METHODS FOR DETECTING A TARGET IN A SAMPLE USING MUTATED NANOBODIES

Abstract

The present invention relates to methods for detecting a target in a sample using mutated nanobodies, wherein an amino acid present in the loop of the FR1 region of framework of the nanobodies is mutated to cysteine.

Claims

1. A mutated nanobody which binds to a target wherein an amino acid present in a loop of an FR1 framework region of the mutated nanobody is mutated to cysteine.

2. The mutated nanobody of claim 1, wherein the amino acid at position 12, 13, 14 or 15, is mutated to cysteine.

3. The mutated nanobody of claim 1, wherein the amino acid at position 13 is mutated to cysteine.

4. The mutated nanobody of claim 1, wherein the target is selected from the group consisting of viral proteins, protein biomarkers, bacteria proteins, membrane proteins, protozoa proteins, fungi proteins, and prion proteins.

5. The mutated nanobody of claim 4, wherein the target originates from infectious agents.

6. The mutated nanobody of claim 4, wherein the target is a viral protein.

7. The mutated nanobody of claim 1, wherein the mutated nanobody is attached to a surface.

8. The mutated nanobody of claim 7, wherein the surface is a gold surface or a surface selected from the group consisting of graphene, reduced graphene oxide and its derivatives, or a metal surface other than gold such as carbon, platinum, nickel, copper, and silver.

9. The mutated nanobody of claim 7, wherein the surface is a working electrode of a biosensor device.

10. The mutated nanobody of claim 7, wherein the mutated nanobody is attached to the surface with a short linker.

11. A biosensor comprising the mutated nanobody and surface of claim 7.

12. A method for detecting a target in a sample, the method comprising: a. providing the biosensor of claim 11, b. contacting the biosensor with the sample, c. measuring a response at the biosensor surface, and d. determining the presence or the absence of the target based on the response measured in measuring step c.

13. The method of claim 12, wherein the sample is selected from the group consisting of nasal or mouth swab, spit, and blood.

14. The mutated nanobody of claim 6, wherein the viral protein is a SARS virus protein, a SARS-CoV-2 virus protein, or a SARS-CoV-2 receptor binding protein (RBP).

15. The mutated nanobody of claim 8, wherein the surface is gold or graphene.

16. The mutated nanobody of claim 10, wherein the short linker is a pyrene-based linker or PEG-aryl radical.

Description

LISTING OF FIGURES

[0056] FIG. 1: This figure corresponds to FIG. 1 (B) from the article Laura S. Mitchell, Lucy J. Colwell “Comparative analysis of nanobody sequence and structure data” Proteins. 2018; 1-10 showing Left: the secondary structure of the nanobody VHH domain which consists of 9 beta sheets separated by loop regions, 3 of which are hypervariable (H1, H2 and H3). Four framework regions (FRs) separate the variable loops.

[0057] FIG. 2: This figure corresponds to FIG. 3 (A) from the article Laura S. Mitchell, Lucy J. Colwell “Comparative analysis of nanobody sequence and structure data” Proteins. 2018; 1-10 showing sequence logo plots that show the amino acid variation between nanobodies.

[0058] FIG. 3: Differential pulse voltammograms of grafted gold electrodes of example 2 VHH C13 (A) and VHH C12 (B). Solution Ferrocenmethanol (FcMeOH, 1 mM) in PBS (0.1M). DPV Parameters: equilibrium time 3s; Estep=0.01V; Epuls=0.06V; T=0.02s; scan rate: 0.06V/s.

[0059] FIG. 4: Change of current as a function of the log concentration of the receptor binding protein: Calibration curve of differential pulse voltammograms of grafted gold electrodes of example 2 VHH C13 according to different concentration of the receptor binding protein of SARS-CoV-2. Solution Ferrocenmethanol (FcMeOH, 1 mM) in PBS (0.1M). DPV Parameters: equilibrium time 3s; Estep=0.01V; Epuls=0.06V; T=0.02s; scan rate: 0.06V/s.

