Surfaces resistant to fouling from biological fluids: towards bioactive surfaces for real applications.

The fouling from four human body fluids - blood plasma, cerebrospinal fluid, urine and saliva - and four animal fluids - foetal bovine and calf sera, egg and milk - relevant to human and veterinary medicine, immunology, biology and diagnostics is assessed on antifouling SAMs and on polymer brushes of oligo(ethylene glycol) methacrylate, 2-hydroxyethyl methacrylate, carboxybetaine acrylamide and N-(2-hydroxypropyl)methacrylamide synthesized via ATRP. While important deposits from the all biofluids are observed on SAMs, a superior resistance is achieved on polymer brushes. Importantly, only poly(CBAA) and poly(HPMA) are capable of resisting the fouling from the most challenging media, blood plasma and eggs.

Controlled/living surface-initiated ATRP of antifouling polymer brushes from gold in PBS and blood sera as a model study for polymer modifications in complex biological media.

The use of PBS, 10% FBS or 10% CS as media for SI-ATRP is reported. Controlled/living SI-ATRP of MeOEGMA in PBS is achieved leading to better control than in water. The livingness is confirmed by chain extension with MeOEGMA or carboxybetaine acrylamide. This technique is successfully adopted for the polymerization of MeOEGMA in 10% FBS or CS as models for complex biological media with reasonable control of the brush growth. All prepared brushes show excellent antifouling properties.

Polymer brushes showing non-fouling in blood plasma challenge the currently accepted design of protein resistant surfaces.

Ultra-low-fouling poly[N-(2-hydroxypropyl) methacrylamide] (poly(HPMA)) brushes have been synthesized for the first time. Similar to the so far only ultra-low-fouling surface, poly(carboxybetaine acrylamide), the level of blood plasma fouling was below the detection limit of surface plasmon resonance (SPR, 0.03 ng·cm(-2)) despite being a hydrogen bond donor and displaying a moderate wettability, thus challenging the currently accepted views for the design of antifouling properties. The antifouling properties were preserved even after two years of storage. To demonstrate the potential of poly(HPMA) brushes for the preparation of bioactive ultra-low fouling surfaces a label-free SPR immunosensor for detection of G Streptococcus was prepared.

Low temperature aqueous living/controlled (RAFT) polymerization of carboxybetaine methacrylamide up to high molecular weights.

Among the class of zwitterionic polymers poly(carboxybetaine)s (poly(CB)s) are unique, emerging as the only ultra-low fouling materials known allowing the preparation of biosensors, fouling resistant nanoparticles, and non-adhesive surfaces for bacteria. Poly(carboxybetaine methacrylate) and poly(carboxybetaine acrylamide) have been prepared via atom transfer radical polymerization (ATRP), however a polymerization with living characteristics has not been achieved yet. Herein, the first successful living/controlled reversible addition fragmentation transfer (RAFT) polymerization of (3-methacryloylamino-propyl)-(2-carboxy-ethyl)-dimethyl-ammonium (carboxybetaine methacrylamide) (CBMAA-3) in acetate buffer (pH 5.2) at 70 and 37 °C is reported. The polymerization afforded very high molecular weight polymers (determined by absolute size exclusion chromatography, close to 250,000 g·mol(-1) in less than 6 h) with low PDI (<1.3) at 70 °C. The polymerization was additionally carried out at 37 °C allowing to achieve yet lower PDIs (1.06 ≤ PDI ≤ 1.15) even at 90% conversion, demonstrating the suitability of the polymerization conditions for bioconjugate grafting. The living character of the polymerization is additionally evidenced by chain extending poly(CBMAA-3) at 70 and 37 °C. Block copolymerization from biologically relevant poly[N-(2-hydroxypropyl)methacrylamide] macroCTAs was additionally performed.

Poly(HEMA) brushes emerging as a new platform for direct detection of food pathogen in milk samples.

Surface plasmon resonance (SPR) biosensors capable of in real time detection of Cronobacter at concentrations down to 106 cells mL⁻¹ in samples of consumer fresh-whole fat milk, powder whole-fat milk preparation, and powder infant formulation were developed for the first time. Antibodies against Cronobacter were covalently attached onto polymer brushes of poly(2-hydroxyethyl methacrylate) (poly(HEMA)) grafted from the SPR chip surface. The lowest detection limit, 104 cells mL⁻¹, was achieved in phosphate buffered saline (pH 7.4) with sensors prepared by covalent immobilization of the same antibodies onto a self assembled monolayer (SAM) of hexa(ethylene glycol) undecanethiol (EG6). However, when the EG6 based sensors were challenged with milk samples the non-specific response due to the deposition of non-targeted compounds from the milk samples was much higher than the specific response to Cronobacter hampering the detection in milk. Similar interfering fouling was observed on antifouling polymer brushes of hydroxy-capped oligoethylene glycol methacrylate and even a 10 times higher fouling was observed on the widely used SAM of mixed hydroxy- and carboxy-terminated alkanethiols. Only poly(HEMA) brushes totally suppressed the fouling from milk samples. The robust well-controlled surface initiated atom transfer radical polymerization of HEMA allowed the preparation of highly dense brushes with a minimal thickness so that the capture of antigens by the antibodies immobilized on the brush layer could take place close to the gold SPR surface to provide a stronger optical response while the fouling was still suppressed. A minimum thickness of 19 nm of poly(HEMA) brush layer was necessary to suppress completely non-specific sensor response to fouling from milk.

Substrate-independent approach for the generation of functional protein resistant surfaces.

A new route for coating various substrates with antifouling polymer layers was developed. It consisted in deposition of an amino-rich adhesion layer by means of RF magnetron sputtering of Nylon 6,6 followed by the well-controlled, surface-initiated atom transfer radical polymerization of antifouling polymer brushes initiated by bromoisobutyrate covalently attached to amino groups present in the adhesion layer. Polymer brushes of hydroxy- and methoxy-capped oligoethyleneglycol methacrylate and carboxybetaine acrylamide were grafted from bromoisobutyrate initiator attached to a 15 nm thick amino-rich adhesion layer deposited on gold, silicon, polypropylene, and titanium-aluminum-vanadium alloy surfaces. Well-controlled polymerization kinetics made it possible to control the thickness of the brushes at a nanometer scale. Zero fouling from single protein solutions and a reduction of more than 90% in the fouling from blood plasma observed on the uncoated surfaces was achieved. The feasibility of functionalization with bioactive compounds was tested by covalent attachment of streptavidin onto poly(oligoethylene glycol methacrylate) brush and subsequent immobilization of model antibodies and oligonucleotides. The procedure is nondestructive and does not require any chemical preactivation or the presence of reactive groups on the substrate surface. Contrary to current antifouling modifications, the developed coating can be built on various classes of substrates and preserves its antifouling properties even in undiluted blood plasma. The new technique might be used for fabrication of biotechnological and biomedical devices with tailor-made functions that will not be impaired by fouling from ambient biological media.