Patent Application
20230258637
The present disclosure relates to methods for detecting a target in a sample using mutated nanobodies.
Epidemic and pan epidemic outbreaks, as the one the world is facing currently with the COVID-19 outbreak, can be better managed with the availability of efficient, portable and cheap diagnostic devices.
The market of “viral biosensors” is still in its infancy and cannot respond to the current demands of screening a large part of the population being infected, neither providing a tool where daily diagnostic can be achieved in an easy and personalized manner. Biosensors hold however great impact to turn current analytical methods into diagnostic strategies by simple restructuring their sensing module for the detection of protein biomarkers and viruses (Dong et al., Bioelectrochemical and Bioenergetics. 1997, 42, 7; Xue et al., Nature Communications. 2014, 5, 4348; Yang et al., Journal of Electroanalytical Chemistry 2001, 516, 10).
Unquestionably, such sensing platforms require continuous updates to address growing challenges in the diagnosis of viruses as these are changed quickly and spread largely from person-to-person, indicating the urgency of early diagnosis. This requires the development of an easily sensing platform, which can be adapted quickly to changing demands. Furthermore, with the rise in personalized medicine and adapted technologies for wireless sensing, current as well as future viral contaminations of millions of people can be controlled and limited. The requirement for weekly confinement can in this manner be limited and even avoided, with unimaginable positive consequences for the economy of a country and the globe.
Currently, the reference diagnosis are real-time reverse transcriptase PCR assays on nasal swabs (effective in early infection with signs of nasopharyngeal involvement) and on tracheal swabs (effective in the event of infection at a slightly later stage with broncho-pneumopathy but more invasive and therefore reserved for severe cases). The time required to obtain RT-PCR results is long, which considerably limits the rapid management measures necessary in the event of a pandemic. In addition, there is the question of mass screening, particularly of asymptomatic patients. One of the major challenges is to gain time in analysis, but also to be highly specific and decrease the amount of false negative responses, as often occurring in RT-PRC analysis.
The technical solution to this problem could therefore be the use of an intelligent portable biosensor based electrochemical read out through the surface immobilization of nanobodies specific to a target of interest for the fast and selective sensing of such target.
Over the years, the possibility of detecting a target substance through antibody immobilization onto a surface, also called, immunosensor or immunobiosensors, has been documented. However, batch-to-batch inconsistency of polyclonal antibodies and extensive production time of monoclonal antibodies, pose additional limitations.
Single domain antibodies, also called camelid-derived antibodies or nanobodies, are the recombinantly expressed binding fragments derived from heavy chain antibodies found in camels and llamas. These unique binding elements offer many desirable properties such as their small size (˜15 kDa) and thermal stability, which makes them attractive alternatives to conventional monoclonal antibodies. These nanobodies have been reported to be inherently unaffected by changes in temperature and retain their structure due to high refolding efficiency (Ingram et al., Exploiting Nanobodies' Singular Traits, 2018; Goldman et al., Enhancing Stability of Camelid and Shark Single Domain Antibodies, 2017; Goldman et al., Analytical Chemistry 2006, 78, 8245). Successful utilization of these nanobodies in sandwich immunoassays immobilized on magnetic microspheres, for example, has been demonstrated effective in detecting various pathogens and toxins (Anderson et al., Analytical Chemistry 2008, 80, 9604; Anderson et al., Analytical Chemistry 2010, 82, 7202.
US2017059561A1 describes single-domain antibodies as the sensing agents immobilized onto a surface of an immunosensing device. However, to facilitate the immobilization of the nanobody, the conductive electrode was chemically functionalized by at least one self-assembled monolayer prior to the immobilization of the nanobody which is time consuming. Further, the orientation of the nanobody to optimize its target recognition capability is not discussed.
The use of nanobodies for the development of materials for specific portable biosensors for rapid reading, for example, has never been reported. There is therefore an unfulfilled need for a portable detection device that is easy to design, cheap to produce while being specific to a target of interest, having a fast reading and possessing good statistic results.
