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Biosensors and Bioelectronics 22 (2006) 336–348
Adsorption of dansylated amino acids on molecularly imprintedsurfaces: A surface plasmon resonance study
Xiao Li, Scott M. Husson ∗Department of Chemical and Biomolecular Engineering, Clemson University, Clemson, SC 29634-0909, United States
Received 8 September 2005; received in revised form 15 February 2006; accepted 4 April 2006Available online 6 June 2006
bstract
Surface plasmon resonance spectroscopy (SPR) was used to measure the adsorption kinetics and isotherms of dansylated amino acids onto surface-onfined molecularly imprinted polymer films (MIP-Fs) and the corresponding non-imprinted polymer control films (NIP-Fs). The surface-confinedolymer films were grafted from flat gold surfaces using atom transfer radical polymerization (ATRP). This approach allowed uniform nanothinlms to be grown, thereby ensuring that the amino acids see a uniform surface during adsorption. N,N′-Didansyl-l-cystine (DDC) and didansyl--lysine (DDK) were used as the template molecules to form the MIP-Fs. Adsorption kinetics data were analyzed using single- and dual-siteangmuir adsorption models. It was found that, within the experimental measurement range, adsorption isotherm data were well described byny of four isotherm models: Langmuir, dual-site Langmuir, Freundlich, or Langmuir–Freundlich (LF). The relatively high heterogeneity indexalues regressed using the Freundlich and LF isotherms suggest the formation of fairly homogeneous MIP-Fs; although Scatchard analysis reveals
inding site heterogeneity does exist. Selectivity studies showed that the MIP-Fs display cross-reactivity between DDC and DDK; nevertheless,IP-Fs prepared against one template showed selectivity for that template. Solution pH and polymer layer thickness were studied as independentarameters to determine their impacts on amino acid adsorption, as monitored by SPR.2006 Elsevier B.V. All rights reserved.
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eywords: Atom transfer radical polymerization (ATRP); Binding kinetics; Gr
. Introduction
Since about the mid-1980s, many biotechnology applica-ions have explored the use of molecularly imprinted polymersMIPs); largely, these have been applications for biosepara-ions and biosensing. For applications in biosensing, MIP-basediosensors possess considerable advantages compared to natu-al receptors, enzymes and antibody assays (e.g., ELISA). Whilehe latter provide high specificities and affinities for binding tar-et molecules, their chemical and thermal instabilities limit theirpplication potential. Replacing natural recognition agents withIPs offers technical, economic, and ethical advantages. They
re easy to prepare at low cost, can withstand environments
hat would destroy natural antibodies, do not require animal-ased production, and can be prepared for compounds (e.g.,mmunosuppressive agents) against which it is difficult to pro-∗ Corresponding author. Tel.: +1 864 656 4502; fax: +1 864 656 0784.E-mail address: [email protected] (S.M. Husson).
ciu
ri(
956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.bios.2006.04.016
lymerization; Molecular imprinting; Surface plasmon resonance (SPR)
uce natural antibodies (Mosbach and Ramstrom, 1996; Piletskyt al., 2001). Results from Lotierzo et al. (2004) even show thatIPs outperform monoclonal antibody natural receptors withwide detection range and long stability. With the enormous
rowth and growth potential for the biosensors market (Piletskyt al., 2001), a feasible technique is required to integrate MIPsith transducers and transform binding events between an MIP
nd its associated analyte into a processable signal. Acousticevices such as quartz crystal microbalances (QCM) and opti-al devices like surface plasmon resonance spectrometers (SPR)how great potential. Nevertheless, QCM requires the analyte toe bound by the surface firmly to meet the Sauerbrey condi-ion (Lieberzeit et al., 2004). Therefore, SPR, which measureshanges in the evanescent field near a metal surface, perhapss suited better for studying adsorption events, as it is largelynaffected by analyte mobility along the surface.
SPR is an optical technique that measures changes in theefractive index of the medium near a metal surface. The sens-ng element comprises a recognition layer coated on a thin∼50 nm) film, typically of gold or silver, deposited on a glass
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X. Li, S.M. Husson / Biosensors a
ubstrate. Monochromatic, p-polarized light is reflected fromhe backside of the glass–gold interface. At a special angle ofncidence known as the resonance angle, Θm, energy from thencident light is used to resonantly excite the surface plasmont the gold–solution interface, which enhances the evanescenteld (Raether, 1977). This event is observed experimentallyhen the reflected light intensity becomes a minimum. Thealue of Θm shifts with changes in the refractive index of thenterfacial region adjacent to the gold surface (within approxi-
ately one-half the wavelength of the incident light (Stenbergt al., 1991)). Therefore, SPR is a very sensitive techniqueor in situ measurement of interactions near the solution–solidnterface. Rapid, label-free, high-sensitivity, real-time measure-
ents are trademarks of SPR, making it a powerful tool foriomolecular interaction characterization. Recent applicationsf this technique include studies of biomolecule adsorptionn self-assembled monolayers (Sigal et al., 1996; Roberts etl., 1998), biomolecule–polymer interactions (Chapman et al.,001; Frazier et al., 2000; Green et al., 1997, 2000; Li et al.,004; Singh and Husson, 2005) and biokinetics (Bunjes et al.,997; Karlsson and Falt, 1997). Recent reports have also high-ighted the application of metallic nanoparticles to enhance theensitivity of sensing by SPR (He et al., 2004; Hutter et al., 2001;yon et al., 1999; Patskovsky et al., 2005).
Although a number of studies have explored the use ofIPs as recognition elements for applications in sensing, fewolecular imprinting studies have applied the SPR technique.
n this work, stable and inexpensive ultrathin MIP films (MIP-s) with recognition properties were prepared on SPR biosen-or chips to create an optical MIP-based biosensor selectiveowards small biomolecules, and impact factors on binding inter-ctions, including equilibration time, solution pH and polymerlm thickness, were studied by SPR.
An important difference between this work and the few previ-usly reported SPR-MIP biosensor studies is that atom transferadical polymerization (ATRP) was used to grow polymer filmsrom the surface by sequential addition of monomer from solu-ion to surface-confined polymer chains. This approach contrastshose previous SPR-MIP biosensor preparations that involvepin coating or other means of physical deposition of mix-ures of template molecule, functional monomer, crosslinker,nd photoinitiator onto the metal substrate, followed by pho-opolymerization (Li et al., 2002). It also differs from thoseorks that used the thiol pre-treatment of gold surfaces followedy spin-casting of imprinting polymer from solution (Taniwakt al., 2003; Yoshikawa et al., 2005). Compared to these physicaloating methods, our “grafting from” approach using ATRP pro-ides advantages: it produces high polymer segment densitiesZhao and Brittain, 2000) that shield the underlying substrate,nd it forms highly uniform polymer surfaces (Huang et al.,998; Li et al., 2004) with controllable layer thicknesses at theanoscale (<15 nm). Since polymer surface chemistry must beeproducible and free of defects for meaningful SPR analysis
Green et al., 2000), these advantages make graft ATRP a desir-ble method for SPR-biosensor preparations. A concern withTRP has been the impact of acidic templates to deactivate theransition metal catalyst used in ATRP (Sankhe et al., 2006;
wlHa
oelectronics 22 (2006) 336–348 337
ei et al., 2005a). However, employment of a stronger cata-yst ligand reduces this interference (Wei et al., 2005a), as doeshe addition of halide salts in cases involving aqueous reactiononditions.
A similar approach to ours was used by Lotierzo et al. (2004)nd Kugimiya and Takeuchi (2001), where gold surfaces wereunctionalized with a self-assembled monolayer in a first step,ut, in these cases, photopolymerization was used instead ofTRP. An AFM root-mean-square roughness (RMS) value of3 nm was reported on an MIP-F with a thickness of 40 nmLotierzo et al., 2004). Comparing this result to our previ-us study (Wei et al., 2005a) (RMS of 1.6 nm on MIP-F withhickness of 5.1 nm), surface-confined ATRP provides thinner,moother surfaces.
