BIORECEPTOR MOLECULES, THE USE OF BIORECEPTOR MOLECULES, SENSORS CONTAINING ELECTRODES MODIFIED WITH THE SAID BIORECEPTOR MOLECULES AND THE DETECTION METHOD OF SARS-COV-2 VIRUS

Abstract

The subject of the invention is a bioreceptor molecule with the formula: R.sub.1-alkyl-C(0)NH—R.sub.2, wherein alkyl is linear or branched alkyl with 2 to 20 C atoms; R.sub.1 is selected from a group comprising thiol group (—SH); disulfide bridge; —S(O)-alkyl, wherein alkyl is linear or branched and contains 1-3 C atoms; thioether, wherein thioether contains 1-3 C atoms; thioacid; thionyl group; R.sub.2 is a peptide with a sequence selected from a group comprising SEQ ID NO 1-8. Another subject of the invention is the use of bioreceptor molecules according to the invention in electrochemical impedance spectroscopy for detecting the SARS-CoV-2 virus. The subject of the invention is also a sensor containing an electrode, whose surface is covered with a layer of metal, characterized in that this layer is modified by bioreceptor molecules according to the invention. Furthermore, the subject of the invention is the method of detecting the SARS-Cov-2 virus by means of electrochemical impedance spectroscopy, including the following steps: a. rinsing and drying of the sensor electrode covered with metal; b. modification of the sensor electrode surface with bioreceptor molecules; c. calibration of the measurement system; d. detection of SARS-Cov-2 virus in a sample by means of a measurement system by observation of impedance changes, characterized in that surface modification of the sensor electrode is carried out using bioreceptor molecules according to the invention, wherein the presence of the virus in the test sample is indicated by a change in impedance of at least 10% in absolute value against the baseline value.

Claims

1. The bioreceptor molecule with the following formula:
R.sub.1-alkyl-C(O)NH—R.sub.2, wherein alkyl is linear or branched alkyl with 2 to 20 C atoms; R.sub.1 is selected from the group comprising thiol group (—SH); disulfide bridge; —S(O)-alkyl, where alkyl is linear or branched and contains 1-3 C atoms; thioether, the thioether contains 1-3 C atoms; thioacid; thionyl group; R.sub.2 is a peptide with a sequence selected from a group comprising SEQ ID NO 1-8.

2. Bioreceptor molecule according to claim 1, wherein R.sub.1 is selected from a group comprising thiol group, disulfide bridge, —S(O)-alkyl, wherein alkyl is linear or branched and contains 1-3 C atoms.

3. The bioreceptor molecule according to claim 2, wherein R.sub.1 is selected from the group comprising thiol group and disulfide bridge.

4. The use of bioreceptor molecules specified in claim 1 in electrochemical impedance spectroscopy for SARS-CoV-2 virus detection.

5. An electrochemical sensor containing an electrode with surface covered with a metal layer, wherein the metal layer modified by bioreceptor molecules defined in claim 1.

6. The electrochemical sensor according to claim 5, wherein the electrode surface is covered with a layer of silver, copper, platinum, chemical, galvanic or evaporated gold.

7. A method of detection of SARS-Cov-2 virus with electrochemical spectroscopy impedance, the method comprising: a. rinsing and drying the metal-coated sensor electrode, b. modification of the sensor electrode surface with bioreceptor molecules, c. calibration of the measurement system, d. detection of SARS-Cov-2 virus in a sample by means of a measurement system by the observation of impedance changes, characterized in that the surface modification of the sensor electrodes are carried out using bioreceptor molecules specified in claim 1, wherein the presence of the virus in the test sample is indicated by a change in impedance of at least 10% in absolute value against the baseline value.

Description

[0037] The advantageous features of the invention are illustrated by the following Figures, supplementing the information contained in the embodiments.

[0038] FIG. 1 shows the chromatogram of HPLC purification of 11-KOD1-NH2 molecule (SEQ ID NO 1)

[0039] FIG. 2 shows the chromatogram of HPLC purification of 11-KOD2-NH2 molecule (SEQ ID NO 2)

[0040] FIG. 3 shows the chromatogram of HPLC purification of 11-KOD5-NH2 molecule (SEQ ID NO 5)

[0041] FIG. 4 shows the chromatogram of HPLC purification of 11-KOD6-NH2 molecule (SEQ ID NO 6)

[0042] FIG. 5 shows the chromatogram of HPLC purification of 8-KOD5-NH2 molecule (SEQ ID NO 5)

[0043] FIG. 6 shows the chromatogram of HPLC purification of 8-KOD-1-NH2 molecule (SEQ ID NO 1)

[0044] FIG. 7 shows the mass spectrometry spectrum for 11-KOD1-NH2 molecule

[0045] FIG. 8 shows the mass spectrometry spectrum for 11-KOD2-NH2 molecule

[0046] FIG. 9 shows the mass spectrometry spectrum for 11-KOD5-NH2 molecule

[0047] FIG. 10 shows the mass spectrometry spectrum for the 8-KOD5-NH2 molecule

[0048] FIG. 11 shows the mass spectrometry spectrum for the 8-KOD1-NH2 molecule

[0049] FIG. 12 shows the Nyquist diagram of the WHN-N protein interaction with the electrode modified with 11-KOD5-NH2 (SEQ ID NO 5). Blank—means the measurement of impedance on the unmodified electrode, incubation—measurement of impedance of the electrode modified with 11-KOD5-NH2, reaction—measurement of the modified electrode's interaction with the WHN-N protein.

