Electroformation of Peptide Self-Assembled Monolayers on Gold

Dec 7, 2011 - Centre for Self-Assembled Chemical Structures (CSACS) ... HHHDD-OH must be deposited at 200 mV to maintain an extended configuration, ...
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Letter pubs.acs.org/Langmuir

Electroformation of Peptide Self-Assembled Monolayers on Gold Olivier R. Bolduc,† Debby Correia-Ledo,†,‡ and Jean-Francois Masson*,†,‡ †

Département de Chimie, Université de Montréal, C. P. 6128 Succ. Centre-Ville, Montréal, QC, Canada H3C 3J7 Centre for Self-Assembled Chemical Structures (CSACS)

‡

S Supporting Information *

ABSTRACT: The application of a potential to deposit a monolayer of 3mercaptopropionic acid-histidinyl-histidinyl-histidinyl-aspartyl-aspartyl (3-MPAHHHDD-OH) controls the density and molecular structure of the peptide monolayer, which results in different wettabilities of the surface, surface density, orientation of the molecule (extended or bent), and nonspecific adsorption of serum proteins. 3-MPAHHHDD-OH must be deposited at 200 mV to maintain an extended configuration, which promoted low biofouling properties.

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INTRODUCTION Peptide-based monolayers are suited for reducing nonspecific interactions with serum proteins.1−3 The level of nonspecific interaction was decreased to a few ng/cm2 on Au substrates for surface plasmon resonance (SPR) biosensing with peptide monolayers. The level of nonspecific adsorption of peptide monolayers is comparable to that on other low-fouling surfaces such as polycarboxybetaine4,5 and lower than that on PEG.6,7 Peptides also offer the advantages of simple synthesis methods and the use of peptide coupling reactions to graft stable proteins, enzymes, or antibodies or to carry out His-tag binding chemistry on a modified peptide monolayer. Although peptide monolayers present many advantages as a chemical layer on SPR sensors, they require overnight deposition to attain monolayer formation and achieve good protein resistance to nonspecific adsorption. Alternative methods for the deposition of the peptide monolayer must be investigated. For example, electrochemical deposition provides control over the orientation and surface coverage of thiolated molecules while increasing the deposition rate on the order of minutes instead of hours. Control of the resistivity between the interfaces was employed to form a monolayer of thiol compounds on Au.8,9 The possibility to influence the kinetics of formation of chemical layers of thiolated molecules was investigated by applying an electric potential at the solid−liquid interface of SPR sensors.10−12 The control of monolayer formation is of great interest from a biosensor conception point of view. It could provide a fast, reliable, and inexpensive method to obtain large-area functionalized surfaces on which to conduct specific assays. By modifying the deposition conditions, surfaces demonstrating different properties can be fabricated, allowing the production of more adapted surfaces depending on their final applications. For example, applying a potential (Eapplied) at the liquid−solid interface of an already-formed monolayer can modulate the conformation of the organic molecule immobilized at the surface depending on their chemical properties.13 In some cases, this change in conformation could be exploited to deploy or hide the detection element or a marker from the solution to be analyzed.14 Therefore, it is important to verify that the © 2011 American Chemical Society

application of a potential while forming a monolayer does not alter the properties of a regular self-assembled monolayer, particularly in this case of low fouling monolayers. This letter investigates the influence of the potential applied to an SPR sensor modified with a peptide monolayer and the influence of the deposition potential on the formation of the monolayer. The formation of monolayers of 3-MPA-HHHDD-OH on SPR biosensors, a monolayer previously shown to reduce nonspecific adsorption significantly,2 is a suitable model system for the investigation of the deposition potential by monitoring the process with SPR sensing in real time. The ability of the resulting SPR biosensors to reduce nonspecific interactions of serum proteins is important. As previously observed for other types of organic monolayers that nonspecific adsorption is a function of the surface density,15 the nonspecific adsorption of bovine serum protein on peptide monolayers with different densities needs to be monitored to evaluate the low fouling properties of electrochemically deposited peptide monolayers.

