In recent years, the emergence of infectious diseases has affected the entire world population, with zoonotic pathogens having the most significant impact (Wan et al., 2020). Climate change, urbanization, and globalization are the main reasons why, in the 21st century, emerging and re-emerging infectious diseases have increased significantly and spread rapidly (Baker et al., 2021) generating a public health problem and a global economic crisis (Rohr et al., 2019). Coronavirus disease 2019 (COVID-19) is an emerging infection produced by the etiologic agent severe acute respiratory syndrome-causing coronavirus type 2 (SARS-CoV-2) that emerged in China in late 2019 (Zhang & Guo, 2020) and then spread worldwide, resulting in the loss of many lives and a devastating impact on the global economy. Due to the tireless efforts of researchers and governments worldwide, the pandemic ended through mass vaccination of the population, early detection of the virus, and proper diagnosis of COVID-19. The diagnosis is made by RT-PCR, which detects the virus's genetic material but requires robust equipment and specialized personnel (Mahapatra and Chandra, 2020, Roberts et al., 2021). And by lateral flow devices (LFID), which were quickly adapted for the timely detection of this deadly virus but have shown low sensitivity (Arbyn et al., 2022).

This pandemic and the rapid evolution of the virus highlighted the need for research and control of infectious diseases and new and improved detection tools that are easily adaptable to respond quickly and in time to the pathogen's spread (Baker et al., 2021). Nanobiosensors have emerged as promising devices in the detection of different biomarkers. These simple, low-cost devices incorporate specific bioreceptors and nanomaterials to produce a rapid, sensitive, and easily interpretable response (Narlawar et al., 2022). They have the potential to overcome the limitations of standard detection techniques and quantify various molecular targets, including biomarkers of infection, holding great promise for precision medicine (Vásquez & Orozco, 2022). In addition, they can be designed with different geometries (Orozco et al., 2007), modified with nanocomposites (Fernández-Sánchez et al., 2009, Mendoza et al., 2008) and nanomaterials (Prakashan et al., 2023, Shahdeo et al., 2022, Vásquez and Orozco, 2023) and easily miniaturized and incorporated into multiparameter probes, with low power consumption and minimal reagent and sample consumption. Yet, to bring these devices to market, clinical validation is vital to increase their technological maturity (Ozkan et al., n.d.).

For detecting SARS-CoV-2, the first clinical validation studies focused on the RT-PCR technique, the gold standard for virus detection and COVID-19 diagnosis. Some authors, such as Sorrelle et al. (SoRelle et al., 2020) performed a clinical validation of RT-PCR for the SARS-CoV-2 nucleocapsid gene with 75 samples (45 positives and 30 negatives), obtaining a LOD of 264 copies/mL and a sensitivity and specificity of 100 %. Fomsgaard et al. (Fomsgaard & Rosenstierne, 2020) compared two commercial RT-PCR extraction kits, the MagNA Pure 96 and the QIAcube Connect system, with which they analyzed 87 patient samples, 65 positives and 22 negatives for SARS-CoV-2, obtaining a sensitivity of 100 % for both kits and a specificity of 100 % and 95.5 %, respectively.

Using RT-PCR as a reference, other authors have validated different rapid antigen tests. For example, Corman et al. (V. M. Corman et al., 2021) validated seven rapid antigen tests with 138 samples (100 positives, 38 negatives). The references included Abbott, RapiGEN, Healgen, Coris Bioconcept, R-Biopharm, nal von Minden, and Roche-SD Biosensor. Five showed a specificity higher than 98.5 %, compared to R-Biopharm, with 94.8 %, and Healgen, with 88.9 %, and a low sensitivity for the RapiGEN test. Igloi et al. (Iglòi et al., 2021) validated the Roche SD Biosensor rapid antigen test with 970 samples, obtaining a sensitivity of 84.5 % and a specificity of 99.5 %. Jegerlehner et al. (Jegerlehner et al., 2021) validated this same device with 1465 samples (141 positives and 1324 negatives) where, compared to Igloi, they obtained a sensitivity of only 65.3 % and a specificity of 99.9 %, and Fernandez et al. (Fernandez-Montero et al., 2021) obtained a sensitivity of 71.43 % and specificity of 99.68 % with 2542 samples from asymptomatic patients. Terpos et al. (Terpos et al., 2021) validated an antigen test based on colloidal gold, in which they analyzed nasal and nasopharyngeal swabs with 109 and 114 positive patients for SARS-CoV-2, respectively, and 250 and 244 negative individuals, respectively, confirmed by PCR, obtaining a sensitivity of over 96 % for samples with cycle threshold (CT) less than 33, and specificity of 99 %.

Other less widely used devices have also been recently clinically validated, such as those based on clustered regularly interspaced short palindromic repeats (CRISPR) with a sensitivity and specificity of 93.8 and 99.0 % for 350 saliva samples (Abugattás Núñez del Prado et al., n.d.), 96 and 100 % for 534 samples (Patchsung et al., 2020), and 96.7 and 95 % for 62 nasopharyngeal swab specimens (Nguyen et al., 2022). Or assays based on reverse transcription recombinase polymerase amplification (RT-RPA), with a sensitivity of 96 % and specificity of 97 % for 91 nasopharyngeal samples (Cherkaoui et al., 2022), and assays based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) with a sensitivity and specificity of 90.2 and 92.4 % from 198 swab samples (Lim et al., 2021)

Since the first immunosensor was reported (Seo et al., 2020), different biosensor formats have been quickly reported to detect SARS-CoV-2. For example, our group has developed immunosensors (Vásquez et al., 2022), genosensors (Cajigas et al., 2022), and peptide-based biosensors (Soto & Orozco, 2022). However, clinically validated biosensors are relatively scarce. Torres et al. (Torres et al., 2021) developed the RAPID 1.0 electrochemical biosensor based on electrochemical impedance spectroscopy to detect the SARS-CoV-2 spike protein. They analyzed 109 positive and 30 negative samples obtained from nasopharyngeal swabs for clinical validation, with a sensitivity of 83.5 % and a specificity of 100 %. In comparison, Bilgin et al. (Ebrem Bilgin et al., 2022) validated a metasurface-enhanced Raman spectroscopy (SERS) biosensor from 36 positive and 33 negative samples, with a sensitivity and specificity of 95.2 %.

This work reports on the clinical validation of an electrochemical immunosensor based on the spike-ACE2 complex developed in previous work (Vásquez et al., 2022). The device was validated with 101 positive and 49 negative samples from people between 0 and 97 years old who attended a health center with respiratory symptoms between November 2022 and April 2023. The samples were collected from nasopharyngeal swabs and tracheal aspiration and were analyzed with the electrochemical immunosensor and by RT-PCR as a standard technique to compare the device performance. After analyzing the 150 samples by chronoamperometry, the resulting current was highly correlated with the CT of most samples, especially those from nasopharyngeal swabs. Finally, the biosensor showed a clinical sensitivity of 96.04 % and a clinical specificity of 87.75 % in detecting SARS-CoV-2 in the samples, demonstrating the potential of electrochemical biosensors for detecting the virus and diagnosing COVID-19 and paving the way for designing them on demand to detect other infectious diseases.