4. ACE2-Based State-of-the-Art SARS-CoV-2 Detection
The ACE2-virus binding event can be translated into different signal outputs depending on the type of signal transduction system used in the experiment (
Figure 4).
Figure 4. ACE2-based biosensing systems for detection of SARS-CoV-2
[51][52][53][54][51,52,53,54] Created with Biorender.
4.1. ACE2-Based Electrochemical Detection of SARS-CoV-2
The electrochemical behavior of the bioreceptor-bioanalyte interaction can be translated into a sensitive electrochemical biosensor. The electrical methods explored for SARS-CoV-2 detection include potentiometry
[55], amperometry
[56], electrochemical impedance spectroscopy (EIS)
[57], differential pulse voltammetry
[58][59][58,59], and square wave voltammetry
[60]. In the amperometric technique, the current generated by the electroactive species is measured and correlated to the analyte concentration
[61]. In voltammetry, the potential difference is measured between the working electrode and the reference electrode. The accumulation of a charge potential at the working electrode is measured compared to the reference electrode in an electrochemical cell when zero or no significant current flows between them
[58][61][58,61]. The bioreceptor-bioanalyte interaction may lead to the change in conductance of the electrolyte solution and can be leveraged as a biosensing signal for developing an impedimetric or conductometric biosensor
[62]. In electrochemical biosensors, the electrode is one of the important components. Since the bioelectrochemical event is generally monitored in the close proximity of the working electrode surface, the electrode functionalization with conductive materials and efficient immobilization of the bioreceptor are immensely important to achieve the desirable performance of the biosensor.
An electrochemical SARS-CoV-2 biosensor is developed by Torres et al.
[51]. The working electrode is functionalized by the drop-casting method. The ACE2 immobilization on the working electrode is achieved by glutaraldehyde cross-linking. Bovine serum albumin (BSA) is used to avoid non-specific adsorption on the electrode surface
(Figure 5). Electrode functionalization is systematically optimized and each fictionalization step is evaluated by cyclic voltammetry and EIS. The CV and Nyquist plots showed a successful functionalization of the working electrode
(Figure 6). A miniaturized handheld device called RAPID (Real-time Accurate Portable Impedimetric Detection) is developed. The binding of the target analyte with ACE2 causes a change in interfacial electron transfer kinetics between the redox probe in solution and conducting electrode sites. Electrochemical impedance spectroscopy (EIS) is employed to detect the sensing signal. The calibration curves obtained with two different samples (spike protein and inactivated virus) show a satisfactory sensitivity and robustness of the method
(Figure 6). The use of Nafion as a protective layer (and high concentration of ACE2) contribute to the sensitivity and robustness of the biosensor. The sensitivity and specificity of the RAPID for nasopharyngeal/oropharyngeal swabs and saliva samples are 85.3% and 100%, and 100% and 86.5% respectively, with an assay volume of 10 µL and a response time of 4 min. The detection limit of the biosensor is 2.18 fg mL
−1 (in buffer) and 1.39 pg mL
−1 for saliva samples.
In another development
[24], the palladium nano thin film electrode (Pd-NTF) is fabricated and functionalized by exploiting Pd-S bond formation between the active Pd surface and ACE2. 1-octadecanethiol is used as blocking agent. The functionalized electrode is subsequently coupled to the EIS to realize a complete biosensor. The practical utility of the platform is demonstrated by screening modulators of SARS-CoV-2 S-protein-ACE2 interaction using a very small assay volume. By using the developed platform, potential pharmacological therapy that leads to suppressed virus binding with ACE2 is also identified.
ACE2 has two hydrophobic regions, the 18-amino-acid signal peptide on the N-terminus and a 22-amino-acid region on the C-terminus of the enzyme. The hydrophobic region facilitates its anchoring to the cell membrane
[63] and allows ACE2 immobilization on amphiphobic structures resembling to cell membrane. Fluorous self-assembled monolayers (SAM) have been used in thin-film transistors (TFTs) to reduce biofouling of material surface
[64]. Based on antibiofouling behavior and possibility of effective enzyme immobilization on electrode surface, Vezza et al.,
[65] deploy a perfluorocarbon self-assembled monolayer of 1H,1H,2H,2H-Perfluorodecanethiol (PFDT) on gold sensor surface to form an amphiphobic surface where ACE2 is immobilized by physisorption on PFDT
(Figure 7). The sensor is challenged to the streptavidin, interleukin 6 (IL6), viral transport medium, and saliva samples to test its specificity. The biosensor shows clinically relevant analytical properties.
A low-cost biosensor, LEAD (Low-cost Electrochemical Advanced Diagnostic) composed of pencil graphite electrodes, is developed by de Lima et al.
[52]. The transducer is modified with gold nanoparticles (AuNPs), cysteamine (cys), and bovine serum albumin (BSA) by using different cross-linkers. The ACE2 is anchored on the modified electrode, resulting in the ACE2-AuNPs-cys
(Figure 8). The use of gold nanoparticles increases the electrode’s sensitivity due to efficient electron transfer between the electrode and the redox probe ([Fe(CN)
6]
−3/−4). The biosensor could detect 229 fg mL
−1 spike protein. Further, the practical utility of the system is demonstrated by detecting virus in clinical samples. Moreover, the biosensor is insensitive to other potential interfering viruses with shelf-life of 5 days. Rapid response time and the use of easily accessible and commercially available materials are the key attributes of the developed biosensor.
