SARS-CoV-2 Diagnostic Using an Electrochemical Aptamer-Based Sensor

Created by: Anastasiya Dudnyk, Bonnie Yang, Gurkaran Chhaggar, Jessica Bensky, Tony Huang

Coronavirus disease 2019 (COVID-19) is caused by a novel coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is the seventh coronavirus known to infect humans. The remaining six are HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, SARS-CoV (which caused severe acute respiratory syndrome) and MERS-CoV (which caused Middle East respiratory syndrome (Li et al., 2020)). Laboratory testing is a key link in the implementation of timely detection, rapid and effective treatment and precise quarantine in COVID-19 prevention and control. The COVID-19 testing methods include nucleic acid amplification, gene sequencing, antigen and antibody testing shown in Figure 1 (Testing, 2021). Up to Feb 24th, 2022, the Canadian government has authorized 52 nucleic acid testing devices, 26 antibody testing devices, 30 antigen testing devices, 28 rapid testing devices, and 10 self-testing devices. The total number of authorized COVID-19 testing devices is 108 (Authorized medical devices for uses related to COVID-19: List of authorized testing devices, 2022).

Figure 1: Scheme showing the comparison of different methods used for COVID-19 diagnosis (PoCT: Point of Care Test (Augustine et al., 2020)).

The main steps of nucleic acid testing include sample collection and processing, nucleic acid extraction, and PCR amplification and detection. The average detection time needs 4 to 6 hours (Covid-19 test collection and analysis, 2021). Because it directly detects the viral nucleic acid in the samples collected, it has strong specificity and relatively high sensitivity, making it the current gold standard detection method. The antibody test: the colloidal gold method had an average detection time of about 15 minutes, and the magnetic particle chemiluminescence method commonly needed 30 to 60 minute. In the early stages of infection, IgM antibodies may not be produced in the body yet, so there is a window period for detection. Therefore, antibody testing can only be used for auxiliary diagnosis and screening of cases with a negative nucleic acid test, but it cannot replace the nucleic acid detection method. Epidemiologists can use the results of antibody tests to estimate the approximate number of asymptomatic infections and whether some degree of herd immunity can be achieved in the near future. Forecasts can help decision-makers decide whether to ease restrictions. Antibody tests can also help test the immunity of people who have recovered from the disease and those who have taken newly developed vaccines (Augustine et al., 2020).

Antigen testing is mainly used for rapid detection of high viral load in acute infection periods and can also be used for rapid triage and management of suspected populations. As the test results may be false-negative due to the sample type and the potential variant strains, they cannot be used in isolation to diagnose SARS-CoV-2 infection (Augustine et al., 2020). Metagenomic next-generation sequencing (mNGS) is mainly used for gene sequence analysis, virus origin, transmission, and evolution. When mNGS is combined with qRT-PCR, they can complement each other and maximize their clinical diagnostic value. qRT-PCR is faster, simpler, and cheaper than mNGS, making it more suitable for mass screening. For patients with highly suspected clinical phenotypes but with a negative qRT-PCR result, mNGS can confirm detection accuracy. mNGS for qRT-PCR positive patients is conducive to monitoring the mutation of SARS-CoV-2, providing valuable information for disease prevention, control, and treatment (Duan et al., 2021). More tests and detection methods will be available and widely used in the future as the understanding and development of the disease progresses.

Because of how easily aptamers can be developed along with their specific binding capabilities, they have been used as an ideal tool for biosensor applications (i.e., sensing technologies). One of the applications involves an optical approach called “aptamer beacons” (Xiao et al., 2005). Covalently bound to the aptamer is a fluorophore. Upon binding to the target (like in the study by idlili et al., 2021), the aptamer goes under a binding-induced conformational change resulting in fluorescence and detection of the target. The drawbacks of this technique include the use of light sources that require lots of power, the high cost, and the large equipment. Because of this researchers wanted to develop a different technique that will overcome those pitfalls and the aptamer-based electrochemical sensor was developed (Xiao et al., 2005). This technique was modified by Idili et al., 2021, which provided insight into a diagnostic technique that can detect SARS-CoV-2 in undiluted biological fluids through a single step mechanism without the use of reagents. This technique provides healthcare workers with a point-of-care diagnostic tool that can help diagnose SARS-CoV-2 infected patients in real time (few seconds to ~5 minutes). Additionally, this novel technique can potentially be used with phones or portable electrochemical setups, making it even easier for users to get their results.

