An Electrochemical Impedance Spectroscopy-Based Aptasensor for the Determination of SARS-CoV-2-RBD Using a Carbon Nanofiber–Gold Nanocomposite Modified Screen-Printed Electrode

Abstract

Worldwide, human health is affected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Hence, the fabrication of the biosensors to diagnose SARS-CoV-2 is critical. In this paper, we report an electrochemical impedance spectroscopy (EIS)-based aptasensor for the determination of the SARS-CoV-2 receptor-binding domain (SARS-CoV-2-RBD). For this purpose, the carbon nanofibers (CNFs) were first decorated with gold nanoparticles (AuNPs). Then, the surface of the carbon-based screen-printed electrode (CSPE) was modified with the CNF–AuNP nanocomposite (CSPE/CNF–AuNP). After that, the thiol-terminal aptamer probe was immobilized on the surface of the CSPE/CNF–AuNP. The surface coverage of the aptamer was calculated to be 52.8 pmol.cm−2. The CSPE/CNF–AuNP/Aptamer was then used for the measurement of SARS-CoV-2-RBD by using the EIS method. The obtained results indicate that the signal had a linear–logarithmic relationship in the range of 0.01–64 nM with a limit of detection of 7.0 pM. The proposed aptasensor had a good selectivity to SARS-CoV-2-RBD in the presence of human serum albumin; human immunoglobulins G, A, and M, hemagglutinin, and neuraminidase. The analytical performance of the aptasensor was studied in human saliva samples. The present study indicates a practical application of the CSPE/CNF-AuNP/Aptamer for the determination of SARS-CoV-2-RBD in human saliva samples with high sensitivity and accuracy.

The critical experimental aspects for developing pathogen electrochemical biosensors: A lesson during the COVID-19 pandemic

“…Apart from the conventional antibody/antigen-based receptors, aptamers (i.e., oligonucleotides) and ACE2 (i.e., human angiotensin-converting enzyme 2) have also been utilized to develop the SARS-CoV-2 electrochemical biosensors. Regarding the former, we found five individual studies that used an aptamer receptor to target different SAR-CoV-2-related biomarkers, including the S1 receptor-binding domain (RDB) [ 52 , 53 ], RNA sequence [ 54 , 55 ], and nucleocapsid protein (NP) [ 27 ] ( Table 2 ). On the other hand, we also found five individual studies that adopted ACE2 as their receptors (or used it as an alternative to the secondary antibodies).…”

“…Indeed, when conceiving an electrochemical aptasensor, the most common strategy is to add thiol groups at the end of aptamer sequences, allowing the formation of the gold-thiol bond to direct attach to either a gold-based sensing surface or signal amplifier [ 61 ]. As shown in Table 2 , three out of five electrochemical aptasensors chose to immobilize their thiolated-aptamers on the AuNPs modified sensing surface through the facile SAMs process [ [52] , [53] , [54] ]. In comparison, only one out of 35 antibody-based biosensors adopted this directly thiolated strategy [ 33 ] (see Table 1 ).…”

“…In many real cases, this feature may become more dispositive than other critical technical performances because the distribution of the point-of-care testing kits depends heavily on precisely coordinated cold-chain logistics, and the less temperature-sensitive aptamer-based biosensors can remarkably ease the burden on the logistical cost. After carefully reading all the SARS-CoV-2 electrochemical aptasensors, we found that four (i.e., 80% of total aptamer-based studies) had undergone sensor stability tests over different time spans at 4 °C (or storage in a refrigerator) [ 27 , 52 , 53 , 55 ]. Among them, Abrego-Martinez et al [ 53 ] reported an outstanding result in the thermal stability test after 21-day storage at 4 °C, in which the impedimetric response of the developed aptasensor only lost 1% of its sensing capability with respect to the freshly prepared one.…”

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Fig. 6 Schematic diagrams of the four strategies of the aptamer-based SARS-CoV-2 electrochemical biosensors: (a) thiolated aptamer label-free detection [ 52 , 53 ], (b) thiolated aptamer duplex association detection [ 54 ], (c) polymers attached aptamer label-free detection [ 27 ], and (d) biotinylated aptamer duplex association with enzyme-labeled detection [ 55 ].

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An electrochemical membrane-based aptasensor for detection of severe acute respiratory syndrome coronavirus-2 receptor-binding domain

“…Hence, several research groups have been working to fabricate the biosensors to detect the SARS-CoV-2 in the early stage of COVID-19. The spike protein [2] , [3] , [4] , [5] , nucleocapsid protein [6] , [7] , [8] , a short fragment of its gene [9] , [10] , [11] , and receptor-binding domain (RBD) [12] , [13] , [14] of the SARS-CoV-2 are the common biomarkers that were detected in the real samples to diagnose the COVID-19.…”