WO2012085320A1 - Immunosensor surface for the direct detection of benzoylecognine by means of surface plasmon resonance - Google Patents

Immunosensor surface for the direct detection of benzoylecognine by means of surface plasmon resonance Download PDF

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WO2012085320A1
WO2012085320A1 PCT/ES2011/070893 ES2011070893W WO2012085320A1 WO 2012085320 A1 WO2012085320 A1 WO 2012085320A1 ES 2011070893 W ES2011070893 W ES 2011070893W WO 2012085320 A1 WO2012085320 A1 WO 2012085320A1
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benzoylecgonine
immunosensor
detection
polymer
antibodies
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PCT/ES2011/070893
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Spanish (es)
French (fr)
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Ricardo Riguera Vega
Eva María MUÑOZ VALENTÍN
Manuel LÓPEZ-RIVADULLA
Óscar QUINTELA JORGE
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Universidade De Santiago De Compostela
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection
    • G01N21/553—Attenuated total reflection and using surface plasmons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/946—CNS-stimulants, e.g. cocaine, amphetamines

Definitions

  • the invention is directed to the detection of benzoylecgonine, the main metabolite of cocaine, more specifically it is directed to the direct detection of benzoylecgonine.
  • Cocaine is a potent central nervous system stimulant. Its consumption causes extreme energy and agitation at the beginning, then shivering. Cocaine is the illicit drug of greater expansion in the world and is generally self-administered by nasal inhalation, intravenous injection and smoked.
  • samples of oral fluid has advantages over other matrices (such as urine or blood) since the collection of the sample is easy, fast and non-invasive.
  • Indirect detection methods are characterized in that they do not directly measure the interaction between the recognition element and the benzoylecgonine of the test sample, but measure another interaction or another stage related to it. In general, they involve performing additional steps of interaction and / or reaction to part of the association between cocaine / benzoylecgonine of the test sample with its recognition element. It also implies, in some designs, the need to use labeled molecules (for example, fluorescent marking). These requirements that characterize the indirect methods translate into a series of drawbacks: 1) an increase in the measurement time of the sensor; 2) a greater probability of making measurement errors during the multiple intermediate stages. 3) industriousness and higher cost of the method when labeled molecules are used.
  • an aptamer is used as a benzoyleegoniga recognition element and consists of three stages: the aptamer-benzoyleegonine junction (stage 1) begins the synthesis of a strand of DNA (stage 2) which in turn interacts with a fluorescent molecule (stage 3).
  • stage 1 begins the synthesis of a strand of DNA
  • stage 2 begins the synthesis of a strand of DNA
  • stage 3 the fluorescent molecule
  • the detector measures changes in fluorescence in the molecule associated with its interaction with DNA and which in turn are related to the presence of benzoylecgonine.
  • This device allows the detection of cocaine in a concentration of 0.6 g L, but in a measurement time of not less than 1 hour.
  • the SPR sensors detect the analyte mass that is associated with the surface of a sensor-chip where its recognition element is immobilized and therefore, the sensitivity of the method is directly proportional to the molecular weight of the analyte. For this reason, small molecules provide a lower SPR signal, making it difficult to perform an accurate measurement.
  • the method consists of two stages: 1) association of the antibody to the drug immobilized on the chip, which is detected by an increase in the SPR signal; 2) the chip is incubated with the sample containing the drug in solution, and that competes with the drug immobilized for its interaction with the antibody. In this step there is a decrease in the SPR signal corresponding to the amount of antibody that was disassociated from the immobilized drug to associate with the drug in solution. This decrease is related to the concentration of drug present in the sample.
  • the invention provides a new immunosensory surface that allows the direct detection of the main metabolite of cocaine, benzoylecgonine, that is, the detection of interaction between a recognition element and the benzoylecgonine of the test sample. In addition, it allows this detection to be carried out, in situ, and in real time.
  • This new immunosensor surface is specially designed so that the detection method is quantitative, precise, selective and sensitive.
  • An additional advantage is that this immunosensor surface can be used by personnel without specific knowledge in the technology, the sample to be analyzed does not need to be previously treated and can be reused. In this way, the invention provides an immunosensor surface suitable for performing cocaine controls in situ with the required accuracy, for example, during roadside controls.
  • the invention is directed to an immunosensor surface comprising a support coated by a metal layer to which a polymer that immobilizes at least one anti-benzoylecgonine antibody binds.
  • the invention is directed to an immunosensor surface comprising a support coated by a metal layer to which a polymer that immobilizes two or more anti-benzoylecgonine antibodies characterized by having different affinity for benzoylecgonine is bound.
  • Another aspect of the invention is directed to an immunosensor comprising an immunosensor surface, as defined above, and a transducer based on the optical phenomenon of surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • Another aspect of the invention is directed to an apparatus for the direct detection and quantification of benzoylecgonine comprising an immunosensor, as defined above, and a device or instrument for processing or conditioning the signal received during the association or dissociation stage.
  • Another aspect of the invention is directed to a kit comprising an immunosensor surface as defined above.
  • Another aspect of the invention is directed to a portable apparatus comprising an immunosensor surface as defined above.
  • Another aspect of the invention is directed to a method of detecting benzoylecgonine and in another aspect to two alternative methods for quantifying benzoylecgonine.
  • a final aspect of the invention is directed to the use of an immunosensor surface as defined above for the direct detection of benzoylecgonine.
  • Figure 1A schematically represents the association of benzoylecgonine with an immunosensor surface: (1) sensor-chip; (2) polymer; (3) antibody; (4) benzoylecgonine.
  • Figure IB represents a sensorgram detailing the stages of association (1) and equilibrium (2) after the beginning of sample injection (ci) and the dissociation stage (3) once the sample injection (fi ).
  • Figure 2A and 2B shows the sensorgrams recorded when injecting samples with different concentration of benzoylecgonine (865, 433, 216, 108, 54, 27, 13.5 and 6.8 nM) on the immunosensor surfaces of examples la and Ib, respectively.
  • Figure 3 shows the calibration curves of example 4 obtained after the analysis of the stages of (A) association and (B) dissociation of the sensorgrams registered with the immunosensor surface of example la.
  • Figure 4 shows the calibration curves of example 4 obtained after the analysis of the stages of (A) association and (B) dissociation of the sensorgrams registered with the immunosensor surface of example Ib.
  • Figure 5 shows the correlation graphs between benzoylecgonine concentration values obtained experimentally with the immunosensor surface II and values calculated with the calibration curves of example 4, obtained after the analysis of the stages of (A) association and (B) dissociation .
  • an immunosensory surface is understood as a biological receptor prepared to specifically detect a substance due to the specificity of the receptor-substance interaction.
  • the anti-benzoylecgonine antibody is capable of specifically recognizing benzoylecgonine in a test sample.
  • the characteristic affinity of each antibody determines the concentration range of benzoylecgonine that can be detected with said antibody. This is defined by upper and lower detection limits corresponding to 10 times and 0.1 times the dissociation constant of the benzoylecgonine-antibody interaction, respectively. Therefore, the use of different anti-benzoylecgonine antibodies on the same immunosensory surface makes it possible to detect benzoylecgonine in different concentration ranges.
  • the immunosensor surface comprises between two and six anti-benzoylecgonine antibodies characterized by having different affinity for benzoylecgonine.
  • the immunosensor surface comprises between two and four anti-benzoylecgonine antibodies characterized by having different affinity for benzoylecgonine.
  • the immunosensor surface comprises an anti-benzoylecgonine antibody.
  • the immunosensor surface further comprises an additional white channel.
  • a white channel or control is understood to be that channel that does not comprise an antibody and thus the signal detection in said channel will correspond to the target or control with which the other measurement signals will be compared. This allows you to discard any noise or interference signals that may be present.
  • the immunosensory surface consists of a support coated by a metal layer to which a polymer that immobilizes at least one anti-benzoylecgonine antibody and a white channel binds.
  • the immunosensor surface consists of a support coated by a metal layer to which a polymer that immobilizes between one and six anti-benzoylecgonine antibodies characterized by having different affinity for benzoylecgonine and a white channel binds.
  • anti-benzoylecgonine antibodies are selected from monoclonal antibodies or polyclonal antibodies.
  • Polyclonal antibodies means those that are produced by a variety of stem cells and are constituted by a natural physiological mixture of structurally distinct antibodies, which bind to different parts of the antigen.
  • “Monoclonal antibodies” means those that are produced by identical stem cells and are constituted by identical antibodies; Its structural homogeneity translates into greater specificity and affinity for the antigen.
  • anti-benzoylecgonine antibodies are monoclonal antibodies.
  • anti-benzoylecgonine monoclonal antibodies examples include antibodies B1077-01, B1077-08, B1077-09, B1077-10, B1077-12 and B1077-15 (these antibodies are marketed by USBiological).
  • immobilization of the antibody is understood as the stable and effective binding of the antibody to the polymer. Stable and effective binding leads to a detection of benzoylecgonine with greater reproducibility and sensitivity, and in this way the problems of the state of the art for the detection of small molecules are solved directly.
  • An immunosensor surface capable of directly detecting and providing Real-time benzoylecgonine and quantify it. It also allows the reuse of the sensor surface. It is possible to achieve stable immobilization, for example, by covalent bonds between the polymer and the antibody.
  • the anti-benzoylecgonine antibodies are bound to the polymer by covalent bonds.
  • covalent bonds are selected from amide bond and carbonyl hydrazine bond.
  • the amide bond can be formed between carboxylic groups of the polymer and amino groups present in amino acids lysine of the antibody.
  • the covalent bond between the polymer and the antibody is a carbonyl hydrazine type bond.
  • the carbonyl hydrazine bond can be formed between carboxylic groups of the polymer modified by chemical transformations to hydrazine groups and hydroxyl groups of the antibody, modified by chemical transformations to aldehyde groups.
  • modified polymer is meant that intermediate polymer obtained by chemical transformations from a carboxylated polymer such as those set forth in Homola, J., 2008.
  • the carbonyl hydrazine type bond can be formed between carboxylic groups of the polymer and hydroxyl groups of cis-diols present in carbohydrates of the antibody.
  • the carbonyl-hydrazine type bond allows a directed immobilization since the cis-diols are in a specific region of the antibody (Fe region) that does not participate in the interaction with benzoylecgonine. In this way, all antibodies are immobilized in an appropriate orientation where the activity of their active centers is maintained.
  • the polymer is selected from a polycarboxylated polymer and a modified polycarboxylated polymer. These polymers can be branched and thus the modified carboxylic or carboxylic groups can have different arrangements in space.
  • the term "effective immobilization" refers to the immobilization of a sufficient number of surface active antibodies; This number should allow obtaining a signal three times higher than the sensitivity of the detector (which is given by background noise) when the association of benzoylecgonine in a sample of concentration equal to the lower limit of detection determined by the antibody is monitored.
  • the amount of antibody that can be immobilized is related to the immobilization capacity of the polymer.
  • the polymer is selected from polymers capable of immobilizing an amount of anti-benzoylecgonine antibodies between 730 pg / mm 2 and 73000 pg / mm 2.
