Bifunctional Dendronized Cellulose Surfaces as Biosensors

Direct-Write Fabrication of Cellulose Nano-Structures via Focused Electron .... Journal of Applied Polymer Science 2015 132 (10.1002/app.v132.23), n/a...
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Bifunctional Dendronized Cellulose Surfaces as Biosensors  † Maria I. Monta~nez,†,‡ Yvonne Hed,† Simon Utsel,† Jarmo Ropponen,† Eva Malmstr€om,† Lars Wagberg, † ,† Anders Hult, and Michael Malkoch* †

KTH Royal Institute of Technology, School of Chemical Engineering, Department of Fibre and Polymer Technology, SE-100 44 Stockholm, Sweden ‡ F-IMABIS-Carlos Haya Hospital, Research Laboratory, 29009 Malaga, Spain

bS Supporting Information ABSTRACT:

Well-defined dendronized cellulose substrates displaying multiple representations of dual-functionality were constructed by taking advantage of the efficiency of the click reaction combined with traditional anhydride chemistry. First, activated cellulose surfaces were decorated with several generations of dendrons, and their peripheral reactive groups were subsequently reacted with a trifunctional orthogonal monomer. The generated substrate tool box was successfully explored by accurately tuning the surface function using a versatile orthogonal dual postfunctionalization approach. In general, the reactions were monitored by using a clickdye reagent or a quartz crystal microbalance (QCM) technique, and the resulting surfaces were well-characterized using XPS, FT-IR, and contact angle measurements. Utilizing this approach two different surfaces have been obtained; that is, triethylenglycol oligomers and amoxicillin molecules were efficiently introduced to the dendritic surface. As a second example, mannose-decorated hydroxyl functional surfaces illustrated their potential as biosensors by multivalent detection of lectin protein at concentration as low as 5 nM.

’ INTRODUCTION Cellulose is one of the most abundant and sustainable resources in nature. Its excellent biocompability, hydrophilicity, and nontoxicity coupled to the high density of free hydroxyl groups make cellulose an invaluable solid substrate that can undergo postfunctionalizations to enter new cutting-edge applications. An example of their potential is in the field of biosensors in which cellulose acts as a solid support for biomolecules immobilization.1,2 In these applications, the protein adsorption behavior to surfaces is of critical importance because nonspecific proteinsurface interactions are often the main cause of low sensitivity or selectivity. Attempts to introduce blocking reagents to reduce nonspecific interactions3 have shown limited protein adsorption control. Therefore, new materials concepts that allow extensive freedom to manipulate and control the function of solid surfaces are ever more important to identify. Parameters, such as number and nature of functional groups, hydrophobicity and hydrophilicity, spatial distribution, and surface topology need to be taken into account for finding optimal conditions to control protein adsorption behavior.4 r 2011 American Chemical Society

To address such grand challenge in which surfaces can in a simple protocol be manipulated to interact with biological molecules, a platform of multifunctional polymeric scaffolds was envisioned. These macromolecular architectures should amplify the functional groups already available at the surface with a precise number of multiple copies. For accomplishing this, a prerequisite is that the polymer platform can undergo facile postfunctionalizations for increased multivalency displayed to biological media. Consequently, the hybridization of cellulose surfaces with branched dendritic entities that improves the sensitivity toward biomolecules is an attractive methodology. This is due to the unprecedented control over the architecture coupled to the possibility of designing a large number of accessible active sites at the periphery of the dendritic scaffolds. PAMAM dendrimers have been chemically attached onto cellulose substrate Received: February 11, 2011 Revised: April 25, 2011 Published: April 27, 2011 2114

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Biomacromolecules to exhibit biofunctional surfaces5,6 for applications involving biomolecular recognitions such as immunoassays.7 Nonetheless, with the introduction of robust and chemoselective reactions, more complex dendritic structures are envisioned to advance beyond simple dendrimers with one kind of functionality distributed homogenously at the periphery.813 The most representative example of such reactions is the copper-catalyzed azidealkyne cycloaddition (CuAAC) reaction, the attractive properties of which include high yield, tolerance to various reaction conditions, and the formation of a single product.14 Other advantages can be considered in surface chemistry applications, such as chemoselectivity and stability of the 1,2,3-triazole moiety linker. The byproducts do not affect the monolayer quality or their functional integrity and can be easily removed from the surface. Therefore, the general characteristics of the click chemistry approach fit very well with the requirements of chemical reactions performed on surfaces.15 This is documented by many reports, which demonstrate the use of click chemistry for the introduction of different functional groups on several surfaces.16 Moreover, because azides and primary alkynes are not biologically available and do not react with biological functionalities, the reaction has often been described as bio-orthogonal; therefore, it is very useful for attaching biomolecules to surfaces. On the basis of the quantitative and irreversible triazol formation, several studies have carried out the introduction of dendrons to cellulose materials as well.6,17 As a result, we herein present a new platform of dendronized cellulose substrates that display multiple orthogonal functionalities. Our overall strategy is shown in Scheme 1, which combines several synthetic methodologies, that is, click chemistry, dendrimer chemistry, and solid-phase synthesis of the dendronized surfaces.

’ EXPERIMENTAL SECTION Full experimental details are contained in the Supporting Information. Materials. All materials were obtained from Sigma-Aldrich. 2,2Bis(methylol)propionic acid (bis-MPA) was kindly donated by Perstorp AB. Flash chromatography was performed using 3264 D 60 Å silica gel from ICN SiliTech (ICN Biomedicals GmbH, Eschwege, Germany). Whatman filter paper (Grade 2 Qualitative) was cut into discs 6 mm in diameter and into 1 cm2 squares and used as cellulose filter paper (CFP-OH). First to fifth generations of acetylene-functionalized core hydroxyl peripheral bis-MPA dendrons were synthesized, according to literature procedures.18,19 The protein used was Concanavalin A (Con A), from Canavalia ensiformis (Jack Bean), type VI. Instrumentation. MALDI-TOF: THF/DHB/Na matrix and THF/9-nitroanthracene/Naþ matrix were used for sample preparation for MALDI-TOF analysis, concentration 1 mg/mL of sample in THF (40 μL Matrix solution/5 μL sample solution). The MALDI-TOF MS spectrum acquisition was conducted on a Bruker UltraFlex MALDITOF MS with SCOUT-MTP Ion Source (Bruker Daltonics, Bremen) equipped with a N2 laser (337 nm), a gridless ion source, and reflector design. All spectra were acquired using a reflector-positive method with an acceleration voltage of 25 kV and a reflector voltage of 26.3 kV. The detector mass range was set to 50010 000 Da to exclude high-intensity peaks from the lower mass range. The laser intensity was set to the lowest value possible to acquire high-resolution spectra. The obtained spectra were analyzed with FlexAnalysis Bruker Daltonics, Bremen, version 2.2. NMR experiments were performed on a Bruker Avance 400 MHz NMR instrument using CDCl3, D2O, or MeOH-d4 as solvents. 1H NMR spectra were acquired with a spectral window of 20 ppm, an acquisition time of 4 s, and a relaxation delay of 1 s. 13C NMR spectra were acquired

