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Measuring and Remediating Nonspecific Modifications of Gold Surfaces Using a Coupled in Situ Electrochemical Fluorescence Microscopic Methodology Zhinan Landis Yu,†,× Cheng Wei Tony Yang,‡,§,× Eleonore Triffaux,∥ Thomas Doneux,∥ Robin F. B. Turner,‡,⊥ and Dan Bizzotto*,† †

AMPEL, Department of Chemistry, The University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada Michael Smith Laboratories, The University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada § Department of Chemical and Biological Engineering, The University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada ∥ Chimie Analytique et Chimie des Interfaces, Faculté des Sciences, Université libre de Bruxelles (ULB), 1050 Bruxelles, Belgium ⊥ Department of Electrical and Computer Engineering, The University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada ‡

S Supporting Information *

ABSTRACT: In surface-based biosensors, the nonspecific or undesired adsorption of the probe is an important characteristic that is typically difficult to measure and therefore to control or eliminate. A methodology for measuring and then minimizing or eliminating this problem on gold surfaces, readily applicable to many common surface modifications is presented. Combining electrochemical perturbation and fluorescence microscopy, we show that the potential at which the adsorbed species is removed can be used as an estimate of the strength of the adsorbate−surface interaction. This desorption potential can be easily measured through an increase in fluorescence intensity as the potential is manipulated. Furthermore, this method can be used to evaluate strategies for preventing or removing nonspecific adsorption. This is demonstrated for a wide variety of surface modifications, from strongly chemisorbed monolayers such as thiol self-assembled monolayers (SAMs) to physisorbed monolayers as well as for complex surface structures like peptide and DNA mixed-component SAMs. The use of a coadsorption strategy or small magnitude potential-step cycles was shown to significantly decrease the amount of nonspecifically or noncovalently bound probe, creating better defined surfaces.

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modified.1,13−16 The type of immobilization method chosen will affect the biosensor performance due to changes in the probe’s coverage, orientation, structure, and biological activity.3,6,16 Therefore, the probe immobilization strategy must be carefully assessed. For example, gold surfaces, often used in electrochemical biosensors, have probes immobilized using physisorption, chemisorption, or covalent immobilization (postfunctionalization).15,17 Physisorption often yields a surface with weak probe attachment and random orientation. Chemisorption typically creates strong and stable probe covalent attachment with some control over the probe orientation (e.g., thiolated peptides or nucleic acids which create a self-assembled monolayer (SAM)). Covalent immobilization or postfunctionalization describes the formation of a covalent bond between the SAM-modified surface and the

n important step in the fabrication of successful surfacebased biosensors is the immobilization (or attachment) onto the surface of the biological capture probe that is selective toward a particular target of interest.1−4 Ideally, the probe would be adsorbed onto the surface with a well-defined orientation and at a surface coverage that yields the maximum number of possible binding events and therefore signal.5−9 This often requires many rinsing steps in addition to using other adsorbates to ensure the optimal probe surface coverage and for passivation of the remaining surface. Measuring the surface coating characteristics (e.g., coverage, orientation, or distribution) is required in order to obtain reproducible and optimal construction of sensitive and selective sensing surfaces. The adsorption of some fraction of the probes in a nonideal orientation or through a nonideal or undesired surface modification or interaction is possible. Its prevention or minimization is important for optimal sensor performance.10−12 Strategies used to immobilize the probes onto a sensor surface depends on the type of probe (e.g., antibody, enzyme, peptide, or nucleic acid) and on the surface to be © XXXX American Chemical Society

Received: October 7, 2016 Accepted: November 24, 2016 Published: November 24, 2016 A

DOI: 10.1021/acs.analchem.6b03953 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry reactive groups present on the probe3,15 providing flexibility in the design of surface-based sensors. One example is the activation of carboxylic acid-terminated SAMs using carbodiimide that facilitates reaction with primary amines on the probe molecule.18 Typically, the efficiency of a particular probe immobilization procedure is evaluated based on free probe−target interaction analysis. In reality, however, the sensing surfaces are often less than ideal. For example, chemisorption and/or covalent immobilization often results in overcrowding of the probes on the surface.7,9 The majority of the probes may be strongly bound to the surface (specific adsorption), while a portion of the probes may be weakly adhered (nonspecific adsorption). Extensive washing with buffer19,20 and competitive replacement with small alkylthiol molecules (creating a mixed SAM) are often used to remove these nonspecifically adsorbed species.21,22 However, other sources of nonspecific adsorption can be present due to defects on the gold surface or strong interadsorbate interactions.23 These subtle variations and imperfections of the sensing surface may affect the probe immobilization and, in turn, the measurement accuracy, specificity, and/or sensitivity. Therefore, the construction of biosensors with robust, reproducible, and functional surfaces requires careful analysis of the interface after probe attachment. Surface analysis of the sensor during each step in the preparation is typically performed ex-situ (e.g., electron spectroscopies, surface-selective infrared spectroscopy, and STM or AFM). Ideally, the detailed surface analysis should be accomplished in situ. Even though experimentally complex, this provides a more realistic evaluation of the surface modification in the aqueous sensing environment. In the case of electrochemical-based sensors, current or charge measurements can be used to characterize its analytical performance.17,24,25 Typically, this approach uses redox current and/or impedance measurements. While useful in the measurement of target recognition events, it provides limited insight into the nonideal or nonspecific adsorption, unless these undesired adsorbates strongly modify the electrochemical signal. Developing sensitive and specific evaluation methods is critical for biosensor optimization.The best method for reducing or eliminating nonspecific adsorption can be determined and then tailored for each type of biosensor. In-situ spectroelectrochemical fluorescence microscopy, a method that couples electrochemistry and fluorescence microscopy capabilities, has been used to interrogate electrode surfaces modified with a variety of fluorophore-labeled adsorbates.26−28 The fluorescence intensity increases as the distance separating the fluorophore from the gold surface increases and provides a clear indication of when adsorbates leave the surface.26,29 This combined technique has been applied to monitor electrochemical adsorption/desorption of nucleic acid and alkylthiol SAMs to/from the Au electrode26−29 through measurement of interfacial capacitance and fluorescence intensity both as a function of applied potential. Changes in the adsorbed layer can be determined through capacitance as well as through changes in fluorescence (most sensitive to the region near but not on the electrode surface). Therefore, perturbing the interface using an electrochemical potential, which modifies the surface energy and changes the electrostatic nature of the interface can result in the removal (displacement by solvent) of all adsorbed species. This desorption will occur at different potentials depending on the strength of adsorbate− surface interactions. This coupled approach enables an in situ

