Two-Dimensional Molecular Imprinting Approach to Produce Optical

Publication Date (Web): October 7, 2006 ... Imprinting of Molecular Recognition Sites through Electropolymerization of Functionalized Au Nanoparticles...
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Langmuir 2006, 22, 9658-9663

Two-Dimensional Molecular Imprinting Approach to Produce Optical Biosensor Recognition Elements Xiao Li and Scott M. Husson* Department of Chemical and Biomolecular Engineering, Clemson UniVersity, Clemson, South Carolina 29634-0909 ReceiVed May 1, 2006. In Final Form: July 12, 2006 This article describes a new two-step methodology for preparing thiol monolayers having artificial recognition sites for dansylated amino acids on gold optical biosensor surfaces. N-Dansyl-L-lysine (DK) was used as the template molecule to form molecularly imprinted monolayers (MIMs). Impact factors that were studied were the concentration of DK in step one (template deposition) and the time and method for thiol monolayer formation in step two (backfilling). Compared to a prior method that used the simultaneous adsorption of the template and thiol from solution, this new approach provides the flexibility to imprint template molecules that have low binding energies on gold. Control over the surface density of imprinting sites can be achieved by this approach, and rebinding studies done using surface plasmon resonance spectroscopy confirmed that the MIMs prepared against DK showed selectivity for that template over didansyl-L-lysine.

Introduction Since about the mid-1990s, significant attention has been paid to developing and applying the molecular imprinting technique to prepare recognition elements for applications in biosensing. Molecularly imprinted sensing materials have been successfully designed to integrate with most types of transduction platforms, including electrochemical,1,2 gravimetric,3,4 and optical devices.5,6 Whereas most work on MI-based biosensors has focused on the 3D molecularly imprinted polymer (MIP) networks, few studies have investigated 2D self-assembled monolayer (SAM) systems, which show great potential advantages such as construction simplicity and fast sensor response times. SAM systems have the additional advantages of being well understood with regard to both the formation mechanism7-10 and structure-property relationships.11,12 Many self-assembly systems have been investigated; however, the most studied and probably the most understood SAM system is that of alkanethiols on gold surfaces. Recently, this SAM system has been used as a foundation layer to produce MIP-based optical biosensors.5,13-15 * To whom correspondence should be addressed. E-mail: shusson@ clemson.edu. Tel: (864) 656-4502. Fax: (864) 656-0784. (1) Panasyuk, T. L.; Mirsky, V. M.; Piletsky, S. A.; Wolfbeis, O. S. Anal. Chem. 1999, 71, 4609. (2) Parmpi, P.; Kofinas, P. Biomaterials 2004, 25, 1969. (3) Liang, C. D.; Peng, H.; Bao, X. Y.; Nie, L. H.; Yao, S. Z. Analyst 1999, 124, 1781. (4) Dickert, F. L.; Hayden, O.; Bindeus, R.; Mann, K. J.; Blaas, D.; Waigmann, E. Anal. Bioanal. Chem. 2004, 378, 1929. (5) Lotierzo, M.; Henry, O. Y. F.; Piletsky, S.; Tothill, I.; Cullen, D.; Kania, M.; Hock, B.; Turner, A. P. F. Biosens. Bioelectron. 2004, 20, 145. (6) Rathbone, D. L.; Bains, A. Biosens. Bioelectron. 2005, 20, 1438. (7) Nuzzo, R. G.; Zegarski, B. R.; Dubois, L. H. J. Am. Chem. Soc. 1987, 109, 733. (8) Nuzzo, R. G.; Fusco, F. A.; Allard, D. L. J. Am. Chem. Soc. 1987, 109, 2358. (9) Bain, C. D.; Troughton, E. B.; Tao, Y. T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321. (10) Pan, W.; Durning, D. J.; Turro, N. J. Langmuir 1996, 12, 4469. (11) Laibinis, P. E.; Whitesides, G. M.; Allara, E. L.; Tao, Y.-T.; Parikh, A. N.; Nuzzo, R. G. J. Am. Chem. Soc. 1991, 113, 7152. (12) Akimoto, K.; Sato, F.; Morikawa, T.; Fujihira, M. Jpn. J. Appl. Phys. 2004, 43, 4492. (13) Yoshikawa, M.; Hotta, N.; Kyoumura, J.; Osagawa, Y.; Aoki, T. Sens. Actuators, B 2005, 104, 282. (14) Wei, X.; Li, X.; Husson, S. M. Biomacromolecules 2005, 6, 1113. (15) Li, X.; Husson, S. M. Biosens. Bioelectron. 2006, 22, 336.