Photopolymerization systems like those used previously gen-rate surface and solution phase radicals simultaneously; there-ore, in some cases, it is difficult to control solution polymeriza-ion. An example is the work of Sellergren and coworkers whosed grafted azoinitiators to grow MIP films on silica (Sulitzkyt al., 2002); this study revealed solution-phase gelation. Withroper selection of initiating species, this problem can be min-mized, as demonstrated by that same group, who used graftedhotoiniferters that form a more stable dithiocarbamate radicaln solution to grow MIP films on silica and polystyrene (Ruckertt al., 2002). ATRP produces no solution phase radical species;herefore, in the absence of chain transfer, solution-phase poly-
erization is avoided.Finally, it is important to compare the kinetics of the graft
olymerization methods. For surface photografting, the graftinginetics are relatively fast. By way of example, the preparation of40 nm thick MIP-F layer by Lotierzo et al. (2004) employedpolymerization time of 30 min. This translates roughly to a
rowth rate of 13 A/min. By comparison, ATRP produces MIP-layers at a rate of roughly 0.2 A/min (Wei et al., 2005a). As
iscussed in this paper, control over ultrathin layer thickness isritical to avoid intralayer diffusional mass transport limitationsn SPR adsorption studies.
. Materials and methods
.1. Materials
Gold substrates were purchased from BIAcore, Inc. (SIA Kitu, BR-1004-05). All chemicals were purchased from Aldrich
nd used as received, unless noted otherwise; they were 11-ercapto-1-undecanol (MUD) (97%), (4-chloromethyl)benzoyl
hloride (97%), 2-vinylpyridine (2-Vpy) (97%), ethylenelycol dimethacrylate (EGDMA) (98%), copper(I) bromide99.995+%), potassium hydrogen phthalate (99.95%) and tris-2-aminoethyl)amine (TREN) (96%). Solvents were purchasedrom Aldrich as ACS reagent grade; they were ethyl alcohol99.5%), anhydrous toluene (99.8%), and acetonitrile (99.9+%).
ater was deionized and filtered through a Millipore Milli-QTM
ater purification system (New Bedford, MA). N,N′-Didansyl--cystine (DDC) (95%), didansyl-l-lysine (DDK) (95%), andEPES (99.5%) were used as received from Sigma. Sulfuric
cid (96%) and hydrogen peroxide (35%) were purchased from
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38 X. Li, S.M. Husson / Biosensors a
cros. All percentages are in wt.%. 2-Vpy and EGDMA wereurified by vacuum distillation at 25 mmHg before use to removehe inhibitor (p-t-butyl catechol and monomethyl ether hydro-uinone, respectively).
.2. Cleaning of biosensor substrates
Prior to use, the gold-coated biosensor substrates (1 cm×cm) were cleaned with fresh Piranha solution (3:1 (v/v) con-entrated H2SO4/H2O2) for 1–2 min, and then rinsed copiouslyith deionized water and absolute ethanol, and dried with nitro-en. (Caution: Piranha solution has very strong oxidizing powernd is extremely dangerous to handle; gloves, goggles, and facehields are needed for protection (Kern, 1993). It is recom-ended strongly to work with small volumes for safety and
isposal reasons.) The time for cleaning was controlled to be2 min in order to prevent gold delamination.
.3. Preparation of initiator-functionalized surfaces andurface-confined polymer films
Experimental details of surface imprinting by ATRPave been reported previously by our group (Wei et al.,005a). Briefly, the gold substrates were modified by ahiol self-assembled monolayer (SAM) of MUD that pro-ides hydroxyl functional groups that were reacted with4-chloromethyl)benzoyl chloride to create an initiator-unctionalized layer. Room temperature molecular imprintingolymerization was carried out from the surface-confined ini-iator sites using 2-Vpy as functional monomer and EGDMAs cross-linking monomer. 2-Vpy was selected because it hasasic functional groups that can interact with acid groups on theansylated amino acids. The catalyst comprised Cu(I)Br andigand tris-(2-(dimethylamino)ethyl)amine (Me6TREN) with a
olar ratio of 1:2. The surfaces were prepared in solutionsith two different concentrations of functional monomer andconcentration of EGDMA of 2 M to yield surfaces with tem-late:2Vpy:EGDMA molar ratios of 0.1:3:40 and 0.1:12:40. Theatios of 2-Vpy to EGDMA were chosen based on our experi-nces from previous studies (Wei et al., 2005a,b). The solventas acetonitrile; the catalyst concentration was 2 mM based onu(I); the total reaction volume used for each surface was 5 mL.he MIP-F surfaces were prepared by adding the template, DDCr DDK, into the polymerization solution at a concentration ofmM. Non-imprinted polymers (NIP-Fs) were prepared as con-
rols; polymerization of NIP-Fs was done under exactly the sameonditions, but with no template in solution. Film thicknessesere manipulated using polymerization time as the dependentarameter.
.4. Ellipsometry
The refractive index and extinction coefficient of bare gold
nd the thickness values for all samples were obtained usingphase-modulated, spectroscopic ellipsometer (PicometerTM,eaglehole Instruments). Details of the experimental measure-ents are given in Supplemental Information.st
i
oelectronics 22 (2006) 336–348
.5. External reflectance Fourier-transform infraredpectroscopy
ER-FT-IR spectra were obtained using a Nicolet Nexus 870T-IR spectrometer equipped with a nitrogen-purged chamber.easurements were done using a resolution of 2 cm−1, 2000
cans, gain of 4, aperture of 10, DTGS-KBr detector, and 80◦ngle of incidence.
.6. Surface plasmon resonance (SPR) spectroscopy
SPR measurements were conducted using a BIAcore Xnstrument (BIAcore, Inc., Sweden) at 25 ◦C. The gold-coatedlass substrate with a thin MIP-F or NIP-F layer was mountedn a plastic cassette. All SPR measurements were conducted onIAcore sensor chips with polymer film side in contact with
he internal flow cell. Prior to analysis, the 10 mM HEPES (pH.0) buffer solution was filtered using Corning cellulose acetateembranes with 0.22 �m pores, and degassed using an Isotemp
acuum oven at 20 mmHg vacuum for 20 min, followed by son-cation for 10 min. All sample solutions of DDC and DDK were
ade in HEPES buffer immediately prior to analysis. Due tohe aqueous solubility difference, the solution concentrations ofDC ranged from 0.1 to 1 mg/mL, and solution concentrationsf DDK ranged from 0.01 to 0.3 mg/mL. For each concentra-ion, at least three injections were done. The multichannel modelas used with a flow rate of 20 �L/min. Each sample injectionas followed by a high flow rate (1000 �L/min) buffer wash
nd regeneration steps to ensure that the baseline recovered tots initial position prior to sample injection. The injection sam-le volume was 40 �L using a normal injection mode, and theegeneration solution was 25 �L of 20% (v/v) acetonitrile ineionized water, which was delivered using a 30 s delay injec-ion mode. The regeneration solution was optimized for volume,njection mode and composition. Complete regeneration of MIP-(NIP-F) surfaces was obtained after injecting the regeneration
olution followed by two rinse steps with buffer at a flow ratef 1500 �L/min. For amino acid samples with high concentra-ion, 2–4 cycles of regeneration were conducted. A successionf short injections of regenerate solution was found to be moreffective than one continuous injection. MIP-Fs experienced nooss of signal even after more than 30 regeneration cycles.
. Results and discussion
.1. Characterization of physical and chemical surfaceroperties
The surface was characterized after each reaction step tonsure its successful completion. Characterization of the phys-cal and chemical properties of the surface was done by ellip-ometry and ER-FT-IR measurements. Polymer growth kineticsere monitored by ellipsometry prior to the adsorption studies
o that the polymer thickness could be designed for the adsorp-ion studies.
Fig. 1 presents typical reflectance FTIR spectra for SAM,nitiator-functionalized SAM, and polymer films. Aliphatic
X. Li, S.M. Husson / Biosensors and Bi
Fig. 1. External reflectance FT-IR spectra on gold substrate: (a) 11-mercapto-undecanol SAM; (b) grafted (4-chloromethyl)benzoyl chloride initiator onSAM; (c) grafted MIP-F poly(2-vinylpyridine-co-EGDMA) (0.1:12:40) layer((o
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70 A); (d) grafted NIP-F poly(2-vinylpyridine-co-EGDMA) (12:40) layer85 A). The template molecule was N,N′-didansyl-l-cystine (DDC) The spectraf SAM and initiator were amplified 5×.