measurement of the various adsorbate−surface interactions by way of an increase in fluorescence due to potential-driven desorption of the adsorbed species. This method enables measurement of nonspecific adsorption and will be used to evaluate the strategy for minimizing or eliminating nonspecific adsorption for various surface modification methods. Shown are examples of physisorbed and chemisorbed monolayers on gold defining strong and weak adsorption characteristics, in addition to a weak chemisorption example. In addition, surfaces that form the basis of biosensors such as monolayers composed of peptide and nucleic acid SAMs adsorbed onto gold are analyzed. The general applicability of this approach is demonstrated through the use of a wide variety of examples covering many aspects of molecular surface modification used in biosensors.

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EXPERIMENTAL SECTION The different surface modifications were characterized similarly, using in situ fluorescence imaging during electrochemical perturbation. The same analysis method was used for all samples, but the type of gold electrode used and the surface preparation method employed differed as detailed below for each adsorbate system studied. Preparing the Au electrodes. Two types of Au electrodes were used in this study: gold bead electrodes and polished gold electrodes. The single crystal Au bead electrodes used were prepared as detailed in ref 26. Before modification, the electrodes were flame-annealed, followed by rinsing with ultrapure water (>18.2 MΩ cm produced by a Milli-Q water purification system (EMD Millipore)) and repeated three times. These electrodes were used for the alkylthiol SAMs, the carboxylated silane adsorbate and the DNA SAMs. The peptide SAMs were prepared on polished gold bead electrodes that followed the procedure in ref 27. Prior to modification with the peptide, each gold electrode was flame annealed, cooled down for a few seconds in air, and then quenched in pure water. The electrodes were then cycled between −0.3 V and +1.5 V in 0.1 M HClO4 until reproducible CVs were obtained to ensure the quality of the substrate. Preparing SAMs and Their Postfunctionalization with a Fluorophore. The alkylthiol SAMs were prepared by immersing the clean Au bead electrode into a 1 mM solution of thiol (11-mercaptoundecanoic acid (MUA; 95% Aldrich) or 6-mercapto-1-hexanol (MCH; 99% Aldrich) prepared in methanol (Certified ACS, Fisher Scientific, Ottawa, ON, Canada)) for 1 h, followed by copious rinsing with methanol before immediate use in the fluorophore modification procedure. The carboxylated silane-modified Au bead electrode was prepared by immersion in aqueous 10% (v/v) TMS-EDTA (N-[(3-trimethoxysilyl)propyl]ethylene-diaminetriacetic acid, Na salt (50% in water from United Chemical Technologies, Bristol, PA)) for 2 h. In order to study the interface using the fluorescence microscopy method, the adsorbates must be labeled with a fluorophore. The prepared MUA SAM or adsorbed TMSEDTA deposited on the electrode surface were chemically modified using carboxyl-amine coupling, accelerated with carbodiimide cross-linking chemistry as described in the Supporting Information. This approach was used to couple Alexa Fluor 488 Cadaverine (Thermo Fisher Scientific, Burlington, ON, Canada) onto the adsorbed monolayers. A series of control experiments were also performed where the carbodiimide activation step was eliminated. A clean and a B


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Analytical Chemistry

Ag/AgCl) was connected to the electrolyte via a salt bridge. In all cases, the electrolyte was deaerated with Ar and maintained in a purged Ar atmosphere. For more detailed information about experimental setup, please refer to the Supporting Information. Reproducibility of the fluorescence intensity measurements depends on the optical efficiency, reflectivity of the substrate, and the amount of fluorophore adsorbed, which can vary since nonspecific adsorption is not well controlled.

