Adsorption of a Carboxylated Silane on Gold: Characterization for Its

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Adsorption of a Carboxylated Silane on Gold: Characterization for its Rational Use in Hybrid Glass/Gold Substrates Cheng Wei Tony Yang, Isaac Martens, Elod L. Gyenge, Robin F. B. Turner, and Dan Bizzotto J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.5b09915 • Publication Date (Web): 19 Jan 2016 Downloaded from http://pubs.acs.org on January 24, 2016

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The Journal of Physical Chemistry

Adsorption of a Carboxylated Silane on Gold: Characterization for its Rational Use in Hybrid Glass/Gold Substrates. Cheng Wei Tony Yang,†,‡ Isaac Martens,¶,§ Elöd L. Gyenge,‡ Robin F.B. Turner,†,§,k and Dan Bizzotto∗,¶,§ †Michael Smith Laboratories, The University of British Columbia, 2185 East Mall, Vancouver, BC, Canada, V6T 1Z4 ‡Department of Chemical and Biological Engineering, The University of British Columbia, 2360 East Mall, Vancouver, BC, Canada, V6T 1Z3 ¶AMPEL, The University of British Columbia, 2355 East Mall, Vancouver, Canada V6T 1Z4 §Department of Chemistry, The University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada, V6T 1Z1 kDepartment of Electrical and Computer Engineering, The University of British Columbia, 2356 Main Mall, Vancouver, BC, Canada, V6T 1Z4 E-mail: [email protected]

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Abstract Integrated sensing and bio-sensing microfluidic systems often require sealing between polysiloxane, glass, and gold interfaces, while maintaining functional support on the gold surface within the cell chamber (e.g. biomolecular interaction analysis). A carboxylated trimethoxysilane (TMS-EDTA) coating has been shown to facilitate the bonding of polydimethylsiloxane (PDMS) to gold and glass slides. In this work, the adsorption of TMS-EDTA onto Au is characterized in order to enable its rational use in hybrid glass/gold substrates. Surface plasmon resonance results suggest that carboxylates are available for streptavidin immobilization. Atomic force microscopy studies indicate that a uniform surface coverage with monolayer thickness is formed. Infrared spectroscopy studies confirm that the carboxyl groups are present. Moreover, there is little evidence of siloxane crosslinking. Electrochemical differential capacitance measurements reveal that the potential-dependent free energies of adsorption are ∼ −20 to −30 kJ/mol (for potentials between −0.5 and 0.2 V ) in the complex electrolyte solution used. Furthermore, at highly negative potentials (∼ −1.1 V ), TMS-EDTA desorbs from the Au surface. The fundamental knowledge obtained about TMS-EDTA adsorption on Au can be applied to construct robust PDMS-based systems with hybrid glass/gold substrates.

Introduction Gold surfaces are frequently integrated into sensors and biosensors of silicon and glass for the quantification of a variety of substances. 1 Developing a simple and easy fabrication method, which leads to sensitive, stable, and reliable integrated systems, still remains a challenge. 2 For example, microfluidic surface plasmon resonance (SPR) imaging biosensors often require the use of glass substrates partially patterned with a microarray of gold spots. 3 It is challenging to form a strong bond 4 between polydimethylsiloxane (PDMS) and glass, while maintaining functional support on Au (inside the PDMS cell chamber) for the covalent immobilization 5

