Stimuli-Responsive Functionalization Strategies to Spatially and

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Stimuli-Responsive Functionalization Strategies to Spatially and Temporally Control Surface Properties: Michael vs. Diels Alder Type Additions Adriana R. Kyvik, Carlos Luque-Corredera, Daniel Pulido, Miriam Royo, Jaume Veciana, Judith Guasch, and Imma Ratera J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b01652 • Publication Date (Web): 04 Apr 2018 Downloaded from http://pubs.acs.org on April 9, 2018

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The Journal of Physical Chemistry

Stimuli-Responsive Functionalization Strategies to Spatially and Temporally Control Surface Properties: Michael vs. Diels-Alder Type Additions

Adriana R. Kyvik,a,b Carlos Luque-Corredera,a,c Daniel Pulido,b,d Miriam Royo,b,d Jaume Veciana,a,b Judith Guascha,b,e and Imma Ratera,a,b,*

a

Institute of Materials Science of Barcelona (ICMAB-CSIC), Campus UAB, 08193, Bellaterra,

Spain b

Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-

BBN), Spain c

Escola Universitària Salesiana de Sarrià, EUSS-UAB, Pg. Sant Joan Bosco 74, 08017,

Barcelona, Spain d

Combinatorial Chemistry Unit, Barcelona Science Park, Baldiri Reixac 10, 08028, Barcelona,

Spain e

Dynamic Biomaterials for Cancer Immunotherapy, Max Planck Partner Group, ICMAB-CSIC,

Campus UAB, 08193, Bellaterra, Spain

1 ACS Paragon Plus Environment

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ABSTRACT: Stimuli-responsive self-assembled monolayers (SAMs) are used to confer switchable physical, chemical, or biological properties to surfaces through the application of external stimuli. To obtain spatially and temporally tunable surfaces, we present microcontact printed SAMs of an hydroquinone molecule that are used as a dynamic interface to immobilize different functional molecules either via Diels-Alder or Michael thiol addition reactions upon the application of a low potential. In spite of the use of such reactions and the potential applicability of the resulting surfaces in different fields ranging from sensing to biomedicine through data storage or clean up, a direct comparison of the two functionalization strategies on a surface has not yet been performed. Although the Michael thiol addition requires molecules that are commercial or easy to synthesize in comparison with the cyclopentadiene derivatives needed for the Diels-Alder reaction, the latter reaction produces more homogeneous coverages in similar experimental conditions.


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INTRODUCTION Self-assembled monolayers (SAMs) are used to modify substrates in order to confer to their surfaces a specific function, such as to enhance/decrease the wettability, friction, adhesion, or biocompatibility, among others.1-2 One of the most studied SAMs are formed by alkanethiolates on gold, since long chain alkanethiolates form densely packed, highly ordered trans-extended monolayers in a rapid and spontaneous manner on gold.3-4 One way to use such SAMs for modifying the surface properties of a substrate is to replace the terminal CH3 units of alkanethiols by a chemical group that will introduce the desired functionality to the surface. This can be done either using directly ω-functionalized alkanethiols to form the SAM or modifying the SAM after its formation. If the terminal functional group responds to a given external stimuli both strategies permit to generate stimuli-responsive surfaces exhibiting switchable physical, chemical, or biological properties, which can be tuned in an accurate and predictable way at any given time. Such smart surfaces can find numerous applications in science and technology ranging from environmental, sensing, or biomedicine to cleanup and data storage. In particular, methods to modify SAMs after their formation are critical to develop such complex smart surfaces.5 For example, a composition change can be caused by the isomerization of an immobilized ligand between a structure that is active and another that is inactive.6 Alternatively, the chemical change can be triggered by activating the substrate, thus allowing the immobilization of ligands present in solution. In some cases, the reaction can even be reversed leading to the release of the ligand from the substrate.7-10 These strategies rely on interfacial chemical reactions initiated by an external non-invasive stimuli, such as light or an electrical potential,3, 11-12 enabling a spatiotemporal control over the surface environment.6,

13

Dynamic

control over the properties of surfaces can be performed by applying an electrical potential to 3 ACS Paragon Plus Environment