MCH SAM modified Au bead electrode were each directly immersed in a solution of the diluted fluorophore as controls to investigate the adsorption of the fluorophore in the absence of covalent bond formation. Prior to the in situ fluorescence imaging analysis, the surfaces were either rinsed with or soaked in phosphate buffered saline, PBS (1× Dulbecco’s PBS pH = 7.4, 0.137 M NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 10 mM KH2PO4, no calcium, no magnesium from Invitrogen catalog no. 21600044), and then analysis was performed in the same buffer. The chemical structures of the adsorbates used in this portion of the work are shown in Figure S1. Preparing the Peptide SAMs. The peptide sequences used to modify the polished gold surfaces were based on a portion of the amino acid sequence expressed in the p53 protein. The design of the peptide sequence is based on the interaction between the tumor suppressor protein p53 and the E3 ubiquitin ligase MDM2, which involves the amino acids 12 to 26 of the p53 protein sequence (PPLSQETFSDLWKLL).30 These two proteins are involved in a regulatory cycle that controls the cell integrity, MDM2 being an inhibitor of p53 activity. The sequences were elongated by three amino acids (PAK) and labeled with 5-carboxyfluorescein (FAM) for fluorescence imaging. In one case, the sequence was modified to include a cysteine residue to enable specific chemical interaction with the gold surface. The adsorbates used were ApCys-p53-FAM (H2N-CPPLSQETFSDLWKLLPAK(5-FAM)CONH2), and Ap-p53-FAM (H2NPPLSQETFSDLWKLLPAK(5-FAM)-CONH2) (from Eurogentec) as well as 4-mercaptobutan-1-ol (MCB; 97%, Fluka). Three types of immobilization have been considered in this work, the adsorption of the Cys modified peptide (Ap-Cys-p53FAM), a one-step coadsorption procedure in which MCB and the Ap-Cys-p53-FAM peptide are simultaneously adsorbed on the gold surface, and the adsorption of an unthiolated peptide probe (Ap-p53-FAM). Surface preparation is described in the Supporting Information. Preparing the DNA SAMs. Gold bead electrodes were modified with dsDNA and MCH. Mixed SAMs were prepared using oligonucleotides containing a fluorophore (AlexaFluor488) on the 3′ end and a hexanethiol modification at the 5′ end. The modified nucleic acid (HS-(CH2)6-CTG-TATTGA-GTT-GTA-TCG-TGT-GGT-GTA-TTT-AlexaFluor, from Eurogentec) was used previously26,28,31 as is lacks stable secondary structures. The preparation of the dsDNA SAM is given in the Supporting Information. The buffer used for the in situ fluorescence imaging studies was 10 mM Tris and 10 mM KNO3(pH = 7.50 ± 0.05). Spectroelectrochemical Measurements. To characterize the surfaces and the potential-dependent desorption of the adsorbed monolayers, in situ spectroelectrochemical fluorescence microscopy was performed as described previously.26−28 Briefly, the Au bead electrode immersed in electrolyte in the spectroelectrochemical cell was positioned at the focal point of an objective (either 5, 20, or 50× ) in an inverted fluorescence microscope. The surface of the Au bead contains a number of (111) facets and was oriented so that a selected facet was in focus. The polished electrode surface used in the peptide SAM studies was also positioned so it was in focus. The fluorescence images were obtained using a filter cube which was appropriate for both AlexaFluor488 and FAM and with one appropriate for AlexaFluor647 (detailed in the Supporting Information). The electrolyte solutions used were as described above. The counter electrode was a Pt coil. A reference electrode (either SCE or

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RESULTS AND DISCUSSION Chemisorbed and Physisorbed Monolayers. The coupled electrochemical - fluorescence approach was first used to investigate a gold surface modified with a well-ordered SAM, exemplifying results which may be achieved from a strong chemisorption system. A MUA SAM was prepared on a gold bead electrode and postfunctionalized a fluorophore (AlexaFluor488 cadaverine). This is a commonly used method for chemically modifying surfaces with a variety of probes18,32 creating functional surfaces through sequential chemical modification. This approach to surface engineering requires a high level of control at each step in the surface functionalization to ensure minimal nonspecific adsorption. After modification, the gold bead electrode was analyzed using fluorescence microscopy in a phosphate buffered saline electrolyte (1× PBS). The fluorescence intensity and the capacitance are shown in parts a and b of Figure 1, respectively. The Au/MUA/AlexaFluor modified surface has a low capacitance, indicating that the surface was modified with a

Figure 1. Average fluorescence intensity (a) and capacitance (b) for potential scans from 0 V to −1.4 V for two SAMs: MUA after modification with NHS/EDC to couple a fluorophore onto the SAM (cadaverine) and MCH after exposure to the same concentration of the fluorophore. The second potential scan (after reductive desorption) is also shown as a dashed line. The change in fluorescence of the MCH SAM in part a is shown after subtracting the background for both the first and second (after desorption) potential scan. Fluorescence images for (c) MUA and (d) MCH (after removing background) taken at the maximum intensity are shown. The dotted line outlines the (111) facet. C


