A method for detecting cytokines and a sensor for their detection

Abstract

The invention relates to an electrochemical impedimetric sensor for interleukin 8 (IL-8) biomarker detection in biological sample based on glass electrode which consists of: • working electrode as a stable platform in the form of a FTO glass substrate modified with nanodiamonds with carboxyl groups (-COOH) present on their surface, • reference electrode, • anti electrode, • electrolyte, • linker in the form of EDC / NHS, detection layer: anti-IL-8 antibodies applied on the solid platform modified with a linker. The invention also provides a method for detecting the IL-8 biomarker in a biological sample using an electrochemical impedance sensor as defined above.

Description

[0001] A method for detecting cytokines and a sensor for their detection.

[0002] The invention relates to an ultrasensitive method for detecting the Interleukin 8 (IL-8) protein and an electrochemical impedance sensor for detecting this protein in a biological sample.

[0003] Interleukin 8 (IL-8) is a proinflammatory cytokine involved in neutrophils, basophils, and lymphocyte T migration to the site of the ongoing inflammation process. IL-8 is produced by many different types of cells including activated monocytes, macrophages, vascular endothelial cells, fibroblasts, and keratinocytes (https: / / pubmed.ncbi.nlm.nih, gov / 1 639201. https: / / pubmed.ncbi.nIm.nih.gov / 7964163 / , https: / / pubmed.ncbi.nIm.nih.gov / 22106907 / , https: / / pubmed.ncbi.nlm.nih.gov / 2648570 / , https: / / pubmed.ncbi.nlm.nih.gov / 7964163 / ). The production of IL-8 is temporary and depends on the activation stimuli such as injury, microbial infection, autoimmune reaction, and others (1639201, 7964163). As a signaling molecule, IL-8 plays a crucial role in neutrophils chemotaxis and degranulation, but also in the proliferation and survival of endothelial cells, and adjustment of extracellular matrix composition via increased production of proteases (https: / / pubmed.ncbi. nlm.nih.gov / 2677047 / , https: / / pubmed.ncbi.nlm.nih.gov / 1639201 / , https: / / pubmed.ncbi.nlm.nih.gov / 15862344 / , https: / / pubmed.ncbi.nlm.nih.gov / 3480540 / , https: / / pubmed.ncbi.nlm.nih.gov / 2824608 / , https: / / pubmed.ncbi.nlm.nih.gov / 3258625 / , https: / / pubmed.ncbi.nlm.nih.gov / 2835419 / , https: / / pubmed.ncbi.nlm.nih.gov / 12626597 / ). Besides the significant function of IL-8 in primary and acute inflammation, the dysregulated production of IL-8 was linked with many pathological conditions such as the development of cancer oorr chemoresistance (https: / / pubmed.ncbi.nlm.nih.gov / 32980444 / , https: / / pubmed.ncbi.nlm.nih.gOv / 30703432 / , https: / / pubmed.ncbi.nlm.nih.gov / 28866366 / , https: / / pubmed.ncbi.nlm.nih.gov / 35582268 / , abnormal angiogenesis

[0004] (https: / / pubmed.ncbi.nlm.nih.gOv / l 5831231 / , https: / / pubmed.ncbi.nlm.nih.gov / 15831231 / ), rheumatoid arthritis (https: / / pubmed.ncbi.nlm.nih.gov / 2209707 / ), asthma (https: / / pubmed.ncbi.nlm.nih.gov / 9196Q89 / , https: / / pubmed.ncbi.nlm.nih.gov / 34453785 / ), cystic fibrosis (https: / / pubmed.ncbi.nlm.nih.gov / 34726115 / ) or psoriasis (https: / / pubmed.ncbi.nlm.nih.gov / 34769465 / ). The most commonly used and commercially available techniques to evaluate the levels of IL-8 protein and / or CXCL-8 (the gene coding IL-8 protein) RNA levels are quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA). Both of the assays show great sensitivity and reproducibility' in the detection of IL-8 protein and / or CXCL-8 RNA in many' publications (https: / / pubmed.ncbi.nlm.nih.gov / 36696085 / , https: / / pubmed.ncbi.nlm.nih.gov / 19726162 / , https: / / pubmed.ncbi.nlm.nih.gov / 2407200Q / , https: / / pubmed.ncbi.nlm.nih.gov / 20577780 / , https: / / pubmed.ncbi.nlm.nih.gov / 17848739 / ). Nevertheless, either qPCR or ELISA require two crucial factors: highly' experienced and well-trained staff to carry out the assays as well as (2) the specifically' equipped lab. Therefore, there is a great need to develop straightforward and rapid biosensor for detection of IL-8 and / or CXCL-8 gene.