The whole premise of this diagnostic technique relies on binding induced conformational change, which results in a change in current (i.e., electron transfer) that can be detected. There is a gold electrode (a conductor that makes contact with a nonmetal) to which the DNA aptamers are covalently attached to (Figure 2A). The aptamers have a redox reporter that can generate a measurable electrochemical signal. The aptamers are designed and selected in a manner that allows them to detect the receptor binding domain (RBD) of SARS-CoV-2 spike (S) protein. When the RBD of the S protein binds to the aptamer, there is a conformational change that takes place (to the aptamer) that changes the position of the redox reporter. The redox reporter is a methylene blue derivative called Atto MB2 (Millipore Sigma, 2022). There is a base level of electron transfer that is occurring that generates a baseline current (this would be in the absence of the S protein). Once the position of the redox reporter is changed upon binding of the S protein to the aptamer, there is a change in the rate of electron transfer (i.e., current) across the gold electrode that can be detected to confirm the presence of SARS-CoV-2 in the sample. Shown in Figure 2B is a graph of current across the electrode in the presence and absence of SARS-CoV-2 S protein.

Figure 2: (A) Electrochemical Aptamer-Based Sensor setup and how the rate of electron transfer changes when the position of the redox reporter is altered. (B) Readout from an electrochemical aptamer-based sensor showing the change in current (i.e., electron transfer) when the S protein binds to the aptamer causing a change in position of the redox reporter relative to the gold electrode (Idili et al., 2021).

Idili et al., 2021 sought to develop an electrochemical aptamer-based sensor for use as a SARS-CoV-2 diagnostic test. They experimented with recently developed DNA aptamers called 1C and 4C. These aptamers have three characteristics which make them ideal candidates for the sensor. Firstly, they have been shown to work in a buffer that mimics physiological conditions, which indicates they can support protein measurements in biological fluids. Secondly, the dissociation constants of the aptamers are similar to commercially available antibodies that bind the S protein. Lastly, the aptamers have been shown to be capable of interacting with the S protein through hydrogen bonds. Specifically, they have two binding interfaces with the 1C variant, but only one with the 4C variant. This suggests that the binding-induced conformational change is stronger for the 1C variant (Idili et al., 2021).

To determine whether the aptamers undergo a conformational change upon interaction with a target, Idili et al., 2021 used fluorescence spectroscopy. They used two targets that are part of SARS-CoV-2: the whole S protein (78 kDa) and its RBD (23 kDa). There are two reasons for choosing these targets. Firstly, they share the same domain recognized by the aptamers. Secondly, detection of the S protein would support the ability of the aptamers to recognize the actual virus (Idili et al., 2021).

Idili et al., 2021 then compared the binding activity of the aptamer variants 1C and 4C for each of the whole S protein and its RBD. They used a fluorophore-quencher pair at the 2 ends of the aptamer for labelling. When the target is not present, the fluorophore and quencher are close to one another, which results in a low signal. When the target binds, the aptamer undergoes a conformational change that separates the fluorophore and quencher, increasing the fluorescent signal. In the graphs in figure 3, the x-axis has the amount of the target protein added, and the y-axis has the percentage change in the fluorescent signal. The 1C aptamer is shown in the top graph and the 4C aptamer is shown in the bottom graph. In each graph, the whole S protein is represented by the red curve, and its receptor binding domain by the black curve. The graphs show that the 1C aptamer has a higher signal change for both of the target proteins tested. For this reason, the 1C aptamer was chosen for the electrochemical aptamer-based sensor. Additionally, both aptamers showed a higher affinity for the whole S protein than only its receptor binding domain, which Idili et al., 2021 explained is due to the larger size of the target.

