WO2023091119A1

WO2023091119A1 - Impedimetric immunosensor for the diagnosis of covid-19

Info

Publication number: WO2023091119A1
Application number: PCT/TR2022/051315
Authority: WO
Inventors: Kadriye Arzum ERDEM GÜRSAN; Muhammed Hüseyin ŞENTÜRK; Esma YILDIZ; Meltem MARAL
Original assignee: Ege Üni̇versi̇tesi̇
Priority date: 2021-11-18
Filing date: 2022-11-18
Publication date: 2023-05-25

Abstract

The invention relates to a method based on the electrochemical impedance spectroscopy method for the sensitive and selective determination of SARS-CoV-2 S1 protein for the diagnosis of COVID-19 disease, an impedimetric immunosensor, and a method for preparation thereof.

Description

IMPEDIMETRIC IMMUNOSENSOR FOR THE DIAGNOSIS OF COVID-19

Technical Field of the Invention

The invention relates to a method based on the electrochemical impedance spectroscopy method for the sensitive and selective determination of SARS-CoV-2 SI protein for the diagnosis of COVID-19 disease, an impedance immunosensor, and a method of preparation thereof.

The developed impedimetric immunosensor has the potential to be applied to the determination of SARS-CoV-2 SI protein in different sample matrices such as serum samples, nasal swabs, throat swabs, etc., as well as saliva samples.

State of the Art of the Invention (Background)

As a result of research on patients with symptoms such as fever, cough, and shortness of breath at the end of December 2019 in Wuhan Province, China, the cause of the disease was defined as "Novel Coronavirus" on 13 January 2020. The World Health Organization (WHO) declared a pandemic within a few weeks after it was announced that the virus was transmitted from animal to human and quickly transmitted from human to human (WHO, 2020). The novel Coronavirus has been officially named SARS-CoV-2 and the name of the pandemic disease caused by it was determined as COVID-19.

After the SARS-CoV-2 virus was identified, the first quantitative polymerase chain reaction (PCR) based diagnostic kit was designed, distributed, and deployed by WHO in January 2020. For measurement with this diagnostic kit, following oral or nasal sampling of a person with suspected illness by a healthcare professional, the nucleic acids (RNA) in the sample are isolated and purified, and the genetic structure of the virus is replicated by reverse transcription-polymerase chain reaction (RT-qPCR) using complementary DNA. A positive/negative result is reached with the signal value based on the optical method taken by quantitative PCR measurement. This first-designed test procedure is known to be complex and expensive, as well as suitable for use in large, centralized diagnostic laboratories. Although it takes 4-6 hours to complete the test, the duration of a diagnosis increases to 24 hours considering the sending of clinical samples to the centers where the test is performed and the logistic need. Thus, although PCR-based test methods are generally known to have high sensitivity and specificity, such nucleic acid-based tests are relatively costly, require specialist personnel, and are time-consuming (Sheridan, 2020).

In Turkey, the diagnostic test was first performed in the National Virology Reference Laboratory of the General Directorate of Public Health and determined Public Health Laboratories. Due to the widespread prevalence of the disease in Turkey and the necessity of performing many tests, many hospitals are now performing COVID-19 diagnostic tests. Apart from PCR-based diagnostic kits for the diagnosis of COVID-19, the existing test kits used in our country are produced by a International Bioeasy Biotechnology company in China. The first of these test kits, the COVID-19 IgG/IgM GICA (Colloidal Gold Immunochromatography Assay) Rapid Coronavirus test, detects the presence of IgG and IgM from blood samples. Second, the COVID-19 POCT Fluorescence Rapid Coronavirus test works with the Fluorescence Immunochromatographic Assay method and has higher sensitivity rates than the GICA rapid test kit. Sputum is used for early infection detection from nasal and throat swab samples (https://bioeasy.com.tr/covid-19-koronavirus-testleri/). In the colloidal gold immunochromatography assay, the use of monoclonal antibodies and colloidal gold increases the cost, and the sensitivity rate is lower than in the fluorescence immunochromatographic assay. The fluorescence measurement device used in the fluorescent immunochromatographic assay is again used in Point-of Care (PoC)— analyses or devices that prevent individual ease of use and are costly. In addition, it has disadvantages such as not being able to perform quantitative analysis with both methods, there may be differences in the interpretation of the results between the users, and exceedingly small volumes being insufficient in the analysis.

