Biofunctional Surfaces Based on Dendronized Cellulose

Jan 8, 2009 - E-mail: [email protected]. Fax: +49 (0) 3641 9 48202., †. Friedrich Schiller University of Jena. , ‡. Thuringian Institute o...
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Biomacromolecules 2009, 10, 382–389

Biofunctional Surfaces Based on Dendronized Cellulose Matthias Pohl,†,‡ Nico Michaelis,† Frank Meister,‡ and Thomas Heinze*,†,‡ Center of Excellence for Polysaccharide Research, Friedrich Schiller University of Jena, Humboldtstraβe 10, D-07743 Jena, Germany, and Thuringian Institute of Textile and Plastics Research, Breitscheidstraβe 97, D-07407 Rudolstadt, Germany Received October 10, 2008; Revised Manuscript Received December 4, 2008

Biofunctionalized surfaces based on dendronized cellulose were prepared either by embedding 6-deoxy-6-(1,2,3triazolo)-4-polyamidoamine (PAMAM) cellulose (degree of substitution, DS 0.25), obtained by homogeneous conversion of 6-deoxy-6-azido cellulose with propargyl-PAMAM dendron via the copper-catalyzed Huisgen reaction, in a cellulose acetate (DS 2.50) matrix or by the heterogeneous functionalization of deoxy-azido cellulose film with the dendron. The amount of amino groups provided by the solid supports was determined and the covalent attachment of enzyme was proven with glucose oxidase as model enzyme after activation with glutardialdehyde. The quality of glucose oxidase immobilization was defined by determining of the specific enzyme activity, coupling efficiency, storage stability, and reproducibility. Although the heterogeneous functionalization of the deoxy-azido film yields a product that binds more enzyme compared to the blend of dendronized cellulose derivative with cellulose acetate, the coupling efficiency is comparatively small. Nevertheless, the different approaches for the preparation of biofunctionalized surfaces based on dendronized cellulose provide an excellent reproducibility and good storage stability.

Introduction The modification of cellulose with dendritic structures is a novel and interesting path to synthesize highly functionalized and unconventional polysaccharide-based products that may be of interest for various applications, for example, as drug delivery systems, catalysts, disinfectants, and as components in cosmetics. Due to the excellent biocompatibility, hydrophilicity, and the nontoxicity, cellulose is also a useful support for the immobilization of enzymes.1 Different types of unmodified cellulose shapes (e.g., fibers and membranes) and various cellulose derivatives were used as support to immobilize enzymes or cells, attached by physical adsorption or ionic interactions.2-6 In this regard, polyelectrolyte complex (symplex) capsules formed from oppositely charged polyelectrolytes, for example, cellulose sulfate and poly(dimethyldiallylammonium) chloride were successfully used to entrap sensitive biological materials including enzymes.7,8 Nevertheless, the introduction of reactive groups into cellulose that may allow a covalent, nonreversible attachment of biomolecules leads to more stable systems.9-12 Quite recently, polyamidoamine (PAMAM) dendrimer-modified cellulose film surfaces were prepared either as a composite material or by covalent binding using 1,3-phenylene diisocyanate as a linker. The ability for enzyme immobilization using laccase as a model enzyme was demonstrated.13 However, there are some problems arising due to the preparation of a composite of a dendrimer embedded in cellulose and the use of bifunctional reagents as linker. Due to noncovalent binding, the dendrimer may be washed out or may diffuse out of the composite during precipitation, washing, and immobilization procedures. Furthermore, the use of bifunctional reagents (linker) may cause side reactions, for example, uncontrolled cross-linking (cellulose* To whom correspondence should be addressed. E-mail: thomas.heinze@ uni-jena.de. Fax: +49 (0) 3641 9 48202. † Friedrich Schiller University of Jena. ‡ Thuringian Institute of Textile and Plastics Research.

cellulose, dendrimer-dendrimer) or multiple bonding between the support matrix and the dendrimer and, hence, a decrease of functional groups for biofunctionalization. To diminish these drawbacks, the immobilization due to covalent linkage (without the use of a linker) of dendrons on the cellulose backbone seems to be a useful approach. In addition, a heterogeneous approach for the covalent binding of dendritic compounds affords a valuable opportunity to gain a high surface functionalization with regard to biofunctionalization. Various attempts are made to synthesize cellulose derivatives that combine the valuable properties of both the biopolymer and the dendron. Recently, Newkome et al. published the synthesis of dendronized cellulose for the first time.14,15 Amino-triester based dendrons with an isocyanate focal moiety were allowed to react with cellulose under homogeneous conditions applying N,N-dimethyl acetamide (DMA)/LiCl as solvent. Furthermore, the preparation of cellulose derivatives decorated with dendrons of different functions in the periphery and with different degrees of substitution could be realized.16-19 In our studies about dendronization of cellulose, the synthetic approach of in situ activation of carboxylic acid moieties of aryl-polyester dendrons with N,N-carbonyldiimidazole was successfully carried out.20,21 In addition, dendritic structures can be introduced by either homogeneous and heterogeneous conversion of 6-deoxy-6-azido cellulose with propargylPAMAM dendrons from first to third generation applying the copper-catalyzed Huisgen reaction in DMSO or ionic liquids as solvents, yielding pure and highly functionalized products modified at the primary position of the cellulose macromolecule only.22,23 It is known that the reaction of p-toluenesulfonic acid ester of cellulose (tosyl cellulose) with diamines may yield soluble 6-deoxy-6-amino cellulose derivatives like 6-deoxy-6-(4-aminophenyl)amino cellulose capable of forming films on various surfaces.24 The amino groups are reactive regarding covalent coupling with oxidoreductase enzymes such as glucose oxidase, lactate oxidase, and peroxidase after activation with bifunctional