[0050] FIG. 13 shows the Nyquist diagram of the Haemophilus influenzae bacteria interaction with the electrode modified with 11-KOD5-NH2. Blank—means the measurement of impedance on the unmodified electrode, incubation—measurement of impedance of the electrode modified with 11-KOD5-NH2 molecule, reaction—measurement of modified electrode's interaction with Haemophilus influenzae.

[0051] FIG. 14 shows the Nyquist diagram of the Streptococcus pyogenes bacteria interaction with the electrode modified with 11-KOD5-NH2 (SEQ ID NO 5). Blank—means the measurement of impedance on the unmodified electrode, incubation—measurement of impedance of the electrode modified with 11-KOD5-NH2 molecule, reaction—measurement of modified electrode's interaction with Streptococcus pyogenes.

[0052] FIG. 15 shows the Nyquist diagram of the Streptococcus pneumonia bacteria interaction with the electrode modified with 11-KOD5-NH2 (SEQ ID NO 5). Blank—means the measurement of impedance on the unmodified electrode, incubation—measurement of impedance of the electrode modified with 11-KOD5-NH2 molecule, reaction—measurement of modified electrode's interaction with Streptococcus pneumonia.

[0053] FIG. 16 shows the Nyquist diagram of the RSV virus interaction with the electrode modified with 11-KOD5-NH2 (SEQ ID NO 5). Blank—means the measurement of impedance on the unmodified electrode, incubation—measurement of impedance of the electrode modified with 11-KOD5-NH2 molecule, reaction—measurement of modified electrode's interaction with the RSV virus.

[0054] FIG. 17 shows the Nyquist diagram of the WHN-N protein virus interaction with the electrode modified with 11-KOD1-NH2 (SEQ ID NO 1). Blank—means the measurement of impedance on the unmodified electrode, incubation—measurement of impedance of the electrode modified with 11-KOD1-NH2 molecule, reaction—measurement of modified electrode's interaction with the WHN-N protein.

[0055] FIG. 18 shows the Nyquist diagram of the Haemophilus influenza bacteria interaction with the electrode modified with 11-KOD1-NH2 (SEQ ID NO 1). Blank—means the measurement of impedance on the unmodified electrode, incubation—measurement of impedance of the electrode modified with 11-KOD1-NH2 molecule, reaction—measurement of modified electrode's interaction with Haemophilus influenza.

[0056] FIG. 19 shows the Nyquist diagram of the Streptococcus pyogenes bacteria interaction with the electrode modified with 11-KOD1-NH2 (SEQ ID NO 1). Blank—means the measurement of impedance on the unmodified electrode, incubation—measurement of impedance of the electrode modified with 11-KOD1-NH2 molecule, reaction—measurement of modified electrode's interaction with Streptococcus pyogenes.

[0057] FIG. 20 shows the Nyquist diagram of the Streptococcus pneumonia bacteria interaction with the electrode modified with 11-KOD1-NH2 (SEQ ID NO 1). Blank—means the measurement of impedance on the unmodified electrode, incubation—measurement of impedance of the electrode modified with 11-KOD1-NH2 molecule, reaction—measurement of modified electrode's interaction with Streptococcus pneumonia.

[0058] FIG. 21 shows the Nyquist diagram of the RSV virus interaction with the electrode modified with 11-KOD1-NH2. Blank—means the measurement of impedance on the unmodified electrode, incubation—measurement of impedance of the electrode modified with 11-KOD1-NH2 molecule, reaction—measurement of modified electrode's interaction with the RSV virus.

[0059] FIG. 22 shows the Nyquist diagram of the WHN-N protein interaction with the electrode modified with 11-KOD7-NH2 (SEQ ID NO 7). Blank—means the measurement of impedance on the unmodified electrode, incubation—measurement of impedance of the electrode modified with 11-KOD7-NH2 molecule, reaction—measurement of modified electrode's interaction with the WHN-N protein.

[0060] FIG. 23 shows the Nyquist diagram of Haemophilus influenzae (A), Streptococcus pneumoniae (B), Streptococcus pyogenes (C), RSV virus (D) and EBV (E) interaction with the electrode modified with 11-KOD7-NH2 (SEQ ID NO 7). Blank—means the measurement of impedance on the unmodified electrode, incubation—measurement of impedance of the electrode modified with 11-KOD7-NH2 molecule, reaction—measurement of modified electrode's interaction with the WHN-N protein.

[0061] FIG. 24 shows the Nyquist diagram for a testing of a swab obtained from a patient infected with SARS-CoV-2, whose infection was confirmed by RT-PCR method, with a sensor modified with 11-KOD1-NH2 molecule (SEQ ID NO 1).

[0062] FIG. 25 shows the Nyquist diagram for a testing of a swab obtained from a patient not infected with SARS-CoV-2, whose absence of infection was confirmed by RT-PCR, with a sensor modified with 11-KOD1-NH2 molecule (SEQ ID NO 1).

[0063] FIG. 26 shows the cumulative results for a testing of a swab obtained from the positive patients infected with SARS-CoV-2 in which following modified sensors have been used: 8-COD1-NH2 (SEQ ID NO 1) (A), 11-KOD3-NH2 (SEQ ID NO 3) (B), 11-KOD4-NH2 (SEQ ID NO 4) (C), 8-KOD5-NH2 (SEQ ID NO 5) (D), 11-KOD6-NH2 (SEQ ID NO 6) (E) and 8-KOD7-NH2 (SEQ ID NO 7) (F).