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potentials, but only when reductively desorbing the MCH SAM. The increase in capacitance indicates desorption starting at −0.7 V accompanied by a fluorescence decrease due to the dilution of desorbed fluorophore into the bulk. The dashed line is the same measurement performed immediately afterward showing no change in the fluorescence signal indicating a complete removal of the adsorbate. These results show that it may be possible for some fluorophore to partition into the MCH SAM, become strongly nonspecifically adsorbed, and only removed with the SAM. This type of adsorption may be present in the MUA SAM surface, but it would be difficult to determine in the presence of the large amount of specifically adsorbed material. Adsorption of the fluorophore onto defective or bare regions in the SAM modified surface is also possible, though not likely in these well-ordered SAMs. Even so, the fluorophore would adsorb through nonspecific physisorption interactions with the surface. This process was mimicked providing an opportunity to study the changes in fluorescence for a more weakly adsorbed species. In this example, the clean Au bead surface was only exposed to the solution of AlexaFluor488 cadaverine for 30 min and then rinsed with Milli-Q water and PBS before spectroelectrochemical analysis in the same buffer. The capacitance and fluorescence (from the (111) facet) are shown in parts a and b of Figure 2, respectively, for two

high-quality SAM. However, even though the SAM was labeled with the fluorophore, the small fluorescence intensity observed at positive potentials was due to quenching when near a metal surface. As the potential is made more negative, the MUAAlexaFluor SAM starts to be reductively desorbed at −0.8 V/ SCE, resulting in a significant increase in both fluorescence and capacitance. Note that the capacitance is indicative of the whole electrode surface, whereas the measured fluorescence intensity comes only from the electrode area which is in the field of view (in the present case it corresponds to a (111) facet). Therefore, the capacitance also increases at potentials more negative than the (111) surface desorption. The SAM is completely removed from the surface at negative potentials, signified by the increase in capacitance to that expected for a uncovered gold surface. At these potentials, the fluorescence intensity also decreases to the background level due to the diffusion of the desorbed fluorophore out of the field of view as demonstrated by repeating the potential sweep in the negative direction (dashed line). An indication of the strength and the homogeneity of the energetics of the MUA SAM bonding is shown by the distinct lack of increase in fluorescence or capacitance between 0 and −0.8 V. A fluorescence increase observed within this potential range would indicate the presence of a more easily displaced adsorbate, one with a weaker bond to the surface. For this surface, the adsorbed MUA-AlexaFluor species are therefore only bound through strong covalent bonds forming the ideal SAM. Fluorescence imaging provides the opportunity to measure the uniformity of the surface modification. Fluorescence images at the maximum intensity are shown for the MUA and MCH SAMs in parts c and d in Figure 1, respectively. The brightfield and other fluorescence images for these modified surfaces are shown in Figure S2 with the Au(111) facet outlined in each image. The fluorescence distribution is generally uniform across the (111) facet before desorption and at the potential of maximum fluorescence intensity, which indicates that modification by NHS/EDC chemistry results in an ideal (111) surface. Some nonuniformity of the fluorophore coupling to the surface is observed around the 111 facet. This indicates that the postfunctionalization process likely depends on the packing density of the carboxyl SAM (which should vary with surface crystallography26), which impacts the dominant mechanism for the NHS/EDC reaction and therefore the surface reaction yield.18,32 These subtle differences in reactivity are easily measured using fluorescence and may be important in the construction and performance of complex surface structures. The ideal Au/MUA/AlexaFluor SAM was reductively desorbed at a single potential indicating a uniformity in the molecular adsorption energy. A control experiment was performed to ensure that the observed fluorescence only results from postfunctionalization of the 11-MUA and is not due to nonspecifically adsorbed unreacted fluorophore. To this end, a MCH SAM covered gold bead electrode was used since the AlexaFluor488 cadaverine will not react with exposed hydroxyl groups. The bead was placed in the fluorophore solution (30 min in the dark) and then characterized using the same conditions as the MUA SAM. Figure 1a shows the change in fluorescence due to the desorption of MCH SAM after removing the linearly increasing background (see data in Figure S3). A significantly lower fluorescence intensity at 0 V shows that only a few fluorophores are physisorbed onto the MCH SAM coated surface and they are not completely quenched. A change in the fluorescence was not observed at small negative

Figure 2. Adsorption of AlexaFluor488 cadaverine onto Au studied using negative-going potential scan (from 0 V to −1.4 V) while measuring fluorescence from the (111) facet (a) and capacitance (b). The modified electrode was either rinsed or soaked in PBS electrolyte before measurement. The capacitance for the Au electrode after desorbing the AlexaFluor488 cadaverine is shown as a dashed line as is the fluorescence intensity after desorption. The intensity of the soaked sample was magnified to be comparable to the rinsed sample for comparison.

treatments of the modified electrode: rinsing or soaking. After rinsing the surface, the capacitance (at 0 V, Figure 1b) is much higher than for the SAM coated electrode but lower than expected for a clean Au surface (dashed line in Figure 1b) indicating some adsorption. Only a small negative potential excursion was required to initiate fluorophore desorption with the majority of the fluorophore removed when the potential reaches ∼−0.8 V since the capacitance merges with that of the clean Au surface (measured in the same electrolyte). Fluorescence was observed from the whole surface (images shown in Figure S4). The intensity remains large at very D


