Peter Berlin

Film-forming aminocellulose derivatives as enzyme-compatible support matrices for biosensor developments

Based on 6(2)-O-tosyl celluloses and 6(2)-O-tosylcellulose derivatives, it has been possible to synthesize a novel soluble aminocellulose type, P-CH2-NH-(X)-NH2 (P=cellulose, (X)=alkylene, aryl, aralkylene or oligoamine) with diamine or oligoamine residues at C6 and solubilizing groups (S) such as acetate, benzoate, carbanilate, methoxy and/or tosylate groups at C2/C3 of the anhydroglucose unit (AGU). Depending on the nature and degree of substitution (DS) of (S), the aminocelluloses are soluble either in DMA and DMSO or in water. They all form transparent films from their solutions. In the case of water-soluble aminocelluloses, for example, an enzyme-specific pH value can be adjusted by protonation of the NH2 end groups at C6. The aminocelluloses apparently form aggregates (on a scale of nanostructures) according to a structure-inherent organization principle. The nanostructures could be imaged on the aminocellulose film surface by atomic force microscopy (AFM) in the form of characteristic topographic structures – as a result of the aggregation of the aminocellulose derivative chains and their interaction with the functionalized film support. In this way, structural and environment-induced factors influencing the nanostructure formation were found. The aminocellulose films can be covalently coupled with biomolecules by bifunctional reaction via NH2-reactive compounds. With the aid of analytically relevant enzymes, e.g. glucose oxidase (GOD), horseradish peroxidase (HRP) and others, it was found that the enzyme parameters can be modified by the interplay of the aminocellulose and coupling structures. A number of new bifunctional enzyme coupling reactions, e.g. via L-ascorbic acid or benzenedisulfonyl chlorides, forming amide or sulfonamide coupling structures led to efficient enzyme activities and long-term stabilities in the case of GOD and HRP coupling to PDA cellulosetosylate films.

A novel soluble aminocellulose derivative type : its transparent film-forming properties and its efficient coupling with enzyme proteins for biosensors

By means of the reaction of 6-tosylcellulose derivatives with diamino compounds, it has been possible to provide a new soluble and film-forming aminocellulose-derivative type with a diamine residue at C-6 of the anhydroglucose unit (AGU) and with so-called solubilizing groups, such as acetate, benzoate, carbanilate, methoxy and/or tosylate groups at C-2/C-3. For example, aminocellulose derivatives were synthesized with aliphatic diamine residues of different alkyl chain lengths ((Ch 2 ) m with m = 2, 4, 6, 8, 12) at C-6. Other new aminocellulose derivatives were those with an aromatic diamine residue, e.g., 1,4-phenylenediamine or benzidine residue and others, at C-6. Depending on the structure of the diamine residue at C-6 and of the substituents at C-2/C-3, the aminocellulose derivatives were soluble in various solvents, mostly in N,N-dimethylacetamide (DMA) or dimethylsulfoxide (DMSO). Aminocelluloses with a diaminoethane or diaminobutane residue at C-6 and methoxy groups at C-2/C-3 were soblue in water. All the amino celluloses synthesized formed transparent films from their solutions. These aminocelluloses apparently form superstructures or so-called supramolecular architectures according to a structure-inherent organization principle, which became visible, for example, in the case of PDA cellulose tosylates (in DMA) as gel-like aggregates after approx. 1 week of storage at 4 °C. The superstructures or aggregates could be imaged on the aminocellulose film surface by atomic force microscopy (AFM) in the form of characteristic topographic structures, e.g. as hole structures. In this way, structural and environment-induced factors influencing the aggregate formation were found. The transparent aminocellulose films were excellently suited for covalent coupling with oxidoreductase enzymes such as glucose oxidase (GOD), lactate oxidase (LOD), peroxidase (POD) via bifunctional compounds. A number of new bifunctional enzyme coupling reactions, e.g. via L-ascorbic acid or benzenedisulfonic dichlorides forming amide or sulfonamide coupling structures led to functionally stable nanostructure building blocks with recognition patterns in the case of GOD and POD coupling to PDA cellulose tosylate films. The PDA cellulose derivatives proved to be promising cellulose structural units because the redox-chromogenic PDA residue at C-6 provides the derivatives with a wide range of reaction possibilities, e.g. diazo coupling reactions, NH 2 - reactive coupling reactions and oxidative coupling reactions to redox dyes.

A novel efficient enzyme‐immobilization reaction on NH2 polymers by means of L‐ascorbic acid

A new enzyme‐immobilization reaction by means of L‐ascorbic acid (ASA) is described using NH2 polymers based on cellulose or poly(vinyl alcohol) with the example of oxidoreductase enzymes. In this way, enzyme proteins such as glucose oxidase (GOD), glutamate oxidase, lactate oxidase, urate oxidase and peroxidase can be covalently fixed with a high surface loading to ultrathin and transparent NH2‐polymer films if their surfaces are previously treated with an ASA solution, in, for example, N,N‐dimethyl acetamide, DMSO or methanol. ASA then obviously reacts like a diketo compound with amino groups of the NH2‐polymer film and enzyme protein, forming dehydroascorbic acid derivatives with neighbouring Schiff’s‐base structures. In a subsequent fragmentation reaction, the latter presumably form stable oxalic acid diamide derivatives as coupling structures between enzyme protein and NH2‐polymer film, as suggested by results from investigations of the ASA reaction with n‐butylamine. The immobilized enzymes can be stored at 4 °C in bidistilled water for at least 1 month without becoming detached from the NH2‐polymer film and without diminished enzyme activity. The apparent Km values of the immobilized enzymes are in part clearly smaller than those of the dissolved enzymes or those found in other immobilization processes such as the diazo coupling or the bifunctional glutardialdehyde reaction. For example, the Km value of the immobilized GOD with different NH2 polymers as the matrix structure is smaller by a factor of approx. 20 than that of the dissolved enzyme.