  • the amount of immobilized anti-benzoylecgonine antibodies is between 2190 pg / mm 2 and 58400 pg / mm 2 . Even more preferably the amount of immobilized anti-benzoylecgonine antibodies is between 7300 pg / mm 2 and 29200 pg / mm 2 .
  • immobilization of the antibody by the selected polymer is monitored in real time, measuring the increase in the SPR signal in uRiU units ("micro-Refractive index Units"), taking into account that 1 uRiU equals 0.73 pg / mm.
  • the polymer is non-stick.
  • non-stick polymer means a polymer that is inert to non-specific interactions between the sample components and the active polymer binding sites.
  • An additional advantage of being inert to non-specific interactions and covering the metal layer is that it protects said metal layer from adhesion or interaction with substances in the sample.
  • the polymer is a non-stick polymer.
  • it is a non-stick polymer for oral fluid samples.
  • the polymer is selected from non-stick polycarboxylated polymers capable of immobilizing between 2190 pg / mm 2 and 58400 pg / mm 2 .
  • non-stick polycarboxylated polymers are polymers of polyethylene glycol and polycarboxylated branched chains (3D hydrogels) used in the commercial sensor-chips HC1000 and HC1500 (Xantec Bioanalitics).
  • the polymer can be attached to the metal layer by known techniques, for example, by joining between gold and sulfur atoms of the polymer, these sulfur atoms being, for example, part of thiol, disulfide or sulfide groups.
  • the metal layer is selected from gold, silver, aluminum and copper; more preferably, between gold and silver.
  • the metal layer has a thickness not exceeding 200 nm, more preferably it has a thickness between 1 nm and 100 nm, even more preferably it has a thickness between 10 nm and 50 nm. In a particular embodiment, the metal layer has a thickness of 50 nm.
  • the support on which said metallic layer is can be made of different materials, for example and without being limiting, it can be made of glass or plastic material. It is also possible that this support is presented in different forms, for example and without limitation, it may have a flat, cylindrical, spherical shape or be part of an optical fiber.
  • the invention is directed to an immunosensor surface comprising a glass support coated with a 50 nm gold layer and the 3D Hydrogel polymer that immobilizes at least one anti-benzoylecgonine monoclonal antibody selected from B1077-08 and B1077 -01.
  • the invention is directed to an immunosensor comprising an immunosensor surface as described above and a transducer based on the optical phenomenon of the SPR.
  • the immunosensor of the invention allows the direct and in situ detection of benzoylecgonine present in a sample thanks to the specific recognition by an anti-benzoylecgonine antibody immobilized on the metal surface of the immunosensor surface, as described above.
  • the transducer based on the optical phenomenon of SPR the mass of benzoylecgonine associated to this surface is monitored in real time through the specific antigen-antibody binding.
  • the invention is directed to an apparatus for the direct detection and quantification of benzoylecgonine comprising an immunosensor as described above and a device or instrument for processing or conditioning the signal received during the association or dissociation stage.
  • this apparatus further comprises a device for performing a calculation.
  • the device or instrument for processing or conditioning the received signal allows its use in a device to perform a calculation.
  • the signal can be read through a digital or analog screen or through a computer.
  • the immunosensor of the invention has high sensitivity and specificity, and allows rapid measurement and reproducibility. It is also reusable, as tested in experiments carried out in the examples.
  • the immunosensor allows real-time detection, its miniaturization is feasible, thus enabling automation and parallel testing, which allows its application in portable devices.
  • the invention relates to an apparatus where the immunosensor is miniaturized and the apparatus is portable.
  • the immunosensor of the invention is a direct detection system without analyte dizziness. This eliminates intermediate steps and indirect measures that add possible errors and lengthen the detection time; (2) the measurement is carried out in real time, with rapid obtaining of the response; (3) it has high specificity, derived from the use of a specific benzoylecgonine antibody as recognition element; (4) it has high sensitivity, which is determined by the antigen-antibody affinity and the number of active antibodies immobilized on the immunosensory surface; (5) it does not exhibit non-specific interaction with benzoylecgonine (or other components of the sample) due to the presence of a non-stick polymer matrix between the metal surface and the antibody; (6) it is feasible to use a reduced sample volume, for example it is feasible to use 100 ⁇ ⁇ ; (7) it is a reusable device since the immunosensory surface can be regenerated for consecutive sample analysis; (8) offers technological advantages such as its easy miniaturization, possibility of automation and parallel testing; (
  • the invention is directed to a method of detecting benzoylecgonine comprising:
  • the immunosensor of the invention or an apparatus incorporating it, as described above comprising the immunosensor surface of the invention.
  • the detection of the antigen-antibody interaction is performed by a SPR spectrometer, and it is possible to incorporate a device for measuring samples in continuous flow. In this way the antibody can be in permanent contact with a regulatory solution that ensures its stability. This solution is replaced by the test sample at the time of injection.
  • the SPR detector records, as a function of time, the increase in the SPR signal associated with the benzoylecgonine mass that specifically binds to the immobilized antibody during the association process. Once the sample injection is finished, the decrease in the SPR signal that occurs as a result of the dissociation process is monitored.
  • the invention provides two alternative methods based on the stages of association and dissociation.
  • the invention is directed to a method of quantification of benzoylecgonine comprising:
  • the SPR (R) response varies linearly with time (t) and the slope (dR / dt) is directly proportional to the concentration of benzoylecgonine in the sample ( Figures 3A and 4A).
  • the corresponding calibration curve relates the slope to the concentration.
  • the invention is directed to an alternative method of quantifying benzoylecgonine comprising:
  • the concentration of benzoylecgonine can be determined through a calibration curve where the SPR response is represented once the injection is finished based on the analyte concentration ( Figures 3B and 4B).
  • the detection and quantification of benzoylecgonine is performed in oral fluid samples, or in mixtures of oral fluid and a regulatory solution; even more preferably in saliva samples or in mixtures of saliva and a regulatory solution.
  • the invention is directed to the use of the immunosensor surface, as described above, for the direct detection of benzoylecgonine.
  • the detection takes place in real time.
  • the detection is carried out in aqueous regulatory solutions, in oral fluid or in mixtures thereof.
  • the regulatory solution may comprise HEPES buffer (2- [4- (2-hydroxyethyl) -l-piperazinyl- (l)] ethanesulfonic acid), phosphate buffer or TRIS buffer (2-Amino-2- hydroxymethyl-propane-l, 3-diol).
  • the materials used are monoclonal antibodies B1077-08 and B1077-01 (USBiological), a HCIOOO sensor chip and an HC1500 sensor chip (Xantec Bioanalytics) consisting of a glass support covered by a 50 nM gold film, In turn covered by a polycarboxylated non-stick polymer matrix (described by the suppliers as 3D hydrogel), the HEPES * regulatory solution was prepared in the laboratory under the following conditions 10 mM HEPES, 150 mM NaCl, pH 7.4.
  • test samples were prepared by diluting a stock solution of benzoylecgonine of lmg / ml concentration in methanol (Cerilliant Corporation) with the HEPES * regulatory solution or with oral fluid from healthy individuals.
  • Monoclonal antibody B 1077-08 is covalently immobilized on the analytical channel of a HCIOOO sensor chip. Immobilization is carried out by formation of amide bonds between surface carboxylic groups and primary amino groups of the antibody (Johnsson, B., Lofas, S., Lindquist, G., 1991. Anal Biochem. 198 (2) , 268-277). During immobilization, a constant flow of HEPES * regulatory solution is circulating on the surface of the sensor-chip, at a flow rate of 10 ⁇ / min. First, the sensor-chip is washed with three consecutive injections of 60 seconds each of an aqueous solution of 0.05 M NaOH / 1M NaCl.
  • the surface is activated with a 10-minute injection of the mixture of N-hydroxysuccinimide (NHS) 0.05 M / N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide (EDC) 0.2 M reagents
  • NHS N-hydroxysuccinimide
  • EDC EDC
  • a solution of antibody 1077-08 B 0.1 mg / ml in a 0.01 M sodium acetate regulatory solution, pH 4.0
  • amino groups of the antibody react spontaneously with the succinimide esters to form amide bonds.
  • the fraction of unreacted succinimide esters is deactivated by injection for 10 minutes of an ethanolamine solution. 1 M, pH 8.5.
  • the immobilization of the antibody was monitored in real time and an increase in the SPR signal of 11000 ⁇ confirmed the immobilization. Taking into account that 1 ⁇ is equivalent to 0.73 pg / mm 2 , in this specific example 8030 pg / mm 2 of antibody was immobilized.
  • control channel is treated with the NHS / EDC mixture followed by ethanolamine under the same experimental conditions as the treatment of the analytical channel, but without immobilizing the antibody.
  • Example Ib Manufacture of the immunosensory surface II with two channels.
  • Monoclonal antibody B 1077-01 is covalently immobilized on the analytical channel of an HC1500 sensor chip.
  • the immobilization is carried out by formation of amide bonds following the same experimental procedure as in example la.
  • the immobilization of the antibody was monitored in real time and an increase in the SPR signal of 10829 ⁇ (7905 pg / mm 2 ) confirmed the immobilization of the antibody.
  • control channel is prepared following the same experimental procedure as in example la.
  • a HEPES * regulatory solution circulates on the immunosensor surface I at a constant flow of 50 ⁇ / min.
  • the sample to be analyzed is injected for 2 minutes on the immunosensory surface at the same flow.
  • the association of benzoylecgonine with the immobilized antibody is monitored in real time through the corresponding sensorgram.
  • the HEPES * regulatory solution circulates again on the surface and the dissociation stage is monitored for 3 minutes.
  • the SPR response recorded in the control channel is subtracted from the response recorded in the analytical channel.
  • a "white" injection of HEPES * regulatory solution without benzoylecgonine is performed under the same experimental conditions as the sample injection, whose response is also subtracted from the sensorgram obtained.
  • This procedure described in literature as a "double reference method” is used to correct small deviations in the sensorgram outside the specific antigen-antibody interaction (Myszka, DG, 1999. J Mol Recognit. 12 (5), 279-284) .
  • the processing of the sensorgrams is done using the SPR Scrubber software (BioLogic vl.lg Software).
  • the recorded sensorgrams are shown in Figure 2A for buffer solutions with different concentrations of benzoylecgonine (865, 433, 216, 108, 54, 27, 13.5 and 6.8 nM). The same experiment is performed with the immunosensory surface II, varying only the injection time of the sample, which in this case is 3 minutes.
  • the recorded sensorgrams are shown in Figure 2B for buffer solutions with different concentrations of benzoylecgonine (865, 433, 216, 108, 54, 27, 13.5 and 6.8 nM).
  • Immunosensor surfaces I and II are regenerated with a 1 minute injection of 0.01 M glycine, pH 2.0. This solution completely eliminates the benzoylecgonine that is still bound to the antibody, without damaging the surface and leaving it ready for the next measurement.