10.1021/bm801149u CCC: $40.75  2009 American Chemical Society Published on Web 01/08/2009

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compounds. However, to get a soluble amino cellulose, crosslinking must be avoided by applying a high molar excess of diamine (up to 25 mol per mol repeating unit). Thus, propargylPAMAM dendrons with terminal amino-groups introduced by the copper-catalyzed Huisgen reaction into the azide-modified cellulose backbone could be an interesting alternative approach for biofunctionalization of cellulose surfaces. Moreover, the 1,2,3-triazole linker formed, the high number of terminal aminogroups providing many reactive sites, and the spacer between the biopolymer matrix and the amino moieties are particular advantages of the functional polymers. In the present paper, we report two different approaches to prepare biofunctionalized cellulose surfaces based on the reaction of 6-deoxy-6-azido cellulose with propargyl-PAMAM dendron containing terminal amino groups (2.5th generation) applying the copper-catalyzed Huisgen reaction. The solid supports were characterized regarding the amount of accessible amino groups provided on the surfaces of the supports. The efficiency of the films was tested after covalent enzyme immobilization using glucose oxidase (GOD) from Aspergillus niger as a model enzyme. Furthermore, coupling efficiency and storage stability were determined in order to get more information of the quality of the glucose oxidase functionalized films.

Experimental Section Materials. Cellulose (Modo 500, Modo Paper Domsjo¨, Sweden, degree of polymerization, DP 521) was dried under vacuum at 110 °C for 3 h before use. Cellulose diacetate was supplied by Rhodia Acetow GmbH Freiburg, Germany, and used as received. p-Toluenesulfonyl chloride was purchased from Merck. N,N-Dimethyl formamide (DMF) and N,N-dimethyl acetamide (DMA) were obtained from Riedel de Hae¨n and sodium azide from Alfa Aesar. Dimethyl sulfoxide (DMSO), CuSO4 · 5H2O, sodium ascorbate, methanol, tetrabutylammonium fluoride trihydrate (TBAF), and glutardialdehyde were Acros products. Glucose oxidase from Aspergillus niger (EC 1.1.3.4), peroxidase from horseradish (EC 1.11.1.7), and 2,2′-azino-bis(3-ethylbenzthiazoline-6sulfonic acid) (ABTS) were purchased from Sigma Aldrich and acid orange 7 from Fluka. All reagents were used without further purification. The p-toluenesulfonic acid esters of cellulose (tosyl cellulose, degree of substitution, DS 0.75 and 1.37) were synthesized homogenously in DMA/LiCl according to ref 26. The deoxy-azido cellulose derivatives (DS 0.75 and 1.32) were obtained by nucleophilic displacement reaction of tosyl cellulose with NaN3 as described in ref 27. The preparation of the propargyl-polyamidoamine (PAMAM) dendron was carried out as described in ref 28. Measurements. FTIR spectra were recorded on a Nicolet Avatar 370 spectrometer using the ATR and the KBr-technique. Elemental analyses (EA) were performed with a Vario EL III from Elementar Analysensysteme, Hanau, Germany. MAS CP/MAS 13C{1H}-NMR (13C: 100.58 MHz) spectra were recorded on a Bruker Avance 400 MHz spectrometer using a 4 mm MAS double resonance probe and ZrO2 rotors. The measurements were carried out at 11 kHz MAS at 343 K. For 13C{1H}-NMR, the cross polarization (CP) contact time was 1.5 ms, 4 k scans were accumulated, and the recycle delay was set to 2 s. Adamantane was used as an external reference.25 SEM measurements were carried out on a Leica S 440i with a tungstencathode. The quantitative determination of the amino groups and the specific enzyme activity measurements were recorded on a Lambda 10 UV/vis spectrometer from Perkin-Elmer. For determination of the immobilized protein amount a fluorescence spectrophotometer from Perkin-Elmer (LS 50B) was used. 6-Deoxy-6-(1,2,3-triazolo)-4-polyamidoamine Cellulose of 2.5th Generation. To a solution of 6-deoxy-6-azido cellulose (DS 0.75, 0.5 g, 2.78 mmol modified anhydroglucose unit, AGU) in 100 mL DMSO, CuSO4 · 5H2O (0.1 g, 0.4 mmol, in 5 mL of water), sodium ascorbate

