Tuning of Surface Charge of Functionalized Poly(Carboxybetaine) Brushes Can Significantly Improve Label‐Free Biosensing in Complex Media

Abstract

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...

Sandwich Immuno-RCA Assay with Single Molecule Counting Readout: The Importance of Biointerface Design

“…It should be noted that in addition to the thiol SAM architectures, we also tested poly­[( N -(2-hydroxypropyl)-methacrylamide)- co -(carboxybetaine methacrylamide)] (HPMA- co -CBMA) brushes for minimizing background signal F b . This polymer coating takes advantage of zwitterionic groups that tightly bind water molecules, where such surfaces have been reported to exhibit ultralow fouling properties, even after the postmodification with protein ligands by using amine coupling . However, the obtained results (see Figure S5a for testing various deactivation agents and Figure S5b for dAb-CS* complex dissolved in different buffer solutions) did not reveal such functionality, and sufficient repelling from unspecific interaction of dAb-CS* conjugate was not achieved when IL-6 immuno-RCA assay was applied.…”

“…This polymer coating takes advantage of zwitterionic groups that tightly bind water molecules, where such surfaces have been reported to exhibit ultralow fouling properties, even after the postmodification with protein ligands by using amine coupling. 24 However, the obtained results (see Figure S5a for testing various deactivation agents and Figure S5b for dAb-CS* complex dissolved in different buffer solutions) did not reveal such functionality, and sufficient repelling from unspecific interaction of dAb-CS* conjugate was not achieved when IL-6 immuno-RCA assay was applied. It was ascribed to unbalanced charge density occurring after the cAb was attached to the surface via the carboxy-betaine groups with the active ester chemistry (leading to net positive charge and possible Coulombic interaction with negatively charged dAb-CS*).…”

A guide to functionalisation and bioconjugation strategies to surface-initiated polymer brushes

“…dimethylaminopyridine) as catalyst 28,40,51–53 Residual active NHS-esters can disrupt the antifouling properties of activated polymer brushes, thus exposure to deactivators, such as amino compound bearing carboxyl or sulfate groups can be carried out. 54 An alternative strategy consists in functionalising amine-bearing brushes with reactive esters. For example, maleimide-NHS esters have been used to functionalize PDMA- co -APMA brushes with maleimide moieties prior to further coupling.…”

Driving Polymer Brushes from Synthesis to Functioning

“…[7][8][9][10] Significant efforts have been made in this field, including the emergence of new concepts to simplify surface polymerizations, and the latest attempt in applications such as biomedicine, [11,12] electronics, [13,14] and biosensing. [15,16] Conventional radical polymerizations are commonly used to synthesize polymer brushes. However, uncontrollable and irreversible termination and transfer processes cause deviation from desired chain lengths and broad molecular weight distribution, limiting the wide application of the polymers.…”

“…The number of publications on polymer brushes is increasing every year, the topics range from polymers, physics, material science, and food science to biology and medical science, clearly demonstrating the importance and growing interest in polymer brushes [7–10] . Significant efforts have been made in this field, including the emergence of new concepts to simplify surface polymerizations, and the latest attempt in applications such as biomedicine, [11, 12] electronics, [13, 14] and biosensing [15, 16] …”