*Article* **Surface Plasmon Resonance Assay for Label-Free and Selective Detection of HIV-1 p24 Protein**

**Lucia Sarcina 1, Giuseppe Felice Mangiatordi 2, Fabrizio Torricelli 3, Paolo Bollella 1, Zahra Gounani 4, Ronald Österbacka 4, Eleonora Macchia 4,\* and Luisa Torsi 1,4,5**



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**Abstract:** The early detection of the human immunodeficiency virus (HIV) is of paramount importance to achieve efficient therapeutic treatment and limit the disease spreading. In this perspective, the assessment of biosensing assay for the HIV-1 p24 capsid protein plays a pivotal role in the timely and selective detection of HIV infections. In this study, multi-parameter-SPR has been used to develop a reliable and label-free detection method for HIV-1 p24 protein. Remarkably, both physical and chemical immobilization of mouse monoclonal antibodies against HIV-1 p24 on the SPR gold detecting surface have been characterized for the first time. The two immobilization techniques returned a capturing antibody surface coverage as high as (7.5 ± 0.3) × 10<sup>11</sup> molecule/cm<sup>2</sup> and (2.4 ± 0.6) × 10<sup>11</sup> molecule/cm2, respectively. However, the covalent binding of the capturing antibodies through a mixed self-assembled monolayer (SAM) of alkanethiols led to a doubling of the p24 binding signal. Moreover, from the modeling of the dose-response curve, an equilibrium dissociation constant KD of 5.30 × 10−<sup>9</sup> M was computed for the assay performed on the SAM modified surface compared to a much larger KD of 7.46 × 10−<sup>5</sup> M extracted for the physisorbed antibodies. The chemically modified system was also characterized in terms of sensitivity and selectivity, reaching a limit of detection of (4.1 ± 0.5) nM and an unprecedented selectivity ratio of 0.02.

**Keywords:** HIV-1 p24 protein; surface plasmon resonance; surface modifications; label-free detection
