Method for developing optimer-based electrochemical biosensor

Abstract

The invention relates to an optimer-based electrochemical biosensor for use in the determination of SARS-CoV-2 S1 protein and Moxifloxacin as well as the method for its operation. The invention also includes an application of said method for the diagnosis of disease and the determination of drugs. In this context, SARS-CoV-2 S1 protein and Moxifloxacin were selected as target analytes and electrochemical analyses of these target analytes were performed using optimer-based procedure and electrochemical impedance spectroscopy technique.

Description

[0001] METHOD FOR DEVELOPING OPTIMER-BASED ELECTROCHEMICAL BIOSENSOR

[0002] Technical Field of the Invention

[0003] The invention relates to an optimer-based electrochemical biosensor for use in the determination of SARS-CoV-2 S1 protein and Moxifloxacin as well as the method for its operation. The invention also includes an application of said method for the diagnosis of disease and the determination of drugs.

[0004] State of the Art

[0005] There are various test protocols for the diagnosis of COVID-19 disease. Among these, the PCR method is defined as the gold standard and is widely used. PCR test is performed with nasopharyngeal swab or sputum samples taken from the individual. Although PCR tests have a high degree of precision and accuracy, they have a drawback in that skilled persons are needed to conduct the test, and the results can vary from a few hours to 1 -2 days. For this reason, various alternative diagnostic methods are being developed that provide much faster results and allow individuals to perform the test on their own without the need for experienced personnel. Antibody tests for the identification of IgG and IgM antibodies are the most used of these alternative test techniques. However, the disadvantage is that antibody tests do not have sufficient sensitivity, can show false negative results, and have difficulty in detecting new virus mutants. For such reasons, it is crucial to develop tests for COVID-19 that are fast, accurate, and easy for people to perform on their own, without the need for any experienced personnel.

[0006] The recent increase in resistance to antibiotics worldwide represents a significant challenge. Avoiding the unnecessary use of broad-spectrum antibiotics, increasing rational drug use, organizing individualized treatment protocols and adjusting drug doses are extremely crucial in terms of preventing resistance to antibiotics and maintaining the therapeutic effects of existing drugs. For this reason, the rapid determination of drugs at the bedside is important in terms of preventing such problems, monitoring treatment, and reducing the burden on hospitals (Heysell et al., 2015; Lee et al., 2013). Optimers are aptamers optimized for a target analyte. The smallest sequence of aptamers that binds to the target analyte with high specificity as a result of various optimization processes is called an optimer. As a result of various optimization steps, optimers have advantages over aptamers such as structural stability, target affinity, synthesis efficiency, lower cost of production, tissue penetration, and access to target epitopes (Aptamer Group, 2021 ).

[0007] In Patent No. WO 2021 / 255479 A1 , Tolley et al. have developed an optimer for the SARS-CoV-2 spike protein. It is stated that the optimer developed in the study also binds to SARS-CoV-2 S1 , S2, and RBD proteins. With the optimer developed in this study, there is no application related to electrochemical biosensor based on an electrochemical determination method to be used in the determination of SARS-CoV-2 S1 protein and Moxifloxacin.

[0008] There are several studies in the literature on the electrochemical determination of moxifloxacin, but there are no optimer-based or aptamer-based electrochemical biosensor studies. Fekry (2017) developed an electrochemical sensor for the determination of moxifloxacin. In the study, the oxidation of Moxifloxacin at different pH values in Brittion Robinson buffer with carbon paste electrode modified with silver nanoparticles was investigated. The application of the developed sensor was carried out in pharmaceutical tablets and human urine samples. In another study by the same team (Fekry et al. 2021 ), the carbon paste electrode was modified with Nation, graphene oxide, and zeolite. Analysis was performed in phosphate buffer (PBS, pH 7.4) medium by alternating voltammetry (CV) technique. In addition, urine samples were applied. Neither study is optimer-based. Hammam et al. (2018) have developed a molecularly imprinted polymer (MlP)-based electrochemical sensor for the determination of moxifloxacin. The synthesized molecularly imprinted polymer was used as a recognition material on polyvinyl chloride (PVC) membrane, and Moxifloxacin was determined by potentiometry technique. With the developed method, application was made in pharmaceutical tablets and urine samples. As should be clear, this study is not optimerbased, and the determination of Moxifloxacin is carried out by the MIP method. Jiang et al. (2016) have developed a molecularly imprinted polymer (MlP)-based electrochemical sensor for the determination of moxifloxacin. A molecularly imprinted polymer was immobilized on the surface of a graphene-modified glassy carbon electrode (GCE). In the analyses performed with the differential pulse voltammetry technique, the method was applied in pharmaceutical tablets and urine samples. In this study, which was carried out without the use of optimer, the determination of Moxifloxacin is performed by MIP method, the electrode surface used is modified, and differential pulse voltammetry is used as a measurement technique. In addition, there are no patents involving an optimerbased biosensor study for the determination of Moxifloxacin in the state of the art.