CH2 peaks at 2921 and 2853 cm−1 (spectrum a) confirm theormation of a SAM layer. Appearance of the carbonyl peakt 1735 cm−1 (spectrum b) confirms initiator immobilization.ollowing polymerization, two significant peaks at 1735 and168 cm−1 (spectra c and d), which correspond to C O and
O C stretching modes, respectively, support the successfulrowth of MIP-F and NIP-F. Characteristic peaks for poly(2-inylpyridine) (P2-Vpy) are difficult to see in Spectra 1c andd due to the low molar amount of 2-Vpy relative to EGDMAn the polymer films and lower peak sensitivities for the 2-Vpyunctional groups relative to the carbonyl group in EGDMA.owever, similar results were seen in our previous work wherePS results confirmed that 2-Vpy was incorporated in the sur-
ace films (Wei et al., 2005a).Previous XPS results have shown that the NIP and MIP films
ave the same surface molar percentages of 2-Vpy when pre-ared from 2-Vpy:EGDMA solutions with molar ratios of 3:40nd 12:40 (Wei et al., 2005a), which are the polymerizationonditions used in this study. In addition, AFM indicated thathe both MIP-F and NIP-F surfaces were smooth and uniformith equivalent root-mean-square roughnesses (RMS) within
xperimental uncertainty (Wei et al., 2005a). The similarities ofIP-F and NIP-F in both surface composition and topography
re important for our study on dansylated amino acid adsorp-ion. Since the template molecules will see comparably the sameurfaces in rebinding experiments, any enhancement seen inemplate binding capacity or affinity for the MIP-F cannot bettributed to differences in the surface composition or rough-ess, but, rather, differences must be attributed to an imprintingffect.
.2. Characterization of adsorption by surface plasmon
esonance (SPR) spectroscopyBecause SRP signals depend on the thickness of the goldlm, all adsorption measurements were conducted on one SPR
slwb
oelectronics 22 (2006) 336–348 339
hip; therefore, the surface properties were the same for differ-nt concentration studies. Upon injection of analyte-containingolution over the surface, there was a rapid signal response dueo differences in the refractive indices of the buffer and analyteolution (1000 RU is a change in resonance angle of 0.1◦ andorresponds to a change in the index of refraction in solutionf 0.0011 (BIAcore AB, 1997)), followed by a slower increases the DDK adsorbed to the surface, eventually approachinghe equilibrium binding amount. By reintroduction of buffernto the flow cell, there was an immediate drop in the SPRignal due to refractive index switching, as well as the des-rption of template molecules from the surface. An exampleensorgram is provided in Supplemental Information that shows
complete cycle of sample injection and surface regenera-ion. In all measurements, the response returned to its originalaseline value after high-speed buffer wash and regeneration;herefore, it is possible to remove the template molecule fromhe polymer with 100% efficiency. Findings for DDC adsorp-ion indicate that no measurable amount of DDC, which has aisulfide group, was able to interact with the MIP-F-coated goldurface. This finding suggests that the film provided uniform,ense coverage of the substrate over the region that was sampledy SPR.
.3. Characterization of binding kinetics by SPR
Binding kinetics are of particular interest in real-time analy-is of biomolecular adsorption on immobilized surfaces. N,N′-idansyl-l-cystine (DDC) and its analogue, N,N′-didansyl-l-
ysine (DDK), were chosen as the model templates for theseeasons: They have high enough molecular masses to be studiedy SPR (analysis by the BIAcore X SPR instrument imposes aetection limit of ∼200 Da), have high enough solubility in theolymerization solution, contain functional groups that allowharacterization of surface adsorption by fluorometry (previoustudy (Wei et al., 2005a)), and are available commercially. Inhis paper, kinetics analysis by SPR was conducted by injec-ions of DDC at concentrations of 0.1–0.6 mg/mL over theopolymer surfaces. Data were exported from SPR sensorgramverlay using BIAevaluation software 3.1, where blank bufferesponses were subtracted, and analyzed using Microsoft Excelnd SigmaPlot (SPSS, Inc.) software, where bulk shift responsesere subtracted.The entire adsorption process of template molecules onto a
IP-F (NIP-F) surface involves template external mass transfero the solution–film interface, intrafilm mass transfer to bindingites, and site-binding (“reaction”) on the surface. In Fig. 2, thenitial binding responses obtained in SPR measurements at dif-erent flow rates were used to examine the diffusion of templateolecules to the copolymer surface. The choice of flow ratesas based on typical flow rates used in polymer-SPR biosen-
or studies, 5–30 �L (Bjork et al., 2005; Lotierzo et al., 2004).e found that the binding rate was independent of flow rate,
uggesting that the DDC–MIP-F (NIP-F) interaction was notimited by external mass transfer to the film surface. To testhether adsorption was controlled by the reaction kinetics ory intrafilm mass transport, we estimated the time constants for
340 X. Li, S.M. Husson / Biosensors and Bi
Fig. 2. SPR sensorgram of initial binding response for 0.3 mg/mL of N,N′-didansyl-l-cystine (DDC) adsorbing to the MIP-F surface (70 A) prepared witht5b
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emplate (DDC):2-Vpy:EGDMA molar ratio of 0.1:12:40 at different flow rates:�L/min (dash line) and 20 �L/min (solid line). The solvent was 10 mM HEPESuffer at pH 7.0. Buffer shift has been subtracted.
ntrafilm diffusion and binding using Eq. (1) (Comyn, 1985) and2):
D = h2
6D(1)
B = 1
ka[C](2)
here h is film thickness, D the intrafilm diffusion coefficient,a the association (reaction) rate constant and [C] is the con-entration of dansylated amino acid. If reactive binding is theimiting step, τD should be much smaller than τB. Due to thenavailability of diffusion coefficient data for similar diffusants
n highly crosslinked polymers, we estimated a value for the dif-usion coefficient that would give a time constant for intrafilmiffusion that was two orders of magnitude lower than that foreactive binding. From Eq. (2), τB was calculated using ka valuesDAav
ig. 3. Binding kinetics of N,N′-didansyl-l-cystine (DDC) on MIP-F surfaces (70 Arst 20 s (t0 = 140 s in the experiments described). Data (symbols) were fit to a singdsorption model (solid curves). The concentrations of free template molecules DDC0 �L/min. Buffer shift and bulk response were subtracted from original sensorgram
oelectronics 22 (2006) 336–348
btained by fitting SPR data to Langmuir models (one-to-onend dual-site adsorption models). For τD to be two orders ofagnitude lower than τB, D estimated using Eq. (1) would
ave to be ≤5× 10−12 cm2/s in our system. Accordingly, forhe time scales to be equivalent, the value of D would have to be5× 10−14 cm2/s. Such a low value of D suggests that reactive
inding is the limiting step in our adsorption studies. This analy-is demonstrates the importance of creating ultrathin layers, withhicknesses <10 nm, to avoid intrafilm diffusional mass transferimitations in real-time biosensing applications. For example,n increase in film thickness to just 100 nm would increase thealculated D by 100-fold to 10−10 cm2/s, which is equal to theagnitude for diffusion of small molecules in solids. So, for
elatively larger molecules diffusing in our crosslinked film, weould likely be diffusion limited at 100 nm.This result supports that a simple bimolecular interaction
odel, in terms of an integrated reaction rate equation, is appro-riate to describe the DDC–MIP-F (NIP-F) interaction. Theinding kinetics data were first fitted to a simple single-siteangmuir adsorption model (Fig. 3, dotted curves). Due to theifferent concentration conditions of analyte during associationnd dissociation steps, binding kinetics were fitted separatelyith association and dissociation rate equations. Eqs. (3) and
4) show the simple bimolecular interaction and the correspond-ng overall rate equation:
+ Ska←→kd
AS (3)
d[AS]
dt= ka[A][S]− kd[AS] (4)
n these equations, A represents the template molecule (e.g.,
DC), S the free interaction sites in the copolymer surface films,S the complex formed by adsorption of template molecule ton interaction site, and square-brackets indicate concentrationalues. ka and kd are the first-order rate constants for adsorption
) using SPR. (Left) association over the first 80 s; (right) dissociation over thele-site Langmuir adsorption model (dotted curves) and a dual-site Langmuir(in mg/mL) were 0.1 (�), 0.2 (♦), 0.3 (�), 0.5 (©), and 0.6 (+). Flow rate was
. The solvent was 10 mM HEPES buffer at pH 7.0.