  • the immunosensors comprising the manufactured immunosensor surfaces I and II have similar detection limits (LOD) and quantification (LOQ) of 6.8 nM (2 ⁇ ⁇ ) and 13.5 nM (4 ⁇ ⁇ ) respectively and they operate in a linear concentration range of 865-6.8 nM (250-2)
  • LOD detection limits
  • LOQ quantification
  • a second calibration curve is performed where the SPR response is represented after the end of the injection (during the dissociation stage) as a function of the concentration of benzoylecgonine ( Figures 3B and 4B)
  • the calibration curve follows a linear behavior in the range of concentrations 54 - 6.8 nM (15.6 - 2 ⁇ g / L) and the LOD values are maintained and LOQ measured during the association stage in both immunosensors.
  • Intra-assay reproducibility is verified by duplicate sample analysis where the average value of coefficient of variation calculated between values corresponding to the same concentration is 8.4% and 10.9% for immunosensor surfaces I and II, respectively. These values are satisfactory and are within the range of coefficient of variation values calculated in the determination of cocaine by liquid chromatography in combination with mass spectrometry (Concheiro, M., Gray, TR, Shakleya, DM, Huestis, MA, 2010 Bioanal Chem Anal. 398 (2), 915-924).
  • the inter-assay reproducibility of the longest lasting immunosensor is verified by analyzing two sets of samples of known concentration of benzoylecgonine prepared. The first set is analyzed and the corresponding calibration curve is generated.
  • the second set is analyzed 8 days later and the values obtained are compared with the theoretical values obtained from the calibration line prepared with the first set of samples (Figure 5).
  • the excellent correlation between these values (r ⁇ 0.99; pending ⁇ 1.00) shows the remarkable inter-assay reproducibility.

Abstract

The invention relates to an immunosensor surface for the direct detection of benzoylecgonine by means of surface plasmon resonance. The invention is characterized in that it provides a novel immunosensor surface that enables direct real time detection of benzoylecognine, the main metabolite of cocaine. The detection of benzoylecognine is carried out by means of surface plasmon resonance. Two alternative methods are provided for analyzing the SPR data collected to determine the concentration of benzoylecognine.

Description

SUPERFICIE INMUNOSENSORA PARA LA DETECCIÓN DIRECTA DE B ENZOILECGONIN A MEDIANTE RESONANCIA DE PLASMÓN SUPERFICIAL  IMMUNOSENSOR SURFACE FOR DIRECT DETECTION OF B ENZOILECGONIN THROUGH SURFACE PLASMON RESONANCE
Campo técnico de la Invención  Technical Field of the Invention
La invención se dirige a la detección de benzoilecgonina, el principal metabolito de la cocaína, más en concreto se dirige a la detección directa de benzoilecgonina.  The invention is directed to the detection of benzoylecgonine, the main metabolite of cocaine, more specifically it is directed to the direct detection of benzoylecgonine.
Antecedentes de la invención  Background of the invention
La cocaína es un potente estimulante del sistema nervioso central. Su consumo provoca una energía extrema y agitación en un inicio, pasando después a temblores. La cocaína es la droga ilícita de mayor expansión en el mundo y generalmente es autoadministrada por inhalación nasal, inyección intravenosa y fumada.  Cocaine is a potent central nervous system stimulant. Its consumption causes extreme energy and agitation at the beginning, then shivering. Cocaine is the illicit drug of greater expansion in the world and is generally self-administered by nasal inhalation, intravenous injection and smoked.
Esta droga es la causante de un número importante de accidentes de tráfico cada año This drug is the cause of a significant number of traffic accidents every year
(Walsh, J.M., de Gier, J.J., Christopherson, A.S., Verstraete, A.G., 2004. Traffic Inj Prev.(Walsh, J.M., de Gier, J.J., Christopherson, A.S., Verstraete, A.G., 2004. Traffic Inj Prev.
5(3), 241-253) poniendo de manifiesto la necesidad del desarrollo de técnicas de identificación rápida para controles de droga en carretera. 5 (3), 241-253) highlighting the need for the development of rapid identification techniques for roadside drug controls.
En este sentido, el análisis de muestras de fluido oral presenta ventajas frente a otras matrices (como orina o sangre) ya que la recogida de muestra es fácil, rápida y no invasiva.In this sense, the analysis of samples of oral fluid has advantages over other matrices (such as urine or blood) since the collection of the sample is easy, fast and non-invasive.
Además, la cocaína y sus metabolitos difunden rápidamente al fluido oral desde el plasma, permitiendo la detección de consumo reciente de la droga. In addition, cocaine and its metabolites rapidly diffuse the oral fluid from the plasma, allowing the detection of recent drug use.
En la actualidad, el método de análisis más sensible de cocaína/benzoilecgonina emplea la técnica de cromatografía líquida en combinación con la espectrometría de masas (Concheiro, M., de Castro, A., Quíntela, O., Cruz, A., Lopez-Rivadulla, M., 2008. Anal Bioanal Chem. 391(6), 2329-2338; Concheiro, M., Gray, T.R., Shakleya, D.M., Huestis, M.A., 2010. Anal Bioanal Chem. 398(2), 915-924). Así, este método permite detectar benzoilecgonina en concentraciones por debajo del límite recomendado por la administración de "Substance Abuse and Mental Health Services Administration" (SAMHSA) y que está en 20 g L. Su desventaja radica en que se requiere el traslado de la muestra a un laboratorio haciendo más costoso y largo el proceso de identificación.  Currently, the most sensitive method of analysis of cocaine / benzoylecgonine uses the liquid chromatography technique in combination with mass spectrometry (Concheiro, M., de Castro, A., Quintela, O., Cruz, A., Lopez -Rivadulla, M., 2008. Bioanal Chem Anal. 391 (6), 2329-2338; Concheiro, M., Gray, TR, Shakleya, DM, Huestis, MA, 2010. Bioanal Chem Anal. 398 (2), 915 -924). Thus, this method makes it possible to detect benzoylecgonine in concentrations below the limit recommended by the administration of "Substance Abuse and Mental Health Services Administration" (SAMHSA) and that is at 20 g L. Its disadvantage is that the transfer of the sample is required to a laboratory making the identification process more expensive and lengthy.
Por otro lado, existen sistemas de detección in situ de cocaína/benzoilecgonina en el mercado pero muchos carecen de precisión y ofrecen un análisis meramente cualitativo (Crouch, D.J., Walsh, J.M., Cangianelli, L., Quíntela, O., 2008. Ther Drug Monit. 30(2), 188-195; Walsh, J.M., Crouch, D.J., Danaceau, J.P., Cangianelli, L., Liddicoat, L., Adkins, R., 2007. J Anal Toxicol. 31(1), 44-54). Existe un creciente número de trabajos sobre diseño de sensores de cocaína cuyo objetivo es que sean sensibles y de rápida aplicación, sin embargo la mayoría de ellos siguen siendo métodos indirectos de detección. Los métodos indirectos de detección se caracterizan porque no miden directamente la interacción entre el elemento de reconocimiento y la benzoilecgonina de la muestra problema, sino que miden otra interacción u otra etapa relacionada con esta. En general, implican la realización de pasos adicionales de interacción y/o reacción a parte de la asociación entre cocaína/benzoilecgonina de la muestra problema con su elemento de reconocimiento. También implica, en algunos diseños, la necesidad de utilizar moléculas marcadas (por ejemplo, mareaje fluorescente). Estos requerimientos que caracterizan los métodos indirectos se traducen en una serie de inconvenientes: 1) un aumento en el tiempo de medida del sensor; 2) una mayor probabilidad de cometer errores de medida durante las múltiples etapas intermedias. 3) laboriosidad y encarecimiento del método cuando se emplean moléculas marcadas. On the other hand, there are cocaine / benzoylecgonine in situ detection systems on the market but many lack precision and offer a purely qualitative analysis (Crouch, DJ, Walsh, JM, Cangianelli, L., Quintela, O., 2008. Ther Drug Monit. 30 (2), 188-195; Walsh, JM, Crouch, DJ, Danaceau, JP, Cangianelli, L., Liddicoat, L., Adkins, R., 2007. J Anal Toxicol. 31 (1), 44-54). There is a growing number of works on the design of cocaine sensors whose objective is to be sensitive and quick to apply, however most of them remain indirect methods of detection. Indirect detection methods are characterized in that they do not directly measure the interaction between the recognition element and the benzoylecgonine of the test sample, but measure another interaction or another stage related to it. In general, they involve performing additional steps of interaction and / or reaction to part of the association between cocaine / benzoylecgonine of the test sample with its recognition element. It also implies, in some designs, the need to use labeled molecules (for example, fluorescent marking). These requirements that characterize the indirect methods translate into a series of drawbacks: 1) an increase in the measurement time of the sensor; 2) a greater probability of making measurement errors during the multiple intermediate stages. 3) industriousness and higher cost of the method when labeled molecules are used.
Un ejemplo de método indirecto está descrito en He, J.L., Wu, Z.S., Zhou, H., Wang, H.Q., Jiang, J.H., Shen, G.L., Yu, R.Q., 2010. Anal Chem. 82(4), 1358-1364, donde se emplea un aptámero como elemento de reconocimiento de benzoileegoniga y consta de tres etapas: la unión aptámero-benzoileegonina (etapa 1) inicia la síntesis de una hebra de ADN (etapa 2) que a su vez interacciona con una molécula fluorescente (etapa 3). El detector mide cambios de fluorescencia en la molécula asociados a la su interacción con ADN y que se relacionan a su vez con la presencia de benzoilecgonina. Este dispositivo permite la detección de cocaína en concentración de 0,6 g L, pero en un tiempo de medida no menor de 1 hora.  An example of an indirect method is described in He, JL, Wu, ZS, Zhou, H., Wang, HQ, Jiang, JH, Shen, GL, Yu, RQ, 2010. Anal Chem. 82 (4), 1358-1364 , where an aptamer is used as a benzoyleegoniga recognition element and consists of three stages: the aptamer-benzoyleegonine junction (stage 1) begins the synthesis of a strand of DNA (stage 2) which in turn interacts with a fluorescent molecule (stage 3). The detector measures changes in fluorescence in the molecule associated with its interaction with DNA and which in turn are related to the presence of benzoylecgonine. This device allows the detection of cocaine in a concentration of 0.6 g L, but in a measurement time of not less than 1 hour.
Recientemente, se ha diseñado un sensor electroquímico de detección directa, in situ, de cocaína que utiliza un aptámero como elemento de reconocimiento de la droga (Swensen, J.S., Xiao, Y., Ferguson, B.S., Lubin, A.A., Lai, R.Y., Heeger, A.J., Plaxco, K.W., Soh, H.T., 2009. J Am Chem Soc. 131(12), 4262-4266). Sin embargo, con este dispositivo sólo se han detectado concentraciones de cocaína por encima de 2800 g L.  Recently, an electrochemical direct detection, in situ, cocaine sensor has been designed that uses an aptamer as a drug recognition element (Swensen, JS, Xiao, Y., Ferguson, BS, Lubin, AA, Lai, RY, Heeger, AJ, Plaxco, KW, Soh, HT, 2009. J Am Chem Soc. 131 (12), 4262-4266). However, only cocaine concentrations above 2800 g L. have been detected with this device.
