ε3, and ε4 are the dielectric constant of the SAM, the bulk solution, and the target. C1 and Cdif are the capacitance of the SAM and the diffuse layer.
4. Discussion It has been shown by Smith and White4 that the formal potential of the immobilized redox active species is a linear function of the potential at the plane of electron transfer (PET). Figure 4a illustrates schematically our redox SAM. The Fc molecules are indicated by open circles at the plane of electron transfer (PET), which is separated from the electrode surface by a dielectric made of carbon chains (zigzag lines) with dielectric constant ε1. The interfacial potential profile is represented by a solid line. The potential drop is linear across the dielectric region between the metal and the PET. Beyond the PET, the potential profile within the bulk solution follows the diffuse layer behavior described by the GuyChapman model. Although more accurate treatments have been developed, this model is employed here because of its simplicity and ability to explain the main features of our experiment, particularly the principle of the detection method. 4.1. Model. The relevant equations4 are given below. We introduce an extra term σ in the equation to represent the charge per unit surface associated with charged head groups or targets that are assumed, for simplicity, to be located at the plane of electron transfer. The potential applied to the electrode, E, can be written as
E ) E°' +
ln ( RT nF ) [ Γ ] ΓO R
where
(1)
]
1-f ln ( RT nF ) ( f )]
In eq 1, E° and φref are the standard electrode potential and the potential of the reference electrode, respectively. R, T, n, and F are the molar gas constant, the absolute temperature, the number of electrons involved in the redox reaction, and Faraday’s constant. ΓT, ΓO, and ΓR are the total, oxidized, and reduced redox molecule surface densities, f ) ΓO/ΓT, zO and zR are the charge of the oxidized and reduced redox molecules, cel is the electrolyte concentration, σM and σPET are the charge densities on the electrode and PET, and E°′ is the formal potential. Figure 5 shows a comparison between experimental and theoretical E°′ as a function of the electrolyte ionic strength for the SAM 1:5 Fc:HHT. The experimental data are fitted to eq 1 using E° as a fitting parameter. Input parameters are ε1 ) 2.6, ε3 ) 78.5, and (φS - φref) ) -0.85 V vs Hg/Hg2SO4. ΓT ) 1.38 × 10-10 mol cm-2 is obtained from the peak current value of the AC voltammogram, according to eq 2 below.22 Eac and ν are the voltage amplitude and frequency of the ac excitation. Ntotal is the total moles of redox species in the surface layer. In order to account for the fact that the electrolyte is not a 1:1 electrolyte in our case, the results are plotted vs ionic strength, instead of concentration. The agreement between theory and experiment is good. The fit gives a E° value of about -0.22 V vs Hg/Hg2SO4. This procedure provides a simple way to characterize a redox SAM.
Ipk ) 2nνFNtotal
sinh(nFEac /RT) cosh(nFEac /RT) + 1
(2)
4.2. Effect of Molecular Interaction on Formal Potential. The results in sections 3.1 and 3.2, where the neutralization of positively charged and negatively charged SAM caused shifts in E°′, can be interpreted according to the above model. The neutralization of -NH3+ causes a decrease in σ, lowering the potential at PET, thus reducing φPET - φS. Since E°′ ) E° + (φPET - φS), a negative shift is observed and vice versa for the neutralization of the -COO-. As illustrated in Figure 4b, interaction with the target molecules causes a modification of the interfacial potential profile. For the biotin-streptavidin system, the net charge of streptavidin depends strongly on the pH of the surrounding solution. The charged groups in the amino acids that make up streptavidin undergo protonation for decreasing pH and the calculated net charge per molecule vs pH is shown in Figure 6 (solid curve) using the software Proteine M.M., pI, composition, titrage program (ABIM, France: http://www.iutarles.up.univ-mrs.fr/w3bb/d_abim/). This program considers only the amino acid sequence, ignoring protein folding or the position of the amino acid side-chains. This will inevitably introduce error in the prediction, as the charges buried inside the protein will have little contribution to the total net charge. At pH 2.5, the positively charged streptavidin causes an increase in charge densities at the PET; therefore, a positive shift in E°′ was observed in section 3.3.
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Ho et al. rocene SAM provide an excellent platform for future experimentation with a range of probe-target combinations. Acknowledgment. We acknowledge the financial support for this work by the Defence Science and Technology Laboratory. References and Notes
Figure 5. Experimental and theoretical dependence of formal potential upon electrolyte ionic strength for a SAM composition of 1:5 Fc:HHT.
Figure 6. Investigation of the effect of buffer pH on the formal potential of the streptavidin-biotin SAM; the titration curve for streptavidin (solid) is calculated using the software Proteine M.M., pI, composition, titrage program (ABIM, France: http://www.iut-arles. up.univ-mrs.fr/w3bb/d_abim/).
The streptavidin-reacted electrodes were then measured in buffers of different pH, and the measured shifts in E°′ are also shown in Figure 6. It is important to emphasize here that in our SAM the redox potential is pH-independent,24 since hydrogen ions do not participate in the redox process. Therefore, the shifts in E°′ vs pH can only be due to the change in σPET. This can result from protonation of the -OH group of HUT25 and the streptavidin charge. The trend in the shift in formal potential agrees well with the titration curve of streptavidin, suggesting that the dominant effect on σPET is due to the streptavidin charge rather than the -OH group. It should be pointed out that the redox characteristics of the Fc can also be affected by ion pairing. Therefore, in order to keep the same ionic environment, the pH titration experiment was done in the same type of buffer (phosphate buffer), which only offers a limited pH range. 5. Conclusion This paper has presented a study by AC voltammetry of the effect of molecular interactions on the electrochemical properties of mixed ferrocene-terminated SAMs. Our results on the neutralization of charged head groups and on pH titration for the biotin-streptavidin system indicate that the main effect of the interaction is the modification of the electrostatic potential at the plane of electron transfer, resulting in a directional shift of the formal potential. Our work indicates that the use of mixed ferrocene-terminated SAMs deserves consideration for the development of a generic detection platform for molecular interactions. These SAMs can in principle be functionalized with a variety of capture probes such as nucleic acids, antibodies, peptides, and proteins. The detection relies on the intrinsic charge of the target molecules, eliminating the need for labels. The ease in fabrication, reproducibility, and robustness of fer-
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