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of biomolecules. Similarly, microfluidic electrochemical devices often utilize glass substrates decorated with sputtered Au electrodes. 6 For example, Figure 1A shows a hypothetical PDMS-based electrochemical cell (with two gold electrodes sputtered on glass). Figure 1B shows the top view of the device with the working electrode (WE) and the secondary electrode (SE). During analysis, an aqueous solution of analyte(s) is injected into the PDMS microfluidic cell chamber. Without treating the gold electrodes, however, leakages can occur at the PDMS-Au interface and can compromise the device performance. 7 Therefore, studying substances that can self-organize onto silicon, glass, and gold surfaces is required so as to address some of these issues, and to pave the way for new technological advances. 8 A carboxylated silane (N -[(3-trimethoxysilyl)propyl]ethylene-diamine triacetic acid, or TMS-EDTA) has been widely used to modify oxide-, silanol-, and hydroxyl-containing surfaces and nanoparticles for fuel cell applications, 9 heavy metal ion chelation, 10 PET and MR imaging, 11 rare-earth ion adsorption and separation, 12,13 and bacteriophage immobilization. 14 Recently, TMS-EDTA has been used to modify glass slides for carboxyl-amine bonding with PDMS blocks modified with (3-aminopropyl)-trimethoxysilane (3-APTMS). 15 Interestingly, this carboxylated silane has also been shown to modify Au slides for a similar type of bonding with PDMS. Shear tests showed that TMS-EDTA coated Au slides produced a comparable PDMS bond strength as the traditional thiol-based surface modification strategies (such as 11-mercaptoundecanoic acid, or 11-MUA). Consequently, TMS-EDTA was applied to assist in the development of an integrated optical microfluidic biosensor (i.e. to ensure a strong PDMS-Au bond and to provide functional support on the Au surface for the immobilization of antibodies). 16 Due to its ability to modify both glass and Au, TMS-EDTA may offer distinct advantages over 11-MUA, which can only modify Au but not glass. More specifically, the above-mentioned experimental results suggest that TMS-EDTA can potentially be used in a one-step, solution-based modification strategy for glass substrates that have been partially patterned with gold electrodes (to assist in both bio-sensing and sensor fabrication). To

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better explain this concept, Figure 1C shows the cross-sectional view of the hybrid substrate across the WE inside the PDMS chamber. Functional carboxylates are potentially available on the Au electrode (from TMS-EDTA modification). This surface may then be used to immobilize biomolecules for bio-sensing applications. Moreover, Figure 1D shows the crosssectional view of the hybrid device across all Au electrodes, illustrating how TMS-EDTA can potentially be used to chemically modify both glass and gold surfaces for carboxylamine bonding with 3-APTMS-modified PDMS. Specifically, the PDMS-Au bonding formed could create a tight seal around the cell chamber, and prevent solution leakages via the gold electrodes. The adsorption mechanism of silanes onto metal substrates is not well understood. 17 To the best knowledge of the authors, the surface modification of Au by TMS-EDTA has only been analyzed by water contact angle and X-ray photoelectron spectroscopy measurements. 15 To extend this fabrication technology for use in hybrid glass/gold substrates, the adsorption of TMS-EDTA onto Au needs to be characterized (i.e. ability to capture biomolecules, surface uniformity and thickness, chemical surface structure, and strength of adsorption). Therefore, the current work characterizes TMS-EDTA coated Au using four techniques: 1) surface plasmon resonance (SPR) to test the amount and stability of immobilized biomolecules (using streptavidin as a model protein), 2) atomic force microscopy (AFM) to reveal surface uniformity, roughness, and thickness, 3) infrared (IR) spectroscopy to elucidate the chemical structure of surface-adsorbed species and the extent of siloxane crosslinking, and 4) electrochemical differential capacitance to determine the strength of TMS-EDTA adsorption onto Au.

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Experimental Section General Materials and Gold Surface Preparation Detailed lists of the specific materials and reagents used are given below in each of the corresponding experimental sections. Here, the common materials and surface preparatory procedures are described. All chemicals and reagents were obtained from commercial sources: N -[(3-trimethoxysilyl)propyl]ethylene-diamine triacetic acid, Na salt (TMS-EDTA, pH ∼ 11; 50-60 wt.% water, 32-38 wt.% TMS-EDTA salt, and 8-12 wt.% sodium chloride) was from United Chemical Technologies (Bristol, PA); acetone (ACS reagent, ≥99.5%) and 2-Propanol (ACS reagent, ≥99.5%) were from Sigma-Aldrich (Oakville, ON, Canada). For all studies, ultrapure water (∼ 18.2 M Ω-cm) produced by a Milli-Q water purification system (EMD Millipore) was used. Planar Au slides for SPR, AFM, and IR experiments (see details in the corresponding experimental sections below) were cleaned by immersion in acetone, 2-propanol, and ultrapure water (repeated three times), followed by blow drying in an argon stream (Ultra High Purity 5.0, Praxair Canada Inc.). Subsequently, these planar Au surfaces were immediately immersed into different solutions (specified below) to create the desired surface modifications. For electrochemical differential capacitance studies, polycrystalline Au bead electrodes (diameter ∼= 2 mm) were cleaned and flame annealed by heating in butane flame until red hot, followed by rinsing with ultrapure water (repeated three times).