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EXPERIMENTAL SECTION

Electrochemical SPR Measurements. An electrochemical SPR combining dove-prism-based SPR instrumentation16 with a custom 1 mL electrochemical cell and a potentiostat (Biologic SP-150) was constructed to allow the simultaneous real-time monitoring of the electrochemical formation of the peptide monolayer. Bare SPR sensors consisting of gold-coated glass slides were prepared as previously described2 before being mounted on the instrument. The sensitivity of the SPR instrumentation around the refractive index of a 1 M KOH solution in ethanol (n = 1.3726) was 4400 ± 100 nm/RIU, which was measured using sucrose solutions ranging from 1.365 to 1.380 RIU. Thin copper strips covered with electrolyte glue assured the electrical connection with the gold surface used as the working electrode. A freshly cleaned Pt counter electrode and a freshly prepared Ag/AgCl reference electrode were employed for every run to avoid any alteration of the electrodes due to 3-MPA-HHHDD-OH. Received: September 5, 2011 Revised: November 24, 2011 Published: December 7, 2011 22

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Potentiostatic Formation of 3-MPA-HHHDD-OH Layers. 3MPA-HHHDD-OH was synthesized as previously described.2 A 10 mM solution of 3-MPA-HHHDD-OH in a 1 M KOH ethanol solution was freshly prepared before use. Each solution was sparged with nitrogen before the experiment. A volume of 0.50 mL of 1 M KOH in ethanol solution was injected into the electrochemical cell to acquire the s-polarized light used as a reference for SPR measurements. A constant ΔE was applied in amperometric monitoring mode immediately before acquiring p-polarized light (SPR-active). The SPR and amperometric signals were both acquired at 1 Hz for 90 min. Optical measurements were acquired after applying the potential difference to avoid a change in the SPR signal due to the influence of the application of the electrical potential, as previously observed for the application of an oscillating potential on SPR sensors.17 Following the application of a potential difference to the surface for 30 s, 0.50 mL of 10 mM 3-MPA-HHHDD-OH was injected into the electrochemical cell to monitor the formation of the 3-MPA-HHHDD-OH layer. Data was generated in triplicate for each potential investigated. Characterization of 3-MPA-HHHDD-OH Layers. SPR measurements provided information about the density of the layer formed. Contact angle measurements were acquired by depositing 300 μL of 1× phosphate-buffered saline (PBS) on the SPR sensors. A photograph of the PBS droplet was acquired and processed with Image J (NIH freeware). XPS was performed on an ESCALAB 3 MKII with a Mg Kα source operated at 206 W. The mid-IR spectrum was obtained using a Ge-attenuated total reflection (GATR-FTIR) instrument. Peptide monolayers electroformed at different potentials were exposed to bovine serum to measure the amount of nonspecifically bound proteins. This experiment was conducted on the SPR sensors by exposing them to PBS for 5 min to monitor the stable baseline, to crude bovine serum for 20 min, and to PBS for 5 min. The amount of nonspecifically adsorbed proteins was calculated using the Jung equation,18 as previously described for the nonspecific adsorption of serum proteins.19

identical to a self-assembled monolayer deposited overnight at open circuit potential were obtained (Table 1). As expected, Table 1. Electroformation of 3-MPA-HHHDD-OH Layers Eapplied/mV 0 50 100 150 200 250 300 350 400 overnight preparation

time to full monolayer/103 s 3.2 ± 0.2 2.4 ± 0.1 2.1 ± 0.1 1.3 ± 0.1 0.35 ± 0.05 0.67 ± 0.03 0.66 ± 0.03 0.37 ± 0.04 0.38 ± 0.02 16 h

Γ reached after 90 min/ (1015molecules/cm2) 0.22 0.21 0.23 0.22 0.24 a

± ± ± ± ±

0.03 0.05 0.02 0.05 0.04

a a a

0.23 ± 0.04

a

Significantly exceeds the SPR response expected for the surface coverage of a full monolayer. (Measurements in triplicate and the error represent two standard deviations of the mean.)

the formation of the monolayer occurred faster by increasing the potential in that same window (Table 1). Indeed, the surface density achieved by overnight deposition and with electroformation at potentials below 200 mV varies slightly between 0.21 × 1015 and 0.24 × 1015 molecules/cm2. This value is close to the one expected for a fully standing peptide monolayer assuming a standard 0.8 nm diameter for a peptide, which result in 0.2 × 1015 molecules/cm2. For applied potentials higher than 200 mV, the SPR response of 3-MPAHHHDD-OH exceeded the one expected for a full monolayer (Table 1). The shape of the SPR curves for sensorgrams of the formation of a monolayer at applied potentials of 250 to 400 mV suggests a different mechanism of immobilization. By applying a potential of greater than 200 mV to the surface, it takes a slightly longer time to reach equilibrium than at 200 mV but the SPR response stabilizes over a short period of time near that of a full monolayer before rapidly increasing beyond that value until the end of the experiment. Although SPR provides a good measure of the surface concentration, it is also influenced by the orientation of the molecules at the surface. For example, phase transitions where the self-assembled molecules of an alkylthiol chain melts by becoming disorganized with gauche defects creates a thinner monolayer, which results in larger SPR shifts.20 This increase in the SPR shift at high potential may be indicative of the monolayer collapsing to the surface, increasing the local refractive index close to the SPR sensor. However, complementary methods are necessary to elucidate the mechanism of monolayer formation at potentials greater than 200 mV. The electrochemical adsorption of molecules can be observed from the current measured during the formation of the monolayer. Figure 2 shows that increased ΔE induces a faster immobilization of 3-MPA-HHHDD-OH on the surface of the SPR sensor, which is visible as the increased values of initial current (i(t)) and by the shorter time required to reach saturation (a current near 0 mA). For example, the time required to form a monolayer is nearly an hour at 0 mV, whereas it took less than 6 min at 200 mV. This result is in agreement with that observed by Lennox’s group for alkanethiols.10 The amperometric curves suggest that the initial value of i(t) is nearly proportional to the ΔE applied at the interface of the sensors within the range investigated in this study (Figure S2 in the Supporting Information). The shape of