Por otro lado, en la última década, los inmunosensores de Resonancia de Plasmón Superficial (SPR) han cobrado gran importancia como dispositivos para la cuantificación de compuestos (Mullett, W.M., Lai, E.P., Yeung, J.M., 2000. Methods. 22(1), 77-91; Shankaran, D.R., Gobi, K.V., Miura, N., 2007. Sens. Actuators, B FIELD Full Journal Title:Sensors and Actuators, B: Chemical. B121(l), 158-177). Este tipo de sensores permite la detección precisa de analitos en matrices complejas, por la alta especificidad antígeno- anticuerpo. Además, son altamente sensibles gracias a la detección óptica basada en el fenónemo de SPR (Homola, J., 2008. Chem Rev. 108(2), 462-493). También tienen el atractivo de ser dispositivos sencillos, de bajo coste de fabricación, y compatibles con las tecnologías de la microfabricación (Hoa, X.D., Kirk, A.G., Tabrizian, M., 2007. Biosens Bioelectron. 23(2), 151-160). On the other hand, in the last decade, Superficial Plasma Resonance Immunosensors (SPR) have become very important as devices for the quantification of compounds (Mullett, WM, Lai, EP, Yeung, JM, 2000. Methods. 22 (1 ), 77-91; Shankaran, DR, Gobi, KV, Miura, N., 2007. Sens. Actuators, B FIELD Full Journal Title: Sensors and Actuators, B: Chemical. B121 (l), 158-177). This type of sensors allows the precise detection of analytes in complex matrices, due to the high antigen-antibody specificity. In addition, they are highly sensitive thanks to the optical detection based on the SPR phenomenon (Homola, J., 2008. Chem Rev. 108 (2), 462-493). They also have the attractive to be simple devices, low manufacturing cost, and compatible with microfabrication technologies (Hoa, XD, Kirk, AG, Tabrizian, M., 2007. Biosens Bioelectron. 23 (2), 151-160).
La detección directa por SPR ha estado limitada hasta hace pocos años a compuestos de peso molecular igual o superior a 1000 Dalton y en la actualidad, es posible detectar compuestos de peso molecular de alrededor de 200 Dalton. Sin embargo, para la mayoría de los casos la aplicación de SPR para la detección directa de moléculas pequeñas sigue siendo una metodología laboriosa y de baja sensibilidad (de Mol, N.J., 2010. Affinity constants for small molecules from SPR competition experiments. In: Springerlink (Ed.), Methods Mol Biol: surface plasmon resonance, pp. 101-111, 1 ed. Humana Press; Mitchell, J.S., Wu, Y., 2010. Methods Mol Biol. 627, 113-129). Los sensores de SPR detectan la masa de analito que se asocia a la superficie de un sensor-chip donde se encuentra inmovilizado su elemento de reconocimiento y por lo tanto, la sensibilidad del método es directamente proporcional al peso molecular del analito. Por esta razón las moléculas pequeñas proporcionan una señal de SPR menor, dificultando la realización de una medida precisa.  Direct detection by SPR has been limited until a few years ago to compounds of molecular weight equal to or greater than 1000 Dalton and at present, it is possible to detect compounds of molecular weight of about 200 Dalton. However, for most cases the application of SPR for the direct detection of small molecules remains a laborious and low sensitivity methodology (from Mol, NJ, 2010. Affinity constants for small molecules from SPR competition experiments. In: Springerlink (Ed.), Methods Mol Biol: surface plasmon resonance, pp. 101-111, 1 ed. Human Press; Mitchell, JS, Wu, Y., 2010. Methods Mol Biol. 627, 113-129). The SPR sensors detect the analyte mass that is associated with the surface of a sensor-chip where its recognition element is immobilized and therefore, the sensitivity of the method is directly proportional to the molecular weight of the analyte. For this reason, small molecules provide a lower SPR signal, making it difficult to perform an accurate measurement.
La escasez de resultados es un buen indicativo de las dificultades para desarrollar métodos directos en SPR para detectar moléculas pequeñas. Y de hecho, los avances científicos para la aplicación de SPR para la detección de drogas de bajo peso molecular siguen apuntando a metodologías indirectas. Por ejemplo, en el caso de Klenkar, G., Liedberg, B., 2008. Anal Bioanal Chem. 391(5), 1679-1688, se describe un sensor de detección indirecta de drogas, entre ellas la cocaína, que utiliza un sensor-chip en donde es la droga la que se encuentra inmovilizada en su superficie y a la que se une un anticuerpo como elemento de reconocimiento. El método consta de dos etapas: 1) asociación del anticuerpo a la droga inmovilizada en el chip, que se detecta mediante un incremento en la señal de SPR; 2) el chip se incuba con la muestra que contiene la droga en disolución, y que compite con la droga inmovilizada por su interacción con el anticuerpo. En este paso se produce una disminución de la señal de SPR correspondiente a la cantidad de anticuerpo que se desasoció de la droga inmovilizada para asociarse a la droga en disolución. Esta disminución está relacionada con la concentración de droga presente en la muestra.  The shortage of results is a good indication of the difficulties in developing direct methods in SPR to detect small molecules. And in fact, scientific advances for the application of SPR for the detection of low molecular weight drugs continue to point to indirect methodologies. For example, in the case of Klenkar, G., Liedberg, B., 2008. Anal Bioanal Chem. 391 (5), 1679-1688, an indirect drug detection sensor is described, including cocaine, which uses a sensor-chip where it is the drug that is immobilized on its surface and to which an antibody binds as a recognition element. The method consists of two stages: 1) association of the antibody to the drug immobilized on the chip, which is detected by an increase in the SPR signal; 2) the chip is incubated with the sample containing the drug in solution, and that competes with the drug immobilized for its interaction with the antibody. In this step there is a decrease in the SPR signal corresponding to the amount of antibody that was disassociated from the immobilized drug to associate with the drug in solution. This decrease is related to the concentration of drug present in the sample.
Descripción de la invención Description of the invention
La invención proporciona una nueva superficie inmunosensora que permite la detección directa del principal metabolito de cocaína, benzoilecgonina, es decir, se detecta la interacción entre un elemento de reconocimiento y la benzoilecgonina de la muestra problema. Además, permite que esta detección se realice además, in situ, y en tiempo real. Esta nueva superficie inmunosensora está especialmente diseñada para que el método de detección sea cuantitativo, preciso, selectivo y sensible. Una ventaja adicional es que esta superficie inmunosensora se puede utilizar por personal sin conocimientos específicos en la tecnología, la muestra a analizar no necesita ser tratada previamente y se puede reutilizar. De este modo, la invención proporciona una superficie inmunosensora adecuada para realizar controles de cocaína in situ con la precisión requerida, por ejemplo, durante controles en carretera. The invention provides a new immunosensory surface that allows the direct detection of the main metabolite of cocaine, benzoylecgonine, that is, the detection of interaction between a recognition element and the benzoylecgonine of the test sample. In addition, it allows this detection to be carried out, in situ, and in real time. This new immunosensor surface is specially designed so that the detection method is quantitative, precise, selective and sensitive. An additional advantage is that this immunosensor surface can be used by personnel without specific knowledge in the technology, the sample to be analyzed does not need to be previously treated and can be reused. In this way, the invention provides an immunosensor surface suitable for performing cocaine controls in situ with the required accuracy, for example, during roadside controls.
Así, en un aspecto la invención se dirige a una superficie inmunosensora que comprende un soporte recubierto por una capa metálica a la que se une un polímero que inmoviliza al menos un anticuerpo anti-benzoilecgonina. Thus, in one aspect the invention is directed to an immunosensor surface comprising a support coated by a metal layer to which a polymer that immobilizes at least one anti-benzoylecgonine antibody binds.
En otro aspecto, la invención se dirige a una superficie inmunosensora que comprende un soporte recubierto por una capa metálica a la que se une un polímero que inmoviliza dos ó más anticuerpos anti-benzoilecgonina caracterizados por presentar diferente afinidad por benzoilecgonina.  In another aspect, the invention is directed to an immunosensor surface comprising a support coated by a metal layer to which a polymer that immobilizes two or more anti-benzoylecgonine antibodies characterized by having different affinity for benzoylecgonine is bound.
Otro aspecto de la invención se dirige a un inmunosensor que comprende una superficie inmunosensora, como se definió anteriormente, y un transductor basado en el fenómeno óptico de la resonancia de plasmón superficial (SPR).  Another aspect of the invention is directed to an immunosensor comprising an immunosensor surface, as defined above, and a transducer based on the optical phenomenon of surface plasmon resonance (SPR).
Otro aspecto de la invención se dirige a un aparato para la detección y cuantificación directa de benzoilecgonina que comprende un inmunosensor, como se definió anteriormente, y un dispositivo o instrumento para procesar o acondicionar la señal recibida durante la etapa de asociación o de disociación. Another aspect of the invention is directed to an apparatus for the direct detection and quantification of benzoylecgonine comprising an immunosensor, as defined above, and a device or instrument for processing or conditioning the signal received during the association or dissociation stage.
Otro aspecto de la invención se dirige a un kit que comprende una superficie inmunosensora como se definió anteriormente.  Another aspect of the invention is directed to a kit comprising an immunosensor surface as defined above.
Otro aspecto de la invención se dirige a un aparato portátil que comprende una superficie inmunosensora como se definió anteriormente.  Another aspect of the invention is directed to a portable apparatus comprising an immunosensor surface as defined above.
Otro aspecto de la invención se dirige a un método de detección de benzoilecgonina y en otro aspecto a dos métodos alternativos para cuantificar benzoilecgonina.  Another aspect of the invention is directed to a method of detecting benzoylecgonine and in another aspect to two alternative methods for quantifying benzoylecgonine.
Un último aspecto de la invención se dirige al uso de una superficie inmunosensora como se definió anteriormente para la detección directa de benzoilecgonina. Breve descripción de las figuras A final aspect of the invention is directed to the use of an immunosensor surface as defined above for the direct detection of benzoylecgonine. Brief description of the figures
Figura 1A: representa de forma esquemática la asociación de benzoilecgonina a una superficie inmunosensora: (1) sensor-chip; (2) polímero; (3) anticuerpo; (4) benzoilecgonina.  Figure 1A: schematically represents the association of benzoylecgonine with an immunosensor surface: (1) sensor-chip; (2) polymer; (3) antibody; (4) benzoylecgonine.
Figura IB: representa un sensorgrama donde se detallan las etapas de asociación (1) y equilibrio (2) tras el comienzo de inyección de la muestra (ci) y la etapa de disociación (3) una vez ha finalizada la inyección de muestra (fi). Figure IB: represents a sensorgram detailing the stages of association (1) and equilibrium (2) after the beginning of sample injection (ci) and the dissociation stage (3) once the sample injection (fi ).