[0060] FIG. 5: Dilution testing of grafted gold electrodes of example 2 compared to RT-PCR. White column corresponds to RT-PCR, light grey corresponds to VHH 72 biotin, dark grey corresponds to VHH C13 of the present invention and black corresponds to non-mutated VHH 72.

[0061] FIG. 6: Results of three different sensing interfaces modified with non-mutated VHH 72, biotin-modified VHH 72 and cystein modified nanobody VHH C13 using samples from patients where RT-PCR gave Covid-19 positive or negative results. In the case of VHH C13 results concordance between RT-PCR and the mutated SARS-CoV-2 nanobody of the present invention is made on the basis of 100 PCR negative samples (dark grey bullets) and 73 PCR positive samples (light grey bullets).

[0062] FIG. 7: Change of current as a function of the log of the concentration of HSV-1 virus.

[0063] FIG. 8: Change of current as a function of the log concentration cultured SARS-CoV-2 viral particles of example 2 VHH C12 (A) and VHH C13 (B). Solution Ferrocenmethanol (FcMeOH, 1 mM) in PBS (0.1M). DPV Parameters: equilibrium time 3s; Estep=0.01V; Epuls=0.06V; T=0.02s; scan rate: 0.06V/s

EXAMPLES

Example 1: Production of Non-Mutated and Mutated Nanobody Specific to the Receptor Binding Protein of the SARS-Cov-2 Virus

[0064] 1. Production of the Non-Mutated Nanobody Specific to the Receptor Binding Protein of the SARS-Cov-2 Virus: VHH 72

[0065] The sequence of the non-mutated nanobody specific to the receptor binding protein of the SARS-Cov-2 virus is as follows:

TABLE-US-00001 (SEQ ID NO: 1) QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVA TISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAA AGLGTVVSEWDYDYDYWGQGTQVTVSSGSHHHHHH

[0066] The nanobody contains a C-terminal polyhistidine tag in order to facilitate the purification.

[0067] This non-mutated nanobody was produced in T7 Express Escherichia coli cells (NEB) cultured in Turbo Broth medium (Athena) at 37° C. for 4 h. At this stage, the expression was induced with 0.3 mM IPTG and the temperature was decreased to 17° C. and the cells were grown for an additional 18 h. Cells were pelleted by centrifugation for 10 min, 5000 g, at 4° C. The pellets were flash freezed in liquid nitrogen then thawed at room temperature. The pellets were then resuspended in lysis buffer (50 mM Tris, 300 mM NaCl, 5% glycerol, 0.1% Triton, 5 mM Imidazole, 20 ug/ml DNase, 0.1M PMSF, 0.1 mg/ml lysozyme) and put under agitation for 45 minutes at 4° C. The cells were sonicated at 50% amplitude for 3 rounds of 30 seconds. The lysat was centrifugated at 13000 g for 30 minutes at 4° C. and the supernatant was then purified on a 5 ml Ni-NTA column (GE Healthcare) in 50 mM Tris, 5% glycerol, 5 mM Imidazole, 300 mM NaCl, pH 8.0. The fractions eluted in 250 mM imidazole were concentrated by centrifugation using an Amicon Ultra 10 kDa cutoff concentrator prior to being loaded onto a HiLoad 16/60 Superdex 75 pg gel filtration column (GE Healthcare) equilibrated in phosphate buffered saline (PBS). The purified nanobodies were concentrated by centrifugation; their concentration was determined by measuring the absorbance at 280 nm with a NanoDrop 2000 (Thermo Scientific).

[0068] 2. Production of the Mutated Nanobody Specific to the Receptor Binding Protein of the SARS-Cov-2 Virus of the Present Invention VHH C13

[0069] The non-mutated nanobody of point 1 was mutated at position 13 in order to replace glutamine (Q) by a cysteine (C) following the procedure hereafter. A synthetic gene encoding the mutated protein has been ordered from Twist bioscience. The mutated nanobody was produced following the same protocol as described in point 1 for non-mutated nanobody.