[0064] FIG. 27 shows the cumulative results for a testing of a swab obtained from the negative patients (not infected with SARS-CoV-2) in which following modified sensors have been used: 8-COD1-NH2 (SEQ ID NO 1) (A), 11-KOD3-NH2 (SEQ ID NO 3) (B), 11-KOD4-NH2 (SEQ ID NO 4) (C), 8-KOD5-NH2 (SEQ ID NO 5) (D), 11-KOD6-NH2 (SEQ ID NO 6) (E) and 8-KOD7-NH2 (SEQ ID NO 7) (F).

[0065] FIG. 28 shows the schematic time of positive swab measurement (max. 5 minutes from adding the sample to obtaining the result).

[0066] FIG. 29 shows a schematic result of the difference between the reference value (incubation) and the tested sample (reaction) indicating a positive result for SARS-CoV-2, i.e. an impedance change ‘Δ’ greater than 10%.

EMBODIMENTS

Example 1

Selection Procedure for Peptide Sequences

[0067] For the selection of SARS-CoV-2 virus-specific binding sequences, the nucleocapsid N protein was selected, hereinafter referred to as WHN-N protein.

[0068] The peptide selection was carried out with the M13 phage library according to the standard procedure. 15 μg of WHN-N biomarker in TBS buffer was applied to microtiter plates and incubated at 4° C. overnight. Surfaces of wells were then blocked for 1 hour at 4° C. with 0.5% BSA diluted in TBS. Subsequently, approximately 1×10.sup.11 phage forming units (pfu) were diluted in 100 μl TBS buffer with 0.1% TWEEN® 20 for 1 hour at room temperature with agitation. After incubation, wells were washed ten times with TBS buffer with 0.5% Tween-20. Bacteriophages were eluted with 0.2 M glycin-HCl, 0.1% BSA (pH 2.2) and amplified by host cell infection with E. coli ER2738. After 4.5 hours of growth at 37° C. the multiplied bacteriophages were separated from bacterial cells by centrifugion. The phages present in the supernatant were precipitated by addition of ⅙ volume of PEG/NaCl solution (20% w/v polyethylene glycol-8000; 2.5 M NaCl) and incubated for 16 hours at 4° C. The solution was centrifuged and the sediment was suspended again in 1 mL TBS buffer and titrated to determine the phage concentration. The procedure was repeated 3 times, after which the phages were plated and random plaques were selected. After amplification, the phage was purified by precipitation in PEG/NaCl and then suspended in 1/50 of the original volume in TBS buffer. Single-stranded DNA was isolated by incubation of bacteriophages in iodide buffer (4 M NaI, 1 mM EDTA in 10 mM Tris-HCl, pH 8.0) in order to denature the phage protein shell. The released DNA was then precipitated in 70% ethanol. The purified DNA was sequenced by the Genomed company (Poland).

Example 2

Synthesis and Purification of the Bioreceptor Molecule

[0069] Peptides were obtained using an automatic synthesizer with a pipetting arm, using the solid phase peptide synthesis (SPPS) method, using the Fmoc/tBu.sup.t procedure. The syntheses were performed using Rink Amide AM resin (Deposition degree: 0.7 mmol/g). All reagents used had a high degree of purity (>95%, >97%, >98%, or analytical grade) and were purchased from the following manufacturers: Sigma Aldrich, VWR Chemicals, POCH S.A., P.P.H Stanlab, Iris Biotech GmbH, Alfa Aesar, Acros Organics, Thermo Fisher Scientific.

[0070] The synthesis was carried out using a module allowing for simultaneous synthesis of 8 independent peptide sequences with the use of disposable synthesis columns equipped with a sinter enabling drainage of the resin from the synthesis mixture.

[0071] Prior placing in the synthesizer, the resin was swelled for 30 minutes by cyclic rinsing 3×DMF, 3×DCM, 3×DMF. After that time the columns containing the resin were placed in the synthesizer in order to carry out the automatic synthesis cycles.

[0072] The automatic synthesis consisted of 7 to 12 (depending on the sequence) repeated steps of Fmoc protection group deprotection from α-amino group, rinsing and attachment of another protected amino acid derivative. During the deprotection step, Fmoc protection groups were removed with 20% piperidine solution in DMF.

[0073] In order to synthesize the indicated sequences, the 150 μmol scale method was used, with 4 times excess of acylating reagents. The reaction was carried out at 40° C. The acyclic mixture consisted of uniform amounts of Fmoc-AA: TBTU: HOBt: NMM dissolved in DMF.

[0074] A record of a single synthetic cycle is shown below: [0075] Deprotection (2500 μl 20% piperidine in DMF) 8 min×1 [0076] Deprotection (2500 μl 20% piperidine in DMF) 12 min×1 [0077] Rinsing (2210 μl DMF) 1 min×4 [0078] Acylation (1560 μl of TBTU/HOBt mixture in DMF+390 μl NMM+10 μl NMP+1638 μl Fmoc-AA) 30 min×3 [0079] Rinsing (2210 μl DMF) 1 min×5

[0080] The last cycle of synthesis was followed by the final step of deprotection, rinsing and drying of the resin, carried out as described below: [0081] Deprotection (2500 μl 20%/piperidine in DMF) 8 min×1 [0082] Deprotection (2500 μl 20%/piperidine in DMF) 12 min×1 [0083] Drying (Solvent extraction with the use of a vacuum pump) 30s×1 [0084] Rinsing (2210 μl DMF) 1 min×6 [0085] Rinsing (2210 μl EtOH) 1 min×5 [0086] Drying (Solvent extraction with the use of a vacuum pump) 300s×1

[0087] After completion of the last final cycle of automatic synthesis, the resin columns were removed from the unit and the resin was rinsed again with 15 ml of diethyl ether and left in a vacuum desiccator until the next step of synthesis—the linker attachment.