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Analytical Chemistry negative potentials, which is a consequence of the desorbed fluorophore remaining in the field of view. Soaking the fluorophore-exposed Au bead in PBS buffer for 90 min before analysis results in a decreased fluorescence intensity, though this is difficult to quantify. The PBS electrolyte likely displaces some of the fluorophore due to competitive adsorption of phosphate and chloride onto the Au electrode.33,34 The first fluorescence peak (around −0.35 V) is a result of desorption from regions around the 111 facet influencing the intensity at the center of the facet. The fluorescence maximum at −0.6 V/SCE is due to desorption from the (111) facet which quickly decreases to the background level at −0.8 V/SCE. Fluorescence images for the surfaces at the initial potential and from the maximum intensity image are shown in Figure S3 revealing that the adsorption of the fluorophore is quite uniform. These adsorbed species require a desorption potential that is much less negative as compared to the MUA SAM and represent the potential dependence characteristic of nonspecifically adsorbed species. Thus, the potential at the start of the increase in fluorescence intensity enables an estimate of the strength of the fluorophore−gold interaction. The changes observed in the capacitance are less obvious than in the MUA SAM case but still shows desorption of the adsorbed fluorophore at −0.8 V. Other changes in the adsorbed layer observed using fluorescence (e.g., peak at −0.4 V) are not clearly delinated in capacitance, illustrating the need for this complementary optical method. Carboxylated Silane-Modified Au. Comparing the impact of increasingly negative potentials on the chemisorbed and physisorbed species using fluorescence intensity and capacitance showed that strongly adsorbed species require more negative potentials to be removed from the surface. This approach can be used to investigate the distribution of adsorbate−surface interactions as a way of distinguishing and evaluating the type of adsorbed species on the gold surface (e.g., specific or nonspecific). In some cases, the capacitance can be used to define this desorption potential, but it is less reliable for those adsorbates with lower adsorption strengths or when present as a small fraction of the total species adsorbed. Therefore, this approach can be particularly useful for adsorbates that fall between the two extremes of either physiand strongly chemi-sorbed monolayers. A carboxylated silane (TMS-EDTA) is a particularly good example of these types of species. The adsorption of this molecule onto gold has been recently investigated for use in microfluidic applications.35 TMS-EDTA adsorption is strong enough to compete with Cl− and OH− for the gold surface, but it is less strongly adsorbed than a thiol SAM. TMS-EDTA possesses three carboxyl groups and it was used to modify a gold surface using NHS/EDC modification of the carboxyls for strepavidin immobilization in a SPR flow cell.35 To study this interface using the coupled electrochemical-fluorescence method, the adsorbed TMSEDTA was postfunctionalized with the AlexaFluor488 cadaverine fluorophore using NHS/EDC chemistry as was done for MUA SAM. It is expected that some of the TMS-EDTA carboxyls exposed to solution would become fluorophore labeled. The fluorescence intensity and capacitance for TMS-EDTA modified gold after fluorophore labeling with AlexaFluor488 cadaverine are shown in parts a and b of Figure 3, respectively. Two TMS-EDTA modified gold surfaces were analyzed, one rinsed immediately after preparation, the second after soaking for 90 min in the PBS buffer solution (as done in the clean Au

Figure 3. Fluorescence intensity (a) and capacitance (b) measured during a negative going potential scan (0 V to −1.4 V) for a TMSEDTA-modified Au electrode after NHS/EDC treatment and labeling with fluorophore (AlexaFluor 488 cadaverine). The electrode was either rinsed or soaked in buffer before analysis. The capacitance for a clean gold electrode is shown measured in the same electrolyte (dashed line).

bead study). In both cases, the capacitance confirms the presence of TMS-EDTA on the surface since it is lower than the Au surface with physisorbed fluorophore though substantially larger than the MUA SAM. The capacitance of these two modified surfaces are nearly identical (soaked is slightly larger suggesting a lower surface coverage). Small increases in capacitance are observed at −0.4 V to −0.6 V, which correspond to the increase in fluorescence especially for the rinsed surface. Capacitance also shows that complete desorption from the electrode surface occurs at −0.9 V/SCE. The fluorescence images for these TMS-EDTA modified surfaces (Figure S5) show that the surface modification is again uniform when observed at 0 V. The increase in fluorescence intensity starts at small negative potentials, continuing until −0.8 V which corresponds to the capacitance increase, indicating the start of TMS-EDTA desorption. This desorption potential is significantly more negative than the fluorophore on gold which reveals a more strongly adsorbed layer but not as strong as MUA or MCH, both requiring more negative desorption potentials. In Figure 3a, the slight shoulder observed at −0.5 V for the rinsed surface suggests the electrochemical removal of less strongly adsorbed molecules (e.g., nonspecifically adsorbed material). The source of this type of adsorbate is not clear, it could be unreacted fluorophore (as in the case for the Au) or it could be from the desorption of fluorophore modified TMSEDTA which was destabilized by the chemical treatment. In both cases, desorption will result in a fluorescence increase which can be used to evaluate the methods to remove these undesired adsorbed species. If the TMS-EDTA/AlexaFluor modified surface is soaked in PBS buffer for 90 min, a decrease in the fluorescence signal from 0 to −0.5 V is observed. Competition for the surface with phosphate and chloride ions, which are known to adsorb onto gold,33,34 likely displaces these nonspecifically or weakly adsorbed TMS-EDTA molecules but not those more strongly bound.35 Therefore, for this surface modification, soaking in buffer is more effective than a simple rinsing of the substrate evidenced by the decrease in the amount of adsorbate desorbing at small negative potentials. Peptide SAM-Modified Au Surfaces. The adsorption of large biomolecular probes onto an electrode surface can be E