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generate oxidation or reduction reactions to attached molecules leading to two different redox states. For example, a switch in surface wettability could be obtained by altering the electronic states of single-layered molecules between hydrophilic and hydrophobic states,14 yielding interesting substrates for microfluidic,15 self-cleaning,16 or sensor devices.17 Similarly, surfaces that can dynamically regulate biological functions in response to applied stimuli, can be useful for biological and medical applications, such as to control cell guidance.18-22 SAMs functionalized with hydroquinone (HQ) and its oxidized benzoquinone (BQ), as terminal groups, can be used as a dynamic interface to immobilize different functional molecules on surfaces via stimuli activated Diels-Alder (DA) reactions triggered by the application of a low electrical potential.23 The DA reaction is a reversible cycloaddition occurring between a conjugated diene (in the cis configuration) and an electron-deficient dienophile. Advantageously, this reaction is orthogonal, efficient, atom conservative, and does not require a catalyst. It is also insensitive to the reaction conditions, such as air and humidity, allowing its use under ambient conditions.24 The DA reaction is also crucial for the modification of molecular surfaces such as fullerenes.25 One of the pairs of diene and electron-deficient dienophile most used for DA reactions is cyclopentadiene (Cp) and HQ derivatives, respectively. They react in mild conditions but show some important drawbacks. On one side, the synthesis of the HQ-tagged molecules is complex and not standardized.26 On the other side, it is also very challenging to synthesize the Cp-terminated functional derivatives for the DA reactions due to their low stability, and furthermore, it has been described the low functionalization reproducibility caused by the variability of Cp derivatives on the chemoabsorption process previous to the DA reaction.27 In order to avoid the use of Cp or other less favorable dienes, Michael thiol addition (MA) reactions can be considered as an alternative to the Diels-Alder (DA) reactions for SAM


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derivatization in spite of the differences in their reaction mechanisms. MA reactions involve the addition of a nucleophile, which is referred to as the ‘Michael donor,’ to an activated electrophilic alkene, named the ‘Michael acceptor’, resulting in a ‘Michael adduct’. Even though this type of reaction is usually performed with enolate nucleophiles and activated alkenes, there are many other functional groups that possess enough nucleophilicity to undergo this reaction. Indeed, several non-enolate nucleophiles have been previously used, such as amines,28-30 thiols,31 azides,32 and phosphines.33 MA rates of thiols reacting with acrylates or vinyl sulfones depend on the pH of the reaction media and they rely on the particular pKa of each thiol. This is explained by the fact that the reacting species are the thiolates rather than the thiols.34 Interestingly, MA reactions readily occur at physiological conditions and is selective to thiol containing proteins over amine nucleophiles, for example.31 Thus, reactions with lysine residues require a higher pH of 9.3 and their rates are still dramatically slower than the reaction with cysteine at a comparable pH. Another important advantage of MA reactions for SAM derivatization is the fact that the synthesis of functional molecules with thiol terminal groups is easier to perform than that of the Cp derivatives and the resulting compounds are more stable. Nevertheless, there is no comparative study between the interfacial DA and MA reactivity of Cp- and thiol-terminated functional molecules, respectively, onto an electrochemically activated benzoquinone (BQ) SAM. Thus, the differences on the spatial and temporal control given by these two different dynamic surfaces have not yet been determined. Here we will report on the differential interfacial reactivities of SAMs with Cp- and thiolterminated functional molecules and their implications on the modification of properties of gold substrates. To compare the performances of these two surface reactions, DA and MA, two fluorescein derivative molecules were synthesized, one with a Cp (1) and another with a thiol (2)



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termination which were used as fluorescent probes to study the surface functionalization. Both molecules were linked to a readily prepared hydroquinone terminated SAM S1, which offers temporal control through its reversible conversion to a benzoquinone terminated SAM S2, in a redox process that involves two electrons and two protons (Scheme 1a). SAM S1 consisted of electroactive HQ terminal group that was anchored to gold surfaces through thiols, as previously described by Lee et al,35-36 who pioneered the use of the DA reaction for surface functionalization with biological ligands.37-38 Moreover, the SAM strategy offered us the possibility to precisely control the density of electroactive ligands on the surface by mixing two different pegylated alkanethiol molecules, like the electroactive HQ (3) and the hydroxylterminated analogue (4) (Scheme 1b). The alkyl chains of these molecules confer structural stability to the monolayer, through the formation of a compact 2D packing mediated by van der Waals CH2···CH2 interactions, whereas the ethylene glycol linker units give hydrophilicity to the surface and prevent unspecific binding of proteins and cells, if desired.39-40