Synthesis of a HISNHSHHHDIL Molecule Sequence (KOD 1; SEQ ID NO 1)

[0088] For the synthesis of SEQ ID NO 1 peptide, the reaction conditions given in Table 1 below were applied.

TABLE-US-00001 TABLE 1 Molar Weight/volume Quantity of Quantity of Mass Concentra- of substance used solution solution Function Name [g/mol] tion [mol/l] for preperation used prepared Solvent Activator TBTU + HOBt 321.08 + 0.5 19.30 g + 8.12 g 112.20 120 DMF 135.12 Alkali NMM 101.15 4 35.20 ml 67.50 80 DMF Piperidine 20% PIP 85.15 2.02 30 ml 120.10 150 DMF Solvent 1 NMP 99.13 — 5 2.390 5 — Solvent 2 EtOH 46.07 — 50 ml 16 50 ml — Solvent 3 DMF 73.09 — 700 ml 505 700 ml — Derivative 1 Fmoc-Asn(Trt)-OH 596.67 0.5 2.98 g 7.22 10 DMF Derivative 2 Fmoc-Asp(OtBu)- 411.45 0.5 2.06 g 7.22 10 DMF OH Derivative 3 Fmoc-His(Trt)-OH 619.71 0.5 9.30 g 28.08 30 DMF Derivative 4 Fmoc-Ile-OH 353.41 0.5 2.65 g 12.43 15 DMF Derivative 5 Fmoc-Leu-OH 353.41 0.5 1.77 g 7.22 10 DMF Derivative 6 Fmoc-Ser(tBU)- 383.44 0.5 2.88 g 12.43 15 DMF OH

[0089] The remaining peptides were synthesized in a similar way, for example:

Synthesis of a Molecule of the Sequence HMQSHKTHHSQR (KOD 2, SEQ ID NO 2)

[0090] For the synthesis of SEQ ID NO 2 peptide, the reaction conditions given in Table 2 below were applied.

TABLE-US-00002 TABLE 2 Molar Weight/volume Quantity of Quantity of mass Concentra- of substance used solution solution Function Name [g/mol] tion [mol/l] for preperation used prepared Solvent Activator TBTU + HOBt 321.08 + 0.5 19.30 g + 8.12 g 120.20 120 DMF 135.12 Alkali NMM 101.15 4 35.20 ml 67.50 80 DMF Piperidine 20% PIP 85.15 2.02 30 ml 120.10 150 DMF Solvent 1 NMP 99.13 — 5 2.390 5 — Solvent 2 EtOH 46.07 — 50 ml 16 50 ml — Solvent 3 DMF 73.09 — 700 ml 505 700 ml — Derivative 1 Fmoc-Arg (Pbf)- 648.8 0.5 3.24 g 7.22 10 DMF OH Derivative 2 Fmoc-Gln(Trt)-OH 610.7 0.5 4.59 g 12.43 15 DMF Derivative 3 Fmoc-His(Trt)-OH 619.71 0.5 7.75 g 22.86 25 DMF Derivative 4 Fmoe-Lys(Boc)- 468.5 0.5 2.34 g 7.22 10 DMF OH Derivative 5 Fmoc-Thr(tBu)-OH 397.5 0.5 1.99 g 7.22 10 DMF Derivative 6 Fmoc-Ser(tBU)-OH 383.44 0.5 2.88 g 12.43 15 DMF Derivative 7 Fmoc-Met-OH 371.6 0.5 1.86 g 7.22 10 DMF

Synthesis of a Molecule of the Sequence FSLPSTL (KOD 5, SEQ ID NO 5)

[0091] For the synthesis of SEQ ID NO 5 peptide, the reaction conditions given in Table 3 below were applied.

TABLE-US-00003 TABLE 3 Molar Weight/volume Quantity of Quantity of mass Concentra- of substance used solution solution Function Name [g/mol] tion [mol/l] for preperation used prepared Solvent Activator TBTU + HOBt 321.08 + 0.5 16.10 g + 6.77 g 87.40 100 DMF 135.12 Alkali NMM 101.15 4 30.80 ml 61.30 70 DMF Piperidine 20% PIP 85.15 2.02 20 ml 94.20 100 DMF Solvent 1 NMP 99.13 — 5 2.390 5 — Solvent 2 EtOH 46.07 — 20 ml 16 20 ml — Solvent 3 DMF 73.09 — 400 ml 316 400 ml — Derivative 1 Fmoc-Phe-OH 387.4 0.5 1.94 g 7.22 10 DMF Derivative 2 Fmoc-Pro-OH 377.4 0.5 1.69 g 7.22 10 DMF Derivative 3 Fmoc-Thr(tBu)- 397.5 0.5 1.99 g 7.22 10 DMF OH Derivative 4 Fmoc-Leu-OH 353.41 0.5 2.65 g 12.43 15 DMF Derivative 5 Fmoc-Ser(tBu)- 383.44 0.5 2.88 g 12.43 15 DMF OH

[0092] The attachment of the derivative to the peptide chain was carried out each time manually or automatically in the following manner.

