Modification Of SPR Sensing Surfaces Using Hyaluronic Acid As Antifouling Matrix

Hyaluronic acid(HA), as an anionic glycosaminoglycan, has been widely used in biosensors, marine, cosmetics, medical and other fields. In this thesis, we designed and fabricated HA-modified SPR sensor chips via the covalent bonding and mixing/polymerization method, respectively. The resulting HA-modified surfaces were characterized using atomic force microscopy(AFM), contact angle(CA) and other techniques. SPR spectroscopy was further used to measure the nonspecific protein adsorption on the surfaces from single and actual complex protein solutions. Meanwhile, we investigated the kinetics of antigen-antibody interaction and the the regenerability of HA-modified chips.(1) A new protein-resistant surface made through the chemical grafting of easily available hyaluronic acid(HA) onto gold(Au) substrate demonstrates excellent antifouling performance against protein adsorption. AFM images showed the uniform HA layer with a thickness of ?10.5 nm on the Au surface. The water contact angles of Au surfaces decreased from 103° to 12° with the covalent attachment of a carboxylated HA matrix, indicating its high hydrophilicity mainly resulted from carboxyl and amide groups in the HA chains. Using SPR spectroscopy to investigate nonspecific adsorption from single protein solutions(bovine serum albumin(BSA), lysozyme) and complex media(soybean milk, cow milk, orange juice) to an HA matrix, it was found that ultralow or low protein adsorptions of 0.6-16.1 ng/cm2(e.g., soybean milk: 0.67 ng/cm2) were achieved on HA-Au surfaces.(2) Moreover, anti-BSA was chosen as a model recognition molecule to characterize the immobilization capacity and the antifouling performance of antiBSA/HA surfaces. The results showed that anti-BSA/HA sensor surfaces have a high anti-BSA loading of 780 ng/cm2, together with achieving the ultralow(< 3 ng/cm2 for lysozyme and soybean milk) or low(< 17 ng/cm2 for cow milk and 10% blood serum) protein adsorptions. Moreover, using lysozyme and soybean milk with high concentrations as reference proteins, the signal-to-noise ratios for 15 nM BSA were 94.6 and 25.8, respectively, suggesting a very high selectivity for BSA sensing. Additionally, the sensor chips also exhibited a high sensitivity to BSA over a wide range of concentrations from 15 to 700 nM. We also investigated the regenerability of antiBSA sensor chips by injecting 10 mM NaOH, the results showed that the anti-BSA sensor chip had a good performance after 4 cycles with a total time of 4 h.(3) A new class of protein-resistant surfaces made through the polymerization of dopamine(DA) in the presence of hyaluronic acid(HA) onto different substrate(e.g., epoxy resin, polystyrene, glass, Au, steel) demonstrate good antifouling performance against protein adsorption. Specifically, the DA was mixed with HA and then allowed for polymerization on the substrates, leading the formation of PDA/HA hybrid layer. When the mass ratio of DA/HA was 1 and the polymerization time was 2 h, the formed PDA/HA surface had good protein-resistant performance from single protein(eg. BSA and lysozyme, fibrinogen, ?-Lactoglobulin) and complex protein systems(soybean milk, 100% serum). Furthermore, a promising surface regeneration method was developed via the strong alkaline induced depolymerize of PDA. After 10 cycles of regeneration and modificaiton by PDA/HA, the Au surface showed similar SPR curve compared the original one.