Figura 2A y 2B: muestra los sensorgramas registrados al inyectar muestras con diferente concentración de benzoilecgonina (865, 433, 216, 108, 54, 27, 13.5 y 6.8 nM) sobre las superficies inmunosensoras de los ejemplos la y Ib, respectivamente.  Figure 2A and 2B: shows the sensorgrams recorded when injecting samples with different concentration of benzoylecgonine (865, 433, 216, 108, 54, 27, 13.5 and 6.8 nM) on the immunosensor surfaces of examples la and Ib, respectively.
Figura 3: muestra las curvas de calibrado del ejemplo 4 obtenidas tras el análisis de las etapas de (A) asociación y (B) disociación de los sensorgramas registrados con la superficie inmunosensora del ejemplo la.  Figure 3: shows the calibration curves of example 4 obtained after the analysis of the stages of (A) association and (B) dissociation of the sensorgrams registered with the immunosensor surface of example la.
Figura 4: muestra las curvas de calibrado del ejemplo 4 obtenidas tras el análisis de las etapas de (A) asociación y (B) disociación de los sensorgramas registrados con la superficie inmunosensora del ejemplo Ib.  Figure 4: shows the calibration curves of example 4 obtained after the analysis of the stages of (A) association and (B) dissociation of the sensorgrams registered with the immunosensor surface of example Ib.
Figura 5: muestra las gráficas de correlación entre valores de concentración de benzoilecgonina obtenidos experimentalmente con la superficie inmunosensora II y valores calculados con las curvas de calibrado del ejemplo 4, obtenidas tras el análisis de las etapas de (A) asociación y (B) disociación.  Figure 5: shows the correlation graphs between benzoylecgonine concentration values obtained experimentally with the immunosensor surface II and values calculated with the calibration curves of example 4, obtained after the analysis of the stages of (A) association and (B) dissociation .
Descripción detallada de la invención  Detailed description of the invention
En la presente invención se entiende por superficie inmunosensora, un receptor biológico preparado para detectar específicamente una sustancia debido a la especificidad de la interacción receptor-sustancia. Así, el anticuerpo anti-benzoilecgonina es capaz de reconocer específicamente benzoilecgonina en una muestra problema. La afinidad característica de cada anticuerpo determina el rango de concentración de benzoilecgonina que es posible detectar con dicho anticuerpo. Este viene definido por límites superior e inferior de detección correspondientes a 10 veces y 0,1 veces la constante de disociación de la interacción benzoilecgonina-anticuerpo, respectivamente. Por lo tanto la utilización de diferentes anticuerpos anti-benzoilecgonina en una misma superficie inmunosensora permite detectar benzoilecgonina en diferentes rangos de concentración. Sobre el sensor- chip se pueden definir varios canales independientes donde es posible inmovilizar anticuerpos diferentes que operen en rangos de concentración distintos. En una realización particular, la superficie inmunosensora comprende entre dos y seis anticuerpos anti- benzoilecgonina caracterizados por presentar diferente afinidad por benzoilecgonina. En una realización más particular, la superficie inmunosensora comprende entre dos y cuatro anticuerpos anti-benzoilecgonina caracterizados por presentar diferente afinidad por benzoilecgonina. En otra realización particular, la superficie inmunosensora comprende un anticuerpo anti-benzoilecgonina. In the present invention, an immunosensory surface is understood as a biological receptor prepared to specifically detect a substance due to the specificity of the receptor-substance interaction. Thus, the anti-benzoylecgonine antibody is capable of specifically recognizing benzoylecgonine in a test sample. The characteristic affinity of each antibody determines the concentration range of benzoylecgonine that can be detected with said antibody. This is defined by upper and lower detection limits corresponding to 10 times and 0.1 times the dissociation constant of the benzoylecgonine-antibody interaction, respectively. Therefore, the use of different anti-benzoylecgonine antibodies on the same immunosensory surface makes it possible to detect benzoylecgonine in different concentration ranges. Several independent channels can be defined on the sensor-chip where it is possible to immobilize different antibodies that operate in different concentration ranges. In one embodiment In particular, the immunosensor surface comprises between two and six anti-benzoylecgonine antibodies characterized by having different affinity for benzoylecgonine. In a more particular embodiment, the immunosensor surface comprises between two and four anti-benzoylecgonine antibodies characterized by having different affinity for benzoylecgonine. In another particular embodiment, the immunosensor surface comprises an anti-benzoylecgonine antibody.
En una realización particular, la superficie inmunosensora comprende además un canal adicional blanco. Se entiende por canal blanco o control, aquel canal que no comprende un anticuerpo y así la detección de señal en dicho canal corresponderá al blanco o control con la que se compararán las demás señales de medida. Esto permite descartar las señales de ruido o interferencia que pudiera haber.  In a particular embodiment, the immunosensor surface further comprises an additional white channel. A white channel or control is understood to be that channel that does not comprise an antibody and thus the signal detection in said channel will correspond to the target or control with which the other measurement signals will be compared. This allows you to discard any noise or interference signals that may be present.
En una realización particular, la superficie inmunosensora consiste en un soporte recubierto por una capa metálica a la que se une un polímero que inmoviliza al menos un anticuerpo anti-benzoilecgonina y un canal blanco. En una realización más particular, la superficie inmunosensora consiste en un soporte recubierto por una capa metálica a la que se une un polímero que inmoviliza entre uno y seis anticuerpos anti-benzoilecgonina caracterizados por presentar diferente afinidad por benzoilecgonina y un canal blanco.  In a particular embodiment, the immunosensory surface consists of a support coated by a metal layer to which a polymer that immobilizes at least one anti-benzoylecgonine antibody and a white channel binds. In a more particular embodiment, the immunosensor surface consists of a support coated by a metal layer to which a polymer that immobilizes between one and six anti-benzoylecgonine antibodies characterized by having different affinity for benzoylecgonine and a white channel binds.
En una realización preferida de la invención, los anticuerpos anti-benzoilecgonina se seleccionan entre anticuerpos monoclonales o anticuerpos policlonales. Se entiende por "anticuerpos policlonales" aquellos que son producidos por una variedad de células madre y están constituidos por una mezcla fisiológica natural de anticuerpos estructuralmente distintos, que se unen a distintas partes del antígeno. Se entiende por "anticuerpos monoclonales" aquellos que son producidos por células madre idénticas y están constituidos por anticuerpos idénticos; su homogeneidad estructural se traduce en una mayor especificidad y afinidad por el antígeno. En una realización más preferida de la invención los anticuerpos anti-benzoilecgonina son anticuerpos monoclonales. Ejemplos de anticuerpos monoclonales anti-benzoilecgonina, aunque la invención no se limita a los mismos, son los anticuerpos B1077-01, B1077-08, B1077-09, B1077-10, B1077-12 y B1077-15 (estos anticuerpos están comercializados por USBiological).  In a preferred embodiment of the invention, anti-benzoylecgonine antibodies are selected from monoclonal antibodies or polyclonal antibodies. "Polyclonal antibodies" means those that are produced by a variety of stem cells and are constituted by a natural physiological mixture of structurally distinct antibodies, which bind to different parts of the antigen. "Monoclonal antibodies" means those that are produced by identical stem cells and are constituted by identical antibodies; Its structural homogeneity translates into greater specificity and affinity for the antigen. In a more preferred embodiment of the invention, anti-benzoylecgonine antibodies are monoclonal antibodies. Examples of anti-benzoylecgonine monoclonal antibodies, although the invention is not limited thereto, are antibodies B1077-01, B1077-08, B1077-09, B1077-10, B1077-12 and B1077-15 (these antibodies are marketed by USBiological).
En la presente invención se entiende por "inmovilización del anticuerpo" la unión estable y efectiva del anticuerpo al polímero. La unión estable y efectiva conduce a una detección de benzoilecgonina con mayor reproducibihdad y sensibilidad, y de este modo se solucionan los problemas del estado de la técnica para la detección de moléculas pequeñas de forma directa. Se proporciona una superficie inmunosensora capaz de detectar de forma directa y en tiempo real la benzoilecgonina y cuantificarla. Además permite la reutilización de la superficie sensora. Es posible conseguir una inmovilización estable, por ejemplo, mediante enlaces covalentes entre el polímero y el anticuerpo. Así, en una realización particular, los anticuerpos anti-benzoilecgonina se unen al polímero mediante enlaces covalentes. En una realización más particular, los enlaces covalentes se seleccionan entre enlace amida y enlace tipo carbonilo-hidrazina. El enlace amida se puede formar entre grupos carboxílicos del polímero y grupos amino presentes en amino ácidos lisina del anticuerpo. En una realización preferida de la invención, el enlace covalente entre el polímero y el anticuerpo es un enlace tipo carbonilo-hidrazina. El enlace carbonilo-hidrazina se puede formar entre grupos carboxílicos del polímero modificados mediante transformaciones químicas a grupos hidrazina y grupos hidroxilo del anticuerpo, modificados mediante transformaciones químicas a grupos aldehido. Se entiende por "polímero modificado" aquel polímero intermedio obtenido mediante transformaciones químicas a partir de un polímero carboxilado como las que se recogen en Homola, J., 2008. Chem Rev. 108(2), 462-493. El enlace tipo carbonilo-hidrazina se puede formar entre grupos carboxílicos del polímero y grupos hidroxilo de cis-dioles presentes en carbohidratos del anticuerpo. El enlace tipo carbonilo-hidrazina permite una inmovilización dirigida ya que los cis-dioles se encuentran en una región específica del anticuerpo (región Fe) que no participa en la interacción con benzoilecgonina. De esta manera, todos los anticuerpos se inmovilizan en una orientación adecuada donde se mantiene la actividad de sus centros activos. En una realización particular el polímero se selecciona entre un polímero policarboxilado y un polímero policarboxilado modificado. Estos polímeros pueden estar ramificados y así los grupos carboxílicos o carboxílicos modificados pueden tener diferentes disposiciones en el espacio. In the present invention, "immobilization of the antibody" is understood as the stable and effective binding of the antibody to the polymer. Stable and effective binding leads to a detection of benzoylecgonine with greater reproducibility and sensitivity, and in this way the problems of the state of the art for the detection of small molecules are solved directly. An immunosensor surface capable of directly detecting and providing Real-time benzoylecgonine and quantify it. It also allows the reuse of the sensor surface. It is possible to achieve stable immobilization, for example, by covalent bonds between the polymer and the antibody. Thus, in a particular embodiment, the anti-benzoylecgonine antibodies are bound to the polymer by covalent bonds. In a more particular embodiment, covalent bonds are selected from amide bond and carbonyl hydrazine bond. The amide bond can be formed between carboxylic groups of the polymer and amino groups present in amino acids lysine of the antibody. In a preferred embodiment of the invention, the covalent bond between the polymer and the antibody is a carbonyl hydrazine type bond. The carbonyl hydrazine bond can be formed between carboxylic groups of the polymer modified by chemical transformations to hydrazine groups and hydroxyl groups of the antibody, modified by chemical transformations to aldehyde groups. By "modified polymer" is meant that intermediate polymer obtained by chemical transformations from a carboxylated polymer such as those set forth in Homola, J., 2008. Chem Rev. 108 (2), 462-493. The carbonyl hydrazine type bond can be formed between carboxylic groups of the polymer and hydroxyl groups of cis-diols present in carbohydrates of the antibody. The carbonyl-hydrazine type bond allows a directed immobilization since the cis-diols are in a specific region of the antibody (Fe region) that does not participate in the interaction with benzoylecgonine. In this way, all antibodies are immobilized in an appropriate orientation where the activity of their active centers is maintained. In a particular embodiment the polymer is selected from a polycarboxylated polymer and a modified polycarboxylated polymer. These polymers can be branched and thus the modified carboxylic or carboxylic groups can have different arrangements in space.