Surface Plasmon Resonance (SPR) For SPR studies, planar Au sensor chips (SIA Kit Au) from GE Healthcare (Mississauga, ON, Canada) were cleaned as previously described. Two Au chips were separately immersed in: 1) aqueous solution of 10% TMS-EDTA (v/v), and 2) ultrapure water (serving as reference surface for physical immobilization). After 2-hour immersion, the surfaces were rinsed with water and blown dry with argon gas, and then glued onto the cassettes according to 6


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the manufacturer’s instructions. All SPR experiments were performed at 25 degrees C in an SPR biosensor (BIACORE 3000™ apparatus) using these in-house assembled sensor chips. 1X PBS, pH 7.4, was prepared using Dulbecco’s Phosphate Buffered Saline (powder, no calcium, no magnesium) from Life Technologies (Burlington, ON, Canada), and used as the running buffer. N -(3-Dimethylaminopropyl)-N ’-ethylcarbodiimide hydrochloride (EDC; 98%) and N -hydroxysuccinimide (NHS; 98%) were from Sigma-Aldrich (Oakville, ON, Canada). Streptavidin purified (S203) from Leinco Technologies, Inc. (St. Louis, MO) was used as the model protein. For the Au chip immersed in pure water, streptavidin (100 µg/mL, diluted in PBS) was injected over the surface for 10 minutes. For TMS-EDTA modified Au chip, carboxyl-groups were first activated with an aqueous mixture of 50 mM NHS and 200 mM EDC for 7 minutes, and streptavidin was injected over the surface for 10 minutes. Stability of both streptavidin-covered surfaces was investigated by introducing five short injections (3 minutes each) of a stringent regeneration buffer (50 mM NaOH). The NaOH regeneration buffer (NaOH 50) was from GE Healthcare. All buffer and sample injections were conducted at a flow rate of 10 µL/min.

Atomic Force Microscopy (AFM) For AFM studies, cleaned planar Au slides (TA134, 5 nm Ti and 100 nm Au) from Evaporated Metal Films (Ithaca, NY) were used. Two types of surfaces were created. The first type was prepared by immersing the Au slide in an aqueous solution of 10% TMS-EDTA (v/v), and the second type was prepared by immersing the Au slide in ultrapure water (serving as Au substrate). After 2-hour immersion, the surfaces were rinsed with ultrapure water, blown dry with argon gas, and analyzed immediately using AFM. An Agilent 5500 AFM equipped with a 90 µm scanner and SiN Nanoprobe tips (Digital Instruments, spring constant of 0.8 N/m nominal) were used for contact-mode imaging of the adsorbed layers. For imaging, a 512×512 grid defined over an area of 3×3 µm2 was measured at 1 Hz with integral and proportional gains of 0.5 and 1, respectively (with a setpoint in the attrac7



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tive regime). Topography and friction measurements were obtained for both TMS-EDTA coated Au and clean Au substrate. Data processing was performed in Gwyddion 2.3.4 (Czech Metrology Institute) with median height matching and second order polynomial background subtraction.

Infrared Spectroscopy (PM-IRRAS) For polarization modulation-infrared reflection-adsorption spectroscopy (PM-IRRAS) studies, planar Au slides (TA134, 5 nm Ti and 100 nm Au) from Evaporated Metal Films were cleaned using the protocol described above. Three types of Au substrates were created by overnight immersion in: 1) aqueous solution of 10% TMS-EDTA (v/v), 2) aqueous solution of 10% 3-APTMS (v/v), and 3) ethanolic solution of 10 mM 11-MUA. The Au slide was blown dry with argon gas, and stored in a desiccator to remove excess moisture (at least two days prior to the experiments). (3-Aminopropyl)-trimethoxysilane (3-APTMS; 97%) and 11-mercaptoundecanoic acid (11-MUA; 97%) were from Sigma-Aldrich (Oakville, ON, Canada). Ethyl alcohol (denatured) was purchased from UBC Zoology Stores (Vancouver, BC). PM-IRRAS was carried out using a Bruker-55 spectrometer with an external PMA 50 accessory. The IR beam, after passing through a ZnSe grid polarizer and a ZnSe photoelastic modulator (HINDS Instruments, PEM-90, modulation frequency of 50 kHz), was focused on the sample at an incident angle of 80-85º. The light reflected from the sample was then focused onto an MCT detector (model D313/x1, Infrared Associates Inc. Stuart, Fl, USA.). The PM-IRRAS signal is given by the differential reflectivity