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Scheme 1. (a) Schematic representation of the electroactivated interfacial DA and MA reactions. SAM S1 was electrochemically oxidized to SAM S2, which reacted with molecules functionalized either with ω-terminated-Cp or thiol groups to produce SAMs S3 and S4, respectively. (b) Molecular structures of fluorescein molecules functionalized with Cp 1 and



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thiol, 2-, terminating groups, as well as the thiolated HQ derivatives, 3-, and its analogous hydroxyl derivative 4. All these molecules contain a polyethylene chain bridging the ωterminated group and the anchoring unit. In such molecules the alkyl chain provides structural order to get compact molecular monolayers, whereas the oligo(ethylene glycol) chain prevents unspecific protein and cell adsorption. As mentioned above, some of the main disadvantages of the DA reaction when using HQ as the dienophile have been attributed to synthetic difficulties26-27 to achieve the desired molecules and to the low chemoabsorption reproducibility of the process,23 which hinder its extended use for a wide range of applications. In the present study we first optimized the synthesis of the HQ derivative 3 to form the electroactive SAM S1 and a complete explanation of the synthetic route is provided. It is known that molecular packing can impact to the physical properties of SAMs.4143

Thus, in this work we have used a multitechnique approach for the surface characterization,

which includes X-ray photoelectron spectroscopy (XPS), cyclic voltammetry (CV), and contact angle measurements for both the reduced S1 and oxidized S2 SAMs. Then, we analyzed the performance of the DA and MA reactions as surface functionalization strategies to obtain spatial and temporal stimuli-responsive SAMs. Specifically, electroactivated SAM S2 with BQ terminal group was conjugated with the newly synthesized fluorescein terminated molecules 1 and 2, with Cp and thiol anchoring groups respectively, to demonstrate temporal control over the functionalization of the surfaces. To achieve a spatial control, the microcontact printing technique was used,44 thereby obtaining a pattern with inert areas consisting of hydrophilic monolayers of hydroxyl molecules 4, resistant to non-specific adsorption of proteins and cells, surrounded by areas functionalized with SAMs of HQ derivative 3. Due to the different lengths of molecules 3 and 4 the HQ groups expose the OH groups on these patterned surfaces allowing


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the specific interactions of BQ groups on the surface of triggered SAMs to study the interfacial reactions on the surface.

EXPERIMENTAL Synthesis. All the chemicals were purchased from Sigma Aldrich (US) and used without further purification. Compound 4 consisting of an alkanethiol functionalized with a tetra(ethylene glycol) group, the 1-mercaptoundec-11-yl-tetra(ethylene glycol), was synthetized through a three step procedure (Scheme 2a). The synthesis of the hydroquinone-tetra(ethylene glycol)alkanethiol 3 consisted of the subsequent modification of the alkanethiol 4 with the hydroquinone moiety 8 (Scheme 2b) resulting in a six step procedure (Scheme 2a). Briefly, treatment of tetra(ethylene glycol) with 11-bromo-1-undecene in a saturated aqueous NaOH solution afforded the alkene 5. Subsequent addition of thiolacetic acid in the presence of a radical initiator gave the thioacetate 6, and the final hydrolysis afforded thiol 4 (OH-SH). Compound 8 was prepared in two synthetic steps from commercially available materials (Scheme 2b). The first reaction is based on the addition of dihydropyran to hydroquinone to provide the protected di-tetrahydropyranehydroquinone 7, which was purified via recrystallization in hexane after completion of the reaction (monitored with TLC). Deprotonation of 7 with t-butyllithium followed by addition of dibromobutane provided the di-tetrahydropyran-hydroquinone-butylbromide 8. Purification step was successfully optimized using an unusual solvent mixture of chloroform and dichloromethane that gave better results compared with the previously described hexane-ethyl acetate mixture.9 Precursor 8 was then used to convert thiol 4 into the hydroquinone derivative 3 (HQ-SH). First,



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thiol 4 was protected with triphenylmethyl chloride, and the subsequent addition of the masked hydroquinone 8, gave the intermediate 10, which after treatment with TFA afforded 3 (HQ-SH). This second synthetic route may be shortened, since the hydrolysis of the thioacetate 6 to obtain 4, and subsequent protection with triphenylmethyl chloride to obtain 9, can be carried out in a one-pot sequence (for more details see SI).