As described previously,15 control over the recognition-layer thickness is crucial to avoiding intralayer diffusional mass-transfer limitations in real-time biosensing applications. Using controlled polymerization techniques to form polymeric MIP nanolayers14,15 has led to significant improvements relative to conventional formation methods that yield thick materials with slow apparent binding kinetics due to the diffusion of the template into and out of the cavities.16 Along similar lines of reasoning, building molecularly imprinted monolayers (MIMs) as biosensor recognition elements would minimize any problems associated with slow mass transfer to recognition sites on the biosensor surface. SAM formation usually results in a well-packed monolayer film with angstrom-level thickness. With such a low thickness, the template can be extracted completely following the creation of the imprint sites during the imprinting step. Also, the template molecules can reach the imprint sites easily and quickly during the rebinding step, which is important for real-time sensing. Furthermore, SAMs provide highly uniform surfaces9,17 that allow us to conduct meaningful SPR analysis, where surface chemistry should be reproducible and preferably defect-free.18 Compared to 3D MIP systems, the surface-binding capacity of this simplified 2D MIM may be much lower; nevertheless, this problem can be overcome by the adsorption of gold nanoparticles onto the optical sensor platform.19,20 Since the binding sites are easily accessible, this approach seems promising for developing molecular recognition agents to detect large template molecules such as proteins, which have been difficult to prepare using conventional imprinting methods. This article discusses the preparation of simplified 2D, molecularly imprinted monolayers with recognition properties. These 2D MIMs were prepared on SPR biosensor chips to create an optical biosensor. The first difference between this work and the few previously reported 2D molecular imprinting approaches is that an optical transduction platform was adopted that provides (16) Yilmaz, E.; Haupt, K.; Mosbach, K. Angew. Chem. 2000, 39, 2115. (17) Ulman, A. Chem. ReV. 1996, 96, 1533. (18) Green, R. J.; Davies, J.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B. Biomaterials 1997, 18, 405. (19) Yee, C. K.; Ulman, A.; Ruiz, J. D.; Parikh, A.; White, H.; Rafailovich, M. Langmuir 2003, 19, 9450. (20) Tokareva, I.; Minko, S.; Fendler, J. H.; Hutter, E. J. Am. Chem. Soc. 2004, 126, 15950.

10.1021/la0612163 CCC: $33.50 © 2006 American Chemical Society Published on Web 10/07/2006