∆R R

=

(Rp −Rs ) (Rp +Rs )

and the

presented spectra resulted from the sum of 2048 scans recorded with 4 cm−1 resolution. The final step of data processing involved the background subtraction, which was created from the experimental data points using spline interpolation in MATLAB.

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Electrochemical Differential Capacitance Measurements Electrochemical experiments were performed in a three-electrode glass cell using an Autolab electrochemical analyzer (PGSTAT 12, Eco Chemie B.V.) equipped with NOVA 1.8 software. A scan/sweep controller (EG&G PAR 175) and an analog lock-in amplifier (EG&G Model 5210) were used for cyclic voltammetry (CV) and differential capacitance experiments. The working electrode was Au bead; the counter electrode was Pt coil; and the reference electrode was saturated calomel electrode (SCE). The reference electrode was connected to the electrolyte buffer solution via a salt bridge. The working solution was deoxygenated with argon and purged over the surface in all experiments. A buffer solution (100 mM phosphate, 500 mM KCl, and 65 mM KOH, pH 11.6), hereafter called DiffCap buffer, was prepared and used as the electrolyte for all electrochemical differential capacitance measurements. This buffer was formulated to simulate the multi-component surface modification solution used for bonding PDMS to Au and glass substrates. 15 Sodium phosphate dibasic dihydrate (puriss. p.a., buffer substance), potassium chloride (puriss. p.a. ACS), and potassium hydroxide (pellets, 99.99% metals basis, semiconductor grade) were from Sigma-Aldrich (Oakville, ON, Canada). A final working solution volume of 50 mL, measured to 0.1 mL, was used in all experiments (after compensating for volume of solution used in salt bridge). To ensure high reproducibility and cleanliness of the three-electrode electrochemical setup, a reproducible CV of the electrolyte was obtained prior to adding bulk TMS-EDTA concentrations for all experiments (data not shown). Subsequently, incremental concentrations of TMS-EDTA were added to DiffCap buffer, and capacitance was measured for a range of potentials (starting from −1.1 V and stepping positively to 0.2 V ) at each concentration. Specifically, each potential was held for 6 minutes (with stirring) for the system to reach equilibrium prior to measuring capacitance. To desorb the TMS-EDTA layer (true for concentrations less than 50 mM ) and to ensure similar starting conditions, a −1.1 V potential was applied (for 30 seconds without stirring) before proceeding from one potential 9



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to the next. The resulting capacitance was determined assuming a series resistor-capacitor equivalent circuit. MATLAB was used to analyze the concentration- and potential-dependent differential capacitance data for TMS-EDTA adsorption on Au bead electrode. Using the estimation method based on Shepherd et al.’s study, 18 the capacitance measured in DiffCap buffer solution at −0.9 V was normalized to the area of the Au bead electrode (calculated to be ∼ 0.25 cm2 ) and a value of 16.4 µF/cm2 was obtained (see Figure S12 in Supporting Information, SI). All capacitance data were adjusted to account for the Au electrode area to ensure comparability of the results. Subsequently, differential capacitance values were converted to surface coverage (at each applied electrode potential) using the Frumkin parallel plate capacitor model 19 as shown in Eq.1.

(1)

Cθ = Cθ=0 (1 − θ) + Cθ=1 θ

Here, Cθ is the measured differential capacitance value at a particular TMS-EDTA concentration, Cθ=0 is the differential capacitance value of DiffCap buffer (zero TMS-EDTA coverage), Cθ=1 is the minimum differential capacitance value obtained from 200 mM TMSEDTA (maximum TMS-EDTA coverage), and θ is the calculated fractional surface coverage. Next, the concentration-dependent surface coverage at each applied electrode potential was fit to Langmuir or Frumkin adsorption isotherms to obtain potential-dependent free energies of adsorption (−∆Goads ) and Frumkin lateral interaction parameters (α), 20 where positive or negative values of α indicate a repulsive or attractive interaction (between adsorbed molecules), respectively. Langmuir Isotherm: θ = Frumkin Isotherm: θ =