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Scheme 2. (a) Synthesis of thiol 4 (OH-SH) and the hydroquinone derivative 3 (HQ-SH). (b) Synthesis of 8 from hydroquinone and dihydro-2H-pyran.

Fluorescent molecules 1 and 2 were prepared from the common precursor 14 (CF-OEGNH3Cl) which were synthesized in a two-step procedure starting from commercially available 5(6)-carboxyfluorescein (CF), see Scheme 3. CF was activated as the succinimidyl ester and reacted with 1-(tert-butoxycarbonylamino)-4,7,10-trioxa-13-tridecanamine to prepare compound 13 (CF-OEG-Boc). Removal of Boc protecting group afforded derivative 14. The cyclopentadienyl derivative 12 was prepared in a three-step synthesis starting from sodium cyclopentadienylide (NaCp). First, NaCp was alkylated with methyl bromoacetate and the resulting methyl ester was hydrolyzed to obtain 11 (Cp-COOH), which was then converted to the corresponding activated succinimidyl ester 12 (Cp-COOSu). The coupling between 12 and 14 afforded the carboxyfluorescein derivative 1 (CF-OEG-Cp). On the other hand, 3-(tritylthio)propanoic acid was converted to the corresponding succinimidyl ester and reacted with 14 to obtain the thiol-protected derivative 15 (CF-OEG-STrt). Finally, the trityl protecting group was removed to obtain compound 2 (CF-OEG-SH) (for more details see SI).



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Scheme 3. Synthesis of the fluorescein-containing molecules 1 (with Cp) and 2 (with thiol).

These compounds were purified and characterized using 1H-NMR and Electrospray (ESIMS). 1H-NMR experiments were carried out on a Bruker Advance 400 MHz spectrometer. Self-Assembled Monolayer Preparation. Substrates were prepared by evaporation of titanium (50 nm) as adhesion layer and gold (100 nm) onto silicon wafers and cut into substrates of 1x2 cm. These gold substrates were cleaned by sonication in isopropanol, acetone, and ethanol (HPLC grade), exposed to ozone for 20 minutes and immersed in absolute ethanol (HPLC grade) during 30 minutes. Finally, substrates were dried with a stream of nitrogen and immersed in a solution containing molecules 3 and 4 in a 1:1 and 1:99 ratio at 1 mM total concentration in absolute ethanol to obtain SAM S1. They were left immersed in the solution and under an inert


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atmosphere overnight. Before use, SAMs S1 were rinsed with absolute ethanol and dried with a stream of nitrogen. XPS Measurements. X-ray photoelectron spectroscopy measurements on SAMs S1, S2, S3 and S4 were performed with a Phoibos 150 analyzer (SPECS GmbH) in ultra-high vacuum conditions (base pressure 5·10-10 mbar) with a monochromatic aluminium K alpha X-ray source (1486.74 eV). The energy resolution measured by the FWHM of the Ag 3d5/2 peak for a sputtered silver foil was 0.6 eV. The spot size was 3.5 mm by 0.5 mm. Contact Angle Measurements. Contact angle measurements on SAMs S1 and S2 were performed with Milli-Q (MQ) water under ambient temperature and humidity with a Contact Angle Measuring System DSA 100 from KRÜSS. Electrochemical

Measurements.