X. Li, S.M. Husson / Biosensors and Bioelectronics 22 (2006) 336–348 341
Table 1Comparison of the adsorption rate constants and equilibrium adsorption capacities (Qmax) for single- and dual-site Langmuir adsorption models
Surfaces Single-site model Dual-site model
ka
(M−1 s−1)kd (s−1) Qmax
(pmol/cm2 A)R2 ka1
(M−1 s−1)kd1
(s−1)Qmax 1
(pmol/cm2 A)ka2
(M−1 s−1)kd2
(×103 s1)Qmax 2
(pmol/cm2 A)R2
MIP (0.1:3:40) 301.8 0.06 0.93 0.94 401.2 0.6 0.79 17.4 10 0.53 1.0NIP (3:40) 63.2 0.09 1.15 0.93 87.0 0.4 0.98 16.3 4.7 0.53 0.99MIP (0.1:12:40) 204.8 0.05 0.83 0.87 455.2 0.3 0.45 26.7 5.3 0.63 0.99NIP (12:40) 211.7 0.03 0.61 0.92 311.4 0.6 0.22 11.7 3.3 0.31 0.97
T d NIP-F. Template (DDC):2-Vpy:EGDMA molar ratio are 0.1:3:40 (with thicknessesf 0.1:12:40 (with thicknesses for MIP-F (0.1:12:40) and NIP-F (12:40) of 70± 2 and8
(aw
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−
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d
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heterogeneous binding than NIPs (Umpleby et al., 2004;Rushton et al., 2005). The results for the MIP-F/NIP-F set with12:40 functional monomer:crosslinker ratio suggest the same.While the goodness of fit were the same for the MIP-F/NIP-Fset with 3:40 monomer ratio, the fit was improved substantiallyby allowing more than one type of binding site (vide infra).
To improve the binding kinetics model, and to test whetherbinding site heterogeneity might account for lack of fit with theLangmuir model, a dual-site Langmuir adsorption model wasused to describe the experiment data (Fig. 3, solid curves). In thismodel, two binding sites offering different binding interactionswere postulated to exist in the copolymer surface films. Eqs.(11)–(14) give the reactions and the rate expressions for thedual-site Langmuir model:
A+ S1ka1←→kd1
AS1, A+ S2ka2←→kd2
AS2 (11)
d[AS1]
dt= ka1[A][S1]− kd1[AS1],
d[AS2]
dt= ka2[A][S2]− kd2[AS2] (12)
AS1kd1−→A+ S1, AS2
kd2−→A+ S2 (13)
−d[AS1]
dt= kd1[AS1], −d[AS2]
dt= kd2[AS2] (14)
In these equations, S1 represents the free type 1 interaction sitesin the copolymer surface films, AS1 the complex formed byadsorption of template molecule to a type 1 interaction site, S2the free type 2 interaction sites in the copolymer surface films,and AS2 represents the complex formed by adsorption of tem-plate molecule to a type 2 interaction site. ka1, ka2 and kd1, kd2are the rate constants for adsorption (association) and desorption(dissociation) on types 1 and 2 interaction sites. Experimentaladsorption kinetics data were modeled as the sum of two inte-grated rate equations, as shown in Eqs. (15) and (16) for theassociation phase and dissociation phase, respectively:
−([A]ka1+kd1)t
he experiments examined the adsorption of 0.1–0.6 mg/mL DDC on MIP-F anor MIP-F (0.1:3:40) and NIP-F (3:40) of 59± 3 and 57± 2 A, respectively) and5± 3 A, respectively). The solvent was 10 mM HEPES buffer at pH 7.0.
association) and desorption (dissociation). During the dissoci-tion phase, [A] = 0, therefore, from Eqs. (3) and (4) we canrite
Skd−→A+ S (5)
d[AS]
dt= kd[AS] (6)
he concentration of the complex in Eqs. (4) and (6) can beeplaced by the surface uptake measured by SPR (Q). Also, theoncentration of free sites can be expressed as the differenceetween the maximum equilibrium adsorption capacity (Qmax)nd the uptake Q; Eqs. (4) and (6) become
dQ
dt= ka[A]Qmax − (ka[A]+ kd)Q (7)
dQ
dt= kdQ (8)
rom the SPR results, the maximum adsorbed amount ofemplate on a surface was less than 2× 10−12 mol, whereashe amount of template molecule delivered in solution was
6× 10−9 to 6× 10−8 mol. Because the amount of templaten solution was in large excess, the solution concentration ofemplate molecule [A] is treated as a constant during integrationf Eq. (7).
In integrated form, the rate equations are
ssociation : Q = [A]kaQmax[1− e−([A]ka+kd)t]
[A]ka + kd(9)
issociation : Q = Qmax e−kd(t−t0) (10)
here Qmax is the response at start time of dissociation t0.able 1 summarizes the results from data regression using thisingle-site binding model. The multiple regression correlationoefficient, R2, is a measure of the proportion of variabilityxplained by, or due to the regression in a sample of pairedata. It is a number between zero and one and a value closeo zero suggests no correlation by a model. By fitting thexperiment data with the single-site Langmuir binding model,
low correlation coefficient (R2 < 0.9) was observed for
dsorption on MIP-F with high functional monomer content,hile fits were somewhat better to data for NIP-F surfaces andIP-F with low functional monomer content. Past findingsave demonstrated that imprinted materials display more
Q = [A]ka1Qmax 1[1− e ]
[A]ka1 + kd1
+ [A]ka2Qmax 2[1− e−([A]ka2+kd2)t]
[A]ka2 + kd2(15)
342 X. Li, S.M. Husson / Biosensors and Bioelectronics 22 (2006) 336–348
F ts. Lefa te calc 0.6 (
Q
Fhgt(ataafBFtb(
Tacaaebpmbiss
ig. 4. Linear analysis of DDC binding kinetics on MIP-F (70 A) from SPR resulre the absolute values of the slope. (Bottom) ks vs time. Right: dissociation raoncentrations of DDC (in mg/mL) were 0.1 (�), 0.2 (♦), 0.3 (�), 0.5 (©), and
= Qmax1 e−kd(t−t0) +Qmax2 e−kd2(t−t0) (16)
urthermore, linearization analysis was applied to examine theeterogeneity of template MIP-F binding. For an ideal homo-eneous bimolecular interaction, a plot of dQ/dt versus Q forhe association phase would appear linear (Eq. (7)), where ksks = ka[A] + kd) is the slope, and, since ks is only a function ofnalyte concentration, ks would be a constant over the associa-ion phase at a certain constant analyte concentration. However,s shown in Fig. 4, the plot of dQ/dt versus Q is nonlinear atll analyte concentrations, which indicates that ks values areunctions of both analyte concentration and association time.y plotting ks versus time, it is clear that ka depends on time.
or an ideal homogeneous bimolecular interaction, kd would behe slope of plot of ln(Qmax/Qt) versus time (Eq. (10)); it woulde a constant over the dissociation phase, which started at 140 ssee Fig. 2a). However, Fig. 4 shows that kd is not a constant.
tSeu
t: association rate calculation. (Top) dQ/dt vs the response Q; ks (ks = ka[A] + kd)culation. kd were obtained from the slopes of plots of ln(Qmax/Q) vs time. The+). pH was 7.0.
he most important observation is that kd are independent ofnalyte concentration but decrease with time during the disso-iation phase, rapidly during the first 10 s. In this study of DDCdsorption on MIP-F, the time dependence of both associationnd dissociation rate coefficients is another indication of het-rogeneity on the surface. Although a decreasing ka can alsoe explained as the result of lateral interactions between tem-late molecules and functional monomers and among templateolecules (Lahiri et al., 1999), kd changes with time result from
inding site heterogeneity, where, initially, sites with low bind-ng energies release the template rapidly (high kd); thereafter,ites with higher binding energies release the template morelowly (low kd).