When the literature is examined, there are various electrochemical biosensor studies for the determination of COVID-19.

Rahmati et al. (2021) modified the disposable electrode surface with Q12O nanocubes (Q12O NCs) and immobilized ProtA (Protein A) to the modified electrode surface. Then, the IgG antibody was immobilized on the electrode surface and the free parts on the electrode surface were blocked with a 1% BSA (bovine serum albumin) solution. Different concentrations of SARS-CoV-2 spike protein were added to the prepared immunosensor surface and measurements were taken by electrochemical impedance spectroscopy (EIS) method. This sensor was used to detect the SARS-CoV-2 virus without any cross-reactivity when tested with influenza viruses 1 and 2. This study differs from the invention in terms of the difference in process steps as it includes a nanomaterial modification step of the electrode surface and Protein A and IgG antibody immobilization steps.

The procedure used by Rahmati et al. (2021) was also applied in the impedimetric biosensor developed by Ehsan et al. (2021) for the determination of SARS-CoV-2 spike protein. Immobilization of the SARS-CoV-2 spike SI IgG antibody, which is the capture antibody, to the surface was performed in 2 different ways with PBASE and ProtA-mediated binding. SARS-CoV-2 determination was performed on RBD protein. In addition, screen-printed electrodes were prepared by treating them with graphene/carbon ink on a cellulose-fiberbased paper substrate and used in the study. The invention differs from the said study due to differences in the electrode preparation procedure and the selection of RBD protein for the determination of SARS-CoV-2.

Witt et al. (2021) also developed an impedimetric biosensor for the determination of the SARS-CoV-2 SI protein. In the study using boron-doped poly crystalline diamond electrodes, the electrode surface was functionalized with the biotin-streptavidin binding complex and biotinylated anti-SARS CoV-2 SI antibodies. The SARS-CoV-2 SI protein was then incubated on the electrode surface. The use of a boron-doped diamond electrode in this study is a multi-step experimental procedure that differs from the procedure followed in the method of the invention in that the electrode surface is modified with biotin-streptavidin and the biotin-labeled SARS-CoV-2 SI antibody is immobilized on the surface.

Torres et al. (2021) developed an impedimetric biosensor for the determination of the SARS- CoV-2 virus. In the study, Angiotensin-converting enzyme 2 (ACE 2), which was firstly used as a bio-sensing element by using a glutaraldehyde crosslinker, was immobilized on the electrode surface. Then, the electrode surface was blocked with BSA (bovine serum albumin), and a protective membrane was formed on the surface with Nafion. SARS-CoV-2 determination was performed using spike protein. SARS-CoV-2 virus determination was carried out by the electrochemical impedance spectroscopy (EIS) method in the presence of a redox solution. The experimental procedure followed in this study differs from that adopted with the impedimetric immunosensor of the invention due to the development of paper-based electrodes and the use of a different sensing molecule.

In addition to the above-mentioned current publications, there are also studies in the literature using different electrochemical methods and different bio-sensory molecules (IgG, IgM, N protein, DNA, etc.) (Li et al. (2021), Li and Lillehoj (2021), Zhao et al. (2021), Yakoh et al. (2021), Rahmati et al. (2021), Liv (2021), Fabiani et al. (2021)).

When the existing patents for the detection of COVID-19 were examined, Abdohamid et al. developed an electrochemical approach to diagnosing COVID-19 infection in US 11,047,824 B2. With the electrochemical stimulator-analyzer system, the level of reactive oxygen species (ROS) in the sputum sample was measured with the current generated against the applied scanning potential and thus the COVID-19 infection status was determined. The determination of COVID-19 was performed using the electrochemical impedance spectroscopy (EIS) technique depending on the change in the level of reactive oxygen species. The invention differs in this respect depending on the content of the said patent.