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(0.2 g, 1.01 mmol, in 5 mL of water), and 2.5th generation of propargylPAMAM dendron (4.1 g, 5.56 mmol) were added. The mixture was stirred at 25 °C for 24 h. Subsequently, 5 g sodium diethyldithiocarbamate trihydrate was added for complete removal of the coppercatalyst, stirred for 1 h and precipitated in 600 mL of acetone. The polymer was collected by filtration. After washing three times with acetone (250 mL) and drying in vacuum, 2.5th generation of 6-deoxy6-(1,2,3-triazolo)-4-PAMAM cellulose was obtained (3). DS: 0.25 (calculated from N-content determined by EA). FTIR (KBr): ν (cm-1) ) 3384 (OH), 3268 (NH2), 2878-2964 (CH2 and CH), 2106 (N3), 1636 and 1540 (amide), 1026 (C-O). 13C{1H} CP/MAS NMR: δ (ppm) 174.7 (CdO), 144.41, 103.5-61.02 (C1 to C6OH, AGU), 58.0-57.8 (C6triazole, C6azide, AGU), 51.5-29.3 (CH2, dendron). Preparation of Films. Films of Dendronized Cellulose Embedded in Cellulose Acetate Matrix (DCCA). 6-Deoxy-6-(1,2,3triazolo)-4-PAMAM cellulose of 2.5th generation (0.08 g, DS 0.25) was dissolved in DMSO/TBAF · 3H2O according to a described procedure.29 After complete dissolution using sufficient amount of TBAF · 3H2O (3 g), cellulose acetate (2 g, DS 2.50) was added yielding a mixture with 4 wt % of dendronized cellulose derivative on a solvent free basis. The homogenized solution (20.8 wt %) was cast as film on a glass plate using a scraper adjusted to 0.2 mm thickness. The film was precipitated and washed with ethanol and subsequently dried at ambient temperature. Propargyl-PAMAM Dendron-Modified Deoxy-azido Cellulose (DMAC) Films. Deoxy-azido cellulose (2.7 g, DS 1.32) dissolved in N,N-dimethyl acetamide (27 wt %) was cast on a glass plate with a scraper adjusted to 0.2 mm thickness. After precipitation in ethanol, the deoxy-azido cellulose film was washed thoroughly with ethanol and allowed to dry at ambient temperature. The modification with propargyl-PAMAM dendron of 2.5th generation was carried out heterogeneously by the conversion of the deoxy-azido cellulose film in a 5 wt % methanolic propargyl-PAMAM (2.5th generation) solution containing 0.1 g CuSO4 · 5H2O and 0.2 g of sodium ascorbate for 3 h at 25 °C according to ref 19. Subsequently, the film was washed with methanol and with aqueous solution of sodium diethyldithiocarbamate trihydrate (10 wt %) for 1 h to remove copper and was finally washed with acetone. Determination of NH2 Groups Content. Films with an area of 0.25 cm2 (dimension of the original films were ca. 150 cm2) were used for the determination of the amount of amino groups using the acid orange 7 method.30 The films were immersed in 5 mL of a 0.01 g/mL acid orange 7 solution for 14 h at room temperature and subsequently washed thoroughly with bidistilled water followed by 1 mM aqueous HCl solution until no dye-molecules could be detected by UV/vis measurements in the washing solution. The dye ion complexes formed were desorbed by treating the film with 1 M aqueous NaOH and the optical density was measured at the resulting supernatant adjusted to pH 3 with a 1 N aqueous HCl at λ ) 485 nm. The concentration of amino groups was calculated according to the adsorbed acid orange 7 [nmol/ cm2 ) 44.4 nmol mL-1 × Abs485 nm × V (mL)/A (cm2)]. Enzyme Attachment to DCCA and DMAC Films. Prior to the enzyme immobilization procedure, the DCCA and DMAC films were activated with glutardialdehyde.31-34 The films were treated with 4 mL of a 25 wt % aqueous solution of glutardialdehyde for 15 min, subsequently washed with 5 mL bidistilled water (6 times), and used for enzyme immobilization. For the enzyme immobilization, the activated polymer films were immersed with 2 mL (DCCA) or 3 mL (DMAC) of an aqueous glucose oxidase (GOD) solution for 24 h at 4 °C. Subsequently, the films were washed with bidistilled water until no enzyme could be detected in the washing solution by enzyme activity measurements. Determination of Immobilized GOD-Enzyme Activity. The specific activity of GOD immobilized on the films [area: 0.25 cm2] was determined by placing the films in a cuvette containing a solution of the 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) test system; 0.9 mL ABTS per mL of phosphate-buffer solution pH 6 and O2-saturated, 0.16 M glucose, and 2-3 U/mL of peroxidase from