Attachment of a Derivative to a Peptide Chain

[0093] 11-Mercaptooctanoic acid (11-Mrpct) (2 eq relative to the degree of resin deposition) [alternative pathway: 8-Mercaptooctanoic acid (8-Mrpct) or 6-Mercaptohexanoic acid (6-Mrpct)] was dissolved in a small amount of DMF solution, DIC (2 eq) and HOBt (2 eq) were added.

[0094] Then it was all vortexed. The prepared solution was drawn into a syringe containing peptidyl resin and placed on a laboratory bench rocker. The acylation reaction was conducted for 45 minutes. Then the solution was removed from the syringe, a fresh portion of the mixture was drawn and the reaction was repeated. At the end of the reaction the solution was removed from the syringe, and peptidyl resin was rinsed successively with DMF (3×), DCM (3×), DMF (3×) solution.

[0095] In order to assess the effectiveness of acylation, a chloranilic test was performed (red colouring of the grains indicates the attachment of the derivative)

Attachment of Derivative to Peptide Chain Using Microwave Reactor Magnum Nova 10 MW EARTEC Reactor 800 W—an Alternative Method of Introducing a Derivative into the Peptide Chain.

[0096] The peptidyl resin was placed in a synthesis vessel in a microwave reactor and DMF solution was added to swell it. After 30 minutes the solution was removed. 11-Merkaptoundecanoic acid, 11-Mrpct (4 eq towards the degree of resin deposition) was dissolved in a small amount of DMF solution, DIC (4 eq) and HOBt (4 eq) were added, everything was vortexed. The solution of the acyclic mixture prepared in this way was transferred to a vessel containing peptidyl resin. Then the vessel was placed in a microwave reactor. The reaction was carried out for 5 minutes using 7% power and mixing with nitrogen stream. After draining the solution, a fresh portion of the acyclic mixture was introduced into the vessel and the reaction was repeated. The preparation of the acyclic mixture and the conditions of the reaction were identical as described above. At the end of the reaction, the solution was drained and the peptidyl resin was rinsed, consecutively with DMF (3×), DCM (3×), DMF (3×) solution. In order to assess the effectiveness of acylation the chloranilic test was performed. Red colouring of the grains of the resin indicates the attachment of the 11-Mrpct derivative.

[0097] The obtained raw bioreceptor molecule with general formula HS—CH.sub.2(CH.sub.2).sub.8CH.sub.2—CONH-[peptide sequence]-NH.sub.2 and HS—CH.sub.2(CH.sub.2).sub.8CH.sub.2—CONH-[peptide sequence]-NH.sub.2 were purified by reverse phase high-performance liquid chromatography. For purification a preparation column type C18 was used in linear gradient, where the mobile phase is a system of solvents A and B (A—H.sub.2O+0.1% TFA, B—100% ACN+0.1% TFA). Eluates were fractionated and then analysed with the RP-HPLC analytical method with 0-100% B linear gradient (A—H.sub.2O+0.1% TFA, B—100% ACN+0.1% TFA) on C18 type analytical column (FIG. 1-6). The fractions of the highest purity were combined and lyophilized.

[0098] The synthesized and purified compounds were characterized by mass spectrometry. (Table 4, FIGS. 7-11).

TABLE-US-00004 TABLE 4 Reten- Mass tion Theo- Molecule time ret- Ob- name Sequence [min] ical served 11M-K0D1- HS- 12.85 1644.7 1645.5 NH2 CH.sub.2(CH.sub.2).sub.8CH.sub.2C(O)- [M + H].sup.+ HISNHSHHHDIL- NH2 (SEQ ID NO 1) 11M-KOD2- HS- 12.051 1711.1 1712.5 NH.sub.2 CH.sub.2(CH.sub.2).sub.8CH.sub.2C(O)- [M + H]+ HMQSHKTHHSQR- NH.sub.2 (SEQ ID NO 2) 11M-KOD5- HS- 22.322  962.4 985.3 NH.sub.2 CH.sub.2(CH.sub.2).sub.8CH.sub.2C(O)- [M + Na]+ FSLPSTL-NH.sub.2 (SEQ ID NO 5) 11M-KOD6- HS- 16.813 1117.5 1118.9 NH2 CH.sub.2(CH.sub.2).sub.8CH.sub.2C(O)- [M + H]+ SFPVTLQK-NH.sub.2 (SEQ ID NO 6) 11M-K0D7- HS- 16.841 1069.5 1070.4 NH2 CH.sub.2(CH.sub.2).sub.8CH.sub.2C(O)- [M + H]+ TPIYHKL-NH.sub.2 (SEQ ID NO 7) 8M-KOD5- HS- 17.483  920.4  943.3 NH.sub.2 CH2(CH.sub.2).sub.5CH.sub.2 [M + Na]+ C(O)-)- FSLPSTL-NH.sub.2 (SEQ ID NO 5) 8-KOD1- HS-  9.744 1602.7 1603.4 NH2 CH.sub.2(CH.sub.2).sub.5CH.sub.2C(O)- [M + H]+ HISNHSHHHDIL- NH.sub.2 (SEQ ID NO 1)

[0099] In compliance with the content of Examples 1 and 2, the particles shown in table 5 below were obtained.