The inventors of the present invention have discovered that the mutation of a nanobody in a specific region of its sequence makes it possible to graft it directly onto a surface while making it possible to correctly orient the paratope of the nanobody. The resulting sensing platform thus makes it possible to solve the aforementioned technical problems by allowing rapid detection at low costs, while being specific to a target of interest.
The present inventors have found a way to design and produce a “biosensor” based on an electrochemical read out though the surface immobilization of mutated nanobodies specific to a target, for example the SARS-CoV-2 receptor binding domain (RBD) protein.
One first aspect of the invention relates to a mutated nanobody which binds to a target wherein an amino acid present in the loop of the FR1 region of the framework, preferably at position 12, 13, 14 or 15, is mutated to cysteine.
In another aspect, the invention relates to the mutated nanobody of the present invention, wherein the mutated nanobody is attached to a surface.
The present invention further relates to a biosensor comprising the surface of the present invention, for detecting a target in a sample.
Finally, the present invention further relates to a method for detecting a target in a sample, the method comprising:
a. Providing the biosensor of the present invention,
b. Contacting the biosensor with the sample,
c. Measuring the response at the biosensor surface, and
d. Determining the presence or the absence of the target.
Terms as used throughout the invention are hereafter defined.
As used herein, the term “target” refers to a sensing target that is selected from the group consisting of viral proteins, protein biomarkers, bacteria proteins, membrane proteins, protozoa proteins, fungi proteins, and prion proteins. In an embodiment, the target originates from infectious agents. In a preferred embodiment, the target is a viral protein, preferably a SARS virus protein, more preferably SARS-CoV-2 virus protein, and even more preferably SARS-CoV-2 receptor binding protein (RBP). In another preferred embodiment the target is a HSV-1 virus protein. Additional examples of infectious agents are well known to a person of ordinary skill in the art and such embodiments are within the purview of the current invention.
As used herein, the term “sample” refers to biofluids such as nasal swab, mouth swab, spit, blood, amniotic fluid, aqueous humor, vitreous humor, bile, cerebrospinal fluid, chyle, endolymph, perilymph, female ejaculate, male ejaculate, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, sputum, synovial fluid, vaginal secretion. In a preferred embodiment, the sample of the present invention is selected from the group consisting of nasal or mouth swab, spit, and blood. The biofluid can be processed prior to analysis according to the current invention. For example, blood can be processed to make it suitable for analysis by the electrochemical biosensor of the current invention, for example, by centrifugation to removal of the cells or by deproteinization. The person skilled in the art knows how to process the sample depending on its origin, in order to make it suitable for analysis by the biosensor device of the present invention.
If not stated herein otherwise, “about” means±20%, preferably ±10%, more preferably ±5%.
The Mutated Nanobody
Nanobodies are a class of antigen-binding protein derived from camelids comprising only a single 15 kDa variable domain. They exhibit improved stability and are able to bind a large number of epitopes, sometimes even to those not accessible to classical antibodies.
In the present invention we rely on the nomenclature described in the article Laura S. Mitchell, Lucy J. Colwell “Comparative analysis of nanobody sequence and structure data” Proteins. 2018; 1-10 which is hereby incorporated by reference. This article describes the structure of nanobodies, as shown in
Between these three CDR regions there is the framework. This framework is composed of 4 regions called FR1, FR2, FR3 and FR4. Laura S. Mitchell and Lucy J. Colwell article has studied the frequency of mutation from one nanobody to another which is shown in
As demonstrated, nanobodies do not diversify their framework region from one to another to compensate for the loss of the VL domain. Based on the nomenclature of Laura S. Mitchell and Lucy J. Colwell article, the framework from one nanobody to another extends from position 1 to position 25 for FR1, from position 36 to position 49 for FR2, from position 60 to position 97 for FR3 and from position 117 to position 126 for FR4.