[0005] Electrochemical Impedance Spectroscopy

[0006] Precise detection of biomarkers can be also performed with the use of electrochemical impedance spectroscopy (EIS) which is based on the measurement of charge transfer resistance between working and counter electrodes in the widest range of frequency, from 1 mHz to 100 MHz.

[0007] EIS consists in measurement of electrochemical impedance by' applying a low sinusoidal voltage at the specific frequency' (typically from 1 mHz to 1 MHz) to the sensor electrodes and the reading of the current passing through the system. Additionally, electrochemical sensors are polarised by constant voltage (from few to several hundred milivolts).

[0008] Impedimetric biosensors are based on EIS. The change of the charge transfer value of a biosensor occurs when a target substance, e.g. a protein, binds with the receptor molecules immobilised on the surface of impedimetric biosensor electrodes. Difference in the level of charge transfer resistance detected before and after binding of a target substance with the receptor molecules allows for the detection of a target substance in solution.

[0009] The goal of the present invention is a delivery of efficient, easy and fast method of IL-8 protein detection by means of electrochemical impedimetric sensor enabling to distinguish the presence of this marker from others present in analysed biological samples.

[0010] The core of the present invention is the electrochemical impedimetric sensor and the way it functions. The platform of the sensor consists of Fluorine Tin Oxide (FTO) glass substrate modified with nanodiamonds with carboxyl groups (-COOH) on their surface. Anti-IL-8 antibodies are immobilised on the platform and enable specific recognition of the biomarker, IL-8.

[0011] The subject of the invention is an electrochemical impedimetric sensor for Interleukin 8 (IL-8) biomarker detection in biological sample based on the use of a glass electrode which consists of:

[0012] • working electrode as a platform in the form of a glass substrate covered with tin doped fluorine oxide modified with nanodiamonds with -COOH groups on their surface,

[0013] • reference electrode in the form of silver chloride electrode,

[0014] • counter electrode - a platinium wire,

[0015] • electrolyte: 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] / 0.5M KC1 solution,

[0016] • linker in the form of EDC / NHS,

[0017] • detection layer: antibodies immobilised on the electrode surface.

[0018] A method of detection of IL-8 biomarker in biological sample with the use of electrochemical impedance sensor defined above is based on the following:

[0019] • a biological sample solution containing IL-8 protein is incubated over the modified surface of working electrode of an electrochemical impedance spectroscopy (EIS) meter consisting of:

[0020] - APTES layer,

[0021] - layer of nanodiamonds with -COOH groups on their surface,

[0022] - EDC / NHS linker layer,

[0023] - detection layer in the form of anti-IL-8 antibodies diluted 1000 times in PBS,

[0024] • the working platform of EIS sensor is connected to electrolyte and the electrochemical impedance spectroscopy is measured together with measurement data aquisition,

[0025] • measurement data are furhter compared with the EIS measurement registered in the same conditions for the electrode incubated in biological sample solution without IL- 8 biomarker, • finally, measurement data are compared with the EIS data acquired in the same conditions for the electrode incubated with biological sample containing biomarkers other than IL-8,

[0026] • then the obtained measurement results are compared with the results of the EIS measurement recorded under the same conditions for an electrode incubated in a solution of a biological sample containing biomarkers other than IL-8.

[0027] The method in which EIS measurement is performed using alternating current with the amplitude equal to 10 mV.

[0028] The method in which EIS measurement is performed at the frequency range from 10 kHz to 0,1 Hz

[0029] The method in which platinium wire is used as a counter electrode.

[0030] The method in which Ag / AgCl / 3M KC1 is used as a reference electrode.

[0031] The method in which the measurement is performed with the use of 5mM K3[Fe(CN)6] / K4[Fe(CN)6] / 0.5M KC1 as an electrolyte solution.

[0032] The method in which the measurement is performed against a formal potential of redox reaction: Fe2+ / Fe3+

[0033] The method in which incubation of working electrode (in the form of a glass FTO electrode modified with nanodiamonds) with the biological sample is performed in room temperature in a time range from 5 to 30 minutes.