Production of Optical Biosensor Recognition Elements

advantages including nondestructive operation, high sensitivity, absence of electrical interference, and fast response and evaluation. Several approaches have been investigated recently to create 2D electrochemical sensors.21-24 Piletsky et al.21 used hexadecyl mercaptan to form MIMs on Au electrodes with specificity for cholesterol. In this approach, thiol molecules and template molecules (cholesterol) were adsorbed simultaneously on the surface of gold electrode. This seminal work opened the door for 2D MIMs, but the simultaneous adsorption approach does suffer from competitive binding between the thiol and the template during imprinting, which limits control over the density of imprinting sites on the surface. Chou and Liu24 adopted the same MIM system to integrate with an electrochemical sensor prototype. They achieved high detection sensitivity for cholesterol, and the binding kinetics of cholesterol on the sensor were investigated over a 300 s time interval. Unfortunately, no information was given on imprinting selectivity, which is an important consideration if the sensor is to be used for the detection of cholesterol in a mixture of other compounds. Another important contribution of the current study is that we developed a new method to prepare imprint sites. All previous 2D imprinting studies used the simultaneous adsorption of thiol and template molecules.21-24 A drawback to simultaneous adsorption when using biomolecule templates is that the goldthiolate bond strength is much stronger than the interaction between biomolecules and gold, which is mainly based upon van der Waals (hydrophobic) interactions. Therefore, if left to compete for a long enough time, thiols will cover the entire substrate preferentially. We have replaced simultaneous adsorption with a two-step imprinting process involving pretreatment of the gold surface by a solution containing only the template followed by backfilling with thiol. In this way, the gold surface first was covered by an equilibrium deposition of template molecules, and then the thiol chains were formed around the template molecules by backfilling. Removal of the template molecules from the gold substrate using a wash step leaves the thiol layer patterned with cavities that match the template molecules geometrically. In this study, two new procedures have been developed to prepare 2D MIM surfaces. Surface plasmon resonance (SPR) spectroscopy was used to measure the adsorption isotherms of dansylated amino acids onto these MIM surfaces. Impact factors that were studied were the concentration of template molecule in the pretreatment solutions and the time and method for the second step involving thiol backfilling. We discuss solution- and vapor-phase backfilling with thiol. Materials and Methods Materials. Gold substrates were purchased from BIAcore, Inc. (SIA Kit Au, BR-1004-05). These chemicals were used as received from Aldrich with the following weight percentages: 11-mercapto1-undecanol (MUD) (97%), 2-mercaptoethanol (2-ME) (99%), ethyl alcohol (ACS reagent, 99.5%), and water (HPLC grade). N-DansylL-lysine (DK) (95%), N,N′-didansyl-L-lysine (DDK) (95%), and HEPES (99.5%) were used as received from Sigma. Water for surface washing and cleaning was deionized and filtered through a Millipore Milli-Q water purification system (New Bedford, MA). Preparation of Molecularly Imprinted Monolayers (MIMs). Scheme 1 outlines the synthetic pathway to prepare 2D molecularly imprinted, self-assembled monolayers on gold surfaces. The gold(21) Piletsky, S. A.; Piletskaya, E. V.; Sergeyeva, T. A.; Panasyuk, T. L.; El’skaya, A. V. Sens. Actuators, B 1999, 60, 216. (22) Mirsky, V. M.; Hirsch, T.; Piletsky, S. A.; Wolfbeis, O. S. Angew. Chem., Int. Ed. 1999, 38, 1108. (23) Kitano, H.; Taira, Y. Langmuir 2002, 18, 5835. (24) Chou, L. C. S.; Liu, C.-C. Sens. Actuators, B 2005, 110, 204.

Langmuir, Vol. 22, No. 23, 2006 9659 Scheme 1. Schematic Representation of 2D Imprinting with a SAM

coated biosensor substrates were cleaned with fresh Piranha solution. (Details of the cleaning procedures and precautions for the use of Piranha solution are described elsewhere.25) For method I, the cleaned gold substrates were immersed in 5 mL of an ethanol solution containing 1 mM MUD and 3 mM DK overnight (12-20 h) at room temperature. Method I is equivalent to that used by previous investigators.21-24 For method II, the cleaned gold substrates were immersed in 5 mL of a 3 mM DK ethanol solution overnight at room temperature to ensure equilibrium binding of DK to the gold surface. The surfaces were then incubated in 5 mL of a mixed solution of 1 mM MUD and 3 mM DK in ethanol for 5-15 min. In method III, the cleaned gold substrates were pretreated in an ethanol solution of 1 or 3 mM DK, as was done in the first step in method II. Then, the surfaces were put in the setup shown in Scheme 1. A 100 mL round-bottomed flask was charged with 10 mL of pure 2-ME. This flask was connected to a column with a side port where the pretreated gold substrate was placed. The top of the column was covered with an open-top cap with a septum in the center. The whole setup was moved into a vacuum oven at 50 °C and 20 in. Hg for 1-3 h, and a syringe needle provided the open connection between the setup and the inside of the low-pressure oven. The final MIM surfaces were rinsed thoroughly with pure ethanol and deionized water and then dried in a stream of nitrogen. Characterization was done with contact angle, ellipsometry, and ER-FTIR measurements to ensure the successful formation of a thiol monolayer. The cleaned, bare gold surface was used as a reference surface, and the pure thiol monolayer surface was used as a nonimprinted control surface. For a comparison of template adsorption with MIM surfaces obtained using methods I and II, the control surface was prepared by the incubation of Au substrates in 5 mL of 1 mM MUD ethanol solution overnight at room temperature; for comparison with surfaces prepared using method III, the control surface was prepared by the incubation of Au substrates in 5 mL of pure 2-ME overnight at room temperature. Contact Angle Measurements. Static water contact angles were measured with the sessile drop method using a Kru¨ss DSA10-MK2 CA system (Kru¨ss, Germany) at ambient temperature. Five sample spots were taken on each surface using HPLC-grade water as the probe liquid. Ellipsometry. The refractive index and extinction coefficient of bare gold and the thickness values for all samples were obtained using a phase-modulated spectroscopic ellipsometer. Details of the experimental measurements are given in Supporting Information. External Reflectance Fourier-Transform Infrared Spectroscopy. ER-FTIR spectra were obtained using a Nicolet Nexus 870 FTIR spectrometer equipped with a nitrogen-purged chamber and a DTGS-KBr detector. Spectra were collected using 2000 scans at a resolution of 2 cm-1 and an 80° angle of incidence. (25) Li, X.; Wei, X.; Husson, S. M. Biomacromolecules 2004, 5, 869.