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with a spectral window of 240 ppm, an acquisition time of 0.7 s, and a relaxation delay of 2 s. FTIR was conducted on a Perkin-Elmer Spectrum 2000 FTIR equipped with an MKII Golden Gate, single reflection ATR system. Static contact angle measurements were conducted with a KSV instruments CAM 200 equipped with a Basler A602f camera using 5 μL droplets of MiliQ water and a relative humidity of 50%. The contact angles were determinate using the CAM software. X-ray photoelectron spectroscopy (XPS) measurements were performed with the aid of a physical electronics quantum 2000 scanning ESCA microprobe equipped with a monochromatic Al KR X-ray source. For each of the experiments, the pass energy was as follows: 93.90 eV for survey spectra, 23.50 eV for C1s, 29.35 eV for N1s, 46.95 eV for Cl2p, 26.95 eV for Na1s, and 26.95 eV for S2p. Quartz crystal microbalance (QCM) QCM-E4 from Q-Sense AB (V€astra Fr€olunda, Sweden) was used to study the effect of the surface modifications and protein interactions with the modified surfaces. QCM crystals used were AT-cut quartz crystals (5 MHz resonant frequency) with an active surface of sputtered silica. Model cellulose surfaces were prepared on top of these crystals, and the procedure is described separately in this Article. A flow of 100 μL/min was used for all experiments, and all presented data correspond to the normalized frequency of the third overtone. A relationship often utilized to convert a change in frequency into an adsorbed mass per area is the Sauerbrey model.20 This model assumes rigidly attached layers, and the adsorbed amount includes water immobilized with this adsorbed layer. Δf m¼C n

ð1Þ

where m is the adsorbed mass per area (mg/m2), C is the sensitivity constant, 0.177 (mg/(m2 3 Hz)), Δf is the change in resonant frequency (Hz), and n is the overtone number. Nomenclature Description. The two kinds of surfaces employed are called CFP and CQC. CFP stands for cellulose filter paper and CQC stands for quartz crystal cellulose. The unmodified surfaces displaying hydroxyl functionality are called CFP-OH and CQC-OH, respectively, and the activated ones presenting azide groups are named CFP-N3 and CQC-N3, respectively. CFP-[Gx]-(OH)2x is used for referring to the dendronized cellulose filter, in which x represents the generation number of the Bis-MPA dendron attached, (OH) symbolizes the peripheral functionally of the dendron, and 2x represents the number of OH groups displayed for that dendron. Table S1 in the Supporting Information clarifies the number of surface functional groups for each generation. CFP-[G(xþ1)]-(Acet)2x (N3)32x represents the addition of one monomer layer to the previous dendronized surfaces, where (x þ 1) is the final dendron generation reached, displaying 2x acetonide (Acet) and 2x azide (N3) peripheral functional groups. CFP-[G(xþ1)]-(OH)2(2)x (N3)2x surfaces displays 2(2)x hydroxyl (OH) and 2x azide (N3) functional groups. The presence of other peripheral ligands is described with the following nomenclatures: triethylenglycol (TEG), disperse red (Red), amoxicillin (AXO), and mannose (Man). The same nomenclature is applied for CQC surfaces, that is, CQC-[G5]-(OH)32 (Man)16, in which the dendron grows up to fifth generation, presenting 32 hydroxyl groups and 16 mannose ligands.

Preparation of Dendronized Cellulose Filter Paper (Scheme 1). Activation of the Cellulose Filter Paper. Preparation of

CFP-N3. The CFP-OH cellulose filter papers were immersed in a reactive solution (concn 0.18 mM of 1) containing anhydride 1 (1.5 g, 4.4 mmol), TEA (0.91 mL, 6.6 mmol), and DMAP (53 mg, 0.44 mmol) in THF (25 mL). The reaction mixture was shaken at room temperature overnight. 2115

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Scheme 1. (A) Synthetic Approach to Bifunctional Dendronized Cellulose Filter Paper and (B) Post-Functionalization with Orthogonal Reactionsa

a

(a) 1, TEA, DMAP, THF; (b) Acetylene-[G5]-(OH)32, Cu(PPh3)3Br, DIPEA, THF; (c) 2, TEA, DMAP, THF; (d) Dowex, MeOH.

The filter papers were then shaken in THF for 1 h and rinsed with THF, DMF, H2O, THF (50 mL of each), and dried in a vacuum oven at 50 °C. Dendronization of the Cellulose Filter Paper. Preparation of CFP-[Gx]-(OH)2x. Groups of 90 discs of CFP-N3 were immersed in a solution containing acetylene-[Gx]OH (x = 15) dendrons

(0.2 mmol), Cu(PPh3)3Br (37 mg, 0.04 mmol), and DIPEA (34 μL, 0.2 mmol) dissolved in 5 mL of THF (concn 0.04 M of dendron). The suspension was stirred at 40 °C overnight. The filter papers were then shaken in THF for 1 h and rinsed with THF, DMF, and MeOH, and dried in a vacuum oven at 50 °C. 2116

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Biomacromolecules Bifunctionalization of the Cellulose Filter Paper. Preparation of x x CFP-[Gxþ1]-(Acet)2 (N3)2 . The hydroxyl CFP-[Gx]-(OH)2x surfaces were immersed in solution containing anhydride 2 (1.56 g, 2 mmol), TEA (100 μL, 0.75 mmol), and DMAP (6 mg, 0.05 mmol) dissolved in 20 mL of THF (concn 0.1 M of 2). The reaction mixture was shaken at room temperature overnight. The filter papers were then shaken in THF for 1 h and rinsed with DMF, THF, and MeOH and dried in a vacuum oven at 50 °C. Preparation of CFP-[Gxþ1]-(OH)2(2)x(N3)2x. The acetonide CFP-[Gxþ1]-(Acet)2x(N3)2x surfaces were immersed in MeOH containing DOWEX and shaken overnight. The DOWEX resin was removed with a pipet, and the surfaces were then rinsed with MeOH and dried in a vacuum oven at 50 °C.