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The fluorescence images for these modified surfaces recorded at 0 V and at the potential of maximum fluorescence intensity are shown in Figure S6. Evident in these images is that the surface is not homogeneously modified having some small regions with intense fluorescence. These regions have been examined previously27 and were excluded from this fluorescence intensity analysis. Excluding these small regions, the surfaces are generally uniformly covered. Preventing nonspecific adsorption can be accomplished by passivating the gold surface with a small alkylthiol molecule during surface modification.21 Using a codeposition scheme where MCB and Ap-Cys-p53-FAM are allowed to compete for the gold surface results in a significantly different capacitance and fluorescence intensity dependence on potential. At 0 V, the capacitance and the fluorescence intensity are at a minimum. At −0.7 V, the capacitance quickly increases indicating desorption of the layer. The fluorescence intensity also increases at this potential and reaches a maximum at about −0.8 V, just negative of the maximum for the peptide-only surface and slightly positive of the MUA SAM results. In this case, a second potential scan was measured (dotted line) after desorption which shows only the background signal, confirming the complete removal of the adsorbates. The lack of fluorescence at potentials positive of −0.6 V shows that the coadsorption strategy can prevent nonspecific adsorption. The capacitance also verifies the reductive desorption of the adsorbed layer, but since both adsorbed species contribute to the capacitance, distinguishing the MCB from the peptide is not possible. This competitive adsorption strategy will result in a decreased amount of Ap-Cys-p53-FAM on the surface when compared to the single-component approach. A lower fluorescence intensity for this coadsorption layer was observed, but comparing an absolute surface coverage is not simple as different setups were used for the measurements. DNA SAMs on Au. Biosensor surfaces based on thiolmodified DNA SAMs are typically prepared in two steps: a clean gold surface is exposed to a solution of thiol-modified DNA, then it is subsequently immersed in a solution of a small alkylthiol (such as MCH) which acts to displace or remove those DNA molecules that are adsorbed through nonspecific interactions (e.g., interaction of the DNA bases with the gold electrode).21,22 The resulting mixed SAM (DNA/MCH) is routinely used in many nucleic acid biosensing applications. For these sensors, the presence of nonspecifically adsorbed DNA will contribute to the measured background signal, reducing its sensitivity. In the case where the redox reporter on the DNA probes needs to get close to the electrode surface, these nonspecifically adsorbed species can hinder this process, affecting the surface-to-surface reproducibility. As with the previous examples, the combined electrochemistry−fluorescence method was used to assess the nonspecific adsorption of DNA on these surfaces. In addition, these measurements were used to develop a method tailored for removing these nonspecifically adsorbed molecules from the DNA SAM under the conditions where the SAM needs to remain on the surface for sensing. Therefore, a nondestructive potential perturbation (e.g., no reductive desorption) approach for removing the undesired adsorbate is demonstrated. The fluorophore-modified DNA SAMs were prepared with a 30 base pair strand of DNA that contained AlexaFluor647 modifying the distal end with thiol at the other end. The fluorophore in these DNA SAMs may be as far as 10 nm away from the gold surface and therefore not efficiently quenched.

achieved through physisorption or chemisorption and may generate a complex modified surface in terms of the variety of adsorbate−surface interactions. From the examples shown above, this combined fluorescence-electrochemical method could be used as a general approach for investigating these interfaces. This method was used to study the adsorption of a fluorophore(5-FAM)-modified p53 peptide onto a polished gold electrode surface. The peptide SAMs were prepared from unmodified peptide (Ap-p53-FAM) or a peptide modified with cysteine (Ap-Cys-p53-FAM) which introduced a thiol functionality. A comparison of the adsorption of the two peptides onto a planar polished gold electrode surface was performed. Figure 4a,b shows the fluorescence and capacitance, respectively, of the peptide-modified gold surfaces as the

Figure 4. Fluorescence intensity (a) and capacitance (b) measured during a negative going potential scan (0 V to −1.25 V) for the three different methods used to modify the gold electrode by peptide SAM. Also included is the data taken for the MCB/Ap-Cys-p53-FAM mixed SAM after desorption (dotted line).