[0070] The sequence obtained of the mutated nanobody of the present invention VHH C13 is as follows:

TABLE-US-00002 (SEQ ID NO: 2) QVQLQESGGGLVCAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVA TISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAA AGLGTVVSEWDYDYDYWGQGTQVTVSSGSHHHHHH

[0071] 3. Production of the Mutated Nanobody Specific to the Receptor Binding Protein of the SARS-Cov-2 Virus VHH-Biotin

[0072] The non-mutated nanobody of point 1. was labelled with biotin following the procedure hereafter. 1 mg of the non-mutated nanobody at 4 mg/mL in 0.1M bicarbonate buffer pH 9 (100 mL:80 mL H20+0.765 g Na Bicarbonate+0.095 Na carbonate, adjust at 100 mL with H20) was mixed with dye dissolved in DMSO at 10 mg/mL. The mixture was kept for 1 hour under permanent stirring away from light before centrifugation and gel filtration.

[0073] 4. Production of the Mutated Nanobody Specific to the Receptor Binding Protein of the SARS-Cov-2 Virus of the Present Invention VHH C12

[0074] The non-mutated nanobody of point 1 was mutated at position 12 in order to replace valine (V) by a cysteine (C) following the procedure hereafter. A synthetic gene encoding the mutated protein has been ordered from Twist bioscience. The synthetic was inserted in a vector for mammal expression. The mutated nanobodies fused with a FC domain were produced in HEK Expi293 cells cultured in Expi293 expression medium from ThermoFisher at 37° C., 150 rpm until the cells were around 1.10{circumflex over ( )}6 cells/mL. At this stage, cells were transfected with 75 μg of DNA and 225 μg of PEI Max-transfection grade linear (Polysciences) and the cells were grown for an additional 96h. After the first 24h, additives were added on the cells: 0.5 mM of valproic acid, 4 g/L of glucose and 20% tryptone N1. After 96h, cells were pelleted by centrifugation for 10 min, 700 g, at 4° C. and the supernatant was then purified on a 5 ml Ni-NTA column (GE Healthcare) in 50 mM Tris, 5% glycerol, 5 mM Imidazole, 300 mM NaCl, pH 8.0. The fractions eluted in 250 mM imidazole were concentrated by centrifugation using an Amicon Ultra 10 kDa cutoff concentrator prior to being loaded onto a HiLoad 16/60 Superdex 75 pg gel filtration column (GE Healthcare) equilibrated in phosphate buffered saline (PBS). The purified nanobodies-FC were concentrated by centrifugation; their concentration was determined by measuring the absorbance at 280 nm with a NanoDrop 2000 (Thermo Scientific). The VHH and the FC domain were separated by the use of TEV protease with 1:10 ratio. After one night of cleavage at room temperature, the product was loaded onto a HiLoad 16/60 Superdex 75 pg gel filtration column equilibrated in PBS. The purified nanobodies were concentrated by centrifugation using an Amicon Ultra 3 kDa cutoff concentrator, the concentration was determined by measuring the absorbance at 280 nm and the denaturation curve was determined using a Tycho NT.6 from Nanotemper Technologies.

[0075] The sequence obtained of the mutated nanobody of the present invention VHH C12 is as follows:

TABLE-US-00003 (SEQ ID NO: 4) QVQLQESGGGLCQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVA TISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAA AGLGTVVSEWDYDYDYWGQGTQVTVSSGSHHHHHH

Example 2: SARS-CoV-2 Nanobodies Grafting on Gold Electrodes

[0076] Before the grafting of SARS-CoV-2 nanobodies of example 1 on gold electrodes, the gold interfaces were cleaned following the procedure hereafter:

[0077] Electrodes were irradiated with UV-OZONE for 5 min. Then washed with mQ water dried with a dry air. In the second time, electrodes were cleaned electrochemically with 0.5M H.sub.2SO.sub.4 solution. For this, connect the electrodes to the potentiostat (Palmsens, Sensit), deposit H.sub.2SO.sub.4 on the electrodes and start cleaning method (Table.1). When the method is finished, rinse electrodes with mQ water and then dry with a dry air.