Soluble and film-forming cellulose derivatives with redox- chromogenic and enzyme immobilizing 1,4-phenylenediamine groups

Cellulose architectures with redox-chromogenic properties and anchor groups for the immobilization of biomolecules have been prepared by reaction of p-toluenesulfonic acid esters of cellulose (tosylcellulose, DS 2.3) with 1,4-phenylenediamine (PDA) in dimethyl sulfoxide (DMSO) solution at 100°C. The degree of substitution (DS) of the PDA groups and remaining p-toluenesulfonate (tosylate) groups, and thus the properties of the 6-deoxy-6-(4-aminophenyl)amino-2(3)-O-tosylcellulose (PDA cellulose) formed, can be adjusted by varying the PDA molar equivalents and the reaction time. Most of PDA celluloses are soluble in DMSO and N,N-dimethylacetamide (DMA) and form transparent films. The redox-chromogenic properties of PDA cellulose have been demonstrated by oxidation of the PDA groups with H 2 O 2 to the corresponding diimine (λ max = 480 nm) and by oxidative coupling with phenol to the corresponding indophenol (λ max = 595 nm). The suitability of the PDA units as reactive anchor groups was shown by the immobilization of enzymes like glucose oxidase, peroxidase, and lactate oxidase. The highest enzyme activity achieved for peroxidase immobilized with glutardialdehyde on a PDA cellulose film was 0.165 U/cm 2 .

Novel matrices for biosensor applications by structural design of redox-chromogenic aminocellulose esters

The solubility and film forming properties of newly developed cellulose derivatives with 1,4-phenylenediamine (PDA) substituents at position C6 of the anhydroglucose unit (AGU) can be achieved by formation of acetate, benzoate, and carbanilate groups, respectively, preferably in position C2/C3. All derivatives, if soluble in N,N-dimethylacetamide (DMA) or dimethylsulfoxide (DMSO), form transparent films from their solutions. The solubility of these PDA cellulose esters in DMSO or DMA, such as the aging, enzyme immobilization, and redox-chromogenic behaviors of the films, depending on the nature and the degree of substitution of the functional groups were investigated. Regarding the influence of the autoxidation by air-oxygen of the PDA groups on the storage stability, the longest stable derivatives were the PDA cellulose carbanilates. Onto PDA cellulose derivate films glucose oxidase was immobilized via diazo coupling, and by glutardialdehyde and ascorbic acid reactions with an obtained enzyme activity of 45 (diazo coupling, PDA cellulose acetate) to 145 mU/cm2 (ascorbic acid, PDA cellulose carbanilate). The redox-chromogenic properties were demonstrated by oxidative coupling reaction of typical reagents like phenol and chinoline derivatives onto the PDA groups of the cellulose esters in presence of H2O2 and peroxidase. The best coloring results were obtained by using PDA cellulose carbanilates und benzoates.

Designed aliphatic aminocellulose derivatives as transparent and functionalized coatings for enzyme immobilization

The introduction of aliphatic diamine residues into cellulose was realized starting from 6-O-tosylcelluloses containing carbanilate, benzoate, and methoxy groups at the C2/C3 position of the anhydroglucose units (AGU). By nucleophilic substitution reactions of the tosylate groups with alkylene diamines [ADA, H2N-(CH2) n -NH2, n = 2, 4, 6, 8, 12] and monofunctionalization of the ADA it was possible to obtain soluble and film-forming 6-deoxy-6-(ω-aminoalkyl)aminocellulose esters and ethers 1-14. 1,2-Ethylene and 1,4-butylene diamine (EDA and BDA) cellulose carbanilates (1, 2) with a degree of substitution (DS) of the diamino residues of less than 0.4 are soluble in N,N-dimethylacetamide (DMA) and dimethylsulfoxide (DMSO), even 6 months after preparation. The 2,3-di-O-methyl-6-ADA-celluloses are soluble in many different solvents, including water, ethanol, and DMSO. All derivatives form transparent films from their solutions. These films are very smooth and exhibit a roughness of less than 0.5 nm, determined using atomic force microscopy (AFM). Owing to the introduced primary amino groups, the films are well suited for enzyme immobilization. For instance, glucose oxidase (GOD) could be immobilized onto a film of BDA cellulose carbanilate (DSBDA = 0.49, DScarbanilate = 1.50, DStosylate = 0.20) with an activity of 0.2 U/cm2 using benzoquinone as the immobilization reagent.

Novel xylylene diaminocellulose derivatives for enzyme immobilization

In the series of soluble and film-forming aminocelluloses for application as enzyme-compatible support matrices, novel derivatives with xylylene diamine residues selectively at C6 and solubilizing carbanilate groups were synthesized. Synthesis of these aminocelluloses was successful in three reaction steps starting from cellulose (Avicel PH 101) via tosylcellulose with degree of substitution of 0.88 (preferably at C6) and tosylcellulose carbanilate. Using a 10-fold excess of xylylene diamine, only one amino group of the diamine reacts during the nucleophilic substitution of the tosylate groups. In the last step, the influence of the structure of the starting xylylene diamine isomers and of the reaction conditions on the degree of substitution of the tosylate, carbanilate and xylylene diamino groups in the final polymer, on the occurrence of side reactions and on the polymer solubility was studied. All xylylene diamino tosylcellulose carbanilates are soluble in DMA, DMSO and DMF in never dried state and form transparent films on glass surfaces. These films are promising support matrices for enzyme immobilization because of their characteristic topography, as is shown by atomic force microscopy examinations of the film surfaces.