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Scheme 1. Syntheses of 6-Deoxy-6-(1,2,3-triazolo)-4-polyamidoamine (PAMAM) Cellulose of 2.5th Generation Obtained by Copper-Catalyzed Huisgen Reaction (Idealized Structure)

horseradish (POD). The activity measurements (increasing in absorbance at λ ) 405 nm, absorption coefficient  ) 36.8 cm2/µmol) were carried out at room temperature, stirring the test solution in the cuvette. The enzyme activity is specified in international units (U), 1 U ) conversion of 1 µmol substrate per 1 min. Determination of the GOD Protein Value. For the determination of the protein value (quantity immobilized enzyme) of the GODfunctionalized film samples were shaken for 15 min with a K2HPO4/ H2SO4 buffer solution (pH 1.5) to extract the flavin-adenine-dinucleotide (FAD) coenzyme from the film sample according to procedure described in ref 35. Subsequently, the FAD coenzyme content in the buffer solution was measured by fluorescence spectrometry from the light emission intensity at λmax ) 520 nm after fluorescence excitation at λmax ) 460 nm. The protein value resulted from the linear relation protein Value against fluorescence intensity at λmax ) 520 nm, which was determined with the aid of a GOD concentration series (1-15 µg/ mL GOD) in the above buffer solution and the excitation/emission conditions.

Results and Discussion Polymers containing amino groups are efficient materials with regard to reliable biofunctionalization of solid supports. Quite recently, polyamidoamine (PAMAM) dendrimer-modified cellulose films were prepared either as a composite material or by covalent binding of the dendron via 1,3-phenylene diisocyanate as a linker. The ability for enzyme immobilization using laccase as a model enzyme was demonstrated.13 Moreover, it is known that 6-deoxy-6-(4-aminophenyl)amino cellulose forms films on various surfaces and the amino moieties can be used to immobilized biomolecules.24 The homogeneous or heterogeneous conversion of deoxy-azido cellulose with propargylpolyamidoamine (PAMAM) dendron with peripheric amino groups obtained by copper-catalyzed Huisgen reaction are of interest because the product may provide a high density of NH2 moieties on the support surfaces. 6-Deoxy-6-(1,2,3-triazolo)-4-polyamidoamine Cellulose. The 2.5th generation of 6-deoxy-6-(1,2,3-triazolo)-4-PAMAM cellulose was prepared by treating 6-deoxy-6-azido cellulose (1) dissolved in dimethyl sulfoxide (DMSO) with the amino group terminated propargyl-PAMAM dendron (2) in the presence of CuSO4 · 5H2O and sodium ascorbate for 24 h at ambient temperature (Scheme 1). Since the activity of an enzyme like glucose oxidase (GOD) will be influenced or even inhibited by metal ions, the amount of the copper based catalyst must be decreased to a value as low as possible. In addition, to avoid decomposition of the PAMAM-system due to Cu(II), the removal of the catalyst is

required.36,37 Decomposition of PAMAM dendrimers due to the presence of different copper species was shown to occur after several months upon standing.37 Thus, the removal of the copper catalyst after the reaction was completed should avoid fragmentation of the PAMAM dendron. Therefore, to remove the copper ions sodium diethyldithiocarbamate trihydrate, a very efficient complexing agent, was applied. Pure 2.5th generation of 6-deoxy-6-(1,2,3-triazolo)-4-PAMAM cellulose (3) with degree of substitution (DS) of 0.25 was obtained. The removal of the copper based catalyst was proven by ICP-OES analyses (Perkin-Elmer, Optima 2000 DV) possessing a detection limit concerning copper of 5 ppm. Only traces of remaining copper (7 ppm) were found. The amino dendron modified cellulose dissolves neither in common organic solvents, for example, DMSO, N,N-dimethyl acetamide (DMA), and tetrahydrofuran, nor in water in contrast to the second and third generation of PAMAM-triazolo cellulose.22 This cannot be explained with our present knowledge. To describe the molecular geometries and the supramolecular structures including the hydrogen bond system further studies have to be carried out. It may be assumed that in case of 2.5th generation of 6-deoxy-6-(1,2,3-triazolo)-4-PAMAM cellulose the formation of a very intensive hydrogen bond system between the NH and NH2 moieties of the dendron and the OH groups of the cellulose backbone may be formed. This is reasonable due to the fact that typical hydrogen bond breaking solvents, which even dissolve cellulose, like DMA/LiCl and DMSO in combination with tetrabutylammonium fluoride (TBAF) trihydrate dissolve the dendronized cellulose. In addition, to overcome insolubility and to get more information of structure-property relationships, the introduction of carboxymethyl groups into the 6-deoxy-6-(1,2,3-triazolo)-4-PAMAM celluloses was carried out yielding water soluble derivatives up to the third generation. Furthermore, well resolved NMR spectra were obtained showing no impurities, no decompositions, and no indication of the formation of a hydrogen bond system. These spectra will be published elsewhere.38 Nevertheless, the formation of a hydrogen bond system will contribute to a rather stable film, useful for biofunctionalization. Structure elucidation of the 2.5th generation of 6-deoxy-6(1,2,3-triazolo)-4-PAMAM cellulose derivative was carried out by FTIR- (not shown) and solid state NMR spectroscopy. In the FTIR spectrum, new characteristic bands appear at 3268 cm-1 (NH2), 1636, and 1540 cm-1 for the amide moieties due to the dendron introduced. The increased signal intensity at 2964 cm-1 is due to the increasing number of CH2 moieties resulting from the dendritic substituent.