Table 1 shows the global calculated adsorption constants
ogether with the correlation coefficients (R2) obtained by fittingPR data to the Langmuir single- and dual-site adsorption mod-ls. Compared to high affinity protein–ligand binding, wheresually ka > 104 and kd < 10−3 (K > 107 M−1), affinity constants
nd Bioelectronics 22 (2006) 336–348 343
oTrLsmtfbhMpt
irdfitmtiim
3b
baTcR
3
ta
L
d
F
L
Ad
mibpa
Fig. 5. Adsorption isotherms for DDK on NIP-F (©) and MIP-F (0.1:12:40)prepared with DDK (♦) or DDC (�) as the template. Curves represent fits tothe Langmuir (dash), dual-site Langmuir (dash-dot), Freundlich (dotted), andLangmuir–Freundlich (solid) isotherm models. Insert: Scatchard plot. The thick-nesses for MIP-F prepared against DDC, MIP-F prepared against DDK andN ˚
Ht
stnlittiaSiaitcofirssFm(
sl
X. Li, S.M. Husson / Biosensors a
btained here in MIP-F binding are low (K∼ 102–103 M−1).he values of rate constants are consistent with the previous
eports on MIP binding (Cheong et al., 1998; Lahav et al., 2001;ehmann et al., 2004). And as expected, the association and dis-ociation rate constants regressed from the single-site bindingodel fall between the two values of ka or kd regressed from
he dual-site binding model. Comparing the polymers with 3:40unctional monomer:crosslinker ratio, we see that the affinityinding constant, K = ka/kd, appears to be higher for the MIP-F;owever, the NIP-F unexpectedly had higher capacity than theIP-F. We have no explanation for this result. However, for the
olymer set with 12:40 functional monomer:crosslinker ratio,he MIP-F had higher capacity as one would expect.
Fig. 3 demonstrates clearly that the dual-site Langmuir bind-ng model more accurately describes the template moleculeebinding on molecularly imprinted polymer surfaces, likelyue to the heterogeneity of binding sites formed in the polymerlm. Higher-order binding site models or continuous distribu-
ion models, such as the Freundlich or Langmuir–Freundlichodels, are likely to describe the data slightly more accurately
han the dual-site model. However, increasing the number ofndependent binding site types comes with the price of increas-ng the number of adjustable fitting parameters and, for the latter
odels, additional model complexity.
.4. Characterization of equilibrium adsorption propertiesy SPR
The measured equilibrium data were the difference in signaletween points before and after sample injection, with bulk shiftnd blank buffer response subtracted from the overall response.he mass of adsorbed template molecules per unit area wasalculated from the linear correlation between this quantity andU response. (See Li et al. (2004) for additional detail.)
.4.1. Equilibrium isotherm modelsIn this work, four isotherm models were examined to describe
he equilibrium data: Langmuir, dual-site Langmuir, Freundlich,nd Langmuir–Freundlich (LF):
angmuir : Q = Qmax[A]
Kd + [A](17)
ual-site Langmuir : Q = Qmax 1[A]
Kd + [A]+ Qmax 2[A]
Kd + [A](18)
reundlich : Q = Qmax[A]m (19)
angmuir–Freundlich : Q = Qmax[A]m
Kd + [A]m(20)
dditional parameters not defined previously are the equilibriumissociation constants (Kd) and heterogeneity index (m).
The Langmuir model, which is a homogeneous binding siteodel, is used commonly to fit MIP binding isotherms. However,
ncreasingly, studies show that most MIPs have heterogeneousinding sites (Umpleby et al., 2001; Wei et al., 2005b). For exam-le, the dual-site binding model (a.k.a. bi-Langmuir model) waspplied in the slope analysis of Scatchard plots of MIPs binding
bfLm
IP-F were 70± 2, 32± 2 and 85± 3 A, respectively. The solvent is 10 mMEPES buffer at pH 7.8. Surface adsorbed amount (in RU) has been converted
o pmol/cm2 A by dividing the thickness (A) of polymer films.
tudies (Shea et al., 1993; Vlataks et al., 1993). In this model,wo classes of heterogeneous binding sites are formed: a smallumber of high affinity sites, and a large number of sites withow affinity. As shown by a non-linear Scatchard plot inlayedn Fig. 5, MIP-Fs display a heterogeneous binding site charac-eristic in the binding measurements. It is possible to estimatehe Qmax and Kd from a Scatchard plot, where Qmax is the x-ntercept and Kd is the negative reciprocal of the slope. However,ccurate estimates of the independent binding parameters usingcatchard plots can be found only under the condition that affin-
ty constant values differ markedly (Norby et al., 1980). Klotznd Hunston (1971) have derived expressions for the slopes andntercepts of tangents to the upward-curved Scatchard plot. Inheir paper, only when affinity constant values between the twolasses of heterogeneous binding sites differ by at least tworders of magnitude could the errors of graphical parameterstted by Scatchard be reduced to 1%. As estimated using theate constant values from kinetics fitting (Table 1), affinity con-tant values between the two classes of heterogeneous bindingites only differ∼3 times for MIP-Fs and 7–16 times for NIP-Fs.or this reason, the Qmax and Kd values in this paper were deter-ined by nonlinear, least-squares regression using SigmaPlot
SPSS Inc., version 8.02).The Freundlich model is a heterogeneous model. It demon-
trates excellent fit to MIP adsorption isotherm data in theow concentration region (nM–mM) (Umpleby et al., 2004),
ut is incapable of modeling saturation behavior and deviatesrom experimental isotherms in high concentration regions. Theangmuir–Freundlich (LF) model provides heterogeneity infor-ation and is able to model adsorption behavior over the entire
344 X. Li, S.M. Husson / Biosensors and Bioelectronics 22 (2006) 336–348
Table 2Fitted parameters for the Langmuir model determined by regression of MIP-F and NIP-F adsorption isotherm data
Qmax (pmol/cm2 A) K (×104 �M−1) K* (×103 pmol/cm2 A �M) α
Template: N,N′-didansyl-l-cystine (DDC)DDC on MIP–DDC 2.29 6.38 1.46
1.40DDC on NIP 0.86 8.96 0.77DDK on MIP–DDC 3.30 29.66 9.77DDK on NIP 2.82 25.45 7.17
Template: didansyl-l-lysine (DDK)DDK on MIP–DDK 4.53 60.98 27.60DDK on NIP 2.82 25.45 7.17
4.338.96
T K and
cc
toaTSimwt
FTwc
α
t
KmtgadDatmctthci
c
TF
p
D
D
D
D
DDC on MIP–DDK 1.87 1DDC on NIP 0.86
he thicknesses for MIP-F prepared against DDC, MIP-F prepared against DD
oncentration region up to saturation; it is probably the besthoice for fitting the binding isotherm of adsorption on MIP-Fs.
Fig. 5 presents the quantitative results of DDK adsorp-ion on MIP-F and NIP-F surfaces. Curves represent fitsf the data to Langmuir, dual-site Langmuir, Freundlich,nd Langmuir–Freundlich (LF) isotherm models (also seeables 2 and 3 for parameter details). Surprisingly, although thecatchard plots showed surface heterogeneity, the adsorption
sotherms could be well expressed by the simplest Langmuirodel. Similar behavior was reported by Wei et al. (2005b),ho demonstrated that differences in fit were marginal between
he LF model and the Langmuir model.For this set of materials, at all solution concentrations, MIP-
surfaces demonstrate higher binding capacity than NIP-Fs.o test the adsorption selectivity, adsorption studies with DDCere also carried out with these same surfaces. A selectivity
oefficient was defined as
K∗ /K∗
DDK,DDC = DDK,MIP DDK,NIPK∗DDC,MIP/K∗DDC,NIP
where K* represent the initial slope values for the adsorp-ion isotherms. Mathematically, K* = QmaxK, where Qmax and
Camf
able 3itted binding parameters and corresponding correlation coefficient using Langmuir d
H Langmuir Dual-site Langmuir
K* (×103 pmol/cm2 A �M) R2 K* (×103 pmol/cm2 A �M)
DC on MIP (DDC)6.4 3.95 1.0 3.957.0 1.46 0.95 1.467.8 0.74 0.96 0.74
DC on NIP6.4 1.25 0.99 1.257.0 0.77 0.98 0.777.8 0.60 0.96 0.60
DK on MIP (DDK)6.4 38.41 1.0 38.417.0 27.60 0.99 27.607.8 23.42 1.0 23.43
DK on NIP6.4 12.11 1.0 12.117.0 7.165 1.0 7.1657.8 5.985 0.98 5.989
1.112.680.77
NIP-F were 70± 2, 32± 2 and 85± 3 A, respectively. The pH was 7.0.