[0034] The terms which were used above and in the invention description and the patent claims have the following meaning:

[0035] APTES - signifies (3-Aminopropyl)triethoxysilane

[0036] EDC - signifies (l-ethyl-3- (3-dimethylaminopropyl)carbodiimide hydrochloride

[0037] NHS - signifies N-Hydroxysuccinimide

[0038] PBS - signifies phosphate buffered saline

[0039] EIS - signifies electrochemical impedance spectroscopy

[0040] IL-8 - signifies IL-8 protein

[0041] FTO - glass electrode covered with fluorine tin oxide layer

[0042] Reference electrode - Ag / AgCl / 3M KC1 silver chloride electrode

[0043] Anti electrode - platinum wire Electrolyte - means a solution of 5mM K3[Fe(CN)6] / K4[Fe(CN)6] / 0.5M KC1 in which the electrochemical EIS measurement is carried out.

[0044] Description of the figures:

[0045] Fig. 1 - shows a general scheme of the invention.

[0046] Fig. 2 - shows a graph of impedance changes after electrode modification with APTES followed by antibodies immobilisation, and blocking the surface of the electrode with BSA. Fig. 3 - shows a graph of impedance changes after electrode modification with antibodies; at the stage of background reading, i.e. after electrode incubation with BSA; at the stage of IL-8 protein detection.

[0047] Fig. 4 — shows a graph of impedance changes after electrode modification with antibodies; after blocking the surface of the electrode with BSA; after detection of [Lamyloid 1 -40 as a negative control.

[0048] The invention is illustrated by the following examples of implementation, which do not constitute a limitation of the invention

[0049] To the solid platform (FTO glass electrode modified with nanodiamonds) are the antibodies attached which enable detection of IL-8 biomarker.

[0050] Example 1:

[0051] Cleaning of glass electrodes

[0052] Before use FTO glass substrate was cleaned in the ultrasonic cleaner for 5 minutes in acetone, for 5 minutes in anhydrous athanol, and for 15 minutes in deionised water, followed by drying in a stream of argon. Electrodes were then dried in 70°C for 60 minutes. Next, electrodes were incubated for 60 minutes in basic pirahna solution composed of ddH2O : ammonia solution (below 25%) : 30% H2O2at the ratio of 5 : 1 : 1 heated to 60°C. Next, electrodes were washed with deionised water and dried in the stream of argon. Clean electrodes were placed in 5% APTES ((3-aminopropylo)-trietoxysilan) solution in anhydrous ethanol and incubated in room temperature for 1 hour in the dark. Electrodes were then washed with anhydrous ethanol, dried in the stream of argon and placed in 120°C for 20 minutes. Example 2:

[0053] Modification of glass FTO electrode with nanodiamonds

[0054] Prior to nanodiamonds (NDs) immobilisation on the ITO electrode, water suspension of NDs with surface carboxyl groups (-COOH) was diluted 10 times in MES buffer and placed in ultrasonic bath for 15 minutes. Then the suspension was further diluted in MES buffer and mixed with freshly prepared EDC solution in the same buffer at the ratio of 10 : 1. 30 μl of the suspension was immediately placed over the clean FTO electrode, and incubated for 30 minutes in room temperature. The electrodes were then washed with deionised water to remove the unattached NDs and dried in the stream of argon.

[0055] Example 3:

[0056] Functionalization of NDs-modified FTO glass substrate with anti-IL-8 antibodies

[0057] 30 μl of EDC / NHS freshly prepared linker solution in MES buffer was placed over NDs- modified FTO electrode and incubated for 5 minutes in room temperature. Then, the solution was washed away from the electrode with MES and 0.01M PBS pH 7.4 buffers, consecutively. 30 pl of anti-IL-8 antibodies diluted 1000 times in 0.01M PBS pH 7.4 was immediately placed over the linker-activated electrode and incubated in room temperature for 45 minutes. Electrodes were than washed intensively with 0.01M PBS pH 7.4. The excess of the buffer was removed from the electrode and incubated with 30 μl 1% BSA (bovine serum albumin) in 0.01M PBS pH 7.4 for 1 hour in room temperature. Following this electrodes were rinsed with 0.01M PBS pH 7.4. The next stage is analysis of the sensor response with the positive sample in the form of IL-8 protein (suspended in PBS buffer) which is specifically recognised by anti-IL-8 antibodies, and the negative sample without IL-8 protein. Electrochemical measurement is carried out using an alternating current with an amplitude of 10 mV and a frequency range from 10 kHz to 0.1 Hz relative to the formal potential of the redox reaction: Fe2+ / Fe3+. The EIS measurement was performed in a three- electrode system including an FTO electrode as the working electrode, a silver chloride electrode as the reference electrode and a wire platinum as an antielectrode Example 4:

[0058] Analysis of IL-8 protein presence in the sample

[0059] For performing the experiment FTO glass substrate was modified as described above. Positive sample (POS) is a solution of IL-8 protein in PBS buffer. Modified surface of the electrode was covered with 2 ml of electrolyte solution and the first - background - measurement was performed. Next, the electrode was rinsed with PBS buffer and 30 μl of IL-8 protein solution was placed over the modified surface of the electrode. Then, the electrode was rinsed with PBS buffer and the second measurement was performed in 2 ml of the electrolyte solution. As the effect of IL-8 protein presence over the modified surface of the electrode for 5 minutes the change of the charge transfer value was observed.

[0060] Example 5:

[0061] Analysis of sensor response with negative controls

[0062] The proposed sensor was checked for its selectivity. Changes in the value of charge transfer resistance were examined in the presence of selected proteins not binding to anti-IL-8 antibodies present on the surface of a modified FTO glass electrode coated with nanodiamonds, incubated on the electrode surface in the same way as the IL-8 protein. No changes in charge transfer resistances were observed after incubation of the selected negative control proteins compared to the charge transfer values recorded for the background. This means that the sensor does not respond to the analyzed proteins, except for the IL-8 protein. Charts of changes in load transfer resistance are shown in figures 4.

Claims

Claims:

1. Electrochemical impedimetric sensor for interleukin 8 (IL-8) biomarker detection in biological sample based on glass electrode which consists of:• working electrode as a stable platform in the form of a FTO glass substrate modified with nanodiamonds with carboxyl groups (-COOH) present on their surface,• reference electrode,• anti electrode,• electrolyte,• linker in the form of EDC / NHS,• detection layer: anti-IL-8 antibodies applied on the solid platform modified with a linker.

2. Sensor according to claim 1 characterised in that it contains silver chloride electrode (Ag / AgCl / 3M KC1) as reference electrode.

3. Sensor according to claim 1 characterised in that it contains 5mM K3[Fe(CN)6] / K4[Fe(CN)6] / 0.5M KC1 as electrolyte.

4. Sensor according to claim 1 characterised in that it contains a platinium wire as anti electrode.

5. A method of IL-8 biomarker detection in biological sample with the use of electrochemical impedance sensor according to claim 1 is characterised in that:• biological sample solution is incubated over the modified surface of the working electrode of an electrochemical impedance spectroscopy (EIS) meter containing: o APTES layer, o layer of nanodiamonds, o EDC / NHS linker layer, detection layer: anti-IL-8 antibodies diluted 1000 times in PBS;• next, surface of working electrode is connected with electrolyte and EIS reading is performed together with measurement data aquisition.• measurement data are furhter compared with the EIS measurement registered in the same conditions for the electrode incubated in biological sample solution without IL- 8 biomarker,• finally, measurement data are compared with the EIS data acquired in the same conditions for the electrode incubated with biological sample containing biomarkers other than IL-8.

6. The method according to claim 5 characterised in that which EIS measurement is performed using alternating current with the amplitude equal to 10 mV.

7. The method according to claim 5 characterised in that which EIS measurement is performed at the frequency range from 10 kHz to 0,1 Hz.

8. The method according to claim 5 characterised in that the platinium wire is used as an anti electrode.

9. The method according to claim 5 characterised in that Ag / AgCl / 3M KC1 is used as a reference electrode.

10. The method according to claim 5 characterised in that the measurement is performed with the use of 5mM K3[Fe(CN)6] / K4[Fe(CN)6] / 0.5M KC1 as an electrolyte solution.

11. The method according to claim 5 characterised in that the measurement is performed against a formal potential of redox reaction: Fe2+ / Fe3+12. The method according to claim 5 characterised in that the incubation of the working electrode in the form of an FTO glass electrode modified with nanodiamonds with the biological sample is carried out for 5 minutes at room temperature.

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