No abstract

Functional polymer coatings that combine the ability to resist nonspecific fouling from complex media with high biorecognition element (BRE) immobilization capacity represent an emerging class of new functional materials for a number of bioanalytical and biosensor technologies for medical diagnostics, security, and food safety. Here, we report on a random copolymer brush surface - poly(CBMAA-ran-HPMAA) - providing high BRE immobilization capacity while simultaneously exhibiting ultralow-fouling behavior in complex food media. We demonstrate that both the functionalization and fouling resistance capabilities of such copolymer brushes can be tuned by changing the surface contents of the two monomer units: nonionic N-(2-hydroxypropyl) methacrylamide (HPMAA) and carboxy-functional zwitterionic carboxybetaine methacrylamide (CBMAA). It is demonstrated that the resistance to fouling decreases with the surface content of CBMAA; poly(CBMAA-ran-HPMAA) brushes with CBMAA molar content up to 15 mol % maintain excellent resistance to fouling from a variety of homogenized foods (hamburger, cucumber, milk, and lettuce) even after covalent attachment of BREs to carboxy groups of CBMAA. The poly(CBMAA 15 mol %-ran-HPMAA) brushes functionalized with antibodies are demonstrated to exhibit fouling resistance from food samples by up to 3 orders of magnitude better when compared with the widely used low-fouling carboxy-functional oligo(ethylene glycol) (OEG)-based alkanethiolate self-assembled monolayers (AT SAMs) and, furthermore, by up to 2 orders of magnitude better when compared with the most successful ultralow-fouling biorecognition coatings - poly(carboxybetaine acrylamide), poly(CBAA). When model SPR detections of food-borne bacterial pathogens in homogenized foods are used, it is also demonstrated that the antibody-functionalized poly(CBMAA 15 mol %-ran-HPMAA) brush exhibits superior biorecognition properties over the poly(CBAA).

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.

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...

Front Cover: In article 1900351, Vaisocherová‐Lísalová and co‐workers investigate the structural properties of antifouling polymer brush surfaces for use as an efficient tool to control the behavior of living cells. It is shown that antifouling interfaces with a high content of zwitterionic moieties (>65mol%) modulate cell behavior completely differently compared to non‐ionic coatings. These differences are attributed mostly to the surface hydration capabilities. Image credit: https://Neuroncollective.com (Daniel Špaček, Pavel Jirák).

Ultra‐low fouling and functionalizable coatings represent emerging surface platforms for various analytical and biomedical applications such as those involving examination of cellular interactions in their native environments. Ultra‐low fouling surface platforms as advanced interfaces enabling modulation of behavior of living cells via tuning surface physicochemical properties are presented and studied. The state‐of‐art ultra‐low fouling surface‐grafted polymer brushes of zwitterionic poly(carboxybetaine acrylamide), nonionic poly(N‐(2‐hydroxypropyl)methacrylamide), and random copolymers of carboxybetaine methacrylamide (CBMAA) and HPMAA [p(CBMAA‐co‐HPMAA)] with tunable molar contents of CBMAA and HPMAA are employed. Using a model Huh7 cell line, a systematic study of surface wettability, swelling, and charge effects on the cell growth, shape, and cytoskeleton distribution is performed. This study reveals that ultra‐low fouling interfaces with a high content of zwitterionic moieties (>65 mol%) modulate cell behavior in a distinctly different way compared to coatings with a high content of nonionic HPMAA. These differences are attributed mostly to the surface hydration capabilities. The results demonstrate a high potential of carboxybetaine‐rich ultra‐low fouling surfaces with high hydration capabilities and minimum background signal interferences to create next‐generation bioresponsive interfaces for advanced studies of living objects.