El término "inmovilización efectiva" se refiere a la inmovilización de un número suficiente de anticuerpos activos en la superficie; este número debe permitir la obtención de una señal tres veces superior a la sensibilidad del detector (que viene dada por el ruido de fondo) cuando se monitoriza la asociación de benzoilecgonina en una muestra de concentración igual al límite inferior de detección determinado por el anticuerpo. La cantidad de anticuerpo que es posible inmovilizar está relacionada con la capacidad de inmovilización del polímero. En una realización particular, el polímero se selecciona entre polímeros con capacidad de inmovilizar una cantidad de anticuerpos anti-benzoilecgonina comprendida entre 730 pg/mm 2 y 73000 pg/mm 2. Más preferiblemente la cantidad de anticuerpos anti- benzoilecgonina inmovilizados está comprendida entre 2190 pg/mm2 y 58400 pg/mm2. Aún más preferiblemente la cantidad de anticuerpos anti-benzoilecgonina inmovilizados está comprendida entre 7300 pg/mm2 y 29200 pg/mm2. Para determinar la cantidad de anticuerpo anti-benzoilecgonina inmovilizada, se monitoriza en tiempo real la inmovilización del anticuerpo por el polímero seleccionado, midiendo el aumento de la señal de SPR en unidades uRiU ("micro-Refractive índex Units"), teniendo en cuenta que 1 uRiU equivale a 0,73 pg/mm . The term "effective immobilization" refers to the immobilization of a sufficient number of surface active antibodies; This number should allow obtaining a signal three times higher than the sensitivity of the detector (which is given by background noise) when the association of benzoylecgonine in a sample of concentration equal to the lower limit of detection determined by the antibody is monitored. The amount of antibody that can be immobilized is related to the immobilization capacity of the polymer. In a particular embodiment, the polymer is selected from polymers capable of immobilizing an amount of anti-benzoylecgonine antibodies between 730 pg / mm 2 and 73000 pg / mm 2. More preferably the amount of immobilized anti-benzoylecgonine antibodies is between 2190 pg / mm 2 and 58400 pg / mm 2 . Even more preferably the amount of immobilized anti-benzoylecgonine antibodies is between 7300 pg / mm 2 and 29200 pg / mm 2 . To determine the amount of immobilized anti-benzoylecgonine antibody, immobilization of the antibody by the selected polymer is monitored in real time, measuring the increase in the SPR signal in uRiU units ("micro-Refractive index Units"), taking into account that 1 uRiU equals 0.73 pg / mm.
En una realización preferida de la invención, el polímero es antiadherente. En la presente invención se entiende por "polímero antiadherente" aquel polímero que es inerte a interacciones no específicas entre los componentes de la muestra y los sitios activos de unión del polímero. Una ventaja adicional de que sea inerte a interacciones no específicas y se encuentre recubriendo la capa metálica es que protege a dicha capa metálica de la adhesión o interacción con sustancias de la muestra. Así, en una realización preferida, el polímero es un polímero antiadherente. En una realización más preferida, es un polímero antiadherente para muestras de fluidos orales. En una realización particular, el polímero se selecciona entre polímeros policarboxilados antiadherentes con capacidad de inmovilizar entre entre 2190 pg/mm2 y 58400 pg/mm2. Ejemplos de polímeros policarboxilados antiadherentes, aunque la invención no se limita a éstos, son polímeros de polietilenglicol y cadenas ramificadas policarboxiladas (hidrogeles 3D) empleados en los sensor-chip comerciales HC1000 y HC1500 (Xantec Bioanalitics). In a preferred embodiment of the invention, the polymer is non-stick. In the present invention, "non-stick polymer" means a polymer that is inert to non-specific interactions between the sample components and the active polymer binding sites. An additional advantage of being inert to non-specific interactions and covering the metal layer is that it protects said metal layer from adhesion or interaction with substances in the sample. Thus, in a preferred embodiment, the polymer is a non-stick polymer. In a more preferred embodiment, it is a non-stick polymer for oral fluid samples. In a particular embodiment, the polymer is selected from non-stick polycarboxylated polymers capable of immobilizing between 2190 pg / mm 2 and 58400 pg / mm 2 . Examples of non-stick polycarboxylated polymers, although the invention is not limited thereto, are polymers of polyethylene glycol and polycarboxylated branched chains (3D hydrogels) used in the commercial sensor-chips HC1000 and HC1500 (Xantec Bioanalitics).
El polímero puede unirse a la capa metálica mediante técnicas conocidas, por ejemplo, mediante la unión entre el oro y átomos de azufre del polímero, estando estos átomos de azufre formando parte por ejemplo de grupos tiol, disulfuros o sulfuros. En una realización particular, la capa metálica se selecciona entre oro, plata, aluminio y cobre; más preferiblemente, entre oro y plata. En una realización preferida, la capa metálica tiene un grosor no superior a 200 nm, más preferiblemente tiene un grosor de entre 1 nm y 100 nm, aún más preferiblemente tiene un grosor de entre 10 nm y 50 nm. En una realización particular, la capa metálica tiene un grosor de 50 nm.  The polymer can be attached to the metal layer by known techniques, for example, by joining between gold and sulfur atoms of the polymer, these sulfur atoms being, for example, part of thiol, disulfide or sulfide groups. In a particular embodiment, the metal layer is selected from gold, silver, aluminum and copper; more preferably, between gold and silver. In a preferred embodiment, the metal layer has a thickness not exceeding 200 nm, more preferably it has a thickness between 1 nm and 100 nm, even more preferably it has a thickness between 10 nm and 50 nm. In a particular embodiment, the metal layer has a thickness of 50 nm.
El soporte sobre el que está dicha capa metálica puede ser de diferentes materiales, por ejemplo y sin ser limitativos, puede ser de vidrio o de material plástico. También es posible que ese soporte se presente en diferentes formas por ejemplo y sin limitarse, puede presentar forma plana, cilindrica, esférica o formar parte de una fibra óptica.  The support on which said metallic layer is can be made of different materials, for example and without being limiting, it can be made of glass or plastic material. It is also possible that this support is presented in different forms, for example and without limitation, it may have a flat, cylindrical, spherical shape or be part of an optical fiber.
En una realización particular, la invención se dirige a una superficie inmunosensora que comprende un soporte de vidrio recubierto por una capa de oro de 50 nm y el polímero Hidrogel 3D que inmoviliza al menos un anticuerpo monoclonal anti-benzoilecgonina seleccionado entre B1077-08 y B1077-01. En otro aspecto, la invención se dirige a un inmunosensor que comprende una superficie inmunosensora como se describió anteriormente y un transductor basado en el fenómeno óptico de la SPR. El inmunosensor de la invención permite la detección directa e in situ de benzoilecgonina presente en una muestra gracias al reconocimiento específico por un anticuerpo anti-benzoilecgonina inmovilizado sobre la superficie metálica de la superficie inmunosensora, como se describió anteriormente. Por medio del transductor basado en el fenómeno óptico de SPR se monitoriza, en tiempo real, la masa de benzoilecgonina asociada a esta superficie a través de la unión específica antígeno-anticuerpo. In a particular embodiment, the invention is directed to an immunosensor surface comprising a glass support coated with a 50 nm gold layer and the 3D Hydrogel polymer that immobilizes at least one anti-benzoylecgonine monoclonal antibody selected from B1077-08 and B1077 -01. In another aspect, the invention is directed to an immunosensor comprising an immunosensor surface as described above and a transducer based on the optical phenomenon of the SPR. The immunosensor of the invention allows the direct and in situ detection of benzoylecgonine present in a sample thanks to the specific recognition by an anti-benzoylecgonine antibody immobilized on the metal surface of the immunosensor surface, as described above. By means of the transducer based on the optical phenomenon of SPR, the mass of benzoylecgonine associated to this surface is monitored in real time through the specific antigen-antibody binding.
En otro aspecto, la invención se dirige a un aparato para la detección y cuantificación directa de benzoilecgonina que comprende un inmunosensor como se describió anteriormente y un dispositivo o instrumento para procesar o acondicionar la señal recibida durante la etapa de asociación o de disociación. En una realización preferida, este aparato comprende además un dispositivo para realizar un cálculo. El dispositivo o instrumento para procesar o acondicionar la señal recibida permite su utilización en un dispositivo para realizar un cálculo. La lectura de la señal se puede realizar a través de una pantalla, digital o analógica o a través de un ordenador.  In another aspect, the invention is directed to an apparatus for the direct detection and quantification of benzoylecgonine comprising an immunosensor as described above and a device or instrument for processing or conditioning the signal received during the association or dissociation stage. In a preferred embodiment, this apparatus further comprises a device for performing a calculation. The device or instrument for processing or conditioning the received signal allows its use in a device to perform a calculation. The signal can be read through a digital or analog screen or through a computer.
El inmunosensor de la invención presenta elevada sensibilidad y especificidad, y permite rapidez de medida y reproducibilidad. Además es reutilizable, como se probó en experimentos realizados recogidos en los ejemplos.  The immunosensor of the invention has high sensitivity and specificity, and allows rapid measurement and reproducibility. It is also reusable, as tested in experiments carried out in the examples.
Además, el inmunosensor permite la detección en tiempo real, es factible su miniaturización, posibilitando así la automatización y la realización de ensayos en paralelo, lo cual permite su aplicación en aparatos portátiles. Así, en una realización particular, la invención se refiere a un aparato donde el inmunosensor está miniaturizado y el aparato es portátil. In addition, the immunosensor allows real-time detection, its miniaturization is feasible, thus enabling automation and parallel testing, which allows its application in portable devices. Thus, in a particular embodiment, the invention relates to an apparatus where the immunosensor is miniaturized and the apparatus is portable.
