Fouling from complex biological fluids such as blood plasma to biorecognition element (BRE)-functionalized coatings hampers the use of affinity biosensor technologies in medical diagnostics. Here, we report the effects the molecular mechanisms involved in functionalization of low-fouling carboxy-functional coatings have on the BRE capacity and resistance to fouling from blood plasma. The specific mechanisms of EDC/NHS activation of carboxy groups, BRE attachment, and deactivation of residual activated groups on recently developed ultra-low-fouling carboxybetaine polymer and copolymer brushes (pCB) as well as conventional carboxy-terminated oligo(ethylene glycol)-based alkanethiolate self-assembled monolayers (OEG-SAMs) are studied using the polarization modulation infrared reflection/absorption spectroscopy, X-ray photoelectron spectroscopy, and surface plasmon resonance methods. It is shown that the fouling resistance of BRE-functionalized pCB coatings is strongly influenced by a deactivation method affecting the ultra-low-fouling molecular structure of the brush and surface charges. It is revealed that, in contrast to free carboxy-group-terminated OEG-SAMs, only a partial deactivation of EDC/NHS-activated zwitterionic carboxy groups by spontaneous hydrolysis is possible in the pCB brushes. The fouling resistance of activated/BRE-functionalized pCB is shown to be recovered only by covalent attachment of amino acid deactivation agents to residual activated carboxy groups of pCB. The developed deactivation procedure is further combined with ultra-low-fouling brushes of random copolymer carboxybetaine methacrylamide (CBMAA) and N-(2-hydroxypropyl) methacrylamide (HPMAA) with optimized CBMAA content (15%) providing a BRE-functionalized coating with superior fouling resistance over various carboxy-functional low-fouling coatings including homopolymer pCB brushes and OEG-SAMs. The biorecognition capabilities of pHPMAA-CBMAA(15%) are demonstrated via the sensitive label-free detection of a microRNA cancer biomarker (miR-16) in blood plasma.

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Besides the resistance to fouling, the platform should be easily functionalized, i.e., conjugated with molecules having specific biological activity, usually a high affinity for specific targets. [9][10][11][12][13][14][15][16] Applications that require such functionalized antifouling platforms span from rapid detection of chemical and biological species, coatings of nanoparticles used in drug delivery, membranes for separation and cleaning technologies, to scaffolds for tissue engineering.Poly(carboxybetaine) (pCB) brushes are outstanding antifouling platforms allowing facile functionalization with biorecognition elements (BREs) via EDC/NHS (carbodiimide/N-hydroxysuccinimide) chemistry. [17][18][19] Their extraordinary properties stem from their high hydrophilicity and overall electroneutrality, which makes them resistant to hydrophobic as well as electrostatic adsorption from contacted media. However, their net electric charge is pH dependent. The positive charge of quaternary ammonium group is permanent but pKa of pCB carboxyl group is somewhere between 2 and 4. [17,20,21] The measurements of zeta potential of the surface bound poly(carboxybetaine methacrylamide) (pCBMAA), [22] indicated that its isoelectric point (pI) is around 8.5 and thus it is positively charged at lower pH values. The net positive charge of functionalized pCB brushes is further enhanced due to the consumption of betaine carboxyl groups following BRE conjugation reaction (Figure 1). It should be taken into consideration that conjugated BRE may also induce charge shifts and thus an effective optimization of the platform surface charge balance is of importance. [16] The overall reaction scheme for the BRE conjugation via EDC/NHS is shown in Figure 1. The carboxyl group of pCB is converted to active NHS ester which readily reacts with amino group of BRE to create stable amide bond. However, not all NHS esters buried inside pCB brushes are able to react with bulky BREs that cannot penetrate below the surface, but can react nonspecifically with other smaller amino compounds present in complex biological media. Therefore, all residual NHS esters must be eliminated (deactivated) as otherwise they could Poly(carboxybetaine) brushes are excellent antifouling platforms allowing facile functionalization with biorecognition elements via carbodiimide/N-hydroxysuccinimide (EDC/NHS) chemistry. However, residual active NHS esters and the loss of zwitterionic balance after the conjugation may impair initially excellent antifouling properties. This problem has so far been addressed either by using spontaneous hydrolysis or deactivation of residual NHS esters by the reaction with a small amino compound bearing hydroxyl or carboxyl groups. In contrast to this approach, and instead of using a single deactivator, here the use of tailored mixtures of deactivating agents containing carboxyl groups and sulfo or sulfate groups with permanent negative charge that allow to tune surface charge balance is investigated. The approach is applied to poly(carboxybetaine acr...