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Li and Husson Table 1. Static Water Contact Angle Results for MIM Surfaces incubation time (method II)

Figure 1. External reflectance FTIR spectra on a gold substrate: (a) 11-mercapto-undecanol MIM and (b) 2-mercaptoethanol MIM. Surface Plasmon Resonance (SPR) Spectroscopy. SPR measurements were conducted using a BIAcore X instrument at 25 °C. The gold-coated glass substrate with an MIM layer was mounted on a plastic cassette. The analysis methods and conditions were the same as those described in Li and Husson,15 except that the regeneration solution was 25 µL of 25% (v/v) ethanol in deionized water using a normal injection mode. All adsorption (rebinding) measurements were conducted immediately after the preparation of the surfaces. A pH value of 7.0 was chosen to conduct all of the rebinding experiments.

Results and Discussion 11-Mercaptoundecanol (MUD) and 2-mercaptoethanol (2-ME) were chosen as the model thiols to prepare the self-assembled monolayer for two reasons: they have hydroxyl end groups that resist the nonspecific adsorption of biomolecules,26 and they are available commercially. The reason for choosing the short-chain thiol, 2-ME, instead of the long-chain thiol, MUD, for method III is based on the physical properties of these two thiols. 2-ME is a liquid at room temperature (whereas MUD is solid) that has a relatively low normal boiling point of 157-158 °C (53-55 °C at 12 mmHg). In method III (Scheme 1), thiol molecules were adsorbed from the vapor phase onto a gold substrate pretreated with template molecules. Because of the instability of the Au-S bond at temperatures above ∼60-80 °C,9,27 this procedure is best conducted at temperatures lower than 60 °C, and 2-ME can be used to create a thiol vapor under moderate vacuum conditions and low temperature. Previous studies on n-alkanethiols, CH3(CH2)nSH, have shown that long-chain-length monolayers, where n g 5, form a densely packed, crystalline-like assembly with fully extended chains tilted from the surface normal by 20-30°, whereas for the short-chainlength monolayers, where n < 5, as the chain length decreases, the structure becomes increasingly disordered with lower packing densities and coverage.7,28 Therefore, to be more rigorous, the monolayer formed by 2-ME cannot be called self-assembled because of the weak van der Waals interactions between the methylene groups. However, for the convenience of indication, MIM will still be used as the abbreviation for all molecularly imprinted thiol monolayer surfaces prepared by both MUD and 2-ME. Characterization of Physical and Chemical Surface Properties. The ellipsometric thickness measurements for MIM surfaces prepared by methods I and II were between 9 and 12 Å; values were between 1 and 3 Å for MIM surfaces prepared by method III, where 2-ME was used instead of MUD. In the reflectance FTIR spectra (Figure 1), small peaks around 2920 and 2850 cm-1 were observed that can be assigned to the aliphatic -CH2- stretching modes. These results from both ellipsometry (26) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714. (27) Yang, X.; Perry, S. S. Langmuir 2003, 19, 6135. (28) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559.

template concentration (method III)