[0009] There is a need to develop new methods and products to eliminate the disadvantages stated above and in the state of the art.

[0010] Summary and Objects of the Invention

[0011] The invention describes an optimer-based electrochemical biosensor for use in the determination of SARS-CoV-2 S1 protein and Moxifloxacin as well as the method for its operation. The invention also includes an application of said method for the diagnosis of disease and the determination of drugs. In this context, SARS-CoV-2 S1 protein and Moxifloxacin were selected as target analytes and electrochemical analyses of these target analytes were performed using optimer-based procedure and electrochemical impedance spectroscopy technique.

[0012] The optimer-based electrochemical biosensor of the invention for use in the determination of SARS-CoV-2 S1 protein and Moxifloxacin has the potential to be applied to the electrochemical determination of the target analyte by using the appropriate optimer specific to different analytes (protein, toxin, antigen, etc.). There is also the potential of the developed optimer-based electrochemical biosensor to be applied to the determination of different analytes using target analyte-specific optimer in different sample matrices such as serum, urine, saliva, nasal swab, throat swab, sweat, etc.

[0013] By combining the many advantages of biosensors and high affinity of optimers for the target analyte, the study of the invention presents an optimer-based electrochemical biosensor based on electrochemical impedance spectroscopy technique for the determination of different analytes.

[0014] An application with an optimer-based electrochemical biosensor of the invention for the determination of SARS-CoV-2 S1 protein and Moxifloxacin is provided. The invention provides a highly precise, selective, accurate, fast, easy-to-use, cost- effective optimer-based electrochemical biosensor for target analyte determination.

[0015] Description of the Drawings

[0016] The embodiments of the invention, briefly summarized above and discussed in more detail below, can be understood with reference to the example embodiments of the invention described in the accompanying drawings.

[0017] However, it should be noted that the accompanying drawings only illustrate typical embodiments of this invention and are not to be considered as limiting to its scope.

[0018] Figure 1 : A representative flowchart of the procedure followed in the preparation and application of the optimer-based electrochemical biosensor of the invention, showing an application of the electrochemical impedance spectroscopy technique, which is an electrochemical method. Wherein:

[0019] A: Performing the optimer-target analyte interaction,

[0020] B: Immobilization of an optimer target analyte complex on the electrode surface,

[0021] C: Performing electrochemical measurements and obtaining results.

[0022] Figure 2: Nyquist curves obtained from impedimetric measurements of an optimerbased electrochemical biosensor developed for the determination of SARS-CoV-2 S1 protein, taken (a) in the absence of SARS-CoV-2 S1 protein and (b) in the presence of SARS-CoV-2 S1 protein.

[0023] Figure 3: Nyquist curves obtained from impedimetric measurements of an optimerbased electrochemical biosensor developed for the determination of Moxifloxacin, taken (a) in the absence of Moxifloxacin and (b) in the presence of Moxifloxacin.

[0024] Detailed Description of the Invention

[0025] The invention relates to an optimer-based electrochemical biosensor for use in the determination of SARS-CoV-2 S1 protein and Moxifloxacin as well as the method for its operation. The invention also includes an application of said method for the diagnosis of disease and the determination of drugs. In this context, SARS-CoV-2 S1 protein and Moxifloxacin were selected as target analytes and electrochemical analyses of these target analytes were performed using optimer-based procedure and electrochemical impedance spectroscopy technique.