K[A] 1+K[A]

, where K = exp

K[A] exp(−αθ) , 1+K[A] exp(−αθ)

−∆Goads RT

where α = lateral interaction parameter

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Results and Discussion Surface Plasmon Resonance (SPR) SPR experiments were conducted to demonstrate the feasibility of using TMS-EDTA for coupling biomolecules on the Au surface, and to determine its relative stability (compared to bare Au) when a stringent wash buffer was used. The amount of biomolecules captured and the stability of this layer are important factors to be considered when developing a robust optical biosensor. Streptavidin was used as a model protein because it is a common component of chemically selective films for bio-sensing applications. 5 Figure 2A shows the SPR sensorgram that indicates the immobilization of streptavidin on a bare Au chip, followed by five injections of 50 mM NaOH regeneration buffer. Figure 2B shows the immobilization of streptavidin on a TMS-EDTA modified Au chip (using NHS/EDC coupling chemistry), followed by five injections of the same regeneration buffer. As can be seen from Figure 2A, the streptavidin immobilized on the bare gold surface corresponded to a peak value of about 690 RU (Resonance Unit or Response Unit), and the streptavidin washed off of the Au surface after NaOH injection corresponded to a decrease of about 630 RU . Therefore, only about 9.1% of the signal for adsorbed streptavidin remained after five NaOH injections. In contrast, the streptavidin immobilized on a TMS-EDTA modified gold surface (Figure 2B) resulted in a peak value of about 1610 RU , and the streptavidin washed off after NaOH injections resulted in a decrease of about 600 RU . Using a similar calculation, about 62.7% of adsorbed streptavidin remained on the TMS-EDTA modified Au after the stringent NaOH wash. Thus, the TMS-EDTA modified gold surface yielded both a substantial increase in the amount of streptavidin immobilized, and a substantial decrease in the amount of streptavidin removed after NaOH injections. The SPR results suggest that TMS-EDTA can be used to immobilize proteins on Au for bio-sensing applications. However, to acquire a better understanding of TMS-EDTA adsorption on gold for other applications, characterization of the film uniformity and thickness, the 11




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chemical surface structure, and the strength of adsorption is needed. Therefore, results from AFM, IR, and electrochemical differential capacitance measurements are detailed below to better understand the physical, chemical, and electrochemical properties, respectively, of TMS-EDTA adsorption on Au.

Atomic Force Microscopy (AFM) Spatial characteristics such as film uniformity and film thickness are also important for biosensing and biosensor fabrication. For example, the uncontrolled deposition of silanes onto silicon and glass surfaces can result in films with a loose network structure, and the final morphology and thickness (typically 1 to 8 molecular layers) are sensitive to water content, pH, and temperature. 21–24 To reveal more information about the thickness and uniformity of adsorbed TMS-EDTA layer(s) on Au, atomic force microscopy (AFM) was used. Topography (Figure 3A) and lateral friction (Figure 3B) measurements from AFM contact mode imaging of a clean Au substrate and TMS-EDTA modified Au are shown. Histograms of distribution profiles calculated from these topography and friction measurements were also determined (see Figures S1 and S2 in SI). From Figures 3A and S1, it is evident that the topography of the Au surface appears smoother when coated with TMS-EDTA. Similarly, from Figures 3B and S2, the Au surface exhibits higher friction when coated with TMS-EDTA. Subsequently, the same tip was used to scan the clean Au substrate (after scanning the TMS-EDTA modified Au). It is interesting to note that the friction of the bare Au substrate (after TMS-EDTA sample scan) has increased slightly (as compared with the first bare Au substrate scan). This result indicates that the silicon nitride tip may have been contaminated with adsorbed TMSEDTA molecules. Nevertheless, it is evident from Figures 3A and 3B that TMS-EDTA coats Au uniformly as measured over a large surface area (scan size of 3×3 µm2 ). Moreover, TMSEDTA does not form large clumps on the Au surface (i.e. no patchy areas were observed) as commonly observed on silicon-based surfaces. Next, AFM force curves were used to provide a rough estimate of the relative thickness of 13