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Figure 2. SEM pictures (enlargement 3000) of films obtained from (A) cellulose acetate (degree of substitution, DS 2.50) and (B) mixture of cellulose acetate (2 g, DS 2.50) and 6-deoxy-6-(1,2,3-triazolo)-4polyamidoamine cellulose of 2.5th generation (0.08 g, DS 0.25); solvent dimethyl sulfoxide/ tetrabutylammonium fluoride trihydrate, precipitation with ethanol.

Figure 1. CP/MAS 13C{1H}-NMR spectrum of 2.5th generation of 6-deoxy-6-(1,2,3-triazolo)-4-polyamidoamine cellulose (degree of substitution 0.25) [ns ) 4000, tw ) 2 s, tcp ) 1.5 ms, νR ) 11 kHz, recorded at 70 °C], AGU ) anhydroglucose unit (idealized structure).

The structure of 6-deoxy-6-(1,2,3-triazolo)-4-PAMAM cellulose of 2.5th generation was studied by CP/MAS 13C{1H}NMR spectroscopy at high temperature (70 °C). The experiment was carried out with a contact time of 1.5 ms, which has been proven to be the optimum value for the investigation of cellulose and cellulose derivatives. The CP/MAS 13C{1H}-NMR spectrum shows the typical resonances of the anhydroglucose unit (AGU), for carbonyl functions, peripheral CH2 moieties, and for the triazole linker. Surprisingly, the intensities of signals of the substituents are rather low compared to the AGU peaks. It might be assumed that the magnetization transfer was incomplete due to a high mobility of the PAMAM-triazolo cellulose substituents. To improve the spectral resolution of the substituents, the cross polarization contact time could be varied. These experimental variances were not part of these studies, but will be considered for further investigations. Furthermore, it is known that there are some structural problems of the PAMAM dendrimers due to standing, the use of copper and the synthesis itself.36,40,41 In this regard, similar PAMAM functionalized cellulose derivatives from first to third generation as well as carboxymethylated dendronized cellulose derivatives from first to third generation were investigated in previous studies by means of one- and twodimensional NMR spectroscopy techniques showing no structural impurities or side reactions arising from the use of a copper catalyst or the synthesis itself.22,38,39 Despite nonperfect recording conditions, the structural features of the 2.5th generation of 6-deoxy-6-(1,2,3-triazolo)-4PAMAM cellulose can be assigned in the CP/MAS 13C{1H}NMR spectrum (Figure 1). The characteristic signals of the carbon atoms of the modified AGU C1 to C6 are visible in between 105.0 and 64.4 ppm. The resonances for the carbon atoms of the carbonyl functions C12 and C17 of the amide bonds occur at 174.7 ppm. The peaks for the modified position 6 (C6Azide and C6Triazole) are found at 58.5 and 58.0 ppm. The signals of the peripheral CH2 moieties C18 and C19 could be observed at 41.1 and 40.9 ppm. Peaks resulting from further CH2 groups are visible in the range from 51.5 to 31.3 ppm. The carbon atom of C8 of the triazole linker gives a signal at 144.5 ppm. Preparation and Characterization of the Solid Supports. Based on dendritic functionalization of deoxy-azido cellulose with propargyl-PAMAM dendron, providing a high density of amino groups, two different approaches for the preparation of

Figure 3. SEM pictures (enlargement 3000) of films obtained (A) from deoxy-azido cellulose (degree of substitution, DS 1.32); solvent N,Ndimethyl acetamide, precipitation with ethanol and (B) after heterogeneous conversion of the deoxy-azido cellulose film with propargylpolyamidoamine dendron of 2.5th generation (5% solution in methanol) via the copper-catalyzed Huisgen reaction.