General Procedures for Postfunctionalization of the Bifunctional Cellulose Filter Paper. Esterification Reaction with Hydroxyl Compounds (3). The surfaces were immersed in a solution containing anhydride 3 (1.1 g, 2 mmol), TEA (415 μL, 3 mmol), and DMAP (24 mg, 0.2 mmol) in 20 mL of DCM. The reaction mixture was shaken overnight. The surfaces were shaken in THF for 1 h, then rinsed with DCM, MeOH, and THF (50 mL of each), and dried in the vacuum oven. Click Reaction with Nonpolar Acetylene Compounds (4). The surface was immersed in a solution (concn 0.08 mM of acetylene compound) containing acetylene 4, Cu(PPh3)3Br (28 mg, 0.03 mmol), and DIPEA (26 μL, 0.15 mmol) dissolved in 2 mL of THF. The suspension was stirred at 40 °C overnight. The filter papers were then shaken in THF for 1 h, rinsed with THF (4) and dried in a vacuum oven at 50 °C. Click Reaction with Polar Acetylene Compounds (5). The surfaces were immersed in a solution (concn 0.1 M of acetylene compound) containing acetylene 5 (884 mg, 2 mmol), sodium ascorbate (396 mg, 2 mmol), and CuSO4 3 10H2O (100 mg, 0.4 mmol) in 20 mL of water and shaken at room temperature overnight. The reaction mixtures were then shaken in H2O for 1 h and rinsed with H2O, 10% EDTA aqueous solution (2), MeOH (2), H2O (2), and MeOH (2). The filter papers were then dried in a vacuum oven at 50 °C.

Manufacturing of Cellulose Coated QCM Crystals (CQCOH). The preparation of cellulose model surfaces on the QCM crystals was based on a previously described method.21 We dissolved 250 mg of acetone-extracted dissolving pulp (Domsj€o dissolving plus; Domsj€o Fabriker AB, Domsj€o, Sweden) in 12.5 g of N-methylmorpholine-Noxide (NMMO) under stirring at 125 °C. After dissolution, 37.5 g DMSO was added dropwise. This solution was spincoated on polyvinylamine (PVAm)-modified QCM crystals and then precipitated and cleaned in Milli-Q water. The PVAm-modified QCM crystals were prepared as follows. First, the unmodified QCM crystals were rinsed with a Milli-QethanolMilli-Q sequence and then dried with a stream of N2 gas. Subsequently, they were placed in air plasma cleaner (model PDC 002, Harrick Scientific Corporation, Pleasantville, NY) under reduced air pressure for 120 s at high effect (30 W). Finally, they were immersed in a PVAm solution of 1 g/L for 15 min, rinsed with Milli-Q, and dried with a stream of N2 gas.

Functionalization of CQCOH Crystals (Scheme 4, Steps ad). (a) CQC-N3. The CQC-OH crystal was immersed in a solution

that contained anhydride 1 (67 mg, 0.2 mmol), TEA (40 μL, 0.3 mmol), and DMAP (3 mg, 0.02 mmol) in 1 mL of THF at room temperature for 12 h. The crystal was then rinsed with THF, DMF, H2O, and THF, dried under a stream of N2, and placed in the QCM flow through system. (b) CQC-[G4]-(OH)16. Acetylen-[G4]-(OH)16 dendron (80 mg, 0.045 mmol), sodium ascorbate (4 mg, 0.02 mmol), and CuSO4 3 10H2O (2.5 mg, 0.0014 mmol) were dissolved in 10 mL of H2O and passed through a Teflon filter 0.45. Milli-Q was used to establish a baseline in the QCM with the CQC-N3 crystal, and after stabilization the dendron solution was introduced. The reaction proceeded

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for approximately 4 h, whereupon it was stopped by changing to Milli-Q again. (c) CQC-[Gxþ1]-(Acet)2x(N3)32x, (x = 0, 4): The hydroxylfunctionalized crystal was immersed in solution containing anhydride 6 (391 mg, 0.5 mmol), TEA (100 μL, 0.75 mmol), and DMAP (6 mg, 0.05 mmol) dissolved in 1 mL of THF. The crystal was then rinsed with THF, H2O, and MeOH. (d) CQC-[Gxþ1]-(OH)2(2)x(N3)32x, (x = 0, 4): The crystal was immersed in 4 mL of MeOH containing 80 mg of DOWEX for 3 h. The crystal was then rinsed with MeOH, H2O, and THF, dried under a stream of N2, and placed in the QCM flow through system. (e) CQC-[G(xþ1)]-(OH)2(2)x(Man)2x, (x = 0, 4): Acetylenemannose (221 mg, 1 mmol), sodium ascorbate (2 mg, 0.010 mmol), and CuSO4 3 10H2O (8 mg, 0.032 mmol) were dissolved in 30 mL of H2O. After stirring (vortex) of the reagents solution, precipitation in the mixture can be observed. The mixture is passed through a Teflon filter 0.45 until a stable clear solution is observed. Milli-Q was used to establish a baseline in the QCM with the CQC-[G(xþ1)]-(OH)2(2)x(N3)32x crystals, and after stabilization, the mannose solution was introduced. The reaction proceeded for approximately 100 min, whereupon it was stopped by changing to Milli-Q again. SurfaceProtein Interaction Studies. Hepes buffer solution was used to establish a baseline in the QCM with the CQC-[G(xþ1)]-(OH)2(2)x(Man)2x crystal and, after stabilization, the protein solutions with different concentrations were introduced respectively, with cleaning of the surface in between. The treatment proceeded for approximately 60 min, whereupon Hepes buffer solution was introduced again. Cleaning of surfaces was conducted by exposure to mannose cleaning solution and then to buffer solution. Similar experiments were carried out with CQC-[G(xþ1)]-(OH)2(2)x(N3)32x used as controls. Solutions employed for QCM proteinsurface interaction studies: Buffer: 10 mM Hepes (pH 7.4), 300 mM NaCl, 0.9 mM CaCl2, 0.9 mM MnCl2, 0.02% Triton X-100 In Millipore buffer. Protein solutions in Hepes buffer (μM): 1, 0.1, 0.05, 0.01, 0.005. Mannose cleaning solution: Concentration 0.1 M in Hepes buffer. Synthesis of AB2C Anhydride Monomer (2) (Scheme 2). (a). 6-Azidehexanoic Acid (7). 6-Bromohexanoic acid (40.0 g, 0.205 mol) was dissolved in DMSO (250 mL) and heated to 40 °C, followed by stepwise addition of NaN3 (66.7 g, 1.025 mol). The reaction was heated to 80 °C and stirred overnight. The temperature was decreased to 40 °C and HCl concn (85 mL) was added stepwise to the mixture that was left to stir overnight. The product was purified by extraction with diethyl ether (5  200 mL). The ether phases were collected, washed with 10% NaHSO4 aqueous solution (2  200 mL) and water (6  100 mL), dried with MgSO4, filtered, and evaporated to dryness. The product was obtained as a yellow oil (26.71 g, 83%). 1H NMR (CDCl3, 400 MHz, δ): 1.381.46 (m, 2H, CH2CH2CH2CO), 1.571.67 (m, 4H, CH2CH2N3, and  CH2CH2CO), 2.36 (t, J = 7.4 Hz, 2H, CH2CO), 3.27 (t, J = 6.9 Hz, 2H, CH2N3). 13C NMR (CDCl3, 100 MHz, δ): 24.1, 26.1, 28.4, 33.8, 51.1, 180.1. (b). 6-Azidehexanoic Anhydride (8). Acid 7 (53.6 g, 0.341 mol) was dissolved in DCM (100 mL) and cooled down to 0 °C followed by stepwise addition of DCC (35.2 g, 0.170 mol). The reaction was left to reach room temperature overnight and then filtered. The filtrate was collected and evaporated to dryness to yield the pure product as a yellow oil (36.0 g, 71%). 1H NMR (CDCl3, 400 MHz, δ): 1.391.47 (m, 4H, CH2CH2CH2CO), 1.571.72 (m, 8H, CH2CH2N3 and  CH2CH2CO), 2.46 (t, J = 7.3 Hz, 4H, CH2CO), 3.27 (t, J = 6.8 Hz, 4H CH2N3). 13C NMR (CDCl3, 100 MHz, δ): 23.6, 25.9, 28.4, 34.9, 51.0, 169.1. (c). Azide-AcOH (10). The acetonide protected trizma12 9 (26.9 g, 0.167 mol) and TEA (25.3 g, 0.251 mol) were dissolved in an excess of DCM (1.5 L) and cooled to and kept at 0 °C during the whole reaction. Azide anhydride 8 (34.7 g, 0.117 mol) was diluted in DCM (400 mL) then added dropwise to the reaction mixture. The reaction was monitored with 13C NMR until complete disappearance of the azide anhydride. The 2117