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RESULTS AND DISCUSSION SPR and amperometric measurements are complementary techniques. SPR provides information about the reaction kinetics and surface density for the monolayer deposition at the solid−liquid interface, while amperometry provides information about the number of molecules being reduced or oxidized and the density of the layer formed at this interface. The formation of the 3-MPA-HHHDD-OH monolayer for applied potentials of 0, 200, and 400 mV shows distinct behaviors (Figure 1, a complete set of potential is provided in

Figure 1. Overlay of the SPR sensorgrams for the formation of a 3MPA-HHHDD-OH layer for applied potentials of 0, 200, and 400 mV. The dashed line represents the density for a monolayer self-assembled overnight in ethanol at open circuit potential. A complete version of this figure is available as Figure S1 in the Supporting Information.

Figure S1 in the Supporting Information). By applying a potential from 0 to 200 mV to the SPR sensor, surface densities 23

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Figure 3. Mid-IR spectra of electroformed 3-MPA-HHHDD-OH layers for several applied potentials: 0 (solid black line), 200 (solid blue line), and 300 mV (dashed red line).

Figure 2. Amperometric measurements for the formation of a 3-MPAHHHDD-OH layer for ΔE vs Ag/AgCl of 0, 200, and 400 mV. A complete version of this figure is available as Figure S2 in the Supporting Information.

monolayer at potentials higher than 200 mV, the amide bands shifted to 1648 cm−1 for the amide I band and to 1554 cm−1 for the amide II band. These shifts are attributed to disorder in the formed layer and correlate with previous observations using grazing-incidence X-ray diffraction (GIXD).22 This also agrees with the results obtained from the application of a high potential to organic monolayers.23 Mid-IR clearly demonstrates that the peptide monolayer undergoes a phase transition at high potential because of the bending of the peptide monolayer closer to the surface. Indeed, the peptide is negatively charged in pH 7.4 buffers and in the 1 M KOH ethanolic solution used for electroformation. The presence of K in the monolayer as determined by XPS confirms the anionic form of the peptide monolayer. The applied potential-induced bending of the peptide monolayer at potentials greater than 200 mV correlates well with the expected behavior of the effect of the point of zero charge in a classical Gouy−Chapman−Stern model, leading to a compression of the ions closer to the surface at higher potentials. It also correlates with the apparent reduction in monolayer thickness observed in the SPR data because of the electrostatic attraction of the carboxylate end of the monolayer to the anode. This transition induces disorder in the monolayer, by which the molecule comes closer to the surface because of electrostatic attractions, explaining the large SPR response at a high deposition potential. Cyclic voltammetry (CV) was used to characterize redox processes at the interface of the electrode while depositing the peptide. A broad peak that reaches a maximum current at 300 to 400 mV is observed on the anodic sweep, which correlated with the deposition of the peptide monolayer on the SPR sensor. In addition, for 3-MPA-HHHDD-OH monolayers exposed to potentials greater than the phase transition, it was observed that a second oxidation peak appears at a reductive potential of −130 mV (Figure 4). This same peak was observed for potentiostatic conditions for monolayers deposited at 200 mV for 30 min. The peak observed at −130 mV is quite unusual because it displays very sharp geometry that is uncharacteristic of oxidation/reduction reactions. This suggests a change in conformation in layers partially or completely formed that are exposed to a potential of −130 mV. At this cathodic potential, charge repulsion may occur between the carboxylate anion and the electrode (a cathode at this potential), resulting in a phase transition of the monolayer. The peak observed at −1 V is due to the electrochemical reduction of S−Au, leading to the desorption of 3-MPA-HHHDD-OH from the surface of the sensor. The absence of this peak in the blank run indicates that