[0026] The example embodiments are described in more detail below with reference to the accompanying descriptions. Furthermore, the embodiments can be established in different forms and should not be interpreted as being limited to the embodiments specified herein. Rather, these example embodiments are provided so that this description will be thorough, and will fully convey the scope to those skilled in the art.

[0027] Within the scope of the invention, an optimer-based electrochemical biosensor has been introduced, and an application of the developed optimer-based electrochemical biosensor for disease diagnosis and drug determination has been demonstrated. The invention presents an optimer-based electrochemical biosensor based on electrochemical impedance spectroscopy (EIS) technique and demonstrates its application for the electrochemical determination of SARS-CoV-2 S1 protein and Moxifloxacin.

[0028] In the invention, immobilization of the optimer- target analyte complex interacted in the solution phase is achieved on the surface of a pencil graphite electrode. SARS-CoV-2 S1 protein and Moxifloxacin were determined precisely and selectively by measuring the resistance to charge transfer (Ret) value in the redox probe solution with the EIS method.

[0029] It is possible that the optimer-based electrochemical biosensor of the invention is applicable to the determination of target analytes in different sample mediums.

[0030] The electrochemical biosensor for use in the determination of SARS-CoV-2 S1 protein and Moxifloxacin comprises surface-modified, disposable and multi-electrode system- adaptable electrode on which electrochemical measurement is performed, and said biosensor of the invention is based on optimer. Said electrode is pencil graphite electrode, glassy carbon electrode, carbon paste electrode, screen-printed electrode or gold electrode. The surface of said electrode is modified with carbon-based materials, metallic nanoparticles or polymeric materials.

[0031] In an embodiment of the invention, the optimer contained in the electrochemical biosensor of the invention is in the concentration range of 0.01 nM - 100 nM. The procedure followed in the preparation and application of the optimer-based electrochemical biosensor of the invention, showing an application of the electrochemical impedance spectroscopy technique, which is an electrochemical method, is shown in Figure 1.

[0032] The preparation method of an optimer-based electrochemical biosensor of the invention comprises the process steps of; i. performing the optimer-target analyte interaction, ii. immobilizing an optimer-target analyte complex on the electrode surface. Said target analytes are SARS-CoV-2 S1 protein and Moxifloxacin.

[0033] In an embodiment of the invention, the preparation method of an optimer-based electrochemical biosensor of the invention comprises the process steps of; i. interacting the target analyte in the concentration range of 0.01 aM - 500 nM and the specific optimer in the concentration range of 0.01 nM - 100 nM by mixing for 5 - 60 min to perform the optimer-target analyte interaction, ii. immobilizing the optimer-target analyte complex to the electrode surface for 15 - 60 min to perform immobilization of the optimer-target analyte complex to the electrode surface.

[0034] Said target analytes are SARS-CoV-2 S1 protein or Moxifloxacin.

[0035] The operation method of an optimer-based electrochemical biosensor of the invention comprises the process steps of; i. performing electrochemical determination of the target analyte by electrochemical impedance spectroscopy technique, an electrochemical method, in ferricyanide / ferrocyanide [Fe(CN)6]3" / 4-redox probe solution prepared in potassium chloride (KCI) or performing electrochemical determination of the target analyte by voltammetric method by monitoring the change in the redox probe signal.

[0036] Said target analytes are SARS-CoV-2 S1 protein and Moxifloxacin.

[0037] In an embodiment of the invention, the operation method of an optimer-based electrochemical biosensor of the invention comprises the process step of; i. performing electrochemical determination of the target analyte in an amount of 5 - 40 pL by electrochemical impedance spectroscopy, an electrochemical method, in ferricyanide / ferrocyanide [Fe(CN)6]3" / 4-redox probe solution prepared in potassium chloride (KCI) with a concentration in the range of 1 - 5 mM in a frequency range of 1000000 Hz - 0.01 Hz or performing electrochemical determination of the target analyte by voltammetric method by monitoring the change in the redox probe signal.

[0038] Said target analytes are SARS-CoV-2 S1 protein and Moxifloxacin.