Figure 3: The binding activities of DNA aptamers 1C and 4C against the whole SARS-CoV-2 S protein compared to only its RBD (Idili et al., 2021).

Idili et al., 2021 then performed an experiment to test the 1C aptamer in the form of the sensor instead of on its own. They added a redox reporter to enhance the readout, and attached the aptamer to a gold wire electrode. In figure 4, the x-axis of the graph has the amount of target protein added, and the y-axis has the percentage change in the fluorescent signal. The whole S protein is represented by the red curve, and the RBD by the black curve. When the whole S protein was used as the target, once again a higher signal change was seen than for just its RBD. Furthermore, the range of protein added that allowed for a signal change covered the clinically relevant range for the amount of SARS-CoV-2 protein present in infected individuals. This indicates the clinical value of this device as a diagnostic tool (Idili et al., 2021).

Figure 4: The binding activity of DNA aptamer 1C in the form of an electrochemical aptamer-based sensor against the whole SARS-CoV-2 S protein compared to only its RBD (Idili et al., 2021).

Idili et al., 2021 also tested the specificity of the EAB sensors by testing its response to the S protein of 2 previous coronaviruses and a non-viral protein against the S protein of SARS-CoV-2(Figure 5). They found that at 300 Hz and varying concentrations, only the S protein of SARS-CoV-2 was strongly detected and the others did not cause interference (Figure 5 top). At 5 Hz only the SARS-CoV-1 had a similar response to the SARS-CoV-2 (Figure 5 bottom) likely because their target proteins are similar and counter-selection was not performed prior to this testing. This test showed the potential of EAB sensors to discriminate between S protein variants which can help distinguish and separate patients who are infected with a SARS-CoV-2 variant that is known to have a more dangerous effect on the body.

Figure 5: Specificity of the electrochemical aptamer-based sensor compared to other viral proteins.

They also tested the speed at which a signal change occurs with the SARS-CoV-2 S protein (Figure 6 top) and MERS RBD (RBD) (Figure 6 bottom). This was found to be around 15 seconds after the addition of the sample and suggests that EAB sensors could find use in point-of-care (PoC) testing.

Figure 6: Speed of reaction of electrochemical aptamer-based sensor with SARS-CoV-2 S protein and MERS RBD.

Another benefit is that EAB sensors respond best from untreated samples, either in vivo or in vitro. Idili et al., 2021 tested this feature with artificial saliva because it is used widely in SARS-CoV-2 testing, as well as fetal bovine serum (FBS) which allowed them to study how EAB sensors respond to a complex sample. They found that with FBS, the signal change was high enough for clinical relevance (Figure 7A), though it was lower than what was found with buffer solution. They also tested a 50% artificial saliva solution (Figure 7B) and a 100% artificial saliva sample to see if they would yield similar results with a concentration comparable to a real saliva sample. In the 100% condition there was still detection of the protein, but the signal gain was a lot lower (Figure 8). Idili et al., 2021 believe that this may be attributed to the effect that the matrix has on the aptamers conformational change and that optimization of the technique with these types of samples needs to be done before beginning the transition into commercialization.

Figure 7: Response of electrochemical aptamer-based sensors to various types of untreated samples.

Figure 8: Response of electrochemical aptamer-based sensors to 100% untreated artificial saliva.

As COVID-19 continues to spread around the world, widespread testing and SARS-CoV-2 diagnosis are crucial for pandemic control. Unfortunately, many low- and middle-income countries (LMICs) (Figure 9) remain vulnerable to the disease as they struggle to access diagnostic tests. These countries often do not have the domestic capacity to manufacture diagnostic tests and rely heavily on imports. Imports are also limited as manufacturers are unwilling to supply LMICs due to their smaller market when compared to Europe and North America (Batista et al., 2022).

Figure 9: World map of country income groups (The World Bank, 2016).

The EAB sensor is a highly sensitive diagnostic technique that shows potential as a valid alternative to PoC tests. However, when compared to other diagnostic technologies that have been used for decades (e.g. LFA) its commercialization has greatly lagged behind academic output. Technical barriers such as sensitivity and robustness and lack of cost-effective manufacturing methods have hindered its commercialization (Idili et al., 2021). Future efforts must be placed to overcome these barriers in order to provide inexpensive and highly sensitive PoC testing to low resource settings such as LMICs.