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was also assigned to the CH2 deformation. Considering the TMS-EDTA spectra, the CH2 symmetric and asymmetric bands (also evident from ATR-FTIR spectra as shown in Figures S6 and S10 in SI) are also present as expected (2850 and 2928 cm−1 , respectively), but a much weaker signal than 11-MUA is observed. Signatures of CH3 stretch 27 is also evident as a shoulder at 2960 cm−1 , supporting the conclusion that the methyl groups may still be present in the adsorbed layer (a small 2960 cm−1 shoulder is also visible in the 11-MUA spectrum, possibly due to the solvent used). The presence of the absorption at 1680 cm−1 is evidence that the carboxyl groups are still present in the adsorbed TMS-EDTA. These carboxyl groups are also present in the ATR-FTIR spectra of TMS-EDTA (as shown in Figure S11 in SI), and absent in the ATR-FTIR spectra of 3-APTMS (as shown in Figure S9 in SI). The 3-APTMS spectra have distinct features in the 1000−1200 cm−1 region (also evident from Figures S7 and S9 in SI), which are characteristic of Si−O−Si stretching as well as Si−O−C bands 28 due to polysiloxane formation. The extent of siloxane crosslinking for 3-APTMS is quite substantial, as shown by the large peak at 1130 cm−1 and the lack of CH3 features in the alkyl region (see Figures 4B and S8). This is evidence of hydrolysis of the methoxy groups and siloxane crosslinking (creating a significant amount of Si−O−Si absorption). These results are consistent with the previous observation that water plays an important role in the surface attachment of amino-silanes. More specifically, previous results suggest that in the presence of water, the final multilayer film of amino-silanes on Au is composed of a cross-linked, two-dimensional siloxane network (due to complete hydrolysis). 29 On the other hand, the presence of some polysiloxane formation in TMS-EDTA is not too surprising (also evident from Figure S11 in SI), but the extent is significantly less than the 3-APTMS coated Au sample. Furthermore, the intensities of the bands of 3-APTMS spectra are more than 5x that of TMS-EDTA. These results confirm the multilayer formation of 3APTMS on Au, 22,29 and suggest that the amines are adsorbed onto the Au surface and the silane groups are directed away, resulting in extensive crosslinking. 30,31

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These IR results have provided supporting evidence for the previously observed presence of carboxyl groups by X-ray photoelectron spectroscopy. 15 In addition, we showed that TMSEDTA adsorbs on the Au surface with little signs of crosslinking. These results suggest that some of the carboxyl groups may still be oriented away from the surface, which serves to facilitate the coupling reactions that are needed for bonding to amine-modified PDMS and for the immobilization of biomolecules (via NHS/EDC chemistry). Nevertheless, the data presented so far cannot be used to conclusively infer an adsorption strength of TMS-EDTA on Au. Therefore, electrochemical differential capacitance is used to provide this information.

Electrochemical Differential Capacitance Measurements The strength of TMS-EDTA adsorption on Au (in the presence of water and ions) is the final parameter that is explored in this report, due to its importance for PDMS bonding and bio-sensing applications. Electrochemical differential capacitance is one of the best methods to determine the standard free energies of adsorption in a complex aqueous environment, while also elucidating its dependence on the substrate potential. In the previous carboxyl-amine bonding studies 15 and in all of the current SPR, AFM and IR studies, the Au substrates were functionalized with an aqueous 10% TMS-EDTA (v/v) solution (prepared using the commercially available stock TMS-EDTA solution – pH ∼ 11 – containing 50-60 wt.% water, 32-38 wt.% TMS-EDTA salt, and 8-12 wt.% sodium chloride), in the presence of dissolved O2 . The presence of dissolved O2 was found to set the potential of the substrate during the TMS-EDTA modification procedure. Specifically, the open circuit potential was measured to be ∼ 0.05 V and ∼ −0.05 V (vs. SCE) in the presence and absence of O2 , respectively (see Figure S13 in SI). These conditions are important when determining the strength of TMS-EDTA adsorption under the typical wet chemical coating conditions. It is important to note that in order to determine the relevant adsorption strength of TMS-EDTA on Au, the electrolyte buffer must replicate the conditions of aqueous 10% TMS17