The mutated nanobody of the invention is mutated in the framework of the nanobody. To determine the positions of the amino acids in the sequence and to determine the position of the mutated amino acid, we will use in the present invention the nomenclature of Laura S. Mitchell and Lucy J. Colwell article. This framework being conserved from one nanobody to another, the mutation is applicable to all nanobodies. According to the nomenclature of Laura S. Mitchell and Lucy J. Colwell article, the mutation is made in positions which are in the loop of the FR1 region and the mutation consists in replacing the amino acid present at the aforementioned position by a cysteine (Cys or C). This mutation is made at a position that is at the opposite side relative to the paratope, and thus, from the highly variable CDR regions of the nanobody. Indeed, being in such position from the paratope allows to use the mutated nanobody of the present invention to be grafted on a surface in such a way as to orientate the paratope in the opposite direction to the surface while avoiding dimerization. Preferably, the mutation is made in the FR1 region at position 12, 13, 14, or 15.
One first aspect of the invention relates to a mutated nanobody which binds to a target wherein an amino acid present in the loop of the FR1 region, preferably at position 12, 13, 14, or 15, more preferably at position 13 is mutated to cysteine.
Nanobodies are well-known for the person skilled in the art (Serge Muyldermans “Nanobodies: Natural Single-Domain Antibodies” Annu. Rev. Biochem. 2013. 82:775-97; Laura S. Mitchell, Lucy J. Colwell “Comparative analysis of nanobody sequence and structure data” Proteins. 2018; 1-10). Therefore, according to the target to be detected, the person skilled in the art will know how to produce the nanobody specific to said target and thus to transpose the aforementioned technology according to the present invention to any nanobody specific to any target. The process of nanobody generation through llama immunization and phage display is described in Desmyter et al, Curr Opin Struct Biol. 2015; 32:1-8. The present invention is therefore not limited but encompasses all the possibilities of detection of a target of interest by a specific nanobody that is mutated according to the present invention.
Typically, the mutated nanobody of the present invention may comprise a tag sequence, such as for example a polyhistidine tag. Typically, the mutated nanobody of the present invention may be linked to another peptide or polypeptide, such as for example to another nanobody to form diabodies. The other nanobody may bind to the same target as the target of the mutated nanobody of the present invention or to an alternative target.
The Mutated Nanobody Attached to a Surface
Nanobodies have been successfully applied for the development of biosensors. Biosensors are devices where a bioreceptor, in this case a nanobody, is in close contact with a surface, in particular a transducer, that converts the biorecognition event into a measurable signal.
In an embodiment of the present invention, the mutated nanobody is attached to a surface. By “surface” it is referred to in the present invention a conductive substrate that may be flat or curved. In an embodiment, the surface is a gold surface and preferably a working electrode. In another embodiment, the surface is 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, silver. In a preferred embodiment, the surface is graphene surface. Preferably, the surface is a working electrode. In a preferred embodiment, the surface is the working electrode of a biosensor device.
The present invention also relates to a biosensor comprising the surface of the present invention for detecting a target in a sample.
The nanobody modified working electrode became a biosensor capable of analyzing in real time intermolecular interactions on a sensor chip with the use of electrochemical techniques respectively. A gold surface layer or a surface layer selected from the group consisting of graphene, reduced graphene oxide and its derivatives, or a metal surface other than gold is formed on a sensor chip, so that the cysteine-modified nanobody of the present invention can be grafted stably with its high binding capability. If the nanobody interacts with the target in the sample, binding and dissociation can be detected in real time.
Regarding the electrochemical sensor, the changes in the electrochemical properties, for example, electron transfer properties, at the sensing electrode surface upon binding of the target to the nanobody attached to the sensing electrode surface can be measured by a test equipment.
Various electrochemical properties at the sensing electrode surface that can be measured by the test equipment include, but are not limited to, electrical resistance, potential and current changes. In certain embodiments, the test equipment, is a voltmeter, ohmmeter, ammeter, multimeter, or a potentiostat. In a further embodiment, the tests performed with this equipment may be a differential pulse voltammetry, electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), linear sweep voltammetry (LSV) chronoamperometry (CA), or chronopotentiometry.