solid supports were studied. In a first approach, the 2.5th generation of 6-deoxy-6-(1,2,3-triazolo)-4-PAMAM cellulose (3) was embedded in a cellulose acetate matrix. Thus, 3 and cellulose acetate dissolved in DMSO/TBAF · 3H2O were homogenized and a film was cast on a glass plate, which was subsequently precipitated in ethanol. Figure 2 shows scanning electron micrographs of a cellulose acetate film (Figure 2A) compared to a blended film of 2.5th generation of 6-deoxy-6(1,2,3-triazolo)-4-PAMAM cellulose and cellulose acetate (Figure 2B). The cellulose acetate film possesses a curled structuring whereas inclusions of 6-deoxy-6-(1,2,3-triazolo)-4-PAMAM cellulose derivative of 2.5th generation forms a film with a flattened and porous surface. The formation of a film or a membrane is a rather complex process. Thus, the reasons leading to that flattened and porous film due to the presence of the dendronized cellulose derivative could not be explained by our present knowledge. In another approach, a heterogeneous reaction pathway was studied as previously carried out for dendronization of 6-deoxy6-azido cellulose powder.22 A film of deoxy-azido cellulose (DS 1.32) was immersed in a solution of propargyl-PAMAM dendron of 2.5th generation (5 wt%) in methanol ensuring direct surface modification. In order to avoid enzyme inhibition and thus, loss of specific activity by Cu2+ (remaining catalyst), the film was treated with sodium diethyldithiocarbamate trihydrate. Figure 3 shows the scanning electron micrographs of the deoxyazido cellulose film before (Figure 3A) and after the heterogeneous modification with propargyl-PAMAM dendron of 2.5th generation (Figure 3B). As can be seen, the heterogeneous process leads to a complete covering of the film surface with scale-like structures whereas the unmodified deoxy-azido cellulose film possesses a more flattened surface with slight elevations. Thus, it might be assumed that the heterogeneous functionalization of the film surface with propargyl-PAMAM dendron of 2.5th generation via Huisgen reaction will provide

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Pohl et al. Table 1. Number of Moles of Active Surface Amino Groupsa film

NH2 amount (nmol/cm2)

specific activity (mU/cm2)

cellulose acetate DCCA 1 DCCA 2 deoxy-azido cellulose DMAC 1 DMAC 2

1.32 68.54 71.34 1.48 212.12 206.60

3.41 125.20 135.16 0.28 28.73 25.95

a Determined by photometric determination using acid orange 7 method and specific enzyme activity of glucose oxidase after immobilization at films of cellulose acetate (degree of substitution, DS 2.50), 6-deoxy-6(1,2,3-triazolo)-4-polyamidoamine cellulose (DS 0.25) embedded in a cellulose acetate matrix (DCCA), deoxy-azido cellulose (DS 1.33), and heterogeneously functionalized deoxy-azido cellulose film with propargylpolyamidoamine dendron of 2.5th generation via copper-catalyzed Huisgen reaction (DMAC).

Figure 4. ATR/IR spectra of films of (A) deoxy-azido cellulose (degree of substitution 1.33) and (B) after surface modification with propargylPAMAM dendron of 2.5th generation via copper-catalyzed Huisgen reaction.

a higher amount of amino moieties due to roughness the surface (compared to DCCA film). The modification of the surface was clearly determined by ATR/IR spectroscopy implying a high degree of dendronization. Figure 4 shows the comparison of ATR/IR spectra of a deoxyazido cellulose film (DSazide 1.33) and after heterogeneous functionalization with propargyl-PAMAM dendron of 2.5th generation by copper-catalyzed Huisgen reaction. Figure 4A illustrates specific bands from cellulose at 1033 cm-1 (C-O-C) and at 3412 cm-1 (OH) and the very strong signal from the azide moieties at 2100 cm-1. As can be seen in Figure 4B, new bands appear at 3265 (NH2) and at 3083 cm-1 (NH) as well as the signals of the carbonyl function of the amide bond appear at 1631 and at 1542 cm-1 due to introduction of dendron. The strong decrease of the signal at 2107 cm-1 reveals a nearly complete conversion of the azide moieties with the propargylPAMAM dendron forming the 1,2,3-triazole linker in the heterogeneous process. It is well-known that ATR-IR spectroscopy exhibits a diminished resolution compared to KBrtechnique due to diffuse reflections and the problem of the extent of penetration of the IR beam. Thus, crude lines as well as peaksimilar resonances may occur in the spectrum. To get more detailed information of the purity and stability of the PAMAMdendronized cellulose as result of heterogeneous dendronization, FT-IR (KBr-technique) and NMR spectroscopy of similar first to third generation dendronized cellulose derivatives were applied showing no impurities, for example, remaining dendron or structural instability after similar workup procedures.22,39 Biofunctionalization and Characterization of the Support Matrices. Because the amino moiety is most commonly used for biofunctionalization, various colorimetric methods have been developed for its quantitative determination applying, for example, acid orange 7,30 4,4′-dimethoxytrityl chloride,42 fluorescamine,43 bromophenol blue,44 and others.45 Nevertheless, most of these methods include incubation with the dye dissolved in organic solvents like DMA or N,N-dimethyl formamide. Due to the solubility of the solid supports in polar aprotic media, incubation of dye from aqueous solution is required. Thus, acid orange 7 was chosen as dye for colorimetric characterization. The determination of the amino groups was carried out by the

