New analytical techniques that overcome major drawbacks of current routinely used viral infection diagnosis methods, i.e., the long analysis time and laboriousness of real-time reversetranscription polymerase chain reaction (qRT-PCR) and the insufficient sensitivity of "antigen tests", are urgently needed in the context of SARS-CoV-2 and other highly contagious viruses. Here, we report on an antifouling terpolymer-brush biointerface that enables the rapid and sensitive detection of SARS-CoV-2 in untreated clinical samples. The developed biointerface carries a tailored composition of zwitterionic and non-ionic moieties and allows for the significant improvement of antifouling capabilities when postmodified with biorecognition elements and exposed to complex media. When deployed on a surface of piezoelectric sensor and postmodified with human-cell-expressed antibodies specific to the nucleocapsid (N) protein of SARS-CoV-2, it made possible the quantitative analysis of untreated samples by a direct detection assay format without the need of additional amplification steps. Natively occurring N-protein−vRNA complexes, usually disrupted during the sample pre-treatment steps, were detected in the untreated clinical samples. This biosensor design improved the bioassay sensitivity to a clinically relevant limit of detection of 1.3 × 10 4 PFU/mL within a detection time of only 20 min. The high specificity toward N-protein-vRNA complexes was validated both by mass spectrometry and qRT-PCR. The performance characteristics were confirmed by qRT-PCR through a comparative study using a set of clinical nasopharyngeal swab samples. We further demonstrate the extraordinary fouling resistance of this biointerface through exposure to other commonly used crude biological samples (including blood plasma, oropharyngeal, stool, and nasopharyngeal swabs), measured via both the surface plasmon resonance and piezoelectric measurements, which highlights the potential to serve as a generic platform for a wide range of biosensing applications.

Polymer brushes not only represent emerging surface platforms for numerous bioanalytical and biological applications but also create advanced surface-tethered systems to mimic real-life biological processes. In particular, zwitterionic and nonionic polymer brushes have been intensively studied because of their extraordinary resistance to nonspecific adsorption of biomolecules (antifouling characteristics) as well as the ability to be functionalized with bioactive molecules. However, the relation between antifouling behavior in real-world biological media and structural changes of polymer brushes induced by surface preconditioning in different environments remains unexplored. In this work, we use multiple methods to study the structural properties of numerous brushes under variable ionic concentrations and determine the impact of these changes on resistance to fouling from undiluted blood plasma. We describe different mechanisms of swelling, depending on both the polymer brush coating properties and the environmental conditions that affect changes in both hydration levels and thickness. Using both fluorescent and surface plasmon resonance methods, we found that the antifouling behavior of these brushes is strongly dependent on the aforementioned structural changes. Moreover, preconditioning of the brush coatings (incubation at a variable salt concentration or drying) prior to biomolecule interaction may significantly improve the antifouling performance. These results suggest a new simple approach to improve the antifouling behavior of polymer brushes. In addition, the results herein enhance the understanding for improved design of antifouling and bioresponsive brushes employed in biosensor and biomimetic applications.

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...