TABLE-US-00005 TABLE 5 MARK- ING SEQUENCE FORMULA 8-M- KOD-1 8-Mrcpt- HISNHS- HHHDIL- NH.sub.2 (SEQ ID NO 1) 8-Mrcpt-HISNHSHHHDIL-NH2   [00001] 11-M- KOD-1 11-Mrcpt- HISNHS- HHHDIL- NH.sub.2 (SEQ ID NO 1) 11-Mrcpt-HISNHSHHHDIL-NH2   [00002] 8-M- KOD-2 8-Mrcpt- HMQSH- KTHHSQR- NH.sub.2 (SEQ ID NO 2) 8-Mrcpt-HMQSHKTHHSQR-NH2   [00003] 11-M- KOD-2 11-Mrcpt- HMQSH- KTHHSQR- NH.sub.2 (SEQ ID NO 2) 11-Mrcpt-HMQSHKTHHSQR-NH2   [00004] 8-M- KOD-3 8-Mrcpt- HTVHA- HHASHLS- NH.sub.2 (SEQ ID NO 3) 8-Mrcpt-HTVHAHHASHLS-NH2   [00005] 11-M- KOD-3 11-Mrcpt- HTVHA- HHASHLS- NH.sub.2 (SEQ ID NO 3) 11-Mrcpt-HTVHAHHASHLS-NH2   [00006] 8-M- KOD-4 8-Mrcpt- IWGKS- YHIHSLH- NH.sub.2 (SEQ ID NO 4) 8-Mrcpt-IWGKSYHIHSLH-NH2   [00007] 11-M- KOD-4 11-Mrcpt- IWGKS- YHIHSLH- NH.sub.2 (SEQ ID NO 4) 11-Mrcpt-IWGKSYHIHSLH-NH2   [00008] 8-M- KOD-5 8-Mrcpt- FSLPSTL- NH.sub.2 (SEQ ID NO 5) 8-Mrcpt-FSLPSTL-NH2   [00009] 11-M- KOD-5 11-Mrcpt- FSLPSTL- NH.sub.2 (SEQ ID NO 5) 11-Mrcpt-FSLPSTL-NH2   [00010] 8-M- KOD-6 8-Mrcpt- SFPVTLQK- NH.sub.2 (SEQ ID NO 6) 8-Mrcpt-SFPVTLQK-NH2   [00011] 11-M- KOD-6 11-Mrcpt- SFPVTLQK- NH.sub.2 (SEQ ID NO 6) 11-Mrcpt-SFPVTLQK-NH2   [00012] 8-M- KOD-7 8-Mrcpt- TPIYHKL- NH.sub.2 (SEQ ID NO 7) 8-Mrcpt-TPIYHKL-NH2   [00013] 11-M- KOD-7 11-Mrcpt- TPIYHKL- NH.sub.2 (SEQ ID NO 7) 11-Mrcpt-TPIYHKL-NH2   [00014] 8-M- KOD-8 8-Mrcpt- HSMHHRH- NH.sub.2 (SEQ ID NO 8) 8-Mrcpt-HSMHHRH-NH2   [00015] 11-M- KOD-8 11-Mrcpt- HSMHHRH- NH.sub.2 (SEQ ID NO 8) 11-Mrcpt-HSMHHRH-NH2   [00016] [00017]

Example 3

Cleaning of Gold Electrodes

[0100] The gold electrodes on a PCB plate with HDMI output were cleaned before use with NaOH solution and ammonia/hydrogen peroxide mixture diluted with deionized water at a volume ratio of 8:1:1 respectively. The panels with the electrodes were placed in an ultrasonic cleaner and then immersed in 1M NaOH solution for 5 minutes at a temperature above 40° C. After 5 minutes the electrodes were removed from the washing solution and rinsed with deionised water. Then the panel was immersed in the prepared mixture of ammonia with hydrogen peroxide and left for 5 minutes. The electrodes were rinsed with deionized water and then immersed in deionized water for another 5 minutes. The last step of the electrodes washing procedure is to dry them in an argon stream. After this step the electrodes are ready for modifications.

Example 4

[0101] Electrode Surface Modification with 11-KOD5 Bioreceptor Molecules (SEO ID NO 5)

[0102] A solution of peptide 11-KOD5 (SEQ ID NO 5) modified with thiol group was applied to the cleaned gold surface. The sequence (FSLPSTL; SEQ ID NO 5) of the peptide (11-KOD5) is specific for SARS-CoV-2 by recognizing the WHN-N protein. The peptide is dissolved in a mixture of acetonitrile and deionized water at a volumetric ratio of 7:13 (ACN:WDI) to a concentration of 5.20.Math.10.sup.−4 M. The resulting peptide solution was diluted with deionized water up to the concentration of 5.Math.10.sup.−5 M. In order to modify, 2.6 μl of the solution containing peptide was applied to the electrode surface and left in a dark place with 100% humidity, 5-6° C. for 22 h. After this time the unbound peptide was rinsed with deionized water and then the electrode surface was dried in an argon stream. The next step is to test the interaction of the sensor with a positive sample (POZ) in the form of SARS-CoV-2 (WHN-N) capside building N protein suspended in TBS buffer, to which the peptide is sensitive, as well as with negative samples (NEG), which do not contain protein, to which peptide 11-KOD5 of FSLPSTL sequence is specific.

Example 5

Testing the Presence of WHN-N Protein in the Sample

[0103] A modified electrode as described above was used for the experiments. Positive sample (POZ) is a solution of WHN-N protein suspended in TBS buffer. The measurement electrode was placed in HDMI edge connector using a potentiostat containing FRA card for impedance measurements (Autolab M204).