Las ventajas del inmunosensor de la invención se resumen en los siguientes puntos: (1) es un sistema de detección directa y sin mareaje del analito. Esto elimina pasos intermedios y medidas indirectas que añaden posibles errores y alargan el tiempo de detección; (2) la medición se realiza en tiempo real, con obtención rápida de la respuesta; (3) posee alta especificidad, procedente del uso de un anticuerpo específico de benzoilecgonina como elemento de reconocimiento; (4) posee alta sensibilidad, que viene determinada por la afinidad antígeno-anticuerpo y el número de anticuerpos activos inmovilizados en la superficie inmunosensora; (5) no presenta interacción inespecífica con benzoilecgonina (u otros componentes de la muestra) debido a la presencia de una matriz polimérica antiadherente entre la superficie metálica y el anticuerpo; (6) es factible emplear un volumen reducido de muestra, por ejemplo es factible utilizar 100 μΐ^; (7) se trata de un dispositivo reutilizable ya que la superficie inmunosensora puede regenerarse para el análisis consecutivo de muestras; (8) ofrece ventajas tecnológicas como su fácil miniaturización, posibilidad de automatización y la realización de ensayos en paralelo; (10) versatilidad en el diseño del dispositivo inmunosensor ya que la elección del anticuerpo determina el rango de concentración de benzoilecgonina que puede medir. Así, es posible diseñar una superficie inmunosensora multicanal donde se incorporan anticuerpos con diferente afinidad por la benzoilecgonina y que cubren distintos rangos de concentración, como se describió anteriormente. The advantages of the immunosensor of the invention are summarized in the following points: (1) it is a direct detection system without analyte dizziness. This eliminates intermediate steps and indirect measures that add possible errors and lengthen the detection time; (2) the measurement is carried out in real time, with rapid obtaining of the response; (3) it has high specificity, derived from the use of a specific benzoylecgonine antibody as recognition element; (4) it has high sensitivity, which is determined by the antigen-antibody affinity and the number of active antibodies immobilized on the immunosensory surface; (5) it does not exhibit non-specific interaction with benzoylecgonine (or other components of the sample) due to the presence of a non-stick polymer matrix between the metal surface and the antibody; (6) it is feasible to use a reduced sample volume, for example it is feasible to use 100 μΐ ^ ; (7) it is a reusable device since the immunosensory surface can be regenerated for consecutive sample analysis; (8) offers technological advantages such as its easy miniaturization, possibility of automation and parallel testing; (10) versatility in the design of the immunosensor device since the choice of the antibody determines the concentration range of benzoylecgonine that can be measured. Thus, it is possible to design a multichannel immunosensor surface where antibodies with different affinity for benzoylecgonine are incorporated and covering different concentration ranges, as described above.
En otro aspecto, la invención se dirige a un método de detección de benzoilecgonina que comprende: In another aspect, the invention is directed to a method of detecting benzoylecgonine comprising:
i. poner en contacto la muestra problema con la superficie inmunosensora como se describió anteriormente,  i. contacting the test sample with the immunosensory surface as described above,
ii. detectar la señal de SPR durante la etapa de asociación y la etapa de disociación.  ii. Detect the SPR signal during the association stage and the dissociation stage.
Para llevar a cabo la detección de benzoilecgonina es posible emplear el inmunosensor de la invención o un aparato que lo incorpore, tal y como se describió anteriormente que comprenden la superficie inmunosensora de la invención. La detección de la interacción antígeno-anticuerpo se realiza mediante un espectrómetro de SPR, y es posible incorporar un dispositivo para la medida de muestras en flujo continuo. De esta manera el anticuerpo puede estar en contacto permanente con una disolución reguladora que asegura la estabilidad del mismo. Esta disolución es sustituida por la muestra problema en el momento de su inyección. Durante la inyección de la muestra, el detector de SPR registra, en función del tiempo, el aumento de la señal de SPR asociado a la masa de benzoilecgonina que se une específicamente al anticuerpo inmovilizado durante el proceso de asociación. Una vez finalizada la inyección de muestra, se monitoriza la disminución de la señal de SPR que ocurre como consecuencia del proceso de disociación.  To carry out the detection of benzoylecgonine it is possible to employ the immunosensor of the invention or an apparatus incorporating it, as described above comprising the immunosensor surface of the invention. The detection of the antigen-antibody interaction is performed by a SPR spectrometer, and it is possible to incorporate a device for measuring samples in continuous flow. In this way the antibody can be in permanent contact with a regulatory solution that ensures its stability. This solution is replaced by the test sample at the time of injection. During the injection of the sample, the SPR detector records, as a function of time, the increase in the SPR signal associated with the benzoylecgonine mass that specifically binds to the immobilized antibody during the association process. Once the sample injection is finished, the decrease in the SPR signal that occurs as a result of the dissociation process is monitored.
Para la cuantificación de benzoilecgonina en una muestra problema, la invención proporciona dos métodos alternativos basados en las etapas de asociación y de disociación. Así, en un aspecto la invención se dirige a un método de cuantificación de benzoilecgonina que comprende:  For the quantification of benzoylecgonine in a test sample, the invention provides two alternative methods based on the stages of association and dissociation. Thus, in one aspect the invention is directed to a method of quantification of benzoylecgonine comprising:
iii. detección de benzoilecgonina durante la etapa de asociación, según se describió anteriormente, iv. interpolación de la pendiente de la señal (dR/dt) obtenida en la etapa iii) en la recta de calibrado (dR/dt frente a concentración) para obtener el valor de concentración de benzoilecgonina. iii. detection of benzoylecgonine during the association stage, as described above, iv. interpolation of the slope of the signal (dR / dt) obtained in step iii) in the calibration line (dR / dt versus concentration) to obtain the concentration value of benzoylecgonine.
La respuesta de SPR (R) varía linealmente con el tiempo (t) y la pendiente (dR/dt) es directamente proporcional a la concentración en benzoilecgonina en la muestra (Figuras 3A y 4A). La curva de calibrado correspondiente relaciona la pendiente frente a la concentración.  The SPR (R) response varies linearly with time (t) and the slope (dR / dt) is directly proportional to the concentration of benzoylecgonine in the sample (Figures 3A and 4A). The corresponding calibration curve relates the slope to the concentration.
En otro aspecto, la invención se dirige a un método alternativo de cuantificación de benzoilecgonina que comprende:  In another aspect, the invention is directed to an alternative method of quantifying benzoylecgonine comprising:
v. detección de benzoilecgonina durante la etapa de disociación, según se describió anteriormente,  v. benzoylecgonine detection during the dissociation step, as described above,
vi. interpolación de la señal (R) obtenida en la etapa v) en la recta de calibrado (R frente a concentración) para obtener el valor de concentración de benzoilecgonina.  saw. interpolation of the signal (R) obtained in step v) in the calibration line (R versus concentration) to obtain the concentration value of benzoylecgonine.
La concentración de benzoilecgonina se puede determinar a través de una curva de calibrado donde se representa la repuesta de SPR una vez finalizada la inyección en función de la concentración de analito (Figuras 3B y 4B). The concentration of benzoylecgonine can be determined through a calibration curve where the SPR response is represented once the injection is finished based on the analyte concentration (Figures 3B and 4B).
Las curvas de calibrado se obtienen mediante la detección de benzoilecgonina de una serie de muestras patrón de concentración conocida de benzoilecgonina, durante las etapas de asociación y disociación, de forma similar a como se describió la detección para una muestra (Figura IB).  Calibration curves are obtained by detecting benzoylecgonine from a series of standard samples of known concentration of benzoylecgonine, during the association and dissociation stages, similar to how detection was described for a sample (Figure IB).
En una realización preferida de la invención, la detección y cuantificación de benzoilecgonina se realiza en muestras de fluido oral, o en mezclas de fluido oral y una solución reguladora; de forma aún más preferida en muestras de saliva o en mezclas de saliva y una solución reguladora.  In a preferred embodiment of the invention, the detection and quantification of benzoylecgonine is performed in oral fluid samples, or in mixtures of oral fluid and a regulatory solution; even more preferably in saliva samples or in mixtures of saliva and a regulatory solution.
En otro aspecto, la invención se dirige al uso de la superficie inmunosensora, como se describió anteriormente, para la detección directa de benzoilecgonina. En una realización preferida, la detección tiene lugar en tiempo real. En una realización aún más preferida, la detección se realiza en disoluciones reguladoras acuosas, en fluido oral o en mezclas de las mismas. Por ejemplo, y sin limitarse, la disolución reguladora puede comprender tampón HEPES (Ácido 2-[4-(2-hidroxietil)-l-piperacinil-(l)] etanosulfónico), tampón fosfato o tampón TRIS (2-Amino-2-hidroximetil-propano-l,3-diol). Los siguientes ejemplos ilustran la invención y en ningún caso debe de interpretarse como una limitación de la misma. In another aspect, the invention is directed to the use of the immunosensor surface, as described above, for the direct detection of benzoylecgonine. In a preferred embodiment, the detection takes place in real time. In an even more preferred embodiment, the detection is carried out in aqueous regulatory solutions, in oral fluid or in mixtures thereof. For example, and without limitation, the regulatory solution may comprise HEPES buffer (2- [4- (2-hydroxyethyl) -l-piperazinyl- (l)] ethanesulfonic acid), phosphate buffer or TRIS buffer (2-Amino-2- hydroxymethyl-propane-l, 3-diol). The following examples illustrate the invention and in no case should it be construed as a limitation thereof.
Ejemplo de realización de la invención  Example of embodiment of the invention
Los materiales empleados son los anticuerpos monoclonales B1077-08 y B1077-01 (USBiological), un sensor-chip HCIOOO y un sensor-chip HC1500 (Xantec Bioanalytics) que consisten en un soporte de vidrio cubierto por una película de oro de 50 nM, cubierta a su vez por una matriz polimérica antiadherente policarboxilada (descrita por los proveedores como hidrogel 3D), la disolución reguladora HEPES* se preparó en el laboratorio en las siguientes condiciones HEPES 10 mM, NaCl 150 mM, pH 7,4. The materials used are monoclonal antibodies B1077-08 and B1077-01 (USBiological), a HCIOOO sensor chip and an HC1500 sensor chip (Xantec Bioanalytics) consisting of a glass support covered by a 50 nM gold film, In turn covered by a polycarboxylated non-stick polymer matrix (described by the suppliers as 3D hydrogel), the HEPES * regulatory solution was prepared in the laboratory under the following conditions 10 mM HEPES, 150 mM NaCl, pH 7.4.
La detección de benzoilecgonina se realiza en un espectrómetro de Resonancia de Plasmón Superficial Reichert SR7000DC, operando a una temperatura constante de 25 °C.  The detection of benzoylecgonine is performed on a Reichert SR7000DC Superficial Plasmon Resonance Spectrometer, operating at a constant temperature of 25 ° C.
Las muestras problema se prepararon por dilución de una disolución madre de benzoilecgonina de concentración lmg/ml en metanol (Cerilliant Corporation) con la disolución reguladora HEPES* o con fluido oral procedente de individuos sanos.  The test samples were prepared by diluting a stock solution of benzoylecgonine of lmg / ml concentration in methanol (Cerilliant Corporation) with the HEPES * regulatory solution or with oral fluid from healthy individuals.
Ejemplo la. Fabricación de la superficie inmunosensora I con dos canales. Example the. Manufacture of the immunosensor surface I with two channels.
