Development of New Polymeric Materials for the Immobilization of Biomolecules

Abstract This research work is part of a FIRB 2003 MIUR project aimed at the fabrication of novel biochips for the early detection of cancer. Several research groups are involved in this project in order to face and solve the different issues connected with this ambitious goal. In particular, the main objective of this thesis is the development of a suitable substrate for the production of biochips. This substrate has to be able to form chemical bonds with antibodies, in order to achieve a good quality in terms of reproducibility, amount and biological activity of the immobilized antibody. In order to reach this main goal, several problems had to be solved. The first issue entailed testing some materials as inert supports for the deposition of an active polymer layer. These materials had to be inexpensive, commercially available, easy to employ in industrial processes and suitable for the selected coating techniques. Some of the chosen materials were flexible, like aluminium foils, cellulose paper, polyethylene, poly(vinyl chloride) and poly(ethylene terephthalate) sheets, while some others were stiff like glass slides and poly(methyl methacrylate) plates. Diverse deposition methods were put through trial to obtain a uniform polymer layer on the support; in particular, solution casting, dip coating, spray coating and spin coating were tested to verify their performances and their compatibility with the supports. Spin coating turned out to be the best deposition method to cast a uniform layer on flat supports; it is also employed by the electronic industry, so this method should be easily scaled up at industrial level. For the coating of supports, several copolymers were synthesized, extensively characterized by NMR, IR, DSC and SEC analyses and eventually tested. All polymer syntheses were performed according to well–established procedures. The first class of tested materials numbered a series of copolymers of styrene and 4–chloromethyl styrene. These copolymers show a hydrophobicity similar to that of polystyrene (commonly employed in the production of ELISA microwell plates), but the chloromethyl groups were exploited for the reaction with hydrophilic spacers provided with reactive amino groups. A higher hydrophilicity should minimize denaturation of linked proteins and adsorption of undesired species, while the amino groups should form durable chemical bonds with biomolecules. A further activation with glutaraldehyde was performed in order to make the surface able to form covalent bonds with the amino moieties of proteins. The second class of materials comprises copolymers of glycidyl methacrylate with methyl methacrylate, either in absence or in presence of 2–hydroxyethyl methacrylate in order to better modulate their hydrophilic/hydrophobic balance. Although the polymer glycidyl groups should be able to react directly with protein amino groups, all of the above–mentioned activation reactions were also performed on this class of materials in order to facilitate protein binding. Terpolymers of PEG methacrylate with methoxy–PEG methacrylate and either methyl or butyl methacrylate were synthesized to modulate both the hydrophilic/hydrophobic balance and stiffness of the polymer. The side chain hydroxyl groups of PEG methacrylate units were activated by sequential reactions with succinic anhydride and carbodiimide/N–hydroxysuccinimide in order to form activated ester groups. These materials were later discarded as their high hydrophilicity caused problems during the biomolecule deposition phase. Reaction of paper supports with alternating copolymers of maleic anhydride and either methyl or butyl vinyl ether exploited cellulose hydroxyl groups for the covalent binding of the polymer to the substrate and the formation of free carboxylic moieties. Reactive carboxyl groups were also introduced on the surface of poly(methyl methacrylate) plates and poly(ethylene terephthalate) sheets by controlled hydrolysis under suitable conditions. All these materials were then activated by reaction with carbodiimide/N–hydroxysuccinimide. Finally, the sensing element, an anti–human IgM, was deposited on the different supports by using a custom printer developed by Olivetti I–Jet. After optimization of the printing conditions, such as solution concentration, pattern design, and number of coats, the printer was used to produce an antibody matrix on the activated supports. Xeptagen SpA then performed functionality tests to assess the presence and the residual activity of the printed antibody. Among the polymer coatings, the 98:2 styrene/4–chloromethyl¬styrene copolymer activated with α,ω–diaminodiethylene glycol provided the best result. Further activation with glutaric dialdehyde did not improve the performance of the polymer. The synthesized GMA copolymers resulted unsuitable for this application in spite of the large residual antibody activity. Indeed, their high wettability was an issue both during the printing of the antibody and during the treatment with biological samples. On the other hand, activated poly(ethylene terephthalate) sheets turned out to be very well suited for ink–jet printing and showed the highest residual antibody activity