5 min

10 min

15 min

1 mM

3 mM

59 ( 4°

53 ( 3°

41 ( 3°

60 ( 4°

64 ( 5°

and ER-FTIR indicated the successful deposition of thiol molecules on gold surfaces using all three methods. The shifts to higher wavenumber values for -CH2- stretching in the FTIR spectrum for 2-ME compared to that for MUD are indicative of the lower degree of ordering of the short-chain thiol on the gold surface.28,29 Measured water contact angles on surfaces prepared using method I were similar to those measured previously for pure MUD surfaces;25 values were between 30 and 35°. For methods II and III, Table 1 shows that the contact angles ranged from 41 ( 3 to 64 ( 5° depending on the thiol incubation time in method II and the template concentration in method III. These higher values likely result from the partial exposure of the gold surface (which generally exhibits high contact angles of ∼80° 25) or the exposure of hydrophobic methylene chains from thiol molecules that “lie down” at the periphery of imprint cavities, where there is more vacant space. Either way, higher contact angles indicate that surface imprint cavities were formed by methods II and III. By using method II, the contact angle decreased with increasing incubation time, indicating that thiol molecules displace the preadsorbed template molecules. Overall, method III resulted in higher contact values than method II. The surface with higher template concentration in the pretreatment solution of method III had the highest average contact angle among all MIM surfaces. These results are consistent with the expectation that method III improved the persistence of template molecules on the gold substrate by not allowing template displacement from surface to solution. However, it is possible that differences in the packing efficiency of 2-ME and MUD may play some role in the higher contact angles for MIM surfaces prepared by 2-ME. As described previously and seen in the ER-FTIR results, short-chain-length thiols form a relatively disordered monolayer with lower packing densities and coverage, as compared to densely packed, crystalline-like assemblies formed by long-chain-length thiols with -OH end groups extending from the gold surface. More exposure of hydrophobic methylene groups may occur for the short-chainlength thiol monolayer, leading to higher water contact angles. Characterization of Adsorption by Surface Plasmon Resonance (SPR) Spectroscopy. N-Dansyl-L-lysine (DK) and N,N′-didansyl-L-lysine (DDK) were chosen as the model template and analogue, respectively, for these reasons: they have high enough molecular masses to be studied by SPR (analysis by the BIAcore X SPR instrument imposes a detection limit of ∼200 Da), have fair solubility in both ethanol and water, and are available commercially. This last requirement excluded Ndansyl-D-lysine from study. Because SPR signals depend on the property of the gold film, each set of adsorption isotherm data was constructed using one SPR chip; therefore, the surface properties were the same for measurements at different concentrations. Figure 2 shows raw sensorgram examples for DK adsorption on an MIM surface prepared by method III. In all measurements, the response returned to its original baseline value after the high-speed buffer wash and regeneration; therefore, the template molecule could be removed from MIM surfaces with 100% efficiency. From SPR sensorgrams of real time measurements, we observed that the adsorption of template molecules on MIM (29) Snyder, R. G.; Strauss, H. L.; Elliger, C. A. J. Phys. Chem. 1982, 86, 5145.

Production of Optical Biosensor Recognition Elements

Figure 2. SPR sensorgrams of N-dansyl-L-lysine (DK) adsorption on an MIM surface prepared by method III using DK as the template molecule. Sensorgrams were collected at different concentrations of DK (in µM) in pH 7 HEPES buffer: 131.8, 263.5, 527.0, 790.5, 2108.0, and 5270.1. The flow rate was 20 µL/min. The horizontal line is the baseline. All measurements were taken at 25 °C.

Figure 3. Adsorption isotherms at 25 °C for DK on an MIM surface prepared using method I with DK as the template molecule. The solvent was 10 mM HEPES buffer at pH 7.0.

surfaces reached equilibrium well within the 2 min run times; this response was much faster than the 30-45 min response time for 3D molecularly imprinted membrane sensors.30 The measured equilibrium data were the difference in signal between points before and after sample injection, with bulk shift and blank buffer response subtracted from the overall response. The mass of adsorbed template molecules per unit area was calculated from the linear correlation between this quantity and RU response. (See Li et al.25 for additional detail.) Adsorption on MIM Surfaces Prepared by Method I. Figure 3 presents the quantitative adsorption capacity (Q) results of DK adsorption on bare gold, a pure thiol-SAM surface covered by MUD, and an MIM surface with DK as the template molecule prepared by method I, where gold substrates were incubated in a mixed solution of thiol and template molecules. The x axis represents the concentration of template in solution during the rebinding measurements. The data represent average values from two batches, with error bars representing standard deviations. (30) Piletsky, S. A.; Piletskaya, E. V.; Elgersma, A. V.; Yano, K.; Karube, I.; Parhometz, Y. P.; El’skaya, A. V. Biosens. Bioelectron. 1995, 10, 959.