formation of ion pairs of protonated amino groups of 2.5th generation of 6-deoxy-6-(1,2,3-triazolo)-4-PAMAM cellulose with acid orange 7. It is known that the protonation of the primary amino groups of PAMAM dendrimer already occurs at pH value in the range from 7 to 1013. In addition, tertiary amino groups of the dendrimer will also be positively charged due to protonation at pH between 3 and 7.46,47 Thus, a pH value of 7.5 was selected for determination of the amino groups at the surfaces of the solid supports. At this pH the sulfonate group of the dye acid orange 7 is negatively charged and interacts with positively charged amino moieties of dendritic structure ensuring determination of amino groups. After formation of amino dendron modified cellulose-acid orange 7 dye complexes, the amount of the amino groups presented at the surface was determined by UV/vis measurements at a wavelength of 485 nm. In addition, in order to get information about interaction between dye and unmodified solid supports, both pure films of cellulose acetate and deoxy-azido cellulose were studied by the colorimetric characterization method. The quantitative results of the studies with acid orange 7 are summarized in Table 1. As can be seen, the blank values of the unmodified solid supports are rather small (1.32 nmol/cm2 for cellulose acetate and 1.48 nmol/cm2 for deoxy-azido cellulose). The heterogeneously modified deoxy-azido cellulose film provides a high amount of amino groups of up to 212 nmol/cm2 (compare DCCA1 and DMAC1, Table 1). Furthermore, the blend of 2.5th generation of 6-deoxy-6-(1,2,3-triazolo)-4PAMAM cellulose embedded in a cellulose acetate matrix presents a comparatively small amount of amino groups (68.54 nmol/cm2 DCCA1 and 71.34 nmol/cm2 DCCA2). In addition, a second series of DCCA and DMAC films were prepared and characterized, illustrating the high reproducibility with regard to biofunctionalization (compare DCCA1 with DCCA2 and DMAC1 with DMAC2 in Table 1). Characterization of Immobilized Enzyme. For biofunctionalization, glucose oxidase (GOD) from Aspergillus niger was studied that is a typical model enzyme often used. GOD is a dimer glycoprotein with, at least 16% of carbohydrates and a typical molar mass of about 150.000 g/mol. It provides a high specificity against glucose forming D-gluconic acid-5-lactone and H2O2. Thus, the formation of H2O2 can be monitored by oxidation of 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) via horseradish peroxidase (POD) and specific GOD activity was calculated from the intensity of oxidized ABTS dye at a wavelength of λ ) 405 nm of the corresponding UV/ vis spectra. GOD coupling is carried out in two steps; the support film is activated with a bifunctional linker and second immobilization

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Figure 5. Schematic preparation of a blend of 6-deoxy-6-(1,2,3-triazolo)-4-polyamidoamine cellulose (degree of substitution, DS 0.25) and cellulose acetate (DS 2.50) and subsequent surface activation with glutardialdehyde for covalent immobilization of glucose oxidase (GOD; light gray bars ) cellulose acetate, black bars ) 6-deoxy-6-(1,2,3-triazolo)-4-PAMAM cellulose).

Figure 6. Heterogeneous functionalization of deoxy-azido cellulose film with propargyl-polyamidoamine dendron of 2.5th generation via copper-catalyzed Huisgen reaction and subsequent surface activation with glutardialdehyde for covalent immobilization of glucose oxidase (GOD).

of the enzyme in a usual procedure is carried out (Figures 5 and 6).31-34 Due to solubility of the solid supports in organic solvents, glutardialdehyde was used as bifunctional reagent that can be applied in aqueous media. To avoid cross-linking of the

terminal amino groups of the dendronized cellulose derivatives during activation, DCCA and DMAC films were immersed in high excess of the bifunctional reagent (3 mL of a 25 wt % aqueous glutardialdehyde solution per cm2 of film). Subsequently, the films were incubated with aqueous glucose oxidase solution for 24 h at 4 °C to covalently couple the enzyme. The specific enzyme activities of immobilized GOD at DCCA and DMAC films are summarized in Table 1. As expected, the unmodified solid supports show a very low enzyme activity of 3.41 (cellulose acetate) and 0.28 mU/cm2 for (deoxy-azido cellulose) only. The high enzyme activity of 135.16 and 125.20 mU/cm2 was obtained from immobilized GOD on DCCA solid supports. Despite the high number of amino groups present on the support surface of heterogeneously dendronized deoxy-azido cellulose film (DMAC) comparatively low enzyme activity (28.73 and 25.95 mU/cm2) was found. The reproducibility of the values from specific enzyme activity assay of both films is very good, 125.20 and 135.16 mU/cm2 for DCCA films and 28.73 and 25.95 mU/cm2 for DMAC films (Table 1). To get more information about the quality of the GOD functionalized films, the amount of immobilized protein and thus, the coupling efficiency (Arel, in %) was determined. Arel is defined as the quotient of the specific enzyme activities A (in mU/µg protein) of the immobilized and the dissolved enzyme (Arel ) Aimmobilized/Adissolved). Thus, Arel is the specific activities (activity per protein value) of the immobilized and the dissolved enzyme, that is, the higher the percentage value of Arel the less enzyme was inactivated during enzyme coupling. For our purposes, Arel was calculated by determination of protein amount immobilized on the DCCA and DMAC films applying the measurement of light emission in fluorescence excitation of the prosthetic group (flavin-adenin-dinucleotid, FAD) extracted from GOD. Table 2 shows the values of storage stability, immobilized protein amount, and coupling efficiency (Arel) of immobilized GOD on DCCA and DMAC films. As can be seen, the immobilization of GOD on DCCA solid support via glutardialdehyde activation proceeds with an efficiency of 27.2%, representing a better performance compared to results published by other groups.48 Although a high amount of amino groups is