[0039] The analysis was performed by applying the appropriate Randles circuit model to the Nyquist curves obtained as a result of impedimetric measurement and using the fitted data. In the Randles circuit model, the magnitudes of elements of solution resistance (Rs), capacitance (Q), resistance to charge transfer (Ret) representing the semicircle in the Nyquist curve, and Warburg impedance (W) representing the diffusion region of the impedimetric measurement can be measured. Resistance to charge transfer (Ret) values were measured and analyses were performed.

[0040] The target analytes selected as the application of the optimer-based electrochemical biosensor of the invention are SARS-CoV-2 S1 protein and Moxifloxacin. For these analytes, electrochemical analysis is performed with biosensors based on a one-to-one specific optimer, which results in 15 - 45 minutes.

[0041] For the target analytes, SARS-CoV-2 S1 protein and Moxifloxacin, selected as the application of the optimer-based electrochemical biosensor of the invention; the limits of determination in the concentration range of 0.01 aM - 500 nM were calculated separately and representative Nyquist curves are shown in Figure-2 and Figure-3.

[0042] There is also the potential of the optimer-based electrochemical biosensor of the invention to be applied to the determination of different target analytes using targetspecific optimer in different sample matrices such as serum, urine, saliva, nasal swab, throat swab, sweat, etc. The optimer-based electrochemical biosensor of the invention is intended for the determination of different target analytes in different sample matrices such as serum, urine, saliva, nasal swab, throat swab, sweat, etc. by using target-specific optimer is provided by the process step of performing the optimer-target analyte interaction in the preparation method of the electrochemical biosensor. In the optimer-based electrochemical biosensor introduced within the scope of the invention and whose applications for SARS-CoV-2 S1 protein and Moxifloxacin have been demonstrated, the biosensor shows high selectivity to the target analyte, since the appropriate optimer is used to select the target analyte.

[0043] Within the scope of the invention, an optimer-based electrochemical biosensor has been introduced, and an application thereof for disease diagnosis and drug determination has been demonstrated. In this context, SARS-CoV-2 S1 protein and Moxifloxacin were selected as target analytes and electrochemical analyses of these target analytes were performed using optimer-based procedure and electrochemical impedance spectroscopy technique.

[0044] The optimer-based electrochemical biosensors developed for the determination of SARS-CoV-2 S1 protein and Moxifloxacin has the potential to be applied to the electrochemical determination of the target analyte by using the appropriate optimer specific to different analytes (protein, toxin, antigen, etc.). In addition, there is also the potential of the optimer-based electrochemical biosensor of the invention to be applied to the determination of different analytes using target analyte-specific optimer in different sample matrices such as serum, urine, saliva, nasal swab, throat swab, sweat, etc.

[0045] In an optimer-based electrochemical biosensor of the invention for use in the determination of SARS-CoV-2 S1 protein and Moxifloxacin, the optimer-target analyte interaction occurs in the solution phase or on the electrode surface within the scope of the selected target analyte.

[0046] In the optimer-based electrochemical biosensor for use in the determination of SARS- CoV-2 S1 protein and Moxifloxacin, presented within the scope of the invention, the target analyte demonstrates high selectivity in the presence of other interfering species (protein, drug, etc.) since a one-to-one specific optimer is used for the target analyte.

[0047] The operation method of the biosensor of the invention can be integrated into the portable impedance meter device and can be adapted to the determination of different target analytes using a suitable optimer specific to the target analyte.

[0048] Industrial Applicability of the Invention The invention relates to an optimer-based electrochemical biosensor for use in the determination of SARS-CoV-2 S1 protein and Moxifloxacin as well as the method for its operation, and is industrially applicable.

[0049] The invention is not limited to the above descriptions and the person skilled in the art can readily present other different embodiments of the invention. These should be considered within the protection scope of the invention claimed by the claims.

[0050] References

[0051] • Fekry, A. M. 2017. “A new simple electrochemical Moxifloxacin Hydrochloride sensor built on carbon paste modified with silver nanoparticles”, Biosensors and Bioelectronics, 87, 1065-1070.

[0052] • Fekry, A. M., Abdel-Gawad, S. A., Azab, S. M., Walcarius, A. 2021. “A sensitive electrochemical sensor for Moxifloxacin Hydrochloride based on Nafion / graphene oxide / zeolite modified carbon paste electrode”, Electroanalysis, 33(4), 964-974.