Khayamian, Mohammad Ali et al. developed a biosensor system to diagnose COVID-19 infection in Patent No. US 2021/0223201 AL The system comprises a biosensor, an electrochemical stimulant-analyzer, and a processing unit. Charge transfer resistance (Ret) was measured using the electrochemical impedance spectroscopy method and cytokine storm was determined according to the results obtained. The determination of COVID-19 was performed by the electrochemical impedance spectroscopy (EIS) technique depending on the change in Ret values caused by a cytokine storm. The invention differs in this respect depending on the content of the said patent.

Finally, in Patent No. US 11,035,817 Bl, Shimaa et al. developed a cotton tip electrochemical immunosensor design for the detection of coronaviruses. In the study, the screen-printed electrodes are functionalized with the diazonium electrography method and modified with carbon nanofibers. SARS-CoV-2 determination was performed in ferro/ferrocyanide redox probe solution by square wave voltammetry (SWV) method. The said study differs from the invention in that it involves steps of modification of the electrode surface and the technique used in the determination method is different. Both in terms of the experimental procedure followed and the sensing molecule used, the impedimetric immunosensor of the invention is quite different from the three patents mentioned above.

Brief Description and Objects of the Invention

Within the scope of the invention, a procedure has been developed that is selective for SARS- CoV-2 SI protein, high sensitivity, immunosensor platform can be prepared in a shorter time and results can be obtained within the same day, which is applicable to point-of-care diagnostic kits, does not require expert personnel and cost-effective, without the need for modification with any nanomaterials on the electrode surface, compared to the previous method with the developed impedimetric immunosensor.

With the invention, an early diagnosis method based on impedance-based immunosensor technology has been developed for the development of new and national diagnostic kits that can be used in the field for the rapid and precise diagnosis of COVID-19.

Within the scope of the invention, an impedimetric immunosensor was developed based on the electrochemical impedance spectroscopy (EIS) technique for the determination of the SARS-CoV-2 SI protein.

With the invention, the surface of the disposable screen-printed carbon electrodes was chemically activated and an immunosensor was prepared by immobilizing the SARS-CoV-2 SI protein-specific capture antibody on this surface. SARS-CoV-2 SI protein was incubated on the surface of the antibody-immobilized electrode and a label-free immunosensor was developed. By electrochemical impedance spectroscopy (EIS) method, SARS-CoV-2 SI determination was carried out in a sensitive and selective manner by measuring the resistance (Ret) value against the charge transferred in the redox probe solution.

The SARS-CoV-2 SI protein is detected by the invention. In addition, it is applicable for the determination of SARS-CoV-2 Nucleocapsid protein (N), RBD (receptor binding domain) protein, Membrane protein (M), etc. by using the relevant target analyte using the appropriate capture antibody for the determination of SARS-CoV-2 proteins. The applicability of the impedimetric immunosensor of the invention to the sensitive and selective determination of SARS-CoV-2 SI in artificial saliva has been demonstrated.

Definitions of Figures Describing the Invention

The figures and related descriptions required to better understand the invention are as follows.

Figure 1: Flowchart of the experimental procedure followed in the development of the impedimetric-based immunosensor with the demonstrated application for SARS-CoV-2 SI determination

Figure 2: (A) Nyquist diagrams and (B) Calibration graph obtained from impedimetric measurements performed in the absence of SARS-CoV-2 SI Protein (a) on the surface of the impedimetric immunosensor developed for the determination of SARS-CoV-2 SI and in the presence of SARS-CoV-2 SI Protein at different concentrations: (b) 0.5 ng/mL, (c) 1 ng/mL, (d) 2 ng/mL, (e) 5 ng/mL, (f) 7 ng/mL, (g) 10 ng/mL.