TABLE-US-00004 TABLE 1 Cleaning parameters Parameter Value Technique Cycle Voltammetry Current range Full Range (100 pA-100 mA) Pretreatement settings All parameters at 0 Cyclic Voltammetry T equilibration 5 s E begin −0.2 V E vertex 1 1 V E vertex 2 −0.2 V E step 0.001 V Scan Rate 0.1 V/s Number of scans 10

[0078] Once cleaned, 3 types of gold electrodes were produced using the 3 different nanobodies of example 1.

[0079] 1. Gold Electrode Grafting with the Mutated Nanobody of the Present Invention: VHH C13 or VHHC12

[0080] The gold electrode is prepared in two steps: The Au electrode is exposed to 10 μL of an aqueous solution of 3-mercaptoproponic acid (25 mM) for 30 min at room temperature. Then acid-terminated surface is activated with EDC/NHS (1:1 molar ratio, 15 mM) for 20 min, followed by immersion into NH.sub.2-PEG.sub.6-maleimide (10 μL, 0.1 mg/m, in PBS 1×) for 2 h at 4° C. and washed with MQ-water. A solution at 100 μg/mL in PBS 1× of SARS-CoV-2 nanobody VHH C13 of example 1 point 2 or VHHC12 of example 1 point 4 is dropped on electrodes and keeping a 4° C. overnight under humid atmosphere. Surfaces were wash, dry and keep it a 4° C. until use.

[0081] 2. Gold Electrode Grafting with the Non-Mutated Nanobody VHH

[0082] A solution L-Cysteine (SigmaAldrich, France) at 2 mM on PBS 1× were dropped on electrodes and keeping at 4° C. overnight under humid atmosphere. Electrodes were washed, dried and a solution of NHS/EDC (SigmaAldrich, France) at 15 mM was dropped on surfaces (2h at 4° C.) after 2 hours electrodes were washed, dried and a solution at 100 μg/mL of SARS-Cov-2 nanobody VHH of example 1 point 1 was incubated overnight at ° 4° C. under humid atmosphere. Surfaces were wash, dry and keep it a 4° C. until use.

[0083] 3. Gold Electrode Grafting with the Biotin Mutated Nanobody: VHH-Biotin

[0084] A solution L-Cysteine at 2 mM on PBS 1× were dropped on electrodes and keeping at 4° C. overnight under humid atmosphere. Electrodes were washed, dried and the surfaces were incubated 2 hours at 4° C. with NHS/EDC at 15 mM. After 2 hours electrodes were washed, dried and a solution of streptavidine (ThermoFisher, France) was incubated with the surfaces at ° 4° C. under humid atmosphere overnight. Next day, electrodes were washed, dried and a solution of SARS-Cov-2 nanobody VHH-biotin of example 1 point 3 at 100 μg/mL is incubated for 2 hours. Surfaces were wash, dry and keep it a 4° C. until use.

[0085] After the grafting of each gold electrode, differential pulse voltammograms (DPV) were measured for each final electrode. The solution used of the measure is Ferrocenmethanol (FcMeOH, 1 mM) in PBS (0.1M). DPV Parameters are as follows: equilibrium time 3s; Estep=0.01V; Epuls=0.06V; t=0.02s; scan rate: 0.06V/s. The result of the DPV is in FIG. 3. As can be seen, the gold electrode grafting with the mutated nanobody of the present invention VHH C13 (FIG. 3A) and VHH C12 (FIG. 3B) allows having a large current notably by the fact that the mutated nanobody of the present invention blocks less the transfer of electrons from the nanobody to the electrode. This is not the case with the other electrodes.