the i(t) curves also suggests that the adsorption of 3-MPAHHHDD-OH goes through a rapid adsorption phase, which rapidly stabilizes as a current near 0 mA. This result contrasts with the SPR data, which keeps increasing at longer deposition times. The difference in the SPR and amperometric data can be explained by the organization of the monolayer at the surface. Electroformation is performed in ethanol solutions of KOH such that 3-MPA-HHHDD-OH is negatively charged. At high potentials, the carboxylate anions at the C-terminal end of the peptide further away from the surface are strongly attracted to the anode (SPR sensor). This electrostatic attraction may be sufficient to induce a phase transition to the monolayer, from a well-oriented extended chain to a disordered monolayer, with the terminal carboxylic acid bending toward the surface. Larger applied potentials would facilitate this phase transition. Because the phase transition occurs at potentials greater than 200 mV, this potential is thus optimal for the deposition of 3-MPAHHHDD-OH. Surface-potential-induced phase transitions have been reported for 16-mercaptohexadecanoic acid (16-MHA) monolayers of low density.21 Gauche defects were observed for 16MHA monolayers at high potential because of the attractive potential of the carboxylic acid to a polarized surface. The attractive potential caused the monolayer to bend toward the surface, altering the vibrational signature of the monolayer and physical properties such as the contact angle. This prior evidence further supports a phase transition of the peptide monolayer. The peptides are deprotonated under the deposition conditions (1 M KOH) and were subject to anodic potentials by the polarized electrode. Thus, potentials higher than 200 mV versus Ag/AgCl may have triggered the peptide monolayer to bend closer to the surface, leading to larger SPR signals. Using other techniques such as cyclic voltammetry and molecular spectroscopy can elucidate the phase transition and prove the presence of gauche defects or disordering in the monolayer. XPS analysis of monolayers deposited at 0, 200, and 300 mV demonstrated that the surface density of the peptide monolayer is essentially identical. Thus, XPS supports the formation of a monolayer of constant surface density regardless of the potential applied. Mid-IR spectra obtained for 3-MPAHHHDD-OH deposited at potentials of between 0 and 200 mV agree with the extended conformation observed for layers prepared overnight, as reported in our previous work.2 This was confirmed for monolayers deposited at 0 and 200 mV with amide I band at 1675 cm−1 (Figure 3). By depositing the 24

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Table 2. Deposition-Potential-Dependent Properties of Electroformed Peptide Monolayers with the Deposition Time Set at 30 Minutesa Eapplied/mV 0 100 200 300 Overnight preparation

θc/deg 43 41 32 28 35

± ± ± ± ±

4 2 3 5 4

ΓNSB/(ng/cm2) 174 132 25 96 32

± ± ± ± ±

38 19 14 36 5

a

Measurements were made in triplicate; the error represents two standard deviations of the mean.

for 30 min adsorbed 25 ± 14 ng/cm2 of nonspecific proteins, which is a value statistically equivalent to the level observed for a surface prepared by overnight incubation. This value is equivalent to a few % ML, similar to other low fouling coatings (poly(ethylene glycols) and betaines).4,26−28 However, the peptide monolayers were tested in a more complex biofluid (crude bovine serum), which should account for the slightly larger % ML or nonspecifically bound protein concentration (ng/cm2). Interestingly, the disordered surface resulting from deposition at 300 mV was significantly lower-performing, with 96 ± 36 ng/cm2 serum proteins adsorbed to the surface. This higher amount of nonspecific adsorption in relation to monolayers deposited at 200 mV indicated that the ordering and density of the peptide monolayers influence the nonspecific adsorption properties of SPR biosensors exposed to crude blood serum. It is thus very important to control the conditions of formation of chemically or electrochemically adsorbed layers to maximize the performance of the system in use and to avoid any undesired variation influencing the reproducibility of the method used. Advancing contact angles (θc) provide a relative measure of the hydrophilicity or the hydrophobicity of different surfaces. Partially formed layers of 3-MPA-HHHDD-OH for 0 and 100 mV were more hydrophobic in comparison to monolayers deposited at 200 mV and at open circuit potential overnight, which were both statistically identical (Table 2). For monolayers deposited at higher potentials (400 mV), the hydrophilicity of the surface remains statistically identical to the layer deposited at 200 mV. These results indicate that peptide monolayers deposited at 200 mV versus Ag/AgCl exhibit the same molecular orientation, wettability, and low fouling resistance as peptides deposited overnight at open circuit potential.