Detailed Description of the Invention

Figure- 1 illustrates the flowchart for the development of the impedimetric-based immunosensor and the method for detecting the target analyte.

The impedimetric-based method of the invention for the detection of a target analyte in a test sample in the diagnosis of COVID-19 disease comprises the following process steps: a) Chemical activation of the electrode surface with a covalent binding agent for immobilization of capture antibody, b) Immobilization of a target analyte-specific capture antibody on the electrode surface, c) Blocking the electrode surface with a blocking agent, d) Incubation of the antigen, the target analyte, on the immunosensor surface, e) Performing measurements by electrochemical impedance spectroscopy (EIS) technique. Chemical activation of the electrode surface with a covalent binding agent

The surface of the carbon-based electrode was chemically activated using a pair of covalent binding agents [EDC (3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochi oride/NHS (N- Hydroxy succinimide)]. The chemical activation process ensures the immobilization of the capture antibody on the surface of the electrodes of the invention in the next step. In an application of the invention on a carbon-based electrode surface, the activation of the electrodes was studied in the range of 2-100 mM for EDC and 2-100 mM for NHS. The covalent binding agent was incubated on the electrode surface for 30-90 minutes.

A screen-printed carbon electrode was preferred as a carbon-based electrode in the invention. However, the developed impedimetric immunosensor can be applied to other electrodes such as carbon-based pencil graphite electrodes, glassy carbon electrodes, carbon paste electrodes, etc., to carbon-based materials such as graphene, carbon nanotube, carbon nanofiber, etc., to metallic nanoparticles such as silver, platinum, copper nanoparticles, etc., and other electrodes modified with polymeric materials such as dendrimers, conductive polymers, etc.

Immobilization of the capture antibody to the electrode surface

The impedimetric immunosensor developed for the determination of SARS-CoV-2 SI is an application of the invention. The SARS-CoV-2 SI protein-specific capture antibody was immobilized to the chemically activated electrode surface. The effect of the capture antibody, which varies in the concentration range of 0.25 - 10 pg/mL, on the immunosensor response based on the electrochemical method was observed. In this step of the method of the invention, SARS-CoV-2 Nucleocapsid protein (N), RBD protein, Membrane protein (M), etc. can perform the impedimetric determination of the target analyte using the appropriate capture antibody for the determination of SARS-CoV-2 proteins.

Blocking the electrode surface with a blocking agent

It is aimed to bind the SARS-CoV-2 SI protein, which is the target analyte with the highest efficiency, directly to the capture antibody and to prevent non-specific signals by closing the free zones on the electrode surface with the blocking agent ranging in the concentration range of 0.1-500 pg/mL to the immobilized surface of the capture antibody. BSA (bovine serum albumin) was used as a surface-blocking agent in the developed impedimetric immunosensor. It can be adapted to the impedimetric determination of the target analyte using BSA or other blocking agents (such as ethanolamine, casein, etc.) with the appropriate capture antibody for the determination of different SARS-CoV-2 proteins.

Incubation of the antigen, which is the target analyte on the immunosensor surface

The SARS-CoV-2 SI protein incubation, which is the target analyte, has been observed to affect the immunosensor response of the interaction between the capture antibody and the analyte by working at different times ranging from 30 - 90 minutes and temperatures ranging from 25-42°C.

EIS Measurements

Measurement was performed in the frequency range of 100000 Hz - 0.01 Hz in 1-5 mM Fe(CN)6^3-/4'redox probe solution containing KC1 by electrochemical impedance spectroscopy (EIS) method.

The appropriate Randles circuit model was applied to the Nyquist diagrams obtained as a result of the impedimetric measurement and the analysis was carried out using the fit data. In the Randles circuit model, the magnitudes of the elements of solution resistance (Rs), capacitance (Q), charge transfer resistance (Ret) representing the semicircle in the Nyquist diagram, and Warburg impedance (W) representing the diffusion region of the impedance measurement can be measured. The analyses were performed by measuring the resistance (Ret) values against the transferred charge.