Differential pulse voltammetry (DPV) is a voltammetry method used to make electrochemical measurements. It is a derivative of linear sweep voltammetry, with a series of regular voltage pulses superimposed on the potential linear sweep or stairsteps. The current is measured immediately before each potential change, and the current difference is plotted as a function of potential. By sampling the current just before the potential is changed, the effect of the charging current can be decreased in favor of the faradic current making the method highly sensitive.
DPV allows direct analyses down to pM protein concentrations and low bacteria concentrations among others. In the present invention, the redox probe used is ferrocene-methanol (FcMeOH) but can be any other redox couple such as ferroceylamine (Fc-NH2), ferricyanide (Fe(CN)63−] ferrocyanid Fe(CN)64−], ruthenium hexamine (Ru(NH3)6), etc. for the detection of interaction of the target with the grafted nanobody as interaction will considerably decrease the electron charge transfer interaction. The presence of the target acts as a diffusion barrier, hindering the charge transfer from the redox probe to the surface. Addition of the target present in a sample results in a decrease of the redox current as the complex nanobody-target forms a barrier.
Therefore, electrochemical sensor to which the nanobody of the present invention has been bound is useful as a biosensor device for analysis of target-nanobody interactions.
In a further aspect, the present invention relates to a method for detecting a target, preferably a virus, in a sample, the method comprising:
In the method of the current invention the sample containing the target to be measured is contacted with the sensor under conditions that allow the formation of the nanobody-target complexes for a period of time sufficient to allow the formation such complexes. In a preferred embodiment, the sample containing the target to be measured is contacted with the sensor under conditions that allow or promote only specific binding between the paratope of the nanobody of the present invention and the target in the sample. The nanobody of the present invention being specific to the target of interest, non-specific binding between the nanobody and other chemicals in the sample is avoided. A period of time sufficient to allow the formation of the nanobody-target complexes can be about 1 minute to about 60 minutes, about 1 minutes to about 10 minutes, or about 5 minutes.
Grafting of the Nanobody of the Present Invention onto a Surface
The nanobody of the present invention can be attached to the surface in various ways. In certain embodiments, the nanobody is attached to the surface in a covalent manner. Binding in a covalent manner ensures that the nanobody is, for practical purposes, permanently attached to the sensor which avoids the loss of the nanobody, for example, during the washing of the sensor. In another embodiment, the nanobody is attached to the surface in a non-covalent manner.
The immobilization of nanobodies onto the surface is a pivotal step for the construction of the biosensor device of the present invention, as it affects selectively and sensitivity. In an embodiment, the sensor modification is based on the interaction of thiolate groups of the nanobody with gold through the formation of Au—S bond, classically used for gold electrode modification. The strength of the gold-sulphur (Au—S) interaction formed between thiols and gold surfaces provides the basis to fabricate robust self-assembled monolayers for diverse applications. In another embodiment, the sensor modification is based on the interaction of the 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, silver, with the thiolate groups of the nanobody which provides the basis to fabricate robust self-assembled monolayers for diverse applications.
In certain aspects, the sensor modification is based on the interaction of a short linker between the nanobody and 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, silver, preferably gold or graphene, in such a way that the cysteine of the nanobody is at a distance of less than 1 nanometer (1 nm) of said surface. The short distance allows to fabricate robust self-assembled monolayers for diverse applications. In certain aspects, the short linker may be a pyrene-based linker with attached functional ending such as COOH, NH2, or maleimide (MAL), or PEG-aryl radicals such as PEG-aryldiazonium salts, that attacks the surface through the reduction of aryldiazonium ions. Examples of aryldiazoniums salts are 4-[(triisopropylsilyl)ethylenyl]benzenediazonium tetrafluoroborate (TIPS-Eth-ArN2+), 4-nitrobenzene diazonium tetrafluoroborate, 4-Methoxybenzenediazonium tetrafluoroborate, 4-Bromobenzenediazonium tetrafluoroborate, and 4-carboxylicacid benzenediazonium tetrafluoroborate. In an embodiment, the functional groups of the aryldiazonium attached to the 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, silver may be post-functionalized with PEG-maleimide, PEG-iodoacetyl1-pyrenebutyric acid (PBA), 1-pyrenebutanoic acid succinimidyl ester (PBASE), or pyrene-maleimide (Py-MAL), which is linked to the thiolate groups of the nanobody.