Several improvements can be made during the screening and development of aptamers that would dramatically improve aptamer sensitivity and specificity to the target molecule:

1. Implement a modified SELEX called Capillary Electrophoresis SELEX (CE-SELEX). CE-SELEX separates the target bounded sequences from unbound sequences by their difference in electrophoretic mobility (Figure 10). This method enables the selection of aptamer candidates with high affinity, while reducing the selection rounds from 20 (conventional SELEX) to 1-4 (Zhuo et al., 2017).

Figure 10: Schematic of the CE–SELEX process. A random sequence DNA library is incubated with the target. Sequences bound to the target are separated using capillary electrophoresis, PCR amplified and made single stranded, generating a new pool suitable for further rounds of enrichment (Günter, 2009).

1. Introduction of a counter selection step during the SELEX process. The counter selection step incubates the aptamers with molecules that are structurally similar to the target in order to effectively discriminate against non-specific oligonucleotides. This procedure dramatically increases the affinity of the oligonucleotides selected (Idili et al., 2021).
2. The use of buffers with different ionic strengths during SELEX. Using different buffers is important to guarantee the binding to the target in natural conditions, such as saliva which is a highly ionic strength environment (Idili et al., 2021).

By implementing these improvements the sensitivity and specificity of the selected oligonucleotides can be significantly improved. In addition, these optimizations save time and reduce the total cost of aptamer screening.

There are also many ways to improve the manufacturing cost of EAB sensors. The problem with commercially available gold electrodes used in EAB sensors is that it is expensive with units costing upwards of $90 USD each and the reuse of these electrodes presents significant practical challenges. For a truly deployable and scalable approach, the entire sensor must be cheap and disposable. Zakashansky et al., 2021 created an EAB assay that can detect the S1 subunit of SARS-CoV-2 S protein in saliva using the children’s toy Shrinky-Dink© as an electrode. Their low-cost electrodes are implementable at population scales and demonstrate high sensitivity and specificity (Zakashansky et al., 2021).

The use of these sensors goes beyond SARS-CoV-2 detection. These sensors can be modified and used in the detection of:

• Small molecules
• Proteins (e.g. pathogenic proteins)
• Cancer cells
• Microorganisms

Research before Idili et al., 2021 has shows the use of these sensors in the detection of molecules such as heavy metals, dopamine, ATP, different types of drugs, and toxins. Additionally, these sensors have been used in the detection of different cytokines (immune proteins), thrombin (clotting factor), and different growth factors such as, vascular endothelial growth factor (VEGF). Another important application of the sensing capability of aptamers is their use in detecting cancer cells (Shadman et al., 2019). Research has shown these sensors detecting certain cancer and cancer cells, such as leukemic cells, prostate cancer, colorectal cancer, etc. (Shadman et al., 2019). Overall, the use of aptamer-based electrochemical sensors is endless in terms of detecting certain molecules and diseases of the body.

Other Applications of Electrochemical Aptasensors include (Li et al., 2019):

• New drug development
• Bio-imaging
• As therapeutic tool
• Drug discovery
• Disease diagnosis
• Hazard detection
• Food inspection

With many techniques currently being used to help diagnose SARS-CoV-2 infected patients, each comes with its advantages and limitations. To help overcome these limitations, novel research provides scientists with the resources and knowledge to develop new techniques that can possibly overcome the limitations of the previous techniques. Research by Idili et al., 2021 did exactly that, providing the scientific field with a novel SARS-CoV-2 diagnostic technique that can be useful in PoC settings in certain facilities. The turn-around time and ease of use provides the user with quick and accurate results making it easier to control the spread of SARS-CoV-2. Perhaps one of the most important aspects of this technique is that it can be modified to cater to different types of pathogens making it easier to implement electrochemical aptamer-based sensors for the diagnosis of future pathogenic diseases and current diseases, like cancer.

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