The limit of determination in the SARS-CoV-2 SI concentration range of 0.5 ng/mL - 10 ng/mL was calculated with the impedimetric-based immunosensor developed for the determination of SARS-CoV-2 SI, and the Nyquist diagram and the resulting calibration graph are shown in Figure-2.

In order to demonstrate the selectivity of the developed impedimetric immunosensors to the SARS-CoV-2 SI protein, an application was performed in the presence of the Influenza Hemagglutinin (HA) protein and no significant cross-reactivity was observed. The developed immunosensor can be adapted to the determination of SARS-CoV-2 SI selectively even in the presence of proteins such as MERS-CoV, SARS-CoV, etc.

In order to demonstrate the applicability of the immunosensors developed in the study to the real samples, an application was carried out in artificial saliva, and the impedimetric determination of the SARS-CoV-2 SI protein at increasing concentrations was possible with the impedimetric immunosensor of the invention. The developed impedimetric immunosensor can be adapted to the determination of SARS-CoV-2 SI protein in different sample matrices such as saliva samples as well as serum samples, nasal swabs, throat swabs, etc.

With the immunosensor of the invention for the impedimetric determination of SARS-CoV-2 SI protein, analysis can be performed using a sample amount in the range of 5-10 pL.

With the immunosensor platform of the invention, the determination of SARS-CoV-2 SI protein can be adapted by monitoring the change in the redox probe signal by a voltammetric method.

The immunosensor of the invention, the application of which has been demonstrated for the determination of SARS-CoV-2 SI with disposable electrodes, can also be adapted to multielectrode systems. It can also be adapted for the impedimetric determination of the target analyte of interest in multi -el ectrode systems by using a suitable capture antibody for the determination of SARS-CoV-2 related proteins.

The demonstrated immunosensor for SARS-CoV-2 SI determination can be integrated into a portable impedance analyzer and adapted for the impedimetric determination of SARS-CoV-2 SI protein.

Claims

1. An impedimetric-based method for the detection of a target analyte in a test sample for diagnosis of COVID-19 disease, characterized in that it comprises the process steps; a) Chemical activation of the electrode surface with a covalent binding agent for immobilization of the capture antibody, b) Immobilization of a target analyte-specific capture antibody on the electrode surface, c) Blocking the electrode surface with a blocking agent, d) Incubation of the antigen, the target analyte, on the immunosensor surface, e) Performing measurements by electrochemical impedance spectroscopy (EIS) technique.

2. A method according to claim 1, characterized in that the said target analyte is SARS- CoV-2 SI protein and the said analyte-specific capture antibody is a SARS-CoV-2 SI protein-specific capture antibody.

3. A method according to claim 1, characterized in that the test sample is saliva, serum, or nasal and throat swabs.

4. A method according to claims 1 or 3, characterized in that the test sample is 5-10 pL.

5. A method according to claim 1, characterized in that the electrode is a carbon-based electrode.

6. A method according to claim 5, characterized in that the carbon-based electrode is a screen-printed carbon electrode.

7. A method according to claim 5, characterized in that the said carbon-based electrode is a pencil graphite electrode, a glassy carbon electrode, or a carbon paste electrode, etc.

8. A method according to claim 1, characterized in that the said electrode is modified with carbon-based materials, metallic nanoparticles, or polymeric materials, etc.

9. A method according to claim 1, characterized in that the target analyte is SARS-CoV- 2 Nucleocapsid protein (N), RBD protein, or Membrane protein (M).

10. A method according to claim 1, characterized in that the said blocking agent is BSA (bovine serum albumin).

11. A method according to claim 1, characterized in that the said blocking agent is ethanolamine, casein, etc.

12. A method according to claim 1, characterized in that the electrode is disposable or comprises a multi-electrode system.

13. An immunosensor, characterized in that it operates with the method according to claim 1.

14. An immunosensor according to claim 13, characterized in that it can be integrated to the portable impedance analyzer and adaptable to the impedimetric determination of the SARS-CoV-2 SI protein.