[0086] Then, the capacity of each final electrode of specifically binding SARS-CoV-2 receptor binding protein (RBP) and cultured SARS-CoV-2 virus samples was measured by establishing a calibration curve of DPV according to different concentration of SARS-CoV-2 RBP (dilution of stock solution in RBP in PBS (0.1M) and virus samples (dilution of stock solution in SARS-CoV-2 cultured virus in PBS (0.1M)). Incubation for 10 min with the lowest concentration of RBP of cultured virus was started, a DPV signal recorded, and next higher concertation of RBP added. The solution used of the measure is Ferrocenmethanol (FcMeOH, 1 mM) in PBS (0.1M). DPV Parameters are as follows: equilibrium time 3s; Estep=0.01V; Epuls=0.06V; t=0.02s; scan rate: 0.06V/s. The result is in FIG. 4 for RBP and FIGS. 8 (A) and (B) for SARS-CoV-2 virus samples. As can be seen, the mutated nanobody of the present invention VHH C13 binds rapidly at concentrations of 4 to 5 nM of SARS-CoV-2 RBP, whereas for the other electrodes the binding occurs later and at concentrations 4 times and 12 times higher. Similar results were obtained with the SARS-CoV-2 virus samples (see FIGS. 8 (A) and (B)). Therefore, the mutation of the SARS-CoV-2 nanobody at a specific place in its sequence, particularly in position 13, by a cysteine makes it possible to obtain a very sensitive and fast-reading electrode that meets the specifications of a device.

[0087] Different diabodies directed to the SARS-CoV-2 RBP were tested instead of nanobodies, equivalents results have been obtained with binding affinity of 0.28 nM, 0.082 nM and 0.018 nM.

[0088] These diabodies, as well as, the mutated nanobody of the present invention VHH C13 have been tested on SARS-CoV-2 variants, notably UK, South African and Delta variant. Similar results have been obtained.

Example 3: Comparison the Mutated Gold Electrode of the Present Invention to RT-PCR Via Dilution Testing

[0089] Once the efficacy and sensitivity of the sensing electrode of the present invention was established for SARS-CoV-2, it was compared to the gold standard for the diagnosis of SARS-CoV-2 which is PCR.

[0090] Nasopharyngeal specimens were collected from patients in Viral Transport Medium (VTM/UTM) (Yocon®). A strongly positive sample (PCT 18 counts) was 10-fold serially diluted in negative samples.

[0091] Extraction with the MGI Easy Nucleic Acid Extraction Kit on the MGISP-960 Automated Sample Preparation System.

[0092] RT-PCR with TaqPath™ COVID-19 Combo Kit (Multiplex real-time RT-PCR test intended for the presumptive qualitative detection of nucleic acid from SARS-CoV-2 by Thermofischer®) on the QuantStudio 5 Real-Time PCR System.

[0093] The three gold electrodes of example 2 were tested: the non-mutated SARS-CoV-2 nanobody electrode, the biotin mutated SARS-CoV-2 nanobody electrode and the cysteine mutated SARS-Cov-2 nanobody (VHH C13) electrode of the present invention. The result is in FIG. 6. As demonstrated, the RT-PCR detects SARS-CoV-2 virus until the 5.sup.th dilution, non-mutated VHH 72 detects SARS-Cov-2 virus until the 2.sup.nd dilution, VHH-biotin detects SARS-Cov-2 virus until the 4.sup.th dilution and VHH-72-Cys (VHH C13) of the present invention detects SARS-CoV-2 virus until the 7.sup.th dilution. In consequence, the mutated SARS-CoV-2 nanobody of the present invention allows the detection of the SARS-CoV-2 virus at very low concentrations, in a specific way and allows to go beyond the limits of the gold standard diagnostic method by detecting the virus where PCR is no longer able to do so. In consequence, the mutated SARS-Cov-2 nanobody of the present invention can become the new gold standard diagnostic of viruses and particularly SARS-Cov-2 virus.

Example 4: Clinical Trial of the Mutated SARS-Cov-2 Nanobody of the Present Invention Compared to the PCR Gold Standard

[0094] The clinical trial was conducted on the basis of the following protocol.