are obtained from fitting the data to the Langmuir adsorptionodel and represent the maximum surface adsorbed amount and
he affinity constant for adsorption, K = K−1d = ka/kd. Table 2
ives the fitted K* and α values for MIP-F surfaces preparedgainst DDC and DDK at the neutral pH (7.0) adsorption con-ition. The selectivity coefficient is 1.40 for MIP-F surface withDC as the template molecule (MIP–DDC) and 1.11 for DDK
s the template (MIP–DDK). The higher selectivity indicateshat MIP–DDC has stronger specific binding of its own template
olecule than does MIP–DDK. The results also display someross-reactivity between these two molecules. That is, adsorp-ion of DDK onto MIP–DDC was higher than adsorption ontohe NIP-F. Similarly, adsorption of DDC onto MIP–DDK wasigher than adsorption onto the NIP-F. Although the selectivityoefficients are relatively low, the application of these MIP-Fsn sensing arrays should provide enough specific recognition.
Usually, optimum template rebinding occurs under the sameonditions used during the polymerization to produce the MIP.
hanging the environmental conditions impacts cavity shapend the distance between functional groups in the polymeratrix, which may result in loss of specificity of MIP materialsor rebinding their templates. Therefore, we wondered: Would
ual-site Langmuir, Freundlich, and Langmuir–Freundlich (LF) isotherm models
Freundlich LF
R2 m R2 K* (×103 pmol/cm2 A �M) m R2
1.0 0.79 1.0 10.30 0.79 1.00.95 0.74 0.96 5.52 0.74 0.960.96 0.63 0.97 4.93 0.63 0.97
0.99 0.87 0.99 2.29 0.87 0.990.98 0.71 0.99 3.16 0.71 0.990.96 0.67 0.98 3.42 0.67 0.98
1.0 0.53 1.0 70.46 0.80 1.00.99 0.71 1.0 64.92 0.71 1.01.0 0.62 1.0 64.33 0.75 1.0
0.99 0.64 1.0 16.67 0.90 1.00.98 0.85 1.0 8.971 0.93 1.00.96 0.79 0.99 15.69 0.79 0.99
nd Bioelectronics 22 (2006) 336–348 345
tcatsafsa1ewwv
dbfiwtfthoitsdfs
3
fe
Fa
etl
3
oVmcm
F(
X. Li, S.M. Husson / Biosensors a
he aqueous environment used in our rebinding measurementshange the adsorption properties of the imprinted surfaces? Tonswer this question, we compared the adsorption isotherms ofhese two MIP-F surfaces to data from our previous adsorptiontudies (Wei et al., 2005a), which used fluorometry as the char-cterization method and acetonitrile as the rebinding solvent. Weound that selectivities differed by <8% between the two solventystems; but adsorption capacities of DDC were 40% lower inqueous solution, whereas adsorption capacities for DDK were0% higher in aqueous solution. The ability to maintain (andven increase) selective binding capacities in aqueous solutionill benefit the application of such an MIP-F in biosensing,here aqueous environments are used widely for in vivo or initro biomolecule detection systems.
Table 3 compares regression statistics for the Langmuir,ual-site Langmuir, Freundlich and Langmuir–Freundlich (LF)inding models. Overall, R2 values were high (R2 > 0.95) for allour models under all conditions studied. Heterogeneous bind-ng models did show slight improvement for adsorption of DDC,hile R2 remains high (>0.99) for all DDK adsorptions. In con-
rast to the kinetic data regression that showed clear differencesor Langmuir and dual-site Langmuir models, description ofhe equilibrium data was less sensitive to model choice. Oneypothesis that explains this result relates to the heterogeneityf polymer surfaces, as shown in Fig. 4 and the Scatchard plotnlayed in Fig. 5. The inability of the single-site Langmuir modelo describe the kinetics data may be attributed to the fact thatites with low binding energies adsorb/release the template atifferent rates than sites with higher binding energies. There-ore, site heterogeneity may impact kinetics fittings with higherensitivity than equilibrium isotherm fittings.
.5. Factors that affect equilibrium adsorption properties
The binding of template biomolecules on MIP-F/NIP-F sur-aces is complex and is impacted significantly by polymer prop-rties (composition, layer thickness), as well as the measurement
ddbs
ig. 7. Adsorption studies of didansyl-l-cystine (DDC) (a) and didansyl-l-lysine (DDcircle), and 7.8 (triangle). The solvent was 10 mM HEPES buffer adjusted to pH usi
ig. 6. Polymer thickness dependence of DDC adsorption on MIP-F (0.1:12:40)nd NIP-F (12:40) surfaces. The solvent was 10 mM HEPES buffer at pH 7.0.
nvironment (pH, temperature, ionic strength) and interactionime. In this study, our objective was to determine the effects ofayer thickness and solution pH on adsorption properties.
.6. Polymer thickness effect
Fig. 6 shows the adsorption density (uptake per unit volume)f DDC on MIP-F and NIP-F surfaces (template (DDC):2-py:EGDMA molar ratio of 0.1:12:40) with two different poly-er thicknesses. The similar values of adsorption density indi-
ate a linear dependence of template adsorption amount on poly-er thickness. This finding suggests a relatively homogenous
istribution of binding sites exists along the polymer growthirection within the polymer films. Also, this result shows that,y dividing the areal uptake capacity obtained in SPR mea-urements by polymer layer thickness, it is possible to compare
K) (b) on MIP–DDC, MIP–DDK and NIP-F surfaces at pH 6.4 (diamond), 7.0ng NaOH (50% (w/w) in H2O).
346 X. Li, S.M. Husson / Biosensors and Bi
Fa
bntmwi
3
MspbebIcia
cmwisbdpsvteoaH
emftawnTto(
aofoio3dmtbDhatmtibpd
pa2(baabeai
4
pISa
ig. 8. Effect of the pH on the selectivity of DDC and DDK–MIP-F surfaces,nalysis using Langmuir isotherm.
inding capacities of polymer surfaces with various layer thick-esses. We believe that the adsorption density is more represen-ative than areal uptakes, since interactions between template
olecules and imprint cavities happen at the surface and alsoithin the polymer layer. Adsorption densities were presented
n Tables 2 and 3, and plotted in Figs. 5–7.
.7. pH effect
Fig. 7 presents adsorption isotherms for DDC and DDK onIP-F and NIP-F surfaces at three pH values. We consistently
aw >3 times higher surface adsorption densities for DDK com-ared to DDC. The difference in rebinding abilities of these twoiomolecules on polymer surfaces are likely due in part to differ-nces in the activities of these two compounds in water causedy differences in their molecular structures (see Supplementalnformation). DDC is the product of the thiol-disulfide inter-hange reaction of two cysteine molecules, thus it has twoonizable acid groups. Compared to DDK, which only has onecid group, DDC is likely more hydrophilic.
Fig. 8 shows the dependence of binding selectivity coeffi-ient on solution pH for MIP-Fs formed with the two templateolecules. Interestingly, the selectivity of MIP–DDC decreasedith increasing pH value, whereas MIP–DDK increased with
ncreasing pH. Moreover, adsorption of the MIP–DDC is moreensitive to pH than MIP–DDK. A similar observation was madey Sharp and Risley (1999) who reported the dependence ofansyl amino acid retention and resolution on the pH of mobilehase buffer in HPLC measurements. In order to better under-tand the microenvironment in our binding measurements, pKaalues of these two template molecules were measured by poten-iometric titrations covering a pH range of 2.6–12.0. While we
xpected to find two independent pKa values for the acid groupsf DDC, instead we found single inflection points for both DDCnd DDK that gave pKa values of 6.2 for both compounds.ence, under all three pH conditions, most of the biomoleculesnapb
oelectronics 22 (2006) 336–348
xist in their deprotonated form. The approximate fraction ofolecules in the deprotonated form was calculated as 61.3%
or pH 6.4, 86.4% for pH 7.0, and 97.6% for pH 7.8, respec-ively. The pKa for 2-vinylpyridine is ∼4.98 (Perrin, 1965),nd we measured the pKa of poly(2-vinylpyridine) as 5.0± 0.1,hich means that essentially all of the pyridine groups exist inon-protonated, non-ionic form at these solution pH conditions.herefore, under the conditions used in this study, the adsorp-
ion of template on MIP-F (NIP-F) surface could be the resultf hydrophobic (dispersive) interactions and hydrogen bondingat the low pH value).