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Figure 4. Adsorption isotherms at 25 °C for DK on MIM surfaces prepared using method II with DK as the template molecule. The different times reported for MIM surfaces indicate the incubation time in the mixed template-thiol solution after the pretreatment step. The solvent was 10 mM HEPES buffer at pH 7.0.

Having been reported in several studies,21,22,24 this simultaneous adsorption method is a simple and straightforward way to prepare MIM surfaces. However, as Figure 3 shows, method I failed in this case, where the incubation time of 12 h was equivalent to the 12 h used by Pilestky et al.21 and similar to the 16 h used by Chou and Liu.24 The rebinding capacities of template molecules on the MIM surface prepared by this method were similar to those on the surface covered by pure thiol molecules, indicating that there was little-to-no template adsorbed on the gold substrate during the surface preparation process to form cavities. Initial hypotheses for this result were that (a) there may be a difference, possibly in magnitude, in the adsorption kinetics for the template and thiol on Au substrates, where the adsorption rate of the template is much slower than that for the thiol, or (b) the stronger binding energy of thiol on gold relative to that of template on gold may lead to competitive binding between adsorbed template molecules and thiol molecules during simultaneous adsorption. This problem could be common to applying this method to prepare 2D MIM surfaces, where template molecules may have slower adsorption kinetics compared to the fast thiol-gold binding rate, which leads to nearly fully packed monolayers in seconds.10 To overcome this problem and also to test whether it is rate-based or equilibrium-based, we decided to test a two-step process that involved pretreating the gold substrates in a solution containing pure template molecules followed by the deposition of thiol. Adsorption on MIM Surfaces Prepared by Method II. Figure 4 demonstrates the persistence of template molecules on gold substrate during MIM preparation and the successful formation of imprint cavities using method II. The data represent average values from two batches, with error bars representing standard deviations. The rebinding capacities of template molecules on the MIM surface using this method lie between the adsorption capacities on bare gold and pure thiol-SAM surfaces, indicating that there is some area of gold that remains uncovered by MUD onto which the DK can rebind. Furthermore, the area of uncovered gold can be adjusted by the incubation time in the second step. Shown in Figure 4, the imprint site density decreases with increasing incubation time, and the surface was covered almost entirely by thiol molecules after only 15 min of incubation in MUD-template solution. This result again demonstrates that

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the stronger interactions of thiol-gold relative to those of template-gold lead to the replacement of preadsorbed template molecules by thiol molecules. During thiol backfilling, the solution contained the template at the same concentration that was used in the pretreatment step. From a thermodynamic point of view, this provides the same driving force for the adsorption of template on gold from the pretreatment solution and the mixed thiol-template solution. Nevertheless, template adsorption is reversible, and equilibrium is characterized by dynamic exchange of the template between the solution and surface. The thiol-gold bond, however, is covalent. Over time, as template molecules desorb during dynamic exchange, thiol molecules have the opportunity to bind to exposed surface sites. Once bound, the thiol molecules remain on the surface, eventually coating the entire surface. Because the template had been preadsorbed, any changes in the surface functionality must have come about from thermodynamic exchange rather than differences in the adsorption kinetics. Therefore, template replacement by thiol is the reason for the failure of the simultaneous adsorption approach (method I) to produce imprint cavities. The development of method II provided a means to overcome the problem associated with competitive binding between the thiol and template molecules. However, a drawback of this method is that it does not allow fine control over the surface density of imprint sites. As discussed in the previous paragraph, the displacement of adsorbed template by thiol in the second step is controlled solely by the incubation time in step 2 of this method. Because this displacement process is rapid (essentially all template is displaced in about 15 min), controlling the concentration of imprint sites by using method II is challenging and may lead to a lack of reproducibility for capacity data from different batches. That is, it would be preferable for the binding capacities to be controlled (or preserved) by equilibrium binding rather than ratebased displacement. To overcome the drawback of method II, we studied the adsorption of thiol molecules from the vapor phase instead of using thiol self-assembly from a mixed liquid solution; we call this procedure method III. In method III, the displacement of preadsorbed template by thiol into solution is avoided; the highboiling-point template molecule remains on the gold surface. The result is a surface imprint density that is controlled by pretreatment (i.e., preadsorption or predeposition of template), and the volatile 2-ME thiol fills in the gaps between adsorbed template molecules. Adsorption on MIM Surfaces Prepared by Method III. Figure 5 presents the quantitative results of DK adsorption on bare gold, 2-ME covered gold, and the MIM surface prepared by method III with DK as the template. Here, the thiol molecules were deposited from the vapor state (see Scheme 1 for detail) after gold substrates went through the same pretreatment procedure as method II. Relative to the concentrations used in methods I and II (265.5-2635.0 µM), a wider range of solution concentration for DK was covered in this method III, from 131.8 to 5270.1 µM. Figure 5 illustrates the successful deposition of thiol around preadsorbed template using method III to form imprint sites. That Figure also shows that it is possible to adjust the number of imprint sites via the template concentration in the pretreatment solution. By comparing the data collected for the same pretreatment concentration and two different vapor deposition times, it is clear that imprint site density is not affected by thiol vapor deposition times. The data represent average values from two