TABLE-US-00005 TABLE 2 study design of the rapid detection of covid-19 by portable and connected biosensor according to the present invention EXPERIMENTAL This is a proof-of-concept biological diagnostic study carried out PLAN on the first nasopharyngeal swab collected from patients admitted to Lille University Hospital (emergency, hospitalization, resuscitation) for suspected COVID-19 infection. Samples was collected systematically during the classical diagnostic management of covid-19 by first PCR. PCR was performed according to the usual management. The analysis with a biosensor according to the invention was performed on the same first specimen as the PCR. The patient received the usual diagnostic and therapeutic management. Biosensor analyses were ideally performed at the same time as PCR on fresh samples. Investigators and research associates collected data to establish the diagnosis. Statistical analyses were performed when the 200 defined diagnoses (100 positive and 100 negative) based on the gold standard of medical expertise were obtained. This is a research mentioned in 3º of article L. 1121-1 of the public health code because there is no contact by essence with the in vitro diagnostic medical device, nor any change in the diagnostic or therapeutic conduct. There is no risk or constraint associated with the realization of this biological proof of concept. The sampling of the elements was carried out within the framework of the care, for the specific needs of research there is only: An in vitro diagnosis of the samples by the biosensor in parallel with these samples. A prospective collection of medical data (without additional constraints). Measures taken to minimize bias: Consecutive recruitment of biological samples in the event of a continuing epidemic. Blind reading of PCR, biosensor diagnoses and patient characteristics OBJECTIVES To study the concordance between the diagnosis made by a biosensor according to the invention and the diagnosis made by PCR based on nasopharyngeal swabs taken at the patient's admission. EVALUATION Cohen's Kappa Coefficient for concordance for the diagnosis CRITERIA of CoV-2-SARS between PCR and a biosensor according to the invention based on nasopharyngeal swabs taken at patient admission. The analyses will take into account the confirmed (at the outset or after repeated examinations) and probable diagnoses made by the medical team independently of the result of the biosensor analysis. Doubtful atypical cases (no complete diagnostic approach) will be excluded before analysis of this first validation study. CRITERIA OF Male or female or child without age limit INCLUSION Admitted to a Reference Health Establishment (RHS) in an emergency unit, hospitalization or intensive care unit for suspicion of SARS-COV-2 infection, regardless of clinical presentation and degree of severity. Patient to be diagnosed by PCR test on nasopharyngeal swab. Social insured CRITERIA OF Atypical or suspicious cases without a final diagnosis of COVID- NON-INCLUSION 19 positive or negative Refusal of the patient to participate (collection of information, and second use of the sample, collection of saliva by spitting) Pregnant and breastfeeding women Protected Majors NUMBER OF 200 patients: 100 with a positive diagnosis of SARS-COV-2 and PATIENTS 100 with a negative diagnosis of SARS-COV-2 defined by the gold standard by the medical team STATISTICAL Analysis method and strategy ANALYSES The statistical analyses will be carried out using SAS software (version 9.4 or higher). All the statistical tests will be bilateral with a first species risk of 5%. The quantitative variables will be described by the mean and standard deviation in case of Gaussian distribution, or by the median and interquartile (i.e. 25th and 75th percentiles) in the opposite case. The normality of the distributions will be assessed graphically by histograms and by the Shapiro-Wilk test. The qualitative variables will be described by the numbers and percentages of each modality. Concordance between PCR and biosensor To meet secondary objective 1, Cohen's Kappa coefficient between the diagnosis of CoV-2 SARS made by the biosensor device and the PCR will be calculated as well as its bilateral 95% confidence interval. A Kappa value >0.8 will be considered excellent, and a value between 0.6 and 0.8 will be considered good. The percentage of agreement will be reported with its bilateral 95% confidence interval and disagreements will be discussed in comparison to standard gold.