potential is made more negative. Both modified surfaces exhibit a similar capacitance and a similarly small fluorescence intensity at 0 V suggesting that the adsorbed layers are close enough to the surface for the fluorescence to be substantially quenched thereby suggesting monolayer adsorption. As the potential is made negative, an increase in capacitance and in fluorescence intensity for both surfaces is observed at −0.2 V, indicative of the desorption of physisorbed peptide. The fluorescence for Ap-p53-FAM is broad, starts at a less negative potential than Ap-Cys-p53-FAM, reaching a maximum at −0.45 V. The ApCys-p53-FAM adsorbate has a maximum in the fluorescence at −0.7 V which is indicative of chemisorbed species. Previous studies of cysteine36 and cysteine terminated peptide SAMs37 have shown a similar reductive desorption potential in neutral pH electrolyte. Even though a shoulder in the fluorescence− potential curve is observed at −0.4 V, indicative of a small amount of nonspecific adsorption, the presence of a cysteine enables a specific adsorption or interaction with the electrode surface, but it is not sufficient to completely prevent nonspecific or physisorption. For these adsorbed layers, the capacitance indicates that the interface is modified with the peptide at positive potentials and desorbed at potentials negative of −1.0 V, but evidence of the removal of nonspecifically adsorbed species at −0.4 V is not obvious from this capacitance measurement. F


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Figure 5. Fluorescence intensities of a dsDNA SAM after typical preparation from the 111 surface measured for negative going potential step cycles (starting at 0.35 V to −0.4 V) used to remove nonspecifically adsorbed DNA. (a) The first three consecutive cycles, (b) two consecutive potential step cycles performed on the same bead after soaking in IB for a week and then holding at −0.350 V for 10 min. Fluorescence images of the maximum difference in intensity which corresponds to (c) the first set of potential step cycles and (d) the second set of potential treatments as indicated in the figure. The fluorescence images are falsely colored. Note that each image has its own associated intensity bar. The images are 1.580 mm × 1.580 mm.

on the gold electrode is a result of the single crystalline nature of the gold bead electrode surface,26 which contains four (111) facets symmetrically arranged around a (100) facet as labeled in the figure. The regions that show the largest increase in fluorescence are from around the 111 facets (a region with many step edges and terraces) and from the 100 region. Subsequent cycles of potential steps between +0.35 V and −0.40 V (cycles 2 and 3) results in a much smaller fluorescence increase. The applied potential-step cycles perturbs the specifically adsorbed DNA SAM which seems to facilitate the removal of the nonspecifically adsorbed DNA. After this potential treatment, the electrode was soaked in the immobilization buffer for 1 week in an attempt to remove all the nonspecifically adsorbed DNA. The electrode was then treated by holding the potential at −0.35 V/SCE for 10 min. After two of these pulse/hold treatments, the potential stepping cycle was repeated and the fluorescence intensity from the 111 facet is shown in Figure 5b. The increase in fluorescence for the potential hold measurements are shown as images in Figure 5d in addition to the fluorescence increase from the last potential step cycle. Holding the potential at −0.350 V does induce the removal of more nonspecifically adsorbed DNA from the surface, but the change is 10% of what was observed from the as-prepared surface. This shows that even soaking in buffer for many days is not effective in displacing the nonspecifically adsorbed DNA. After these potential/hold treatments, a potential-step cycle was performed which showed only a small repeatable increase in fluorescence at negative potentials (Figure 5b). This small increase in fluorescence is due to the reversible electrostatic repulsion of the negatively charged DNA strand28,38,41−43 from a negatively charged surface. From this series of measurements, the as-prepared surface contains a significant amount of DNA which was removed at potentials more positive than the reductive desorption. As with the other examples where the adsorbate was displaced from the surface using moderately perturbing potentials, this adsorbate can be classified as nonspecifically adsorbed DNA. In addition, the potential pulsing and/or holding procedures demonstrated a method capable of removing this nonspecifically adsorbed DNA and creating a stable DNA SAM interface. The surface presented above is one of the more extreme examples of nonspecific adsorption which we have observed

This incomplete quenching is useful for evaluating the DNA SAM coverage uniformity as well as for studying the DNA orientation change induced by the charge on the electrode surface.31,38−41 The potentials used will be restricted to values more positive than the reductive desorption potential of a DNA/MCH SAM from the gold surface which is around −0.80 V/SCE28 (shown in Figure S7), similar to MUA or MCH SAMs (Figure 1). In this example, a gold bead electrode was modified with a double stranded (ds) DNA/MCH mixed SAM as detailed in the experimental methods. To characterize the SAM, capacitance and fluorescence were measured when the electrode potential was stepped from a positive limit of +0.35 V to increasingly negative potentials (in 25 mV steps), stopping at −0.40 V so as to avoid reductive desorption potentials. In between each negative-going step, the potential was returned to +0.35 V. Only data from the step to the negative potentials are shown in Figure 5a,b (a complete cycle is shown in Figure S8). This process was repeated another two times. Figure 5a shows the potential dependent changes in the average fluorescence intensity measured from the whole surface. The intensity of the as-prepared surface at +0.35 V/SCE is significantly above the background level due to the incomplete quenching of fluorescence from the DNA SAM. When stepping the potential to −0.40 V/SCE, the freshly prepared surface shows a large increase in fluorescence. Repeating this potentialstep cycle treatment (cycles 2 and 3) results in smaller increases in fluorescence. The intensity at the positive potential limit also goes up slightly, but this is due to the desorbed DNA not leaving the optical path before the start of the next measurement. Significantly, the capacitance is constant during this loss of adsorbate (shown in Figure S9), indicating that these changes at the surface are not due to reductive desorption of the thiol, but rather due to an expulsion of nonspecifically adsorbed DNA from the surface, observed since the quenching is no longer effective. As with the other examples, the capacitance appears to be less sensitive to the removal of these weakly adsorbing species. Shown in Figure 5c are images of the increase in fluorescence measured at −0.4 V as compared to the positive potential for each of the three potential step cycles. The fluorescence pattern G