4.2. ACE2-Based Optodiagnostics for COVID-19
Immunodetection systems have garnered substantial research attention due to their certain features, such as rapid response, ease of fabrication, and low cost. The use of an appropriate antibody pair for SARS-CoV-2 detection may improve the sensitivity and reliability of the system. In this regard, a rapid SARS-CoV-2 detection method based on ACE2
(Figure 9) is developed
[53], where ACE2 could form a matched pair with commercial antibodies. The ACE2 is used as a capture probe to selectively bind the SARS-CoV-2 S1 subunit of the S protein. Three different commercially available antibodies (CR3022, F26G19, and S1-mAb) are systematically evaluated to find the best pair with ACE2. Interestingly, the developed assay is more sensitive to the RBD than the S1, indicating the enhanced affinity of ACE2 for RBD. Spike proteins from other related viruses, such as SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV) show no cross-reactivity. The LOD of the developed system is 1.86 × 10
5 copies per milliliter, and this is the first study to detect COVID-19 antigen in a lateral flow format using ACE2 as a capture probe. The study opens avenues to develop mutation-free virus detection assays where bioreceptor-virus interaction can be realized as a detection approach. A förster resonance energy transfer (FRET) phenomenon is exploited to develop a SARS-CoV-2-derived RBD ACE2 biosensor
[66]. The FRET probe is developed by labelling RBD with YPet and Turquoise2-GL as acceptor and donor, respectively. The biosensor could sensitively detect the interaction between hACE2 and the RBD fragment derived from the SARS-CoV-2 spike protein, demonstrating its potential application in drug design.
In another development
[67], a smartphone-assisted ACE2-based optodiagnostic system is designed and developed. Gold nanoparticles and cysteamine are used
[52] and fabricated into a user-friendly sensing system. The cotton swab is functionalized with ACE2, and ACE2 is immobilized on the AuNP-cys. The ACE2-functionalized cotton swab is dipped into the sample where the analyte (spike protein or virus particle) is captured by the ACE2. After that, AuNP-cys-ACE2 is added to the reaction system and a color change is observed. The color change is attributed to the binding of virus/spike protein with ACE2, resulting in a sandwich formation that leads to plasmonic change in the AuNPs. The analytical features of the assay are comparable to the other related reports
[68][69][68,69]. Prolonged incubation (>5 min) of the assay enabled adsorption of the functionalized nanoparticles onto cotton swabs, resulting in a false positive result. The use of blocking agents or a relatively inert support material may decrease the unwanted interactions of the functionalized AuNPs.
4.3. SERS-Based Detection
The development timeline of COVID-19 detection technologies indicates that most of the reported methods are particularly focused on SARS-CoV-2 detection in biological fluids. Waste water-based epidemiology (WWE) has been employed to detect different biomarkers, such as pharmaceuticals, drugs, industrial chemicals, and biologicals in wastewater
[44]. Human viruses have been detected in wastewater
[70][71][70,71]. The proper monitoring of viruses in environmental water can help in pandemic preparedness
[72]. For example, SARS-CoV-2 RNA was detected in sewage waters in the Netherlands
[73] and Australia
[70]. The copy number per mL significantly increased with the increasing prevalence of the virus in the population, indicating the potential of sewage water surveillance as a promising tool to monitor the virus load in a population. The discovery of the enhancement of Raman scattering induced by molecules adsorbed on the surface of nanostructured metals has revolutionized the analytical techniques to achieve desired sensitivities. In the surface-enhanced Raman scattering (SERS) phenomenon, the light scattering is greatly enhanced by molecules adsorbed onto the corrugated metallic surfaces, leading to single-molecule SERS sensing
[74]. The approach has demonstrated the single-molecule sensitivity
[75][76][77][75,76,77].
In an effort to detect SARS-CoV-2 in environmental samples and harness the latent potential of SERS technology to combat the COVID-19 pandemic, a SERS sensor is designed and developed by Yang et al.
[54][54] (Figure 10). The sensor, hACE2 immobilized gold-nanoneedle arrays (GNAs), shows a 10
6-fold virus enrichment attributed to the strong ACE2 binding affinity for SARS-CoV-2 and the unique geometry of the GNAs obstructing virus escape. The strong SARS-CoV-2 binding affinity of the ACE2 and the oblique hierarchical structure of the GNAs, and electromagnetic field effect coupling, synergistically result in a 10
9-fold enhancement of the SERS signal and a detection limit of 80 copies mL
−1. The biosensor integration with machine-learning methods (principal component analysis and discrimination analysis) reliably differentiates the SARS-CoV-2 SERS signal from that of SARS-CoV-1. The practical application of the system is demonstrated by using urine samples spiked with virus, indicating the potential of SERS sensor for virus detection in wastewater.