Nanobodies of the present invention are mutated in order to replace an amino acid of the framework by a cysteine. The sulfide group of the cysteine residue allows to graft directly the mutated nanobody of the present invention onto the gold surface. Thanks to the remote position of the cysteine residue inserted in the sequence with respect to the paratope, the nanobody of the present invention is oriented in such a way that the paratope in the opposite direction to the surface of the sensor. In this way, each nanobody grafted onto the surface is capable of binding the target present in the sample. Further, the direct grafting of the nanobody onto the surface or the grafting through a short linker of the present invention allows to reduce the distance that the electrons have to travel in order to trigger the biosensor signal. In such a way, the mutated nanobody of the present invention allows to be grafted easier, rapidly and to provide a biosensor which is specific to a target of interest, having a fast reading. In an embodiment, the grafting does not use a linker between the nanobody and the surface of the sensor.
In an embodiment, the amount of the mutated nanobody used for the surface grafting is between 0.01 and 20 mg/mL, preferably between 0.05 and 10 mg/mL, and more preferably between 0.1-1 mg/mL. The time for grafting is between 2 and 24 h preferably between 3 and 12 h and more preferable between 9 and 12 h. The solution pH of the PBS 1× dilution is between 5 and 8, preferable between 6 and 8 and more preferable between 7.4 and 8.
1. Production of the Non-Mutated Nanobody Specific to the Receptor Binding Protein of the SARS-Cov-2 Virus: VHH 72
The sequence of the non-mutated nanobody specific to the receptor binding protein of the SARS-Cov-2 virus is as follows:
The nanobody contains a C-terminal polyhistidine tag in order to facilitate the purification.
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).
2. Production of the Mutated Nanobody Specific to the Receptor Binding Protein of the SARS-Cov-2 Virus of the Present Invention VHH C13
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.
The sequence obtained of the mutated nanobody of the present invention VHH C13 is as follows:
3. Production of the Mutated Nanobody Specific to the Receptor Binding Protein of the SARS-Cov-2 Virus VHH-Biotin
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.
4. Production of the Mutated Nanobody Specific to the Receptor Binding Protein of the SARS-Cov-2 Virus of the Present Invention VHH C12
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.
The sequence obtained of the mutated nanobody of the present invention VHH C12 is as follows:
Before the grafting of SARS-CoV-2 nanobodies of example 1 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 H2SO4 solution. For this, connect the electrodes to the potentiostat (Palmsens, Sensit), deposit H2SO4 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.
Once cleaned, 3 types of gold electrodes were produced using the 3 different nanobodies of example 1.
1. Gold Electrode Grafting with the Mutated Nanobody of the Present Invention: VHH C13 or VHHC12
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 NH2-PEG6-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.
2. Gold Electrode Grafting with the Non-Mutated Nanobody VHH
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.
3. Gold Electrode Grafting with the Biotin Mutated Nanobody: VHH-Biotin
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.
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
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
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.
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.
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.
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.
Extraction with the MGI Easy Nucleic Acid Extraction Kit on the MGISP-960 Automated Sample Preparation System.
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.
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
The clinical trial was conducted on the basis of the following protocol.
Results of the clinical trial are in
Example of HSV-1 nanobody: VHH 05 cys
The sequence of VHH 05 cys is as follows:
The nanobody contains a C-terminal polyhistidine tag in order to facilitate the purification.
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 H2SO4 solution. For this, connect the electrodes to the potentiostat (Palmsens, Sensit), deposit H2SO4 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:
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 107 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.
The results are shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 20306199.9 | Oct 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/078184 | 10/12/2021 | WO |