Before performing electrochemical measurements,

substrates with SAM S1 were rinsed with absolute ethanol and dried with a stream of nitrogen. Later on, SAMs were electrochemically characterized using CV with an AUTOLAB 204 and the NOVA 1.9 software. A custom built electrochemical cell with a Pt-wire as counter electrode, a Ag/AgCl electrode as reference electrode, and the SAM S1 on gold as working electrode were used. The electrolyte was a 1 M HClO4 aqueous solution, which was purged with argon before its use. The area exposed to the electrolyte was approximately 1 cm2. Microcontact Printing. Preparation of Stamps. Stamps of poly(dimethylsiloxane) (PDMS; Dow Corning Sylgard 184) were prepared by casting the prepolymer onto photolithographically fabricated master patterns on silicon, which were previously silanized to increase their hydrophobicity.4, 45 Preparation of Dynamic Surfaces with Spatial Control. Micropatterned SAMs were prepared onto gold substrates produced and cleaned as explained above. A PDMS stamp with a pattern of



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20 µm wide lines was used to microcontact print octadecanethiol (1 mM) onto the gold substrate. The stamp was firstly cleaned with ethanol and dried with a nitrogen stream, thereafter it was immersed during 10 seconds in the octadecanethiol solution, dried under a stream of nitrogen, and put into contact with the clean gold surface during 1 minute. The stamp was then peeled off cautiously from the surface and the substrate was transferred into the backfilling solution, a 1 mM ethanolic solution of the synthesized molecules 3 and 4. Ratios of 1:1 and 1:99 of molecules 3:4 were used for the CV characterization and the fluorescein molecule immobilization studies, respectively. The substrates were left immersed in the solution and under an inert atmosphere overnight. Before further use of these SAMs, they were rinsed in absolute ethanol and dried with a stream of nitrogen (see Figure 1).

Figure 1. Scheme of the preparation of a dynamic substrate with spatial (lines of 20 µm width) control of the functionalization. First, microcontact printing was used to pattern hydrophobic areas of octadecanethiol on bare gold. Then, the non-functionalized areas were filled with a mixed SAM of 4 and the electroactive hydroquinone terminated 3.

Stimuli Activated Surface Functionalization. For the DA surface confined reaction, first a voltage of +650 mV for 60 seconds was applied to SAM S1 in order to oxidize all the hydroquinone groups of the SAM. The resulting SAM S2 was then rinsed with MQ-water and immersed in a 20 mM solution of fluorescent Cp-terminated derivative 1 in THF:water (1:1)


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during 30 minutes to give the SAM S3 after proper rinsing with MQ-water and characterized by fluorescence microscopy. For the MA surface confined reaction, the SAM S2 was obtained as explained above. Thereafter, it was immersed in a solution of fluorescent thiolated molecule 2 in a mixture of EtOH:water (9:1) to give the SAM S4. To optimize the homogeneity of the pattern, different experimental conditions were tested. Specifically, the concentration of 2 was varied from 10 µM to 20 mM and the immersion times were ranged from 30 minutes to overnight (SI).32 After functionalization, the substrates were rinsed with MQ-water and characterized by fluorescence microcopy. Fluorescence Microscopy. Fluorescein functionalized substrates were observed with an Olympus BX51 microscope equipped with a CCD camera Olympus DP20.

RESULTS AND DISCUSSION Characterization of the Electroactive Hydroquinone SAM S1. Cyclic voltammogram of mixed SAM S1, prepared with 1:1 molar ratio of 3 and 4 as the working electrode at different scan rates are depicted in Figure 2. Current peaks are found between 0.43-0.47 V for the oxidation and 0.33-0.26 V for the reduction, in agreement with a previous report.35 The linear relationship between the current intensity and the scan rate (linear regression gave R2= 1.000 and R2= 0.996 for anodic and cathodic peaks, respectively) confirmed that the molecules were surface confined. This relationship is usually obtained in rapid reversible redox processes of immobilized redox couples on a surface.23, 46 The surface coverage was estimated by integrating the oxidation peak area observed from the CV in 1 M HClO4, taking a 2e-, 2H+ reaction mechanism, and applying the equation Q = nFAΓ. In this equation, n is the number of electrons



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involved in the redox couple (n=2 for hydroquinone/benzoquinone), F is Faraday’s constant, A is the surface area of the electrode that is in contact with the electrolyte, and Q is the total charge. By integrating the area under the reductive wave, Q was obtained giving place to a density of Γ = 6.21 x 10-12 moles/cm2 for the 50% quinone monolayer and 1.24 10-11 moles/cm2 for the completely covered one, similarly to what has beenwas previously reported.23, 28

Figure 2. CVs of the SAM S1 with a 1:1 molar ratio between 3 and 4. Inset: relation between scanning rate and the reduction and oxidation peak intensity (current) of the SAMs. Contact angle measurements were performed for bare gold and substrates functionalized with 3 and 4 (1:1) before and after hydroquinone oxidation (see experimental part) to the


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benzoquinone derivative (SAM 1 and SAM 2). The values obtained were 66.1, 56.1, and 80.8º, respectively (Figure 3).