[0095] Results of the clinical trial are in FIG. 6. As can be seen, the mutated SARS-CoV-2 nanobody (VHH C13) of the present invention allows to have 95% of concordance with PCR results for negative samples, only 5 PCR negative sample which have been detected positive with the mutated SARS-CoV-2 nanobody of the present invention. Regarding PCR positive samples, 99% of concordance is demonstrated, only 1 PCR positive sample which has been detected negative with the mutated SARS-Cov-2 nanobody of the present invention.

Example 5: Application of the Mutated Nanobody of the Present Invention to HSV-1

[0096] Example of HSV-1 nanobody: VHH 05 cys

[0097] The sequence of VHH 05 cys is as follows:

TABLE-US-00006 (SEQ ID NO: 3) QVQLVESGGGLVCPGGSLTLSCAASGFSFSTTTMKWVRQAPGKGLERVS FINRDSTFTQYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTAVYYCAT ASRITEGADFRGQGTQVTVSSGSHHHHHH

[0098] The nanobody contains a C-terminal polyhistidine tag in order to facilitate the purification.

[0099] Before the grafting of HSV-1 nanobodies of example 2 on gold electrodes, the gold interfaces were cleaned following the procedure hereafter: Electrodes were irradiated with UV-OZONE for 5 min. Then washed with mQ water dried with a dry air. In the second time, electrodes were cleaned electrochemically with 0.5M H.sub.2SO.sub.4 solution. For this, connect the electrodes to the potentiostat (Palmsens, Sensit), deposit H.sub.2SO.sub.4 on the electrodes and start the cleaning method (Table.1). When the method is finished, rinse electrodes with mQ water and then dry with a dry air. Once cleaned, 2 types of gold electrodes were produced using 2 different nanobodies: [0100] 1. Gold electrode grafting with a mutated nanobody of the present invention: VHH 05 cys A solution at 100 μg/mL in PBS 1× of HSV-1 nanobody VHH 05 cys is dropped on electrodes and kept at 4° C. overnight under humid atmosphere. Surfaces were washed, dried and kept at 4° C. until use. [0101] 2. Gold electrode grafting with the non-mutated nanobody (VHH 05) using EDC/NHS chemistry on L-Cysteine modified interfaces. A solution L-Cysteine (SigmaAldrich, France) at 2 mM on PBS 1× were dropped on electrodes and kept at 4° C. overnight under humid atmosphere. Electrodes were washed, dried and a solution of NHS/EDC (SigmaAldrich, France) at 15 mM was dropped on surfaces (2h at 4° C.) after 2 hours electrodes were washed, dried and a solution at 100 μg/mL of HSV-1 nanobody VHH 05 was incubated overnight at 4° C. under humid atmosphere. Surfaces were washed, dried and kept at 4° C. until use.

[0102] After the grafting of each gold electrode, differential pulse voltammograms (DPV) were measured for each final electrode. The solution used of the measure is Ferrocenmethanol (FcMeOH, 1 mM) in PBS (0.1M). DPV Parameters are as follows: equilibrium time 3s; Estep=0.01V; Epuls=0.06V; t=0.02s; scan rate: 0.06V/s. Then, the capacity of each final electrode of specifically binding HSV-1 virus was measured by establishing a calibration curve of DPV according to different concentration of HSV-1 virus (dilution of stock solution of virus 10.sup.7 pfu/mL) in PBS (0.1M). Incubation for 10 min with the lowest concertation of HSV-1 virus was started, a DPV signal recorded, and next higher concentration of HSV-1 virus added. The solution used of the measure is Ferrocenmethanol (FcMeOH, 1 mM) in PBS (0.1M). DPV Parameters are as follows: equilibrium time 3 s; Estep=0.01V; Epuls=0.06V; t=0.02s; scan rate:0.06V/s.

[0103] The results are shown in FIG. 7. As can be seen, the mutated nanobody of the present invention VHH 05 cys binds rapidly to HSV-1 from 10.sup.3 pfu/mL, for the other electrodes no viral binding is observed. Therefore, the mutation of the HSV-1 nanobody at a specific position in its sequence, particularly at position 13, by a cysteine makes it possible to obtain a very sensitive and fast-reading electrode that meets the specifications of a device.