Li and Husson

Figure 5. Adsorption isotherms at 25 °C for DK on MIM surfaces prepared using method III with DK as the template molecule. The different conditions indicated for the MIM surfaces represent the template concentration in the pretreatment solution and the thiol vapor deposition time. The solvent was 10 mM HEPES buffer at pH 7.0. Table 2. Fitted Parameters for the Langmuir Adsorption Model Determined by Regression of MIM and Thiol Monolayer Adsorption Isotherm Dataa

DK on DK-MIM DK on thiol DK on bare gold DDK on DK-MIM DDK on thiol DDK on bare gold

Qmax (pmoles/cm2)

K (104) (µM-1)

K* (pmoles/cm2‚µM)

495.8 270.2 668.4 206.2 132.0 300.2

1.91 1.25 3.42 16.52 13.30 17.26

0.095 0.034 0.228 0.331 0.176 0.483

a The MIM surface was prepared using method III with DK as the template molecule. The DK concentration in the pretreatment solution was 1 mM, and thiol vapor deposition was done for 3 h. For rebinding, the pH was 7.0.

batches, with error bars that represent standard deviations; therefore, the reproducibility is good. To demonstrate the applicability of this method to prepare an MIM surface that is selective toward the rebinding of its own template, selectivity was tested by adsorbing a template analogue, DDK with similar functionality but larger size, on these same surfaces. A selectivity coefficient was defined as

R)

/ / KDK,MI-SAM /KDK,thiol-SAM / / KDDK,MI-SAM /KDDK,thiol-SAM

(1)

where K* represents the initial slope values for the adsorption isotherms. Normalization was carried out relative to the thiolSAM (the control surface) in order to account for differences in nonspecific adsorption of these DK and DDK on the control. Adsorption data were fit using the Langmuir adsorption model. Table 2 gives the fitted K* values for an MIM surface prepared against DK using method III, where the DK concentration in the pretreatment solution was 1 mM and the thiol vapor deposition time was 3 h. Rebinding was done at pH 7.0. The selectivity coefficient was calculated to be 1.48 for this surface. The selectivity indicated by R > 1 suggests that the MIM surface has a specific binding of its own template molecule relative to that

Production of Optical Biosensor Recognition Elements

Langmuir, Vol. 22, No. 23, 2006 9663

for the template analogue. However, there was cross reactivity between these two molecules, indicated by the fact that the adsorption capacity of DDK on the MIM surface was higher than that on the pure thiol-monolayer surface. Despite normalization against the thiol-SAM in order to account for differences in nonspecific adsorption of DK and DDK on the SAM, there may be concern that the calculated selectivity coefficient result might be due to preferred nonspecific binding of DK on gold relative to DDK. To test this possibility, we measured the K* values for DK and DDK on bare gold. Table 2 presents the results, which indicate that nonspecific adsorption of DDK onto gold is preferred relative to DK (i.e, K*(DDK) > K*(DK)). This result provides further evidence that the selectivity is based on differences in the specific binding of DK. The selectivity coefficients were calculated to be 1.64 for MIM surface 2, prepared using a 3 mM DK pretreatment solution and thiol vapor deposition for 3 h, and 1.56 for MIM surface 3, prepared using a 1 mM DK pretreatment solution and thiol vapor deposition for 1 h. The similarity in adsorption selectivity for all three MIM surfaces suggests that the selectivity is a property of the system that is insensitive to the surface density of imprint sites. Equation 2 was used to estimate the percentage of gold surface area left uncovered by SAM molecules after the imprinting process. By this definition, a higher Au % indicates a higher surface coverage by the template during imprinting, and therefore a higher number of surface imprinting sites:

Au % )

Qmax,MI-SAM - Qmax,thiol-SAM × 100% Qmax ,Au - Qmax ,thiol-SAM

(2)

The estimated Au % values calculated from DK adsorption parameters are 57 ( 12, 79 ( 9, and 64 ( 11 for MIM surfaces 1, 2, and 3, respectively. The similar Au % values for surfaces 1 and 3 demonstrate that the number of imprinting sites is not affected (within experimental uncertainties) by the thiol vapor deposition time, which indicates that 1 h is sufficient to completely cover the gold substrate in the exposed spaces between templates. This result also supports the notion that there is no displacement of template by thiol using this method. An analysis of Au % values between surfaces 1 and 2 indicates that the pretreatment template concentration impacts the imprint density. A higher concentration used for surface 2 led to a higher imprint capacity. The Au % values were also calculated from DDK adsorption parameters. According to the definition of Au % in eq 2, if the imprint site occupancies of DK and DDK were equal on the surfaces, then the Au % would be equal. Values calculated from the DDK adsorption parameters given in Table 2 are lower than those calculated using the DK adsorption parameters for all three surfaces, which further suggests that these surfaces are selective toward DK. Limitations of MIM Surfaces. Although this approach provides the benefits mentioned above, it also has limitations. One problem is associated with nonspecific binding. After all,

the mechanism of recognition is based only on differences in the size and shape of template molecules. With no involvement of a specific functional group-template interaction in the current procedure, significant nonspecific binding events are inevitable. Another problem associated with MIM systems is the short storage lifetime that results from the relatively weak (relative to a typical covalent bond) interaction between sulfur and gold, where the S-Au bond energy has been reported to be about 35-40 kcal/ mol.17 We have observed a 30-40% loss of binding capacity only ∼10 days after the surface preparation using 2-ME as the thiol in method III. This loss of capacity possibly is caused by the reorganization of thiol chains on the surface, thus leading to the destruction of the shape-dependent surface imprint cavities. Opportunities exist to prevent the lateral diffusion of these thiol molecules on gold. For example, Mirsky et al.22 developed an approach for preparing a stable 2D imprinted SAM system where the stability against thiol lateral diffusion was achieved by using a molecular spreader-bar. Wang et al.31 discovered that cyclic disulfide functionality can lead to more stable SAMs. Liedberg and colleagues32 compared the thermal stability and chain disordering of SAMs on gold formed by HS(CH2)18-OH and HS(CH2)15-CONH-(CH2CH2O)-H and studied the stability impacts of attaching tail groups such as oligo(ethylene glycol) to alkanethiols through -COO- or -CONH- links. They found that the amide-group-containing SAM displayed a substantial delay of chain disordering and deposition because of lateral hydrogen bonding. In our group, post self-assembly reactions are being considered to improve the stability of the imprint sites in these 2D MIM systems.

Conclusions A simple methodology has been proposed for preparing thiol monolayers having artificial recognition sites for dansylated amino acids on gold optical biosensor surfaces. Compared to a prior method that used the simultaneous adsorption of template and thiol from solution, this new approach provides the flexibility toward various template molecules with low binding energies on gold. In addition, control over the surface density of imprinting sites can be achieved by this approach, and there is ultimate flexibility in the choice of solvent used to deposit template, an important design consideration to minimize template aggregation in the deposition solution. Relative to thin polymeric MIP layers, the synthesis of MIMs is easier, and template rebinding kinetics are faster by an order of magnitude or more. Acknowledgment. We gratefully acknowledge the National Science Foundation (grant CTS-9983737) for funding. Supporting Information Available: Ellipsometry details and the molecular structure of template molecules. This material is available free of charge via the Internet at http://pubs.acs.org. LA0612163 (31) Wang, Y.; Kaifer, A. E. J. Phys. Chem B 1998, 102, 9922. (32) Valiokas, R.; Ostblom, M.; Svedhem, S.; Svensson, S. C. T.; Liedberg, B. J. Phys. Chem B 2002, 106, 10401.