Figure 3. Contact angle images of (a) bare gold, (b) SAM S1, and (c) SAM S2. The bare gold surface showed larger contact angle than the SAM S1 due to hydrophilicity provided by the hydroxyl groups on the surface. On the other hand, SAM S2 presents the highest contact angle, being the most hydrophobic surface, due to the presence of the carbonyl groups.47 The capability of fine tuning the surface tension, thus switching from a hydrophobic to hydrophilic surface due to change in the chemical nature of the functional group facing the solution has been previously reported.48-50 Stimuli Activated SAM Reactivity for Spatial and Temporal Controlled Surface Functionalization. The activation of the SAMs towards the DA and MA interfacial reactions



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was studied by CV. SAMs with a 1:1 molar ratio of 3 and 4 were prepared as previously described and as a preliminary study, solutions of either 15 mM of commercially available hexyl-Cp and hexanethiol in THF:PBS (1:1) were used as simple target ligands to react with the electroactivated surface. Thus, SAM S1 was oxidized to benzoquinone by applying a potential of +650 mV to obtain SAM S2. In order to see how much time is needed to apply the potential for a complete oxidation of SAM S1 to S2, the potential was applied during 20 seconds three consecutive times. After every 20 seconds of oxidation, the SAM S2 was immersed in the target ligand solution during 30 min. After every oxidation and immersion (reaction) step, a CV was performed in order to check the different evolution of the DA or MA interfacial reactions. As seen in Figure 4, the redox peak during the DA reaction with the hexanethiol target ligand disappears after a total 60 seconds of oxidation, as expected, since the cycloadduct formed is not electroactive anymore. On the contrary, the redox peaks of the MA reaction with the hexyl-Cp were only slightly decreased and shifted since in this case, the product of the reaction is still electroactive and thus, the redox signal is not lost.28 S3 and S4 substrates where also analyzed by C1s XPS spectra and compared with the precursor surfaces; S1 and S2 (see SI). Both S1 and S2 (Figure S1) presents two peaks, one at 285.0 eV that corresponds to the bond energy of aliphatic carbons and another at 286.7 eV corresponding to the C-O bond but S2 present and extra peak observed at 289.2 eV which is assigned to the oxidized C=O of the benzoquinone moiety. After the DA reaction with Cp-hexyl, S3, there is a clear increase of the intensity of the peak associated to the aliphatic carbon due to the DA covalent reaction with the Cp, which confirms the covalent anchoring of the Cp-hexyl onto the surface. After the MA reaction with hexanethiol, S4, an increase in the C aliphatic peak


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is also observed and also the peak attributed to C=O disappears, confirming that the reaction takes place (see SI).

Figure 4. DA and MA interfacial reactions using 1 M HClO4 as electrolyte for the oxidation and ligand solutions (hexyl-Cp and hexanethiol) of 15 mM in THF:PBS (1:1) for the interfacial reactions. Cyclic voltammograms monitoring the three oxidation steps of 20 seconds followed by 30 min immersion in the ligand solution.

In order to include not only a temporal control, but also a selective spatial functionalization upon a certain external stimulus (applied potential), the microcontact printing technique was