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EDTA (v/v) solution. Moreover, the electrolyte composition must also be held constant for the accurate characterization of this adsorption. However, with every increase in TMS-EDTA concentration, [Cl− ] also increased (the molar ratio of sodium chloride to TMS-EDTA salt of the stock solution was calculated to be ∼ 2.26). To minimize the effects of chloride competitive adsorption (contributed from the incremental addition of stock TMS-EDTA solution), 500 mM KCl was added to the 100 mM phosphate buffer. Furthermore, 0.2 g of KOH was added to the phosphate and chloride buffer solution to replicate the highly basic state of 10% TMS-EDTA (v/v) aqueous solution (pH = 10.88±0.03). Consequently, DiffCap buffer (an electrolyte containing 100 mM phosphate, 500 mM KCl, and 65 mM KOH at pH 11.6) was prepared and used in these electrochemical differential capacitance studies (the influence of each component on capacitance measurements is shown in Figure S14 in SI). The capacitance in the DiffCap buffer was measured at the start of each experiment showing high reproducibility (see Figures S15 and S16 in SI). To perform these measurements, incremental concentrations of TMS-EDTA (1 µM to 200 mM ) were added to the DiffCap buffer. At each concentration, a series of electrode potentials was applied (changing from −1.1 to 0.2 V ), and differential capacitance was measured after each applied electrode potential was held for 6 minutes with stirring. Triplicates of lower concentrations of TMS-EDTA were conducted and small differences were observed (see Figure S17 in SI). Due to the long duration of differential capacitance measurements, further tests were conducted to test the stability of TMS-EDTA coated Au electrode in DiffCap buffer over the course of several hours. When compared with a bare Au electrode, the capacitance of TMS-EDTA modified electrode appeared to be very stable in DiffCap buffer for the duration of the experiments (see Figure S18 in SI). Figure 5A shows a selection of capacitance-potential curves of increasing TMS-EDTA concentrations in DiffCap buffer. The curve labeled “Buffer” represents the capacitance of a bare Au bead electrode in DiffCap buffer (i.e. in the absence of TMS-EDTA). In general, as the concentration of TMS-EDTA increased, the measured capacitance value decreased, due

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potentials less than −0.6 V , pseudocapacitance artifacts were observed, hence this range of potentials did not fit the parallel plate model as stated in Eq.1 and these potentials were not analyzed (see Figure S19 in SI). Therefore, the surface coverage was calculated in the limited potential window between −0.5 and 0.2 V (Figure 5B). In general, as the concentration of TMS-EDTA increased, the calculated surface coverage increased (within a range between 0 and 1). For potentials between −0.2 and 0 V , pseudocapacitance features due to Cl – or OH – adsorption influenced the capacitance and the calculation of coverage. With surface coverage data, the free energies of TMS-EDTA adsorption at each applied electrode potential were calculated by fitting to a Langmuir (dashed curve) or a Frumkin (solid curve) adsorption isotherm. The results at applied electrode potentials of −0.5 V (Figure 6A), −0.25 V (Figure 6B), and 0.1 V (Figure 6C) are shown. It was observed that at negative electrode potentials, both Langmuir and Frumkin isotherms described the data adequately. Surface excess was likely smaller at more negative potentials since TMS-EDTA is negatively charged at high pH (see Figure S20 in SI). However, as the electrode potential became more positive, the Langmuir isotherm failed to adequately fit the experimental data. The surface excess is greater at more positive potentials resulting in increased repulsion among the adsorbed TMS-EDTA molecules, for which the Frumkin isotherm better describes the experimental data. Potential-dependent free energies of adsorption (Figure 7A) and lateral interaction parameters (Figure 7B) derived from Frumkin fitting are shown. Figure 7A reveals that the strength of TMS-EDTA adsorption on Au electrode increases as the applied potential becomes more positive. Potential-dependent free energies of adsorption was determined to be ∼ −20 to −30 kJ/mol for the potential window shown (−0.5 to 0.2 V ). A positive Frumkin lateral interaction parameter from Figure 7B indicates repulsion among adsorbed TMSEDTA, and that this repulsion increases as the applied potential becomes more positive. These results are expected because surface excess is likely greater at more positive potentials, resulting in increased repulsion among the negatively charged TMS-EDTA molecules.