Figure 4. Cyclic voltammogram of the electroformation of a 3-MPAHHHDD-OH layer from −2 to 0.8 V at a rate of 10 mV/s starting at 0 V. The dashed red line represents a blank run in the absence of 3MPA-HHHDD-OH. The dotted blue line represents a CV obtained for a larger range of ΔE, allowing the reductive desorption of 3-MPAHHHDD-OH. Black arrows represent the starting point of each run.

the capacitive current dominates the response and that this transition is not due to an artifact from the SPR sensor’s surface but to the presence of a layer of 3-MPA-HHHDD-OH at the liquid−solid interface (Figure 3). These results strongly suggest that the monolayer undergoes a reversible phase transition depending on the potential applied to the SPR sensor. Mid-IR, CV, and SPR clearly demonstrated that 3-MPA-HHHDD-OH undergoes a reversible phase transition at potentials greater than 200 mV because the electrostatic attractions of the monolayer to the anode SPR sensor create disorder in the monolayer. Such a reversible nature of the reorientation of selfassembled monolayers with cathodic and anodic potentials was observed for 16-MHA monolayers.21 Thus, the peptide monolayer returns to the extended conformation at low potentials. SPR monitors the quantity of nonspecifically adsorbed proteins at the interface. To verify that electroformed peptide monolayers could be used as low fouling surfaces for biosensing in crude serum, electrodeposited monolayers were exposed to crude bovine serum. These experiments were performed at open circuit potential for monolayers deposited for 30 min at various applied potentials. Thereby, a range of monolayer surface coverages was obtained. The resistance to nonspecific adsorption is reported here in ng/cm2, which is a measure related to the total quantity of proteins adsorbed. Bovine serum is a complex mixture of proteins, and the composition of proteins adsorbing to the surface is relatively unknown although albumin is suspected to adsorb along with IgG and fibrinogen.24,25 As a comparison, the seminal work of Whitesides et al. reported the adsorption of pure solutions of a single protein, in % monolayer (% ML). Low fouling surfaces achieved about 1% ML with diluted protein solutions at 1 mg/mL,4,26,27 whereas 100% ML leads to a change in the SPR response of approximately 4 to 5 kRU (1 RU ≈ 0.1 ng/cm2), thus 400−500 ng/cm2. In comparison, other low fouling surfaces lead to the adsorption of serum proteins on the order of single digit ng/cm2.28 Partially formed monolayers of 3-MPA-HHHDD-OH provide poorer resistance to the nonspecific adsorption of serum proteins (Table 2). Indeed, monolayers deposited at 0 and 100 mV nonspecifically adsorbed 174 ± 38 and 132 ± 19 ng/cm2 of proteins, respectively, far from the performance of the monolayer deposited at open circuit potential, which adsorbed 32 ± 5 ng/cm2. The layer electroformed at 200 mV

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CONCLUSIONS

This study showed that the electroformation of 3-MPAHHHDD-OH monolayers at 200 mV significantly reduces the preparation time (less than 6 min) required to obtain a monolayer offering the same properties as a layer selfassembled by overnight incubation. It was shown that the peptide monolayer adopts an extended or bent conformation depending on the potential applied to the surface, which influenced the low biofouling properties of the monolayer. Advancing contact angles and mid-IR and SPR measurements showed that the electroformation of peptide-based layers could lead to surfaces with different properties using the same molecule by varying the difference in potential applied. 25

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(24) Green, R. J.; Davies, J.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B. Biomaterials 1997, 18, 405−413. (25) Green, R. J.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B. Biomaterials 1999, 20, 385−391. (26) Holmlin, R. E.; Chen, X. X.; Chapman, R. G.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17, 2841−2850. (27) Ostuni, E.; Chapman, R. G.; Holmlin, R. E.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17, 5605−5620. (28) Vaisocherova, H.; Yang, W.; Zhang, Z.; Cao, Z. Q.; Cheng, G.; Piliarik, M.; Homola, J.; Jiang, S. Y. Anal. Chem. 2008, 80, 7894−7901.

ASSOCIATED CONTENT S Supporting Information * Complete data set for the electroformation of 3-MPAHHHDD-OH. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Tel: 1-514-343-7342. Fax: 1-514-343-7586.

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ACKNOWLEDGMENTS Financial support was provided by the Canada Foundation for Innovation (CFI), the National Sciences and Engineering Research Council of Canada (NSERC), the Fonds Québécois de la Recherche sur la Nature et les Technologies (FQRNT), and the Centre for Self-Assembled Chemical Structures (CSACS).

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REFERENCES

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