Given the similarity in structures between DDC and DDKnd the apparent lack of measurably distinct pKa values for eachf the two acid groups on DDC, we have no clear explanationor the observed differences in pH dependency for adsorptionf DDC and DDK on their corresponding MIPs (or NIP). Whats clear from measured pKa values is that about 40% of DDCr DDK molecules are unionized at pH 6.4, while only about% are unionized at pH 7.8. Therefore, the differences in pHependency for adsorption of DDC and DDK on their MIP-Fsay result from differences in the primary mode of interac-
ion. While speculative, for example, H-bonding interactionsetween template and MIP-F may play a greater role in theDC system; whereas, the DDK system may depend more onydrophobic interaction, which is consistent with the argumentbove that DDK is more hydrophobic. In this way, increasinghe pH from 6.4 to 7.8 would disrupt the primary interaction
ode for the DDC system but would have a lesser effect onhe DDK system. In addition, DDC, as a di-acid, may formntramolecular hydrogen bonds through rotation about the S Sond. Perhaps that geometry is favored during the imprintingrocess. For pH > pKa, the intramolecular hydrogen bonds areisrupted, and this favored geometry is compromised.
A few reports have described pH effects on rebinding tem-late with MIPs, including those that describe pH-dependentffinity of imprinted metal complex binding sites (Subat et al.,004; Rachkov et al., 2004) or intelligent imprinted hydrogelsByrne et al., 2002). In this work, we demonstrated that SPR cane used to measure the pH-dependence of adsorption of chargednalytes on ultrathin MIP films. Given the results of our study,nd studies that have shown the influence of pH on the swellingehavior of MIP materials and its influence on recognition prop-rties (Turner et al., 2004), we believe that pH changes representpromising way to tune the selectivity of aqueous MIP systems
nvolving ionizable analytes and/or monomer units.
. Conclusions
ATRP graft polymerization was used to prepare uniformolymer surfaces for biomolecule adsorption studies by SPR.mprinted and non-imprinted polymer films were grown fromPR sensing elements (gold). The controllable nature of ATRPllowed the growth of uniform MIP films with adjustable thick-
esses, preventing diffusional mass transfer limitations fromffecting the kinetic analysis. ATRP therefore can be used torepare MIP films as recognition elements for applications iniosensors.
nd Bi
fifrMwibpfssa
mflls
A
Wa9oC
A
i
R
BB
B
B
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C
C
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K
K
K
L
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L
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X. Li, S.M. Husson / Biosensors a
SPR measured higher adsorption capacities for imprintedlms relative to non-imprinted films when high percentage ofunctional monomer was used in polymer synthesis. Cross-eactivity was seen between similar template compounds, but
IP-Fs showed selective binding, and the selectivity changedith solution pH values. Kinetic models were compared that
ncorporated first-order interactions with single- and dual-siteinding. Impact factors that were studied included solution pH,olymer thickness and interaction time. Adsorption capacity wasound to scale with polymer layer thickness, i.e., adsorption den-ity was found to be independent of layer thickness. This resultuggests that films of different thicknesses can be compared byppropriate normalization.
While this study used fluorescently labeled analytes, theethodology used to prepare the films applies also to non-uorescent templates of interest in many environmental or bio-
ogical samples. This methodology can also be extended to otherubstrate geometries and chemistries.
cknowledgments
We thank Xiaolin Wei for supplying the Me6TREN ligand.e thank Dr. Robert Latour for advice with SPR. We gratefully
cknowledge the National Science Foundation (Grant Nos. CTS-983737, DMR-0315487, DMR-0215881) and the Departmentf Commerce through the National Textile Center (Grant No.05-CL01) for funding.
ppendix A. Supplementary data
Supplementary data associated with this article can be found,n the online version, at doi:10.1016/j.bios.2006.04.016.
eferences
IAcore AB, 1997. BIAtechnology Handbook. BIAcore AB, Uppsala, Sweden.jork, A.P., Persson, N.-K., Peter, K., Nilsson, R., Asberg, P., Inganas, O.,
2005. Dynamics of complex formation between biological and luminescentconjugated polyelectrolytes—a surface plasmon resonance study. Biosens.Bioelectron. 20, 1764–1771.
unjes, N., Schmidt, E.K., Jonczyk, Rippmann, A.F., Beyer, D., Ringsdorf, H.,Graber, P., Knoll, W., Naumann, R., 1997. Thiopeptide-supported lipid layerson solid substrates. Langmuir 13, 6188–6194.
yrne, M.E., Park, K., Peppas, N.A., 2002. Molecular imprinting within hydro-gels. Adv. Drug Delivery Rev. 54, 149–161.
hapman, R.G., Ostuni, E., Liang, M.N., Meluleni, G., Kim, E., Yan, L.,Pier, G., Warren, H.S., Whitesides, G.M., 2001. Polymeric thin films thatresist the adsorption of proteins and the adhesion of bacteria. Langmuir 17,1225–1233.
heong, S., Rachkov, A.E., Park, J., Yano, K., Karube, I., 1998. Synthesisand binding properties of a noncovalent molecularly imprinted testosterone-specific polymer. J. Polym. Sci. A 36, 1725–1732.
omyn, J., 1985. Polymer Permeability. Kluwer Academic Publishers, TheNetherlands (Chapter 1).
razier, R.A., Matthijs, G., Davies, M.C., Roberts, C.J., Schacht, E., Tendler,
S.J.B., 2000. Characterization of protein-resistant dextran monolayers. Bio-materials 21, 957–966.reen, R.J., Davies, J., Davies, M.C., Roberts, C.J., Tendler, S.J.B., 1997. Sur-face plasmon resonance for real time in situ analysis of protein adsorptionto polymer surfaces. Biomaterials 18, 405–413.
R
R
oelectronics 22 (2006) 336–348 347
reen, R.J., Frazier, R.A., Shakesheff, K.M., Davies, M.C., Roberts, C.J.,Tendler, S.J.B., 2000. Surface plasmon resonance analysis of dynamic bio-logical interactions with biomaterials. Biomaterials 21, 1823–1835.
e, L., Smith, E.A., Natan, M.J., Keating, C.D., 2004. The distance-dependenceof colloidal Au-amplified surface plasmon resonance. J. Phys. Chem. B 108,10973–10980.
uang, X., Doneski, L.J., Wirth, M.J., 1998. Make ultrathin films using surface-confined living radical polymerization. CHEMTECH, 19–25.
utter, E., Cha, S., Liu, J.F., Park, J., Yi, J., Fendler, J.H., Roy, D., 2001. Roleof substrate metal in gold nanoparticle enhanced surface plasmon resonanceimaging. J. Phys. Chem. B 105, 8–12.
arlsson, R., Falt, A., 1997. Experimental design for kinetic analysis ofprotein–protein interactions with surface plasmon resonance biosensors. J.Immuno. Meth. 200, 121–133.
ern, W., 1993. In: Kern, W. (Ed.), Handbook of Semiconductor Wafer CleaningTechnology. Noyes Publications, Park Ridge, NJ.
lotz, I.M., Hunston, D.L., 1971. Properties of graphical representations ofmultiple classes of binding sites. Biochemistry 10, 3065–3069.
ugimiya, A., Takeuchi, T., 2001. Surface plasmon resonance sensor usingmolecularly imprinted polymer for detection of sialic acid. Biosens. Bio-electron. 16, 1059–1062.