[0053] • Hammam, M. A., Wagdy, H. A., El Nashar, R. M. 2018. “Moxifloxacin hydrochloride electrochemical detection based on newly designed molecularly imprinted polymer”, Sensors and Actuators B: Chemical, 275, 127-136.

[0054] • Heysell, S. K., Moore, J. L., Peloquin, C. A., Ashkin, D., Houpt, E. R. 2015. “Outcomes and use of therapeutic drug monitoring in multidrug-resistant tuberculosis patients treated in Virginia, 2009-2014”, Tuberculosis and respiratory diseases, 78(2), 78-84.

[0055] • Home Page - 2023. Aptamer Group. (2023, September 25). https: / / aptamergroup.com /

[0056] • Jiang, Z., Li, G., Zhang, M. 2016. “A novel sensor based on bifunctional monomer molecularly imprinted film at graphene modified glassy carbon electrode for detecting traces of moxifloxacin”, RSC advances, 6(39), 32915-32921 . • Lee, C. R., Cho, I. H., Jeong, B. C., Lee, S. H. 2013. “Strategies to minimize antibiotic resistance”, International journal of environmental research and public health, 10(9), 4274-4305.

Claims

CLAIMS1 . An electrochemical biosensor for use in the determination of SARS-CoV-2 S1 protein and Moxifloxacin, characterized in that it comprises a surface- modified electrode on which the electrochemical measurement is performed, and is optimer-based.

2. An electrochemical biosensor according to claim 1 , characterized in that said electrode is a pencil graphite electrode, a glassy carbon electrode, a carbon paste electrode, a screen-printed electrode or a gold electrode.

3. An electrochemical biosensor according to claims 1 or 2, characterized in that the surface of said electrode is in a modified form with carbon-based materials, metallic nanoparticles or polymeric materials.

4. An electrochemical biosensor according to claim 1 , characterized in that said optimer is in a concentration range of 0.01 nM - 100 nM.

5. A preparation method of an electrochemical biosensor, characterized in that it comprises the process steps of; i. performing the optimer-target analyte interaction ii. immobilization of an optimer-target analyte complex on the electrode surface.

6. A preparation method according to claim 5, characterized in that it comprises the process steps of; i. interacting the target analyte in the concentration range of 0.01 aM - 500 nM and the specific optimer in the concentration range of 0.01 nM - 100 nM by mixing for 5 - 60 min in order to perform the optimer-target analyte interaction, ii. immobilizing the optimer-target analyte complex to the electrode surface for 15 — 60 min to perform immobilization of the optimer-target analyte complex to the electrode surface.

7. A preparation method according to any one of claims 5 or 6, characterized in that said target analyte is SARS-CoV-2 S1 protein or Moxifloxacin.

8. A preparation method according to any one of claims 5 or 6, characterized in that said electrode in process step ii is a pencil graphite electrode, a glassy carbon electrode, a carbon paste electrode, a screen-printed electrode or a gold electrode.

9. A preparation method according to any one of claims 5 or 6, characterized in that the surface of said electrode in process step ii is in a modified form with carbon-based materials, metallic nanoparticles or polymeric materials.

10. An operation method of an optimer-based electrochemical biosensor, characterized in that it comprises the process step of; i. performing electrochemical determination of the target analyte by electrochemical impedance spectroscopy technique, an electrochemical method, in ferricyanide / ferrocyanide [Fe(CN)6]3" / 4-redox probe solution prepared in potassium chloride (KCI) or performing electrochemical determination of the target analyte by voltammetric method by monitoring the change in the redox probe signal.1 1 . An operation method according to claim 10, characterized in that it comprises the process step of; i. performing electrochemical determination of the target analyte in an amount of 5 - 40 pL by electrochemical impedance spectroscopy, an electrochemical method, in ferricyanide / ferrocyanide [Fe(CN)6]3" / 4-redox probe solution prepared in potassium chloride (KCI) with a concentration in the range of 1 - 5 mM in a frequency range of 1000000 Hz - 0.01 Hz or performing electrochemical determination of the target analyte by voltammetric method by monitoring the change in the redox probe signal.

12. An operation method according to any one of claims 10 or 11 , characterized in that said target analyte is SARS-CoV-2 S1 protein or Moxifloxacin.

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