**4. Conclusions**

In conclusion, the characterization of two biofunctionalization strategies for gold surfaces was performed through a multi-parameter SPR assay. The physisorption of antibodies against HIV-1 p24 (anti-p24) directly on the bare gold detecting surface led to the immobilization of (7.5 ± 0.3) × 10<sup>11</sup> molecule/cm2. The covalent binding of anti-p24 on a mixed SAM of alkanethiols brings a decreased surface coverage of (2.4 ± 0.6) × 10<sup>11</sup> molecule/cm2, thus being 68% lower than the one registered with physisorbed capturing antibodies.

However, the chemical immobilization endows the detecting interface with a reduced steric hindrance between the closest neighbor biorecognition elements, providing enhanced capturing efficacy toward the target analyte. Indeed, a doubled response was recorded for the latter assay. In addition, compared to the physisorbed antibodies, the covalently bound anti-p24 also resulted in a lower dissociation constant. In fact, KD values of 7.46 × 10−<sup>5</sup> M and 5.30 × 10−<sup>9</sup> M were measured, respectively, highlighting a better analyte/antibody binding affinity for the assay on anti-p24 modified SAM.

The biosensing assay of both covalently bound and physisorbed anti-p24 were also characterized in terms of selectivity and sensitivity. The modified surfaces were tested against the exposure to a non-binding protein, the human C-reactive protein (CRP), for the first time and the response was compared to the p24 signal in the same range of concentrations. This allowed for the estimation of a selectivity ratio as low as 0.02, and a limit of detection of (4.1 ± 0.5) nM for the covalently bound antibodies, being one order of magnitude lower than the state-of-the-art limit of detection of 40 nM reported for the direct SPR assays and comparable to the label needing ELISA gold standard. This study thus represents a proof of principle of the early detection of HIV infection. Meanwhile, a selectivity ratio of 0.08 and a limit of detection of (27 ± 1) nM were found for the physisorbed anti-p24 assay. Moreover, this SPR characterization could pave the way toward developing reliable bio-electronic platforms in which the gold sensing electrode can be modified following the biofunctionalization strategy assessed in the present study.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/bios11060180/s1, Figure S1: SPR sensogram recorded for the real-time exposure of the HIV-1 anti-p24 modified SAM to the target protein p24. The protein concentration ranged from 500 pM to 1uM. The baseline level was established in PBS; Figure S2: SPR sensogram of the assay of HIV-1 p24 protein at increasing concentrations (0.8 nM–350 nM), performed on the physisorbed anti-p24 antibodies on gold.

**Author Contributions:** Conceptualization, E.M. and L.T.; methodology, E.M., Z.G. and L.S.; validation, L.S. and E.M.; formal analysis, L.S.; investigation, R.Ö., P.B., G.F.M., and F.T.; data curation, L.S., E.M. and P.B.; writing—original draft preparation, E.M. and L.S.; writing—review and editing, L.T., R.Ö., F.T. and P.B.; supervision, E.M.; funding acquisition, E.M. and L.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** Academy of Finland projects #316881, #316883 "Spatiotemporal control of Cell Functions", #332106 "ProSiT—Protein Detection at the Single-Molecule Limit with a Self-powered Organic Transistor for HIV early diagnosis"; Biosensori analitici usa-e getta a base di transistori organici auto-alimentati per la rivelazione di biomarcatori proteomici alla singola molecola per la diagnostica decentrata dell'HIV (6CDD3786); Research for Innovation REFIN—Regione Puglia POR PUGLIA FESR-FSE 2014/2020; Dottorati innovativi con caratterizzazione industrial—PON R&I 2014–2020; "Sensore bio-elettronico usa-e-getta per l'HIV autoalimentato da una cella a combustibile biologica" (BioElSens&Fuel); SiMBiT—Single molecule bio-electronic smart system array for clinical testing (Grant agreemen<sup>t</sup> ID: 824946); PMGB—Sviluppo di piattaforme meccatroniche, genomiche e bioinformatiche per l'oncologia di precisione—ARS01\_01195-PON "RICERCA E INNOVAZIONE" 2014–2020; Åbo Akademi University CoE "Bioelectronic activation of cell functions"; and CSGI are acknowledged for partial financial support.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are openly available in https:// www.fairdata.fi/en/ (accessed on 2 June 2021). Repository Research data storage service IDA (ida.fairdata.fi).

**Acknowledgments:** Rosaria Anna Picca and Kyriaki Manoli are acknowledged for their useful discussions.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