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Table 2. Storage Stability, Protein Amount, and Coupling Efficiency (Arel) of Immobilized Glucose Oxidasea film

determined after

specific activity (mU/cm2)

protein amount (µg/cm2)

Arel (%)

DCCA 2 DCCA 2 DMAC 1 DMAC 1

15 min 2 months 15 min 2 months

135.16 71.41 28.73 17.24

4.34 3.12 12.89 8.16

27.2 27.5 2.2 2.5

a At films of a blend (DCCA) of 6-deoxy-6-(1,2,3-triazolo)-4-polyamidoamine cellulose (degree of substitution, DS 0.25) and cellulose acetate (DS 2.50) and heterogeneously functionalized deoxy-azido cellulose film with propargyl-polyamidoamine dendron of 2.5th generation via coppercatalyzed Huisgen reaction (DMAC).

present on the solid support of DMAC films and, consequently, a high amount of immobilized protein was realized, a coupling efficiency of only 2.2% could be achieved. The advantage of covalent enzyme immobilization in their active conformation frequently involves a high inactivation rate or the complete loss of activity. Thus, activity loss may be caused by groups involved in the coupling reaction, which are essential for the activity of the enzyme. Furthermore, enzyme coupling may lead to protein denaturation, or the enzyme loses its active conformation due to the fixation. Finally, the positioning of the active center of the enzyme in relation to the film surface plays an essential role in enabling unrestricted substrate access. Similar observations have already been reported by other researchers.49,50 In addition, the storage stability (stability of the immobilized enzyme under storage conditions) has been of interest. To evaluate long-term stability, the samples were stored in bidistilled water and their enzyme activity was measured after 60 days. Thus, GOD immobilized at DCCA solid support shows 53% of remaining activity after 2 months of storage. In case of covalently bound GOD on DMAC films 60% of specific enzyme activity remained (Table 2). It should be pointed out that longterm stability can be improved by the use of other linkers, for example, isophthaloyl chloride, 1,3-benzenesulfonyl chloride.4 However, due to the solubility of the solid supports in polar aprotic solvents (DMA, DMSO, etc.), an aqueous medium is required and, thus, the variety of bifunctional compounds is restricted to glutardialdehyde. Furthermore, the reasons leading to decreased enzyme activity after 2 months of storage may be of different nature and not only attributed due to enzyme attachment, for example, chemical inactivation (hydrolyses of enzyme).

Conclusions Novel solid supports were prepared either by the embedding of 6-deoxy-6-(1,2,3-triazolo)-4-polyamidoamine cellulose, obtained by homogeneous conversion of 6-deoxy-6-azido cellulose with propargyl-polyamidoamine dendron of 2.5th generation via the copper-catalyzed Huisgen reaction, into a cellulose acetate matrix or by means of a heterogeneous functionalization of a previously prepared deoxy-azido cellulose film with the dendron. The number of accessible amino groups determined by acid orange 7 method indicates that the heterogeneously functionalized cellulose solid support provides the higher amount of amino groups. The feasibility for enzyme immobilization on the dendronized cellulose films after activation with glutardialdehyde has been demonstrated using GOD as a model enzyme. The quality of enzyme coupling on the solid supports was determined by specific enzyme activity, reproducibility, coupling efficiency, and storage stability. Although the heterogeneous functionalized dendronized cellulose solid support provides the

higher amount of amino groups, the specific enzyme activity of immobilized GOD (28.73 mU/cm2) and the coupling efficiency (2.2%) were rather small compared to the blend of dendronized cellulose embedded in cellulose acetate (135.16 mU/cm2, 27.2%). Nevertheless, the heterogeneous approach of dendronization with propargyl-polyamidoamine dendron of 2.5th generation affords an interesting possibility for biofunctionalized surfaces and thus, protein attachment. Based on this concept, further studies regarding choice of enzyme, increasing of coupling efficiency, and storage stability have to be carried out. However, the biofunctionalization presented in this work provides a valuable method for various interesting applications that can be envisioned with these fascinating cellulose derivatives with different dendritic structures, for example, in field of biosensors, catalysts, and drug delivery systems. Acknowledgment. The authors thank Dr. Stephanie HesseErtelt (Center of Excellence for Polysaccharide Research, Friedrich Schiller University of Jena) for the measurement of the 13C{1H} CP/MAS NMR spectra. T.H. thanks the German Science Foundation (Project HE 2054/8-1) and the “Fonds der Chemischen Industrie” for financial support of these studies.

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