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is accompanied by competition from water molecules, hydroxide ions, and chloride ions for the Au surface. In this complex system, adsorption is a displacement process; therefore, the adsorption free energy of TMS-EDTA on Au is likely more negative than what we measure. Finally, recall that the typical wet chemical modification was achieved by immersing the Au substrate in a 10% TMS-EDTA (v/v) solution with dissolved oxygen. The open circuit potential (OCP) of the Au substrate under these conditions (see Figure S13 in SI) was ∼ 0.05 V (vs. SCE). The free energy of adsorption at this deposition potential (obtained from Figure 7A) suggests that TMS-EDTA strongly modifies the Au surface at OCP. These results support the previously reported observation that a relatively strong bond was achieved between TMS-EDTA-modified Au slides and amine-modified PDMS blocks. 15 Finally, at highly negative potentials (∼ −1.1 V ), TMS-EDTA is desorbed. This result suggests that the TMS-EDTA layer can be removed by applying negative potentials (i.e. a bare Au surface can be recovered after the PDMS bonding procedure).

Conclusions The adsorption of a carboxylated silane (TMS-EDTA) onto Au has been characterized using SPR, AFM, IR and electrochemical methods. The results from SPR experiments indicate that free carboxylates on TMS-EDTA modified Au are available for the immobilization of streptavidin, following activation by NHS/EDC. Furthermore, this unconventional surface chemistry can withstand stringent regeneration conditions – a quality important for developing robust biosensors. AFM imaging strongly suggests that a thin and uniform coverage of TMS-EDTA on the gold surface is obtained. In IR studies, the TMS-EDTA coated gold surface shows a significant presence of carboxyl groups (similar to 11-MUA coated Au) and a lack of polysiloxane formation (in contrast to 3-APTMS coated Au). Electrochemical differential capacitance measurements reveal that TMS-EDTA adsorption on Au is potential-dependent. At very negative potentials (∼ −1.1 V ), there is minimal adsorption. For potentials between

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−0.5 and 0.2 V , the potential-dependent free energies of adsorption were determined to be ∼ −20 to −30 kJ/mol in the complex electrolyte solution (measured under relevant conditions for use in future sensor and biosensor fabrication and applications). These results suggest that at more positive potentials (i.e. at the potential typical for surface modification), strong adsorption is thermodynamically favorable. Due to its negative charge, there is more repulsive lateral interaction among the adsorbed TMS-EDTA at these potentials. The fundamental knowledge obtained about TMS-EDTA adsorption on Au is valuable towards the rational design of integrated sensors and biosensors. Our results suggest that TMS-EDTA can potentially be used in a hybrid glass/gold substrate to achieve: 1) the bonding between 3-APTMS-modified PDMS and Au to create a leak-free PDMS microfluidic chamber (Figure 1D), 2) the immobilization of biomolecules on Au inside the PDMS cell chamber (Figure 1C), and 3) the electrochemical desorption of TMS-EDTA layer to recover clean Au for subsequent sensing and bio-sensing applications. With the results obtained, current efforts are being directed towards applying and using this fabrication technology for constructing robust electrochemical and optical integrated systems.

Acknowledgement The authors would like to thank Dr. Eric T. Lagally for pioneering the idea behind this work, and Drs. Charles Haynes and Louise Creagh for the use of SPR biosensor (BIAcore 3000™), and other supplies and equipment in Haynes Laboratory at Michael Smith Laboratories. The funding for this work is provided by NSERC Discovery and Equipment Grants, CFI, Michael Smith Laboratories, and UBC Department of Chemical and Biological Engineering.

Supporting Information Available Histograms of distribution profiles from AFM topography and friction measurements, AFM force curves, background subtraction example for PM-IRRAS analysis, ATR-FTIR spectra 24


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for samples, Au bead area normalization procedure, OCP measurements, components of DiffCap Buffer, reproducibility of DiffCap buffer (averaged values and standard deviations), reproducibility of low concentration of TMS-EDTA in DiffCap Buffer, stability measurements, surface coverage for all potentials, and titration curve of aqueous 10% TMS-EDTA (v/v) solution. This material is available free of charge via the Internet at http://pubs.acs.org/.

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Adsorption of a Carboxylated Silane on Gold: Characterization for Its

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