ahav, M., Katz, E., Willner, I., 2001. Photochemical imprint of molecu-lar recognition sites in two-dimensional monolayers assembled on Auelectrodes: effects of the monolayer structures on the binding affinitiesand association kinetics to the imprinted interfaces. Langmuir 17, 7387–7395.
ahiri, J., Isaacs, L., Grzybowski, B., Carbeck, J.D., Whitesides, G.M., 1999.Biospecific binding of carbonic anhydrase to mixed SAMs presenting ben-zenesulfonamide ligands: a model system for studying lateral steric effects.Langmuir 15, 7186–7198.
ehmann, M., Dettling, M., Brunner, H., Tovar, G.E.M., 2004. Affinity parame-ters of amino acid derivative binding to molecularly imprinted nanospheresconsisting of poly[(ethylene glycol dimethacrylate)-co-(methacrylic acid)].J. Chromatogr. B 808, 43–50.
i, P., Huang, Y., Hu, J., Yuan, C., Lin, B., 2002. Surface plasmon resonancestudies on molecular imprinting. Sensors 2, 35–40.
i, X., Wei, X., Husson, S.M., 2004. Thermodynamic studies on the adsorptionof fibronectin adhesion-promoting peptide on nanothin films of poly(2-vinylpyridine) by SPR. Biomacromolecules 5, 869–876.
ieberzeit, P.A., Schirk, C., Glanznig, G., Gazda-Miarecka, S., Bindeus, R.,Nannen, H., Kauling, J., Dickert, F.L., 2004. From nanopatterning tofunctionality-surface and bulk imprinting for analytical purposes. Super-lattice Microstruct. 36, 133–142.
otierzo, M., Henry, O.Y.F., Piletsky, S., Tothill, I., Cullen, D., Kania, M., Hock,B., Turner, A.P.F., 2004. Surface plasmon resonance sensor for domoicacid based on grafted imprinted polymer. Biosens. Bioelectron. 20, 145–152.
yon, L.A., Musick, M.D., Smith, P.C., Reiss, B.D., Pena, D.J., Natan, M.J.,1999. Surface plasmon resonance of colloidal Au-modified gold films. Sens.Actuator B 54, 118–124.
osbach, K., Ramstrom, O., 1996. The emerging technique of molecularimprinting and its future impact on biotechnology. Nat. Biotechnol. 14,163–170.
orby, J.G., Ottolenghi, P., Jensen, J., 1980. Scatchard plot: common misinter-pretation of binding experiments. Anal. Biochem. 102, 318–320.
atskovsky, S., Kabashin, A., Meunier, M., 2005. Near-infrared surface plasmonresonance sensing on a Si platform with nanoparticle-based signal enhance-ment. Opt. Mater. 27, 1093–1096.
errin, D.D., 1965. Dissociation Constants of Organic Bases in Aqueous Solu-tion. Butterworths, London, p. 176.
iletsky, S.A., Subrahmanyam, S., Turner, A.P.F., 2001. Application of molecu-larly imprinted polymers in sensors for the environment and biotechnology.Sens. Rev. 21, 292–296.
achkov, A., Hu, M., Bulgarevich, E., Matsumoto, T., Minoura, N., 2004.Molecularly imprinted polymer prepared in aqueous solution selective for[Sar1,Ala8]angiotensin II. Anal. Chim. Acta 504, 191–197.
aether, H., 1977. Surface plasma oscillations and their applications. Phys. ThinFilms 9, 145–261.
3 nd Bi
R
R
R
S
S
S
S
S
S
S
S
T
T
U
U
V
W
W
48 X. Li, S.M. Husson / Biosensors a
oberts, C., Chen, C.S., Mrksich, M., Martichonok, V., Ingber, D.E., Whitesides,G.M., 1998. Using mixed self-assembled monolayers presenting RGD and(EG)3OH groups to characterize long-term attachment of bovine capillaryendothelial cells to surfaces. J. Am. Chem. Soc. 120, 6548–6555.
uckert, B., Hall, A.J., Sellergren, B., 2002. Molecularly imprinted compositematerials via iniferter-modified supports. J. Mater. Chem. 12, 2275–2280.
ushton, G.T., Karns, C.L., Shimizu, K.D., 2005. A critical examination ofthe use of the Freundlich isotherm in characterizing molecularly imprintedpolymers (MIPs). Anal. Chim. Acta 528, 107–113.
ankhe, A.Y., Husson, S.M., Kilbey II, S.M., 2006. Polymerization of elec-trolytes by surface-confined atom transfer radical polymerization in aque-ous solutions: effect of catalyst deactivation on polymer growth. Macro-molecules 39, 1376–1383.
harp, V.S., Risley, D.S., 1999. Evaluation of the macrocyclic antibioticLY333328 as a chiral selector when used as a mobile phase additive innarrow bore HPLC. Chirality 11, 75–81.
hea, K.J., Spivak, D.A., Sellergren, B., 1993. Polymer complements tonucleotide bases. Selective binding of adenine derivatives to imprinted poly-mers. J. Am. Chem. Soc. 115, 3368–3369.
igal, G.B., Bamdad, C., Barberis, A., Strominger, J., Whitesides, G.M., 1996.A self-assembled monolayer for the binding and study of histidine-taggedproteins by surface plasmon resonance. Anal. Chem. 68, 490–497.
ingh, N., Husson, S.M., 2005. Thermodynamic measurements and predic-tions of the adsorption of short-chain peptides on nanothin polymer films.Biomacromolecules 6, 9–13.
tenberg, E., Persson, B., Roos, H., 1991. Quantitative determination of surface
concentration of protein with surface plasmon resonance using radiolabeledproteins. J. Colloid Interface Sci. 143, 513–526.ubat, M., Borovik, A.S., Konig, B., 2004. Synthetic creatinine receptor:imprinting of a Lewis acidic Zinc(II)cyclen binding site to shape its molec-ular recognition selectivity. J. Am. Chem. Soc. 126, 3185–3190.
Y
Z
oelectronics 22 (2006) 336–348
ulitzky, C., Ruckert, B., Hall, A.J., Lanza, F., Unger, K., Sellergren, B.,2002. Grafting of molecularly imprinted polymer films on silica sup-ports containing surface-bound free radical initiators. Macromolecules 35,79–91.
aniwak, K., Hyakutake, A., Aoki, T., Yoshikawa, M., Guiver, M.D., Robertson,G.P., 2003. Evaluation of the recognition ability of molecularly imprintedmaterials by surface plasmon resonance (SPR) spectroscopy. Anal. Chim.Acta 489, 191–198.
urner, N.W., Pileska, E.V., Karim, K., Whitsombe, M., Malecha, M., Magan,N., Bagginai, C., Piletsky, S.A., 2004. Effect of the solvent on recogni-tion properties of molecularly imprinted polymer specific for ochratoxin A.Biosens. Bioelectron. 20, 1060–1067.
mpleby, R.J., Baxter, S.C., Chen, Y., Shah, R.N., Shimizu, K.D., 2001. Charac-terization of molecularly imprinted polymers with the Langmuir–Freundlichisotherm. Anal. Chem. 73, 4584–4591.
mpleby, R.J., Baxter, S.C., Rampey, A.M., Rushton, G.T., Chen, Y., Shimizu,K.D., 2004. Characterization of the heterogeneous binding site affinity dis-tributions in molecularly imprinted polymers. J. Chromatogr. B 804, 141–149.
lataks, G., Andersson, L.I., Muller, R., Mosbach, K., 1993. Drug assayusing antibody mimics made by molecular imprinting. Nature 361, 645–647.
ei, X., Li, X., Husson, S.M., 2005a. Surface molecular imprinting by atomtransfer radical polymerization. Biomacromolecules 6, 1113–1121.
ei, X., Samadi, A., Husson, S.M., 2005b. Synthesis and characterization ofmolecularly imprinted polymers for chromatographic separations. Sep. Sci.
Technol. 40, 109–129.oshikawa, M., Guiver, M.D., Robertson, G.P., 2005. Molecularly imprintedfilms derived from Torlon® polyamide–imide. J. Mol. Struct. 739, 41–46.
hao, B., Brittain, W.J., 2000. Polymer brushes: surface-immobilized macro-molecules. Prog. Polym. Sci. 25, 677–710.