[0104] Approximately 150 μl of measurement buffer composed of 100 mM TRIS-HCl, 6.2 mM K.sub.4[Fe(CN).sub.6]×3H.sub.2O, 6.2 mM K.sub.3[Fe(CN).sub.6], 2 M HCl up to pH=7.85, sterile Tween 20 was applied on the electrode surface. The first step of measurement has commenced—electrode calibration. 150 μl of measurement buffer was applied to the electrode, then impedance measurement was performed and the impedances of individual fields on the electrode were checked. During this time, 5 μl of WHN-N protein solution was added to 65 μl of measurement buffer. A solution containing WHN-N protein and measurement buffer was mixed and incubated at room temperature for 1 minute. Then 60 μl of such prepared solution was applied to the electrode adding the solution to the measurement buffer. Impedance measurement was initiated.

[0105] The result was considered as positive when impedance changes were at least 10% of the absolute value in relation to the baseline value (FIG. 12).

Negative Controls:

[0106] The sensor interaction test on gold medium with negative samples (NEG) in the form of night culture of Haemophilus influenzae, Streptocococcus pyogenes Streptococcus pneumoniae, and RSV viruses, is carried out as follows:

[0107] 150 μl of measurement buffer was applied to the individual electrodes modified with 11-KOD5 molecule (SEQ ID NO 5), followed by calibration measurements. Then, onto the electrodes, solutions of Haemophilus influenza, Streptocococcus pyogenes Streptococcus pneumonia bacteria, and RSV virus with a titre of 10.sup.7 CEID50/mL suspended in TBS were applied. Each measurement was carried out on a separate electrode for a single pathogen.

[0108] The results are presented in FIGS. 13-16.

Example 6

[0109] Electrode Modification with a 11-KOD1 Bioreceptor Molecule (SEO ID NO 1)

[0110] A solution of bioreceptor molecule 11-KOD1 (SEQ ID NO 1) was applied on a cleaned gold surface. The sequence (HISNHSHHHDI; SEQ ID NO 1) is specific for the WHN-N protein (SARS-CoV-2 capside N protein). The peptide is dissolved in a mixture of acetonitrile and deionized water in a volume ratio of 4:5 (ACN:WDI) to a concentration of 7.43.Math.10.sup.−4 M. The resulting peptide solution was diluted with deionized water to the concentration of 5.Math.10.sup.−5 M. In order to modify, 2.6 μl of the solution containing the bioreceptor molecule was applied to the electrode surface and left in a dark place with 100% humidity, temperature 5-6° C. for 22 h. After this time the unbound peptide was rinsed with deionised water and then the electrode surface was dried in an argon stream. The next step was to examine the interaction of the sensor with the positive sample (POZ) in the form of the SARS CoV-2 (WHN-N) capside building protein suspended in TBS buffer, to which the peptide is sensitive, as well as with the negative samples (NEG), which do not contain protein to which peptide 11-KOD1 of SEQ ID NO 1 is specific.

Example 7

Testing the Presence of WHN-N Protein in the Sample

[0111] An electrode modified as described above was used for the experiments. The positive test (POZ) is a WHN-N protein solution suspended in TBS buffer at 10 μg/ml. The measurement electrode was placed in HDMI edge connector using a potentiostat containing FRA card for impedance measurements (Autolab M204).

[0112] Approximately 150 μl of measurement buffer of 100 mM TRIS-HCl, 6.2 mM K.sub.4[Fe(CN).sub.6]×3H.sub.2O, 6.2 mM K.sub.3[Fe(CN).sub.6], 0.1% sterile Tween 20. 2M HCl, was added to the surface of the electrode to adjust pH 7.85. The first step of measurement has commenced—electrode calibration. The electrode was covered with 150 μl of measurement buffer, then impedance measurement was performed and impedances of individual fields on the electrode were checked. During this time 5 μl of WHN-N protein suspension was added to 65 μl of measurement buffer. The solution was mixed and incubated at room temperature for 1 minute. Then 60 μl of this solution was applied to the electrode adding the solution to the measurement buffer. Impedance measurement was initiated. The result was considered as positive when impedance changes were at least 10% of the absolute value in relation to the baseline value (FIG. 17).

Negative Controls:

[0113] Sensor interaction on gold base with NEG samples in the form of night culture of Haemophilus influenzae, Streptococcus pyogenes Streptococcus pneumonia bacteria and RSV virus is carried out as follows:

[0114] 150 μl of measurement buffer was applied to the individual electrodes modified with 11-KOD1 molecule, followed by a calibration measurement. Then solutions of Haemophilus influenzae, Streptococcus pyogenes Streptococcus pneumonia bacteria and RSV virus with a titre of 10.sup.7 CEID50/mL suspended in TBS were applied on individual electrodes. Each electrode was measured separately for each pathogen.

[0115] The results are presented on FIGS. 18-21.

Example 8

[0116] Electrode Surface Modification with 11-KOD7 Bioreceptor Molecules (SEO ID NO 7)

[0117] A solution of 11-KOD7 peptide (SEQ ID NO 7) modified with thiol group was applied to the cleaned gold surface. The sequence (TPIYHKL; SEQ ID NO 7) of peptide (11-KOD7) is specific for SARS-CoV-2 virus. The peptide was dissolved in a mixture of acetonitrile and deionized water in a volume ratio of 2:13 (ACN:WDI) to a concentration of 5.98.Math.10.sup.−4 M. The resulting peptide solution was diluted with deionized water to the concentration of 1.Math.10.sup.−5 M. In order to modify, 2.6 μl of the solution containing peptide was applied to the electrode surface and left in a dark place with 100% humidity, 5-6° C. for 22 h. After this time the unbound peptide was rinsed with deionized water and then the electrode surface was dried in an argon stream. The next step is to test the interaction of the sensor with a positive sample (POZ) in the form of SARSCoV-2 (WHN-N) capside building N protein suspended in TBS buffer, to which the peptide is sensitive, as well as with negative samples (NEG), which do not contain protein to which 11-KOD7 peptide (SEQ ID NO 7) is specific.