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Scheme 2. Synthesis of AB2C Reactive Monomera

a

(a) NaN3, DMSO, 80°C; (b) DCC, DCM, 0°C; (c) 1. dimethoxypropane, DMF, p-TSA; 2. TEA; (d) 8, TEA, DCM, 0°C; (e) succinic anhydride, DMAP, TEA, DCM; (f) DCC, DCM, 0°C. mixture was extracted with 10% NaHSO4 (4  300 mL) and Na2CO3 (300 mL). The organic phase was dried with MgSO4, filtered, and evaporated to dryness to yield the product as a white solid (34.1 g, 70%). 1H NMR (CDCl3, 400 MHz, δ): 1.401.47 (m, 8H, CH3 and CH2CH2CH2CO), 1.591.74 (m, 4H, CH2CH2N3, and  CH2CH2CO), 2.30 (t, J = 7.4 Hz, 2H, CH2CO), 3.28 (t, J = 6.8 Hz, 2H, CH2N3), 3.65 (d, J = 5.9 Hz, 2H, CH2OH), 3.83 (s, 4H, CH2O), 5.09 (t, J = 5.9 Hz, 1H, -OH), 6.29 (s, 1H, NH). 13C NMR (CDCl3, 100 MHz, δ): 18.8, 25.2, 26.1, 28.2, 28.6, 36.8, 51.2, 55.0, 64.4, 64.5, 98.9, 174.4. (d). Acid Functional Acetonide-Protected Trizma-Azide (11). The azide functional acetonide-protected trizma 10 (34.1 g, 0.113 mol), DMAP (2.8 g, 0.023 mol), and TEA (16.8 g, 0.159 mol) were dissolved in DCM (250 mL) and placed in an ice bath. Succinic anhydride (15.9 g, 0.159 mol) was added stepwise to the solution, and the reaction was left to react overnight. The residual anhydride was quenched by water (25 mL) overnight. The mixture was extracted with 10% NaHSO4 (4  400 mL) and with brine (4  600 mL), dried over MgSO4, and evaporated to dryness. The pure product was obtained as a slightly yellow solid (40.0 g, 88%). 1H NMR (CDCl3, 400 MHz, δ): 1.331.44 (m, 8H, CH3, and CH2CH2CH2CO), 1.541.64 (m, 4H, CH2CH2N3, and CH2CH2CO), 2.17 (t, J = 7.4 Hz, CH2CONH), 2.602.66 (m, 4H, CH2CO, and CH2COOH), 3.24 (t, J = 6.8 Hz, 2H,  CH2N3), 3.76 (d, J = 12.0 Hz, 2H, CCH2O), 4.18 (d, J = 12.0 Hz, 2H, CCH2O), 4.46 (s, 2H, CCH2OCO), 6.05 (s, 1H, NH). 13 C NMR (CDCl3, 100 MHz, δ): 23.1, 23.9, 24.9, 26.1, 28.5, 28.8, 28.9, 36.7, 51.2, 53.1, 62.3, 63.5, 98.8, 172.3, 174.0, 176.0. (e). Anhydride Functional Acetonide Protected Trizma-Azide (2). The acid functional acetonide-protected trizma-azide 11 (20.0 g, 0.050 mol) was dissolved in DCM (34 mL) and cooled to 0 °C, followed by stepwise addition of DCC (5.2 g, 0.025 mol). The reaction was left to reach room temperature overnight and then filtered. The filtrate was collected and evaporated to dryness to yield the pure product as a yellow highly viscous oil (11.3 g, 58%). 1H NMR (CDCl3, 400 MHz, δ): 1.331.45 (m, 16H, CH3 and CH2CH2CH2CO), 1.541.64 (m, 8H, CH2CH2N3 and CH2CH2CO), 2.15 (t, J = 7.4 Hz,  CH2CONH), 2.652.68 (m, 4H, CH2CO), 2.762.79 (m, 4H, CH2CO), 3.24 (t, J = 6.8 Hz, 2H, CH2N3), 3.77 (d, J = 12.0 Hz, 4H,  CCH2O), 4.16 (d, J = 12.0 Hz, 4H, CCH2O), 4.49 (s, 4H,  CCH2OCO), 5.83 (s, 1H, NH). 13C NMR (CDCl3, 100 MHz, δ): 23.4, 23.5, 24.8, 26.0, 28.3, 28.5, 30.1, 36.6, 51.1, 52.8, 62.4, 63.7, 98.6, 168.0, 171.4, 173.4.