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used to anchor the electroactive molecule 3 only on specific areas of the surface, e.g. in stripes of 20 µm in width. To compare the differences between the DA and the MA surface confined reactions, fluorescence microscopy images were taken after immersion of the resulting SAM S2 in a 20 mM solution of fluorescent ligands 1 and 2 for 30 min. Oxidation of S1 was performed by applying a voltage of 650 mV during 60 seconds to ensure the presence of benzoquinone groups on the surface, yielding S2. For the DA interfacial reaction a ligand solution of 1 in THF:water 1:1 was used, whereas for the MA interfacial reaction a ligand solution of 2 in EtOH:water 9:1 was employed. The luminescent micropattern obtained after the surface confined reactions is shown in Figure 5. In spite of the broad experimental conditions that were tested for the MA reaction (see experimental section) varying the concentration of the fluorescent molecule 2 and the immersion times, as it can be observed in Figure 5b, the MA does not give place to a homogeneous functionalization, but fluorescent patchy areas. The DA reaction has been described to show a rate enhancement in polar solvents and in ethylene glycol (EG) environments due to the stabilization of its transition state.51 On the other hand, the MA, in spite of being considered a powerful click reaction,31 needs mild catalysis (i.e. amounts of a base or a nucleophile in the media) to increase its reaction kinetics and yield, which explains the formation of patchy areas in its absence. In the present conditions, as we compared the two reactions under similar conditions, we did not add any catalyst. Given the considerable variability of the catalytic reaction, which depends on the pKa of the used thiol (ranging from 7 to 11), the strength and concentration of the catalyst


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as well as the steric hindrance between the reactants,31 an optimization of the reaction conditions should be considered for each system.

Figure 5. Fluorescence images of molecules 1 and 2 (20 mM) covalently attached (after 30 min immersion time) on micropatterns consisting of 20 µm wide stripes of electroactivated hydroquinone surface, SAM S1 oxidized to that reacted through the (a) DA reaction and (b) MA reaction, respectively (Scheme 1). The stripe width of the pattern in the left image is narrower than the 20 µm of the PDMS stripes, which can be attributed to the incomplete contact printing of the SAM S1 due to the fact that this process is very pressure dependent. The scale bare corresponds to 10 µm.

CONCLUSIONS A direct comparison of the electroactivated DA and MA functionalization strategies of surface has been performed. To produce spatially and temporally switchable surfaces, we prepared microcontact printed SAMs of HQ that were used as a dynamic interface to immobilize different



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functional molecules via DA or MA reactions upon the application of a low electrical potential. The synthesis of a thiolated HQ derivative containing a bridge with a pegylated chain followed by an alkylated chain is thoroughly described, as well as two novel fluorescein pegylated derivatives tagged with either Cp or thiol groups to compare the interfacial DA and MA reactions. In spite of the different experimental conditions explored, the MA does not give place to a homogeneous functionalization, but patchy areas compared to the fully covered surfaces obtained through the DA interfacial reaction. Given that the synthesis of the Cp derivatives is more complex and the compounds obtained are less stable than the thiol analogues, we can conclude that there is not a clearly advantageous reaction over the other and therefore their use should be decided according to the singularities of each system, such as the difficulty of synthesizing specific Cp-derivatives, the need for fully coated surfaces, or the possibility of adding a catalyst. In summary, the synthesis of the electroactive molecules together with the comparative study between the DA and the MA following a surface science multitechnique strategy, open the way to the rational use of such interfacial reactions with a wide range of potential applications in different fields ranging from sensing to biomedicine through data storage or clean up.

ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. Synthetic procedures and 1H-NMR spectra of compounds, XPS (C, S) of SAMs.


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AUTHOR INFORMATION Corresponding Author *[email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT The research leading to these results received funding from DGI grants (MOTHER MAT2016-80826-R, Dynamo MAT2013-50036EXP and SAF2014-60138-R), the Networking Research Center on Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN) and the Generalitat de Catalunya (grants 2014-SGR-17 and 2014-SGR-137). ICMAB acknowledges support from the Spanish Ministry of Economy and Competitiveness, through the ‘‘Severo Ochoa’’ Programme for Centres of Excellence in R&D (SEV-2015-0496). This work has been developed inside the Materials Science PhD programs of Universitat Autonoma de Barcelona. J. G. kindly acknowledges the People Programme (Marie Curie Actions) of the Seventh Framework Programme of the European Union (FP7/2007-2013) under the TECNIOSpring programme (TECSPR15-1-0015) and the Agency for Competitiveness and Business of the Government of Catalonia, ACCIÓ as well as the Max Planck Society through the Max Planck Institute for Medical Research and the Max Planck Partner Group “Dynamic Biomaterials for Cancer Immunotherapy”.



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TOC GRAPHIC


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