Example 9

Testing the Presence of WHN-N Protein in the Sample

[0118] An electrode modified as described above was used for the experiments. Positive sample (POZ) is a solution of WHN-N protein suspended in TBS buffer. The measurement electrode was placed in HDMI edge connector using a potentiostat containing FRA card for impedance measurements (Autolab M204).

[0119] Approximately 150 μl of measurement buffer composed of 100 mM TRIS-HCl, 6.2 mM K.sub.4[Fe(CN).sub.6]×3H.sub.2O, 6.2 mM K.sub.3[Fe(CN).sub.6], 6 M HCl, 0.1% sterile Tween20 was applied to the electrode surface. The first step of measurement commenced—electrode calibration. 150 μl of measurement buffer was applied onto the electrode, after which the impedance measurement was performed and impedances of individual fields on the electrodes were checked. At that time, 5 μl of WHN-N protein solution was added to 65 μl of measurement buffer. A solution containing WHN-N protein and measurement buffer were mixed and incubated at room temperature for 1 minute. Then 60 μl of the solution was applied to the electrode by adding the solution to the measurement buffer. Impedance measurement was initiated.

[0120] The result was considered as positive when impedance changes were at least 10% of the absolute value in relation to the baseline value (FIG. 22).

Negative Controls:

[0121] The sensor interaction test on gold base with NEG samples in the form of night culture of Haemophilus influenzae, Streptococcus pneumoniae, Streptococcus pyogenes bacteria and RSV, EBV viruses is performed as follows:

[0122] 150 μl of measurement buffer was applied to the individual electrodes modified with 11-KOD7 molecule (SEQ ID NO 7), followed by calibration measurements. Then solutions of Haemophilus influenzae, Streptococcus pneumoniae, Streptococcus pyogenes bacteria as well as RSV and EBV viruses with a titre of 10.sup.7 CEID50/mL suspended in TBS were applied to the individual electrodes.

[0123] Measurements were conducted separately for each pathogen.

[0124] The results are presented in FIG. 23.

Example 10

Testing for SARS-CoV-2 in Patient Swabs

[0125] In order to confirm the sensitivity of the diagnostic test based on selected peptides, the presence of SARS-CoV-2 virus in swabs taken from COVID-19 patients was measured. The presence of the virus in the swab samples was confirmed by the Real-Time PCR molecular method according to the WHO recommendations.

[0126] The swab was taken with a swab stick and dissolved in the buffer composed of: 100 mM TRIS-HCl, 6.2 mM K.sub.4[Fe(CN).sub.6]×3H.sub.2O, 6.2 mM K.sub.3[Fe(CN).sub.6], 0.1% Sterile Tween 20, 2 M HCl and the pH was brought up to 7.85.

[0127] At the same time a single-use sensor (electrode modified with molecule 11-KOD1 of SEQ ID NO 1) and EIS (electrochemical impedance spectrometer) reader MOBI SensDx were prepared. The following instructions were followed: the MOBI SensDx reader was connected to the computer, then the application included in the kit was launched. A single-use sensor was placed in the HDMI socket of the reader. Approximately 200 μl of measurement buffer was applied to the sensor and the measurement was started. After 1 minute, 50 μl of solution was added to the sensor buffer formed by dissolving the swab. The measurement was continued according to the instructions.

[0128] After the measurement was finished, the application showed a positive result (+), which indicated the presence of the virus in the sample. Raw data measured by the MOBI SensDx reader (EU trademark EUTMA-018242325, international patent application PCT/IB2019/050935) is illustrated by FIG. 24.

[0129] Similarly, an experiment was performed using a negative swab (from a patient not infected with COVID-19, which was confirmed by PCR). The result on the MOBI SensDx reader showed no impedance changes on the sensor, as shown in FIG. 25.

[0130] Analogous results were obtained for sensors modified with the remaining molecules. Summary results for positive swabs on sensors modified with KOD1, KOD3, KOD4, KOD5, KOD6 and KOD7 molecules are shown in FIG. 26. The measurement of negative swabs is illustrated in FIG. 27.

[0131] The measurement time is very short and is maximum 5 minutes. This is shown in FIG. 28.

[0132] The result was considered as positive when the difference in resistance between R.sub.CTi and R.sub.CTr is more than Δ>10%, which is schematically shown in FIG. 29.

[0133] A sensor based on peptides modified with a flexible linker can be used to detect SARS-CoV-2 virus in biological samples such as swabs from the throat, nasopharynx, nose, faeces, urine and blood samples as well as in water and food samples as well as from veterinary samples such as tissue, faeces, urine, swabs taken from different surfaces. The examples show how easy it is to modify the gold surface of the electrodes with the obtained bioreactor molecules—the reaction is one-step. The electrodes obtained by the modifications, were used to recognize the N protein (nucleotocapsyde protein) in the tested samples. The above examples show that sensors containing the electrode are capable of detecting selectively SARS-CoV-2 infection. The effectiveness of the test was previously confirmed by the gold standard applied in this type of diagnostics, i.e. RT-PCR (FIGS. 24-25).

[0134] The use of molecules developed in this way in electrochemical impedance spectroscopy allowed to obtain a diagnostic test which is quick and easy to operate, as shown in the above embodiments.