Synthesis of Anhydride 1 (Scheme 3). 4-Azidomethyl Benzoic Acid (12). A solution of 4-chloromethyl benzoic acid (22.3 g, 0.131 mol) and NaN3 (25.5 g, 0.392 mol) in 100 mL of DMSO was stirred for 2 days at 80 °C. After cooling to room temperature, 100 mL of water was added, and the mixture was diluted with 300 mL of Et2O. The organic phase was extracted with water (5  50 mL) and dried over MgSO4 The evaporation of the solvent under reduced pressure provided the pure product as a white solid (21.9 g, 95%). 1H NMR (DMSO-d6, 400 MHz, δ): 7.97 (d, 2H, J = 8.1 Hz, AA0 BB0 ), 7.48 (d, 2H, J = 8.1 Hz, AA0 BB0 ), 4.55 (s, 2H, CH2). 13C NMR (DMSO-d6, 100 MHz, δ): 167.1, 140.6, 130.5, 129.7, 128.4, 53.1. 4-Azidomethyl Benzoic Anhydride (1). Acid 12 (14.6 g, 0.082 mol) was dissolved in DCM (100 mL) and cooled to 0 °C, followed by stepwise addition of DCC (8.5 g, 0.041 mol). The reaction was left to reach room temperature overnight and then filtered. The filtrate was collected and evaporated to dryness to yield the pure product as a white solid (13.0 g, 93%). 1H NMR (CDCl3, 400 MHz, δ): 8.16 (d, 2H, J = 8.1 Hz, AA0 BB0 ), 7.48 (d, 2H, J = 8.1 Hz, AA0 BB0 ), 4.48 (s, 2H, CH2). 13 C NMR (CDCl3, 100 MHz, δ): 161.7, 142.4, 131.1, 129.7, 128.5, 128.3, 54.1. Synthesis of Anhydride 3 (Scheme 3). 4-Oxo-4-(triethylenglycol monoethyl ether)butanoic Acid (13). Triethylene glycol monoethyl ether (50.0 g, 0.280 mol), DMAP (6.8 g, 0.056 mol), and succinic anhydride (35.0 g, 0.340 mol) were dissolved in 200 mL of DCM and left to react overnight. Then, the reaction mixture was quenched with 20 mL of water. Subsequently, the mixture was diluted with 100 mL of DCM and extracted with NaHSO4 10% aqueous solution (3  50 mL) and brine (50 mL). The organic phase was dried over MgSO4, filtered off, and evaporated to dryness to obtain a transparent liquid (63.1 g, 81%). 1H NMR (CDCl3, 400 MHz, δ): 4.26 (t, 2H, J = 8.0 Hz, CH2OCO), 3.693.60 (m, 10H, OCH2), 3.54 (c, 2H, J = 8.1 Hz, CH2CH3), 2.65 (s, 4H, CH2CO), 1.20 (t, 3H, J = 8.1 Hz, CH3). 13C NMR (CDCl3, 100 MHz, δ): 175.8, 171.9, 70.7, 70.5, 70.4, 69.6, 68.9, 66.6, 63.8, 29.3, 29.0, 15.0 . 4-Oxo-4-(triethylenglycol monoethyl ether)butanoic Anhydride (3). Acid 13 (63.1 g, 0.230 mol) was dissolved in DCM (15 mL) and cooled to 0 °C, followed by stepwise addition of DCC (23.7 g, 0.120 mol). The reaction was left to reach room temperature overnight and then filtered. The filtrate was evaporated to dryness to yield the pure product as a transparent oil (50.0 g, 80%). 1H NMR (CDCl3, 400 MHz, δ): 4.25 (t, 2H, J = 8.0 Hz, CH2OCO), 3.69 (t, 2H, J = 8.0 Hz, OCH2), 3.673.62 (m, 6H, OCH2), 3.613.56 (m, 2H, OCH2), 2118

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Scheme 3. Synthesis of Anhydrides (A) 1 and (B) and Acetylene Functionalization of Ligands (C) Disperse Red, (D) Amoxicillin, and (E) D-Mannose

3.52 (c, 2H, J = 8.1 Hz, CH2CH3), 2.79 (t, 2H, J = 8.0 Hz, CH2CO), 2.68 (t, 2H, J = 8.0 Hz, CH2CO), 1.20 (t, 3H, J = 8.1 Hz, CH3). 13C

NMR (CDCl3, 100 MHz, δ): 171.6, 167.8, 70.69, 70.5, 69.8, 68.9, 66.6, 64.1, 30.1, 28.4, 15.1. 2119

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Synthesis of Acetylene Compounds 4, 5 and 6. (Scheme 3). Disperse Red 13-Prop-2-ynyl Succinate (4). A solution of 13 (5.0 g, 14.3 mmol), DMAP (350 mg, 3 mmol), and pyridine (6 mL, 72 mmol) in DCM (50 mL) was prepared in a round-bottomed flask and placed in a ice-cold bath. Then, acetylene anhydride22 14 (5.6 g, 18.6 mmol) dissolved in DCM (20 mL) was added dropwise to the solution. The reaction was stirred overnight at room temperature and monitored by TLC (Hep/EtOAc) until complete disappearance of the starting material. Then, the reaction was quenched with water. Subsequently, the mixture was diluted with 300 mL of DCM and extracted with NaHSO4 10% aqueous solution (5  50 mL), 10% NaHCO3 aqueous solution (2  50 mL), and brine (50 mL). The organic phase was dried over MgSO4, filtered off, and evaporated to dryness to yield the pure product as a red solid (6.4 g, 92%). 1H NMR (400 MHz, CDCl3, δ): 8.38 (d, J = 2.3 Hz, 1H, Ar), 8.15 (dd, J = 2.3 Hz, J = 9.1 Hz, 1H, Ar), 7.94 (d, J = 9.3 Hz, 2H, Ar), 7.77 (d, J = 9.1 Hz, 1H, Ar), 6.80 (d, J = 9.3 Hz, 2H, Ar), 4.70 (d, J = 2,4 Hz, 2H, CH2CCH), 4.33 (t, J = 6.3 Hz, 2H, NCH2CH2-), 3.70 (t, J = 6.3 Hz, 2H, -NCH2CH2), 3.54 (q, J = 7.1 Hz, 2H, CH2CH3), 2.67 (m, 4H, COCH2-), 2.47 (t, 1H, J = 2.4 Hz,  CCH), 1.26 (t, J = 7.1 Hz), 1.26 (t, 3H, 7.1 Hz). 13C NMR (100 MHz, CDCl3, δ): 171.9, 171.3, 153.0, 151.6, 147.2, 144.4, 133.9, 126.9, 126.0, 122.6, 117.4, 111.5, 77.1, 75.0, 61.5, 52.2, 48.7, 45.7, 28.8, 28.7, 12.2. Amoxiciloate Propargyl Amine (5). Amoxicillin trihydrate (419 mg, 1.0 mmol) was dissolved in MeOH (1 mL), and propargylamine (500 μL, 7.2 mmol) was added to the solution. The mixture was stirred overnight, and the solution was left under vacuum. The resulting orange solid was dissolved in water and then freeze-dried to obtain the pure product (400 mg, 95%). 1H NMR (400 MHz, D2O, δ): 7.32 and 6.91 (AA0 BB0 , 4H, J = 7.7 Hz, Ph), 4.98 (d, 1H, J = 8.6 Hz, CHS), 4.69 (s, 1H,  CHPh), 4.34 (d, 1H, J = 8.6 Hz, CHNHCO), 3.95 (dd, 2H, J = 2.5 Hz, J = 2.2 Hz, CH2 CCH), 3.32 (s, 1H,CHCOOH), 2.48 (t, J = 2.5 Hz, 1H, CCH), 1.43 (s, 3H, CH3), 1.18 (s, 3H, CH3). 13C NMR (100 MHz, D2OþCD3OD, δ): 175.9, 173.7, 172.0, 159.3, 130.5, 129.7, 117.0, 80.5, 77.0, 74.1, 67.0, 60.6, 59.9, 59.1, 31.0, 29.8, 27.7. Synthesis of D-Mannose Acetylene (6). D-Mannose Pentaacetate (15). Mannose (10.0 g, 55.5 mmol) was reacted as described in the literature23 to obtain 15 (19.9 g, 92%). D-Mannose Tetraacetate (16). Derived D-mannose 15 (18.0 g, 46.1 mmol) were reacted as described in the literature24 to obtain 16 (14.9 g, 93%). D-Mannose Tetraacetate Trichloro (17). Derived D-mannose 16 (10.0 g, 28.7 mmol) was reacted as described in the literature25 to obtain 17 (10.4 g, 74%). D-Mannose Tetraacetate Acetylene (18). Derived D-mannose 17 (5.3 g, 10.8 mmol) was reacted as described in the literature25 to obtain 18 (3.8 g, 91%). D-Mannose Acetylene (19). Derived D-mannose 18 (3.0 g, 7.8 mmol) was deprotected as described in the literature25 to obtain 19 (1.6 g, 95%).