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alone such as capacitance are not sufficiently sensitive to these undesired adsorbates, while they are easily measured using in situ fluorescence and electrochemical potential perturbation. This type of characterization is important in cases where the adsorbate does not completely cover or block the surface, therefore being unable to prevent these nonspecific interactions. In addition, nonspecific adsorption can result from interactions between surface bound chemisorbed species and those not chemisorbed molecules. Furthermore, this methodology can be used to study a variety of surface modifications and enables the characterization of molecular adsorption onto the surface, whether preparing the surface or chemically modifying the adsorbate. With this method in hand, a careful evaluation of the strategies used to minimize or remove the nonspecific adsorption can be developed. To use this method, a fluorescently labeled adsorbate is required which may limit the type of surface modifications that can be studied. Assuming that the fluorophore does not change the surface modification process significantly, then conditions for removal of the nonspecially adsorbed material can be determined and then applied to the fluorophore-free system.

from many samples of either dsDNA or ssDNA SAMs. Typically, the nonspecifically adsorbed DNA is more uniformly distributed and the fluorescence increase is observed from the whole surface. Another example of the dsDNA/MCH SAM modified electrode is shown in Figures S10 and S11. The same type of potential perturbation approach was applied to remove this nonspecifically DNA, by holding the potential at −0.35 V/ SCE and by either cycling the potential from +0.35 to −0.4 V/ SCE (fluorescence intensity and capacitance changes shown in Figures S10 and S11) and then soaking in buffer overnight or longer. The resulting surface is stable with respect to capacitance and fluorescence intensity changes when perturbed by potential pulsing. Fluorophore labeled DNA SAMs were found to contain weakly bound or physisorbed species that could be driven off the electrode surface with repeated small negative potential excursions, avoiding reductive desorption effects. This nonspecifically adsorbed DNA is most likely adsorbed via interactions with the SAM composed of MCH and dsDNA. Our observations are in line with the results of Erdmann et al.,44 who studied the interaction of duplex DNA (adsorbed onto an AFM tip) with a hydroxyl-terminated SAM (11mercaptoundecanol) on gold in the same potential range as used here. When the potential was positive (vs Ag/AgCl), the dsDNA was adhered to the OH terminated SAM at pH = 8.5 such that a force from the tip was required to displace the dsDNA. For potentials less than 0.0 V/AgAgCl, the dsDNA was easily displaced of the surface with little to no force required. This may explain why a simple rinsing of the DNA/ MCH surface is not sufficient to remove this nonspecifically adsorbed material and that the application of potential cycling within a limited (stable) range was required to facilitate its removal. Changes in the measured capacitance during the potential treatment process did not clearly indicate the removal of this weakly adsorbed DNA, revealing the limitation when relying on electrochemical measurements alone. Nucleic acid biosensors using DNA SAMs rely on hybridization of the target with the immobilized probe on the sensor surface which has been shown to be strongly dependent on many factors (probe density, orientation, or surface charge density) including nonspecific adsorption of the base pairs onto the surface.5,9,14 From these studies, another factor that should be considered is the presence of nonspecifically adsorbed probe on the sensor surface since it would compete with the specifically attached probe for the complementary strand from the solution. Finding the conditions for removing this nonideal adsorbate without compromising the integrity of the surface modification is only possible with an in situ method like the one described. It is capable of reliably measuring these adsorbates in the presence of the specific or desired adsorbate configuration using the potential where fluorescence is observed as the differentiating variable.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b03953. Description of the spectroelectrochemical instrumentation and the procedures followed to prepare the modified electrode surfaces; fluorescence images for each of the systems described and an example of the reductive desorption of a dsDNA/MCH SAM; and another example of the removal of nonspecifically adsorbed DNA (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 604-822-6816. ORCID

Cheng Wei Tony Yang: 0000-0002-1792-1987 Dan Bizzotto: 0000-0002-2176-6799 Author Contributions ×

Z.L.Y. and C.W.T.Y. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge fruitful discussions with Prof. Jeff Shepherd (Laurentian University) at the start of this work. We are also grateful for the efforts of our glassblower (Brian Ditchburn) in making the spectroelectrochemical cells and to the Electrical and Mechanical shops in Chemistry (The University of British Columbia). This work was supported by NSERC (Canada) and through a FRFC grant from the Belgian National Science Foundation.

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CONCLUSION Identification of specific and nonspecific adsorption onto a gold electrode surface can be accomplished using a combination of fluorescence microscopy with electrochemistry for fluorescently labeled adsorbates as demonstrated with many diverse examples of surface modification. The potential where the adsorbate is desorbed or displaced from the electrode surface is indicative of its adsorption strength and can be used to distinguish specific and nonspecific adsorption in cases where strong adsorption to the surface is desired. Unfortunately, electrochemical methods

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