Functionalized Terpolymer-Brush-Based Biointerface with Improved Antifouling Properties for Ultra-Sensitive Direct Detection of Virus in Crude Clinical Samples

Abstract

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.

Evaluation of Dibenzocyclooctyne and Bicyclononyne Click Reaction on Azido‐Functionalized Antifouling Polymer Brushes via Microspotting

“…The use of polymer brushes to encode different functionalities alongside their structure such as stealth properties, functional groups for further functionalization, and many more which include copolymers, [39][40][41][42] block copolymers [43][44][45][46] and terpolymers [47] were reported.…”

Biorecognition antifouling coatings in complex biological fluids: a review of functionalization aspects

“…90 In ref. 91 the composition of the copolymer brush was improved by adding a small amount of pSBMAA bearing permanent negative charges to balance the overall charge of the biointerface after functionalization. For p(SBMAA 3 mol%- co -CBMAA 15 mol%- co -HPMAA 82 mol%) the fouling level from undiluted blood plasma was reported to increase from 5.3 ± 1.1 ng cm −2 to 10.9 ± 3.2 ng cm −2 after anti-bacterial antibody functionalization followed by deactivation with (2-aminoethoxy)acetic acid (AEAA).…”

“…The optimum ratio of antifouling and the BE binding capacity is controlled by varying the ratio of monomer units in the copolymer and their distribution. 30,91…”

“…50,83,[88][89][90] In our previous work, we have described the ATRP copolymerization of functionalizable carboxybetaine methacrylamide with hydroxypropyl methacrylamide, a nonreactive antifouling component yielding outstanding antifouling brushes in which the ratio of both units can be tailored to optimize their BE immobilization capacity. 30,91 Lin et al reported that copolymers of carboxybetaine acrylamide or carboxybetaine methacrylate with a substantial portion of hydrophobic n-butyl methacrylate also retain rather good antifouling characteristics. These copolymers can be deposited by a simple dip-coating on surfaces of hydrophobic supports like polypropylene and polystyrene, which makes them promising for blood-contacting medical devices.…”

The Bioanalytical and Biomedical Applications of Polymer Modified Substrates

“…The polymer brush modified substrates have been employed to construct different biosensors with a high analytical performance [67][68][69][70][71][72][73][74][75][76]. Costantini et al developed an aptasensor for the detection of Ochratoxin A (OTA) in a beer sample by the immobilization of aptamer on the PHEMA brush modified glass substrate [68].…”