’ RESULTS AND DISCUSSION To prepare polymeric platforms that display dual-functionalities, we chose the biocompatible bis-MPA dendrimers because of their facile construction using click reactions2628 coupled to recent reports on the synthesis of orthogonally functionalized bis-MPA dendrimers for biological application.18,29 The robust and chemoselective CuAAC reaction14 was employed to explore the hybridization concept of bis-MPA dendritic moieties to cellulose substrates and to exploit the resulting orthogonal functional surfaces.30 Initially, the hydroxyl groups on the cellulose filter paper (CFP-OH) were activated by the introduction of azide groups through an esterification reaction with anhydride 1 to yield CFP-N3 (Scheme 1A). Anhydride activated chemistry was chosen because it can readily functionalize

Figure 1. Cellulose solid phases treated with Red-Disperse-derived 4 and (CuPPh3)3Br/DIPEA in THF: (A) CFP-OH; (B) CFP-N3; (C) CFP-[Gx]-(OH)2x, x = 15; and (D) CFP-[Gxþ1]-(OH)2(2)x(N3)2x, x = 15. (E) FT-IR spectra of the unmodified, CFP-OH, and modified filter paper: azide-activated CFP-N3, dendronized CFP-[G3]-(OH)8, and bifunctional dendronized CFP-[G4]-(OH)16(N3)8.

constrained polymeric structures under mild conditions.31 To ensure that CFP-N3 contains covalently attached azides, CFP-N3 was reacted with acetylene functionalized Disperse Red 4 as a visual evaluation method. The CuAAC reaction, using (CuPPh3)3Br/DIPEA in THF, and the rinsing with copious amounts of THF, MeOH, and DMF, yielded red surfaces (Figure 1A,B). As a support of reaction efficiency, similar CuAAC condition between 4 and an unmodified cellulose surface CFP-OH was investigated. After the washing procedure, a white surface was obtained, therefore elucidating a covalent attachment of molecules and not only physisorbed to the surface. Such visual control using colorimetric reagent 4 enables its use as sensor to detect the azides presents on a surfaces as well as to determine the success of CuAAC reaction. Following this, a set of bis-MPA dendrons with a single acetylene in the core was synthesized, first to fifth generation, with molecular weights up to 3656 g mol1 and 32 peripheral OH groups. The hydroxyl functional dendrons were successfully attached to the CFP-N3 via CuAAC reaction. Consequently, a library of dendronized cellulose substrates, CFP-[Gx]-(OH)2x (x = 15), were achieved via a convergent solid phase approach allowing bis-MPA dendrons to enhance the available hydroxyl groups at the surfaces from 1 to a maximum of 32 for the fifth generation. The assessment of any residual azides from the CFP-N3 was conducted 2120

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Biomacromolecules with an excess of dye 4 and resulted in no visual coloration (Figure 1C). To extend the functional complexity to a new level and to introduce benignly dual-functionality, we explored controlled hybridization reaction with an additional dendritic layer. Revisiting a previous strategy to bifunctional dendrimers,12 an anhydride-activated AB2C functional monomer 2 was constructed on a 40 g scale bearing a carboxylic group (A), one azide unit (C), and an acetonide-protected diol (B2) (Scheme 2). This type of monomer allows chemoselective orthogonal reactions in which A functionality reacts only with B during the dendritic growth, as in traditional bis-MPA dendrimers, whereas the introduced C click functionality remains dormant during the generation growth. In this context, CFP-[Gx]-(OH)2x (x = 15) substrates were hybridized with AB2C monomer 2 via solid-phase anhydride chemistry, followed by a deprotection step of the acetonide protective groups (Scheme 1A). Consequently, the final substrates were dendronized up to the sixth generation of polyester dendrons decorated with dual-functionality at the periphery and with a resulting increased multivalency, given that each available OH in the cellulosic starting material CFP-OH was transformed to display both 64 dendritic OH and 32 azide functionalities in CFP-[G6]-(OH)64(N3)32, Scheme 1. To clarify the performance of a CuAAC over these bifunctional surfaces, the acetylene functional dye 4 was covalently attached to the present azides on CFP-[G(xþ1)]-(OH)2(2)x(N3)32x (Scheme 1B). The resulting red surfaces, CFP-[G(xþ1)]-(OH)2(2)x(Red)2x, clearly indicated the success of the reaction (Figure 1D). To investigate the features of the dendronized surfaces, we conducted water static contact angle (CA) measurements (Table S2 of the Supporting Information) to monitor the changes in hydrophobicity and hydrophilicity. However, accurate CA values are somewhat difficult to determine because of the inherent roughness and the absorbing nature of the cellulose surfaces,32 especially for contact angles below 90°.33 Therefore, these data can only be taken as qualitative changes of the surface properties. The results revealed a hydrophobic surface (CFP-N3, CA 113°) after the modification of the cellulose filter paper, followed by a decrease in CA for the dendronized CFP-[Gx]-(OH)2x, resulting in the more hydrophilic surfaces when the higher generations are involved. After the reaction with the AB2C monomer 2, the CA increased because of the presence of acetonide groups, and after deprotection, CFP-[G(xþ1)]-(OH)2(2)x(N3)32x, CA drops again to reach a 66° for CFP-[G6]-(OH)64(N3)32. Additionally, FT-IR analysis showed the presence and amplification of the carbonyl stretch at 1730 cm1 with the addition of dendrons to the surfaces, which is not seen for unmodified cellulose (Figure 1E). XPS was further employed to verify the functionalization of the cellulose filter paper (Figure S1 of the Supporting Information). The C 1s spectrum of unmodified cellulose consists of a main peak with a binding energy of 285.5 eV attributed to CO bonds (Figure 2A), whereas for the CFP-N3 surfaces the intensity of this peak decreases because of a higher contribution of the overlapping peaks corresponding to OCdO and CC/CH, demonstrating the presence of esters on the surface. Both latter peaks increase for further modified surfaces CFP-[Gx]-(OH)2x and CFP-[G(xþ1)]-(OH)2(2)x(N3)2x. The highest content of carbonyl carbon is obtained for surfaces decorated with CFP-[Gx]-(OH)2x (12%), which is in agreement with the polyester content of the attached dendrons. Figure 2B shows the N 1s spectra of the successive surfaces. The appearance of the N 1s signal at 399 eV for CFP-N3 showed the introduction

