Publication date: Jul 22, 2025

In label-free biosensors based on graphene, achieving both the specific recognition of target analytes and the suppression of the nonspecific adsorption of interfering substances remains a critical challenge. In this study, a linker molecule possessing a pyrene moiety capable of forming π-π stacking interactions and an active ester group suitable for bioconjugation was employed to construct a selective layer on graphene. Subsequently, a DNA aptamer as the receptor and a phospholipid-mimetic zwitterionic molecule for suppressing nonspecific adsorption were sequentially conjugated to the active ester groups. This approach enabled the design of a biointerface with high selectivity exclusively toward the target molecule. A novel zwitterionic molecule suitable for conjugation with active ester groups was synthesized. The surface modification of the graphene was characterized using X-ray photoelectron spectroscopy, chronocoulometry, and quartz crystal microbalance with dissipation (QCM-D) monitoring. In nonspecific adsorption suppression tests conducted on gold substrates with transferred graphene using QCM-D, the surface modified with zwitterionic molecules exhibited an 83. 6% reduction in bovine serum albumin (BSA) adsorption compared to conventional hydrophilic molecule-modified surfaces. The surface modified with both an aptamer and a zwitterionic molecule exhibited a detection limit of 0. 23 nM for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. This biointerface was capable of specifically detecting the spike protein at a concentration 10,000 times lower than that of BSA (1. 5 μM), demonstrating high selectivity even in the presence of interfering substances. These results demonstrate the potential for the label-free detection of SARS-CoV-2 in respiratory droplets. Graphene modification using aptamers and zwitterionic molecules is expected to be applicable to a wide range of biosensing applications by employing different aptamer sequences.

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