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Figure 2. XPS spectra of the C 1s (A) and N 1s (B) regions and atomic concentration of N 1s and C 1s (C).

of azides groups in the surfaces. However, because the N in the azide exists in two different oxidation states, two XPS peaks are expected,34 and, as a consequence, a smaller peak should be seen at higher energy. This small peak is assigned to the relatively electron-poor middle N atom of the azide group, and its absence could be attributed to the poor stability of azide groups due to prolonged scanning.35 Indeed, the extensive exposure of the X radiation causes degradation of the azide functionality likely with the elimination of molecular nitrogen. XPS of CFP-[Gx]-(OH)2x surfaces shows the N peak slightly shifted to 398.5 eV, with a marked difference in its shape being the single broad peak characteristic of the triazol structure formed after the click reaction. With CFP-[G(xþ1)]-(OH)2(2)x(N3)32x surfaces, the presence of azides could be established by the appearance of a shoulder at 401 eV. Figure 2C shows the relative ratio of 2121

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Scheme 4. Bifunctionalization and Post-Functionalization of Cellulose-Coated Quartz Crystals Resulting in Mono- and Multivalent Surfacesa

a

Dotted arrows indicate that the reaction was performed outside the QCM chamber. Reagents: (a) 1, TEA, DMAP, THF; (b) Acetylene-[G4]-(OH)16, CuSO4, NaAsc., H2O; (c) 2, TEA, DMAP, THF; (d) Dowex, MeOH; (e) 6, CuSO4, NaAsc., H2O.

nitrogen/carbon, which was analyzed by the relative atomic concentration of C 1s and N 1s. From the results, we can conclude the presence of 4-(azidomethyl)benzoate moieties in CFP-N3 and the presence of AB2C monomers including azide in the dendritic surfaces CFP-[G(xþ1)]-(OH)2(2)x(N3)32x. To provide insight into the chemoselective and orthogonal nature of the bifunctional CFP-[G(xþ1)]-(OH)2(2)x(N3)32x surfaces, we chose two different acetylene ligands 5 and 6 (Scheme 1B) for model reactions. Orthogonal postfunctionalization of well-controlled bifunctional dendritic cellulose surfaces have never been reported and need to be exploited to tailor selectively physical and chemical properties of surfaces to obtain sophisticated functional materials. In one postfunctionalization strategy, acetylene-derived amoxicillin 5 ligand was chosen for its ability to introduce haptens at the surfaces, which potentially could act as a biosensor in allergy applications through specific IgE antibodies multivalent interactions. The acetylene functional amoxicillin 5 was reacted using CuSO4/NaAsc under aqueous conditions to yield CFP-[G(xþ1)]-(OH)2(2)x(AXO)2x. To remove the copper salt and excessive reagents, the washing procedure involved immersion of the supports in aqueous EDTA solution.36,37 The amoxicillin-functionalized surfaces were further exposed to reaction with the colorimetric reagent 4 and resulted in noncolored cellulose surfaces, confirming the absence of any remaining azides. As an additional characterization of these CFP-[G(xþ1)]-(OH)2(2)x(R1)32x surfaces, the attachment of 4 and 5 through a triazol was shown by the change of the XPS N 1s peak shape (Figure 2B) and a higher content of nitrogen (Figure 2C). The use of both click catalytic systems, organic and aqueous, demonstrates the versatility of this methodology, allowing the addition of either polar or apolar ligands. To display the tunable nature of these surfaces, triethylene glycol (TEG) 3 was selected as a ligand model to study the addition to the free alcohols. The TEG moieties were successfully

introduced to change in physical properties, and an apparent decrease in CA values was obtained, for example, 54° for CFP-[G6]-(TEG)64(N3)32 (Table S2 of the Supporting Information). Finally, both TEG 3 and amoxicillin 5 were combined to tune the hydrophilic nature of the surface and in parallel incorporate the drug. The introduced dual-functionality is a powerful tool for the design of sophisticated surfaces with promising application in drug allergy testing. To study their potential in biosensor application, we postfunctionalized AB2C-functionalized cellulose-coated quartz crystals with mannose ligand 6 and evaluated them in situ with QCM38 through a specific protein interaction. Lectin recognition on carbohydrates presenting surfaces has been previously evaluated through QCM;3941 however, the covalent attachment of dendritic structures or subsequent ligands has never been reported, at least to the knowledge of the authors. Our goal is not only to study how the use of dendrons can increase the density of ligands on the surface but also to evaluate the specific recognition and affinity for lectins through multivalent interactions on solid supports. Moreover, the tunability of the surface due to the orthogonal bifunctionality should influence the potential of a biosensor because the surface could be designed to avoid nonspecific binding of protein. Two kinds of bifunctional surfaces CQC-[G(xþ1)]-(OH)2(2)x(N3)32x were prepared involving either only AB2C monomers (x = 0) or multivalent dendritic structures functionalized with AB2C monomers (x = 4). Both methodologies are outlined in Scheme 4. The cellulose-coated quartz crystal CQC-OH was functionalized outside the chamber with azide groups by reaction with 1. This was followed by a click reaction with the fourth generation dendron performed in the QCM using a flow of aqueous solution containing the acetylene dendron and CuI. The covalent binding was monitored by measuring the decrease in frequency (Figure 3A) corresponding to an increase in sensed mass on the surface (eq 1). 2122

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Figure 4. Complete experiment of QCM response to protein interaction: Con A 0.01 μM  CQC-[G5]-(OH)32(Man)16 with cleaning step. The protein interaction was left to proceed for at least 60 min in all cases. After this, the surface was rinsed with buffer solution, followed by mannose solution, and finally buffer solution again to regenerate the surface.

Figure 3. QCM curves monitoring surface reactions. (A) Step (b) in Scheme 4: Dendron click reaction to obtain CQC-[G4]-(OH)16. (B) Step (e) in Scheme 4 on the monovalent surface: Mannose reaction to obtain CQC-[G1]-(OH)2Man. (C) Step (e) of Scheme 4 on the multivalent surface: Mannose reaction to obtain CQC-[G5]-(OH)32(Man)16.

The frequency drops immediately upon injection of reactive solution, showing a typical high initial reaction rate, which is decreasing as the reaction sites are consumed until equilibration is reached. The flow of reaction solution was maintained until stabilization of the frequency was obtained, indicating that there is no functional group left on the surface available to react and consequently ensuring a complete functionalization of the surface. The following rinsing step with water increased the frequency from 60 to 50, removing the noncovalently attached dendrons from the surface. Both hydroxylfunctionalized surfaces, CQC-OH and CQC-[G4]-(OH)16, were modified outside the chamber by reaction with AB2C anhydride 2 and further deprotection of acetonide (steps c and d, Scheme 3) to yield surfaces presenting dual-functionality, both hydroxyl and azide groups. The final functionalization of the surfaces with mannose acetylene 6 via the CuAAC reaction was performed in the QCM. The results show a frequency decrease in both surfaces after injection of the reagents (Figure 3B,C). A continuous flow of fresh

reaction solution was introduced until complete functionalization of the surface was obtained, followed by a washing procedure with water to exclude noncovalently attached reagents on the surface. Frequency values decreased around 16 and 120 Hz for CQC-[G1](OH)2(N3) and CQC-[G5]-(OH)32(N3)16, respectively, meaning that CQC-[G5]-(OH)32(Man)16 surfaces contain approximately 7.5 times more mannose ligands than CQC-[G1]-(OH)2(Man) surfaces, assuming that the associated water content in the attached layers is equal. The biorecognition properties of the surfaces were evaluated by the adsorption of Con A onto the surfaces. A representation of a single experiment is shown for the multivalent surface CQC[G5]-(OH)32(Man)16 (Figure 4). When a solution of Con A in Hepes buffer is injected, the frequency drops immediately upon injection as a consequence of the molecular recognition process, and the adsorption proceeds for ∼60 min. The subsequent rinsing with buffer solution increased the frequency slightly, removing loosely bound protein from the surface. However, after rinsing with a highly concentrated mannose solution in buffer, followed by a rinsing with pure buffer solution, the frequency increased to a level close to that of the baseline before the protein adsorption, indicating an almost complete desorption of protein from the surface and thereby regeneration of it. In more detail, the curve obtained between mannose addition and buffer addition is a result of both bulk effect and surface effect. Upon the addition of mannose in buffer, there is a sharp decrease in frequency of the QCM. The sharp drop is a bulk effect that comes immediately after addition due to a relatively high amount of mannose in the solution which increases the viscosity of the solution. This is measured by the instrument as an increase in mass on the surface although it is not. The counter effect, that is, the subsequent increase in frequency (or decrease in mass), comes from the desorption of protein from the surface. Then, when the surface is rinsed with buffer solution in the next step, the bulk viscosity goes back to the same value as that in the beginning of the measurement, showing the real effect on the surface; that is, the proteins are desorbed. Desorption of protein from the surface when the mannose solution is introduced indicated specific interaction between Con A and mannose. The system was found to be reversible through several onoff cycles. This straightforward process enables elimination of signal artifacts due to nonspecific binding to 2123

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Figure 5. QCM response during Con A adsorption using different concentrations onto different mannose functionalized surfaces: (A) CQC-[G1]-(OH)2(Man) and (B) CQC-[G5]-(OH)32(Man)16.

control substrates. In this case, protein exposure to control nonmannosylated surfaces (CQC-[G(xþ1)]-(OH)2(2)x(N3)32x) elucidated no binding. Therefore, the results indicate that the binding is specific between the mannose-binding receptors on the lectin protein and the mannose presented on the surface, whereas the OH dendritic functionalization provides a suitable background to avoid unspecific interactions with the surface. In case of showing nonspecific binding, an easy modification of the surface by inserting TEG moieties to OH groups could improve the sensitivity because the addition of PEG chains to peripheral alcohols in dendronized surfaces has already been reported to reduce protein adsorption.42 Because QCM is sensitive not only to the mass of protein but also to the mass of water associated with it, adsorbed amounts of protein can be directly compared only if the water content in the adsorbed layers is assumed to be constant in the different measurements. Figure 5 displays frequency-response graphs recorded for Con A-binding at different concentrations of the protein on both monovalent and multivalent mannose surfaces CQC-[G(xþ1)]-(OH)2(2)x(Man)2x, x = 0 and x = 4, respectively. Δf is associated with the protein binding increase with increased mannose density on the surface. Interestingly, one order of magnitude increased in concentration of protein was required for monomannosylated surface to reach the same Δf response obtained for the dendronized surface. Furthermore, the detection limit was found at 0.05 μM for the monomeric surface, showing a very small but significant adsorption, whereas the lower detection was improved for the multivalent mannose surface, found at 5 nM, meaning the dendronized surface to be sensitive to 10-fold diluted protein solution compared to the monomeric one. Whereas the dendritic components do not represent a major part of the total volume of the cellulose filter paper, their involvement is more complex than investigated here. In fact, their presence can significantly alter the support properties, imparting entirely new features and functions on the hybrid

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composites. The introduction of dendritic scaffolds with unprecedented control over structure, size and chemical functionality, allows a high degree of surface manipulations. When addressed to biosensor applications, the well-defined dendritic structures generate surfaces with increased reproducibility and their subsequent determination of biomolecules. Additionally, dendronized surfaces displaying multiple copies of ligands, such as mannose or amoxicillin derivatives, enable multivalency with increased sensitivity and selectivity. Of special importance is the control of nonspecific protein adsorption, usually limiting the sensitivity of assays. Whereas a few papers report studies concerning the impact of density of bioligand on polymerized39 or dendronized6 surfaces, to our knowledge, there is no example of surfaces presenting more than one active functional group to both attach the desired ligand and tune the properties of the remaining surface to avoid nonspecific interactions. In the developed lectin biosensor surface, the mannosyl units were selectively detected over the hydroxyl functional surface background without unspecific interactions. Moreover, these multivalent hydroxyl functional dendronized surfaces showed an increased sensitivity compared with the monomannosyl alternative. Other molecules, as oligosaccharides, can probably also be used to post-modify the dendronized surfaces, and thus this approach allows for a great flexibility in developing sensing surfaces from a common starting platform.

’ CONCLUSIONS The described methodology delivers a new tool box for the design of sophisticated biosensors, with advantages such as low detection limit, versatility, and suppression of nonspecific interactions. The study combines defined dendrons, robust click chemistry, and dual-functionality to provide highly sophisticated cellulose surfaces with unprecedented tunability. Engineered dendronized surfaces delivered multiple representation of dualfunctionality, which exhibited tailored hydrophilicity and in conjugation of penicillin haptens. Furthermore, mannosylated dendronized substrates were fabricated, and their subsequent specific lectin binding through QCM revealed a 10-fold stronger multivalency to Con A compared with the monomeric one. ’ ASSOCIATED CONTENT

bS

Supporting Information. Survey XPS spectra of prepared surfaces and complete experiments of protein interaction with QCM curves are shown. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT M.I.M. acknowledges the ISCIII (IS Carlos III) grant. M.M. and Y.H. acknowledge Swedish Research Council Grants 20063617 and 2009-3259. S.U. also acknowledges BiMaC Innovation at KTH Royal Institute of Technology for financial support. ’ REFERENCES (1) Vigo, T. L. Polym. Adv. Technol. 1998, 9, 539–548. 2124

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