El anticuerpo monoclonal B 1077-08 se inmoviliza covalentemente sobre el canal analítico de un sensor-chip HCIOOO. La inmovilización se lleva a cabo por formación de enlaces tipo amida entre los grupos carboxílicos de la superficie y grupos amino primarios del anticuerpo (Johnsson, B., Lofas, S., Lindquist, G., 1991. Anal Biochem. 198(2), 268-277). Durante la inmovilización, un flujo constante de disolución reguladora HEPES* se encuentra circulando sobre la superficie del sensor-chip, a un caudal de 10 μΐ/min. En primer lugar, el sensor-chip se lava con tres inyecciones consecutivas de 60 segundos cada una de una disolución acuosa de NaOH 0,05 M / NaCl 1 M. Luego, la superficie se activa con una inyección de 10 minutos de la mezcla de reactivos N-hidroxisuccinimida (NHS) 0,05 M / N-etil-N'-(3-dimetilaminopropil)carbodiimida (EDC) 0,2 M. De esta manera los grupos carboxílicos del sensor-chip son transformados en ésteres activos de succinimida. A continuación, se inyecta durante 10 minutos una disolución de anticuerpo B 1077-08 (0,1 mg/ml en una disolución reguladora de acetato sódico 0,01 M, pH 4,0). Durante este paso, grupos amino del anticuerpo reaccionan espontáneamente con los ésteres de succinimida para formar enlaces tipo amida. Por último, la fracción de ésteres de succinimida que no reacciona se desactiva por inyección durante 10 minutos de una disolución de etanolamina 1 M, pH 8,5. La inmovilización del anticuerpo se monitorizó en tiempo real y un aumento de la señal de SPR de 11000 μΚίΙΙβ confirmó la inmovilización. Teniendo en cuenta que 1 μΚίυ equivale a 0,73 pg/mm2, en este ejemplo concreto se inmovilizaron 8030 pg/mm2 de anticuerpo. Monoclonal antibody B 1077-08 is covalently immobilized on the analytical channel of a HCIOOO sensor chip. Immobilization is carried out by formation of amide bonds between surface carboxylic groups and primary amino groups of the antibody (Johnsson, B., Lofas, S., Lindquist, G., 1991. Anal Biochem. 198 (2) , 268-277). During immobilization, a constant flow of HEPES * regulatory solution is circulating on the surface of the sensor-chip, at a flow rate of 10 μΐ / min. First, the sensor-chip is washed with three consecutive injections of 60 seconds each of an aqueous solution of 0.05 M NaOH / 1M NaCl. Then, the surface is activated with a 10-minute injection of the mixture of N-hydroxysuccinimide (NHS) 0.05 M / N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide (EDC) 0.2 M reagents In this way the sensor-chip carboxylic groups are transformed into active succinimide esters . Next, a solution of antibody 1077-08 B (0.1 mg / ml in a 0.01 M sodium acetate regulatory solution, pH 4.0) is injected for 10 minutes. During this step, amino groups of the antibody react spontaneously with the succinimide esters to form amide bonds. Finally, the fraction of unreacted succinimide esters is deactivated by injection for 10 minutes of an ethanolamine solution. 1 M, pH 8.5. The immobilization of the antibody was monitored in real time and an increase in the SPR signal of 11000 μΚίΙΙβ confirmed the immobilization. Taking into account that 1 μΚίυ is equivalent to 0.73 pg / mm 2 , in this specific example 8030 pg / mm 2 of antibody was immobilized.
El canal control se trata con la mezcla NHS/EDC seguido de etanolamina bajo las mismas condiciones experimentales que el tratamiento del canal analítico, pero sin inmovilizar el anticuerpo. The control channel is treated with the NHS / EDC mixture followed by ethanolamine under the same experimental conditions as the treatment of the analytical channel, but without immobilizing the antibody.
Ejemplo Ib. Fabricación de la superficie inmunosensora II con dos canales.  Example Ib. Manufacture of the immunosensory surface II with two channels.
El anticuerpo monoclonal B 1077-01 se inmoviliza covalentemente sobre el canal analítico de un sensor-chip HC1500. La inmovilización se lleva a cabo por formación de enlaces tipo amida siguiendo el mismo procedimiento experimental que en el ejemplo la. La inmovilización del anticuerpo se monitorizó en tiempo real y un aumento de la señal de SPR de 10829 μΚίΙΙβ (7905 pg/mm2) confirmó la inmovilización del anticuerpo. Monoclonal antibody B 1077-01 is covalently immobilized on the analytical channel of an HC1500 sensor chip. The immobilization is carried out by formation of amide bonds following the same experimental procedure as in example la. The immobilization of the antibody was monitored in real time and an increase in the SPR signal of 10829 μΚίΙΙβ (7905 pg / mm 2 ) confirmed the immobilization of the antibody.
El canal control se prepara siguiendo el mismo procedimiento experimental que en el ejemplo la.  The control channel is prepared following the same experimental procedure as in example la.
Ejemplo 2. Detección directa de bezoilecgonina.  Example 2. Direct detection of bezoylecgonine.
Una disolución reguladora HEPES* circula sobre la superficie inmunosensora I a un flujo constante de 50 μΐ/min. La muestra que se quiere analizar se inyecta durante 2 minutos sobre la superficie inmunosensora al mismo flujo. La asociación de benzoilecgonina al anticuerpo inmovilizado se monitoriza en tiempo real a través del sensorgrama correspondiente. Tras la inyección de muestra, la disolución reguladora HEPES* vuelve a circular sobre la superficie y la etapa de disociación se monitoriza durante 3 minutos.  A HEPES * regulatory solution circulates on the immunosensor surface I at a constant flow of 50 μΐ / min. The sample to be analyzed is injected for 2 minutes on the immunosensory surface at the same flow. The association of benzoylecgonine with the immobilized antibody is monitored in real time through the corresponding sensorgram. After injection of the sample, the HEPES * regulatory solution circulates again on the surface and the dissociation stage is monitored for 3 minutes.
La respuesta de SPR registrada en el canal control se resta de la respuesta registrada en el canal analítico. Además, se realiza una inyección "blanco" de disolución reguladora HEPES* sin benzoilecgonina en las mismas condiciones experimentales que la inyección de muestra, cuya respuesta también se resta del sensorgrama obtenido. Este procedimiento descrito en bibliografía como "método de doble referencia" se utiliza para corregir pequeñas desviaciones en el sensorgrama ajenas a la interacción específica antí geno- anticuerpo (Myszka, D.G., 1999. J Mol Recognit. 12(5), 279-284). El procesado de los sensorgramas se realiza mediante el software de SPR Scrubber (BioLogic vl.lg Software). Los sensorgramas registrados se muestran en la figura 2A para disoluciones tampón con distintas concentraciones de benzoilecgonina (865, 433, 216, 108, 54, 27, 13.5 y 6.8 nM). El mismo experimento se realiza con la superficie inmunosensora II, variando únicamente el tiempo de inyección de muestra que en este caso es de 3 minutos. Los sensorgramas registrados se muestran en la figura 2B para disoluciones tampón con distintas concentraciones de benzoilecgonina (865, 433, 216, 108, 54, 27, 13.5 y 6.8 nM). The SPR response recorded in the control channel is subtracted from the response recorded in the analytical channel. In addition, a "white" injection of HEPES * regulatory solution without benzoylecgonine is performed under the same experimental conditions as the sample injection, whose response is also subtracted from the sensorgram obtained. This procedure described in literature as a "double reference method" is used to correct small deviations in the sensorgram outside the specific antigen-antibody interaction (Myszka, DG, 1999. J Mol Recognit. 12 (5), 279-284) . The processing of the sensorgrams is done using the SPR Scrubber software (BioLogic vl.lg Software). The recorded sensorgrams are shown in Figure 2A for buffer solutions with different concentrations of benzoylecgonine (865, 433, 216, 108, 54, 27, 13.5 and 6.8 nM). The same experiment is performed with the immunosensory surface II, varying only the injection time of the sample, which in this case is 3 minutes. The recorded sensorgrams are shown in Figure 2B for buffer solutions with different concentrations of benzoylecgonine (865, 433, 216, 108, 54, 27, 13.5 and 6.8 nM).
Ejemplo 3. Regeneración de la superficie inmunosensora. Example 3. Regeneration of the immunosensory surface.
La superficies inmunosensoras I y II se regeneran con una inyección de 1 minuto de glicina 0,01 M, pH 2,0. Esta disolución elimina por completo la benzoilecgonina que aún queda unida al anticuerpo, sin dañar la superficie y dejándola preparada para la siguiente medida.  Immunosensor surfaces I and II are regenerated with a 1 minute injection of 0.01 M glycine, pH 2.0. This solution completely eliminates the benzoylecgonine that is still bound to the antibody, without damaging the surface and leaving it ready for the next measurement.
Ejemplo 4. Obtención de las curvas de calibrado. Example 4. Obtaining the calibration curves.
Una serie de muestras de concentración conocida de benzoilecgonina (865, 433, 216, 108, 54, 27, 13,5 y 6,8 nM en HEPES*) se inyectan por duplicado sobre las superficies inmunosensoras I y II de acuerdo con el procedimiento detallado en el ejemplo 2. Los sensorgramas obtenidos se caracterizan por una etapa de asociación donde la respuesta de SPR varía linealmente con el tiempo y la pendiente (dR/dt) es directamente proporcional a la concentración en benzoilecgonina en la muestra (Figura 2). Con estos datos se obtiene una curva de calibrado donde se representa la pendiente en función de la concentración de benzoilecgonina (Figuras 3A y 4A). Con este método de análisis los inmunosensores que comprenden las superficies inmunosensoras I y II fabricadas presentan límites de detección (LOD) y cuantificación (LOQ) análogos, de 6,8 nM (2 μ^) y 13,5 nM (4 μ^) respectivamente y operan en un rango lineal de concentración de 865 - 6,8 nM (250 - 2 Alternativamente, se realiza una segunda curva de calibrado donde se representa la respuesta de SPR tras finalizar la inyección (durante la etapa de disociación) en función de la concentración de benzoilecgonina (Figuras 3B y 4B). En este caso la curva de calibrado sigue un comportamiento lineal en el rango de concentraciones 54 - 6,8 nM (15,6 - 2 μg/L) y se mantienen los valores de LOD y LOQ medidos durante la etapa de asociación en ambos inmunosensores. A continuación, se describe una serie de ensayos realizados en estos ejemplos que ponen de manifiesto la reproducibilidad y estabilidad de las superficies inmunosensoras así como su utilidad para detectar benzoilecgonina en fluido oral: A series of samples of known concentration of benzoylecgonine (865, 433, 216, 108, 54, 27, 13.5 and 6.8 nM in HEPES *) are injected in duplicate on immunosensor surfaces I and II according to the procedure detailed in example 2. The sensorgrams obtained are characterized by an association stage where the SPR response varies linearly with time and the slope (dR / dt) is directly proportional to the concentration of benzoylecgonine in the sample (Figure 2). With these data, a calibration curve is obtained where the slope is represented as a function of the concentration of benzoylecgonine (Figures 3A and 4A). With this method of analysis, the immunosensors comprising the manufactured immunosensor surfaces I and II have similar detection limits (LOD) and quantification (LOQ) of 6.8 nM (2 μ ^) and 13.5 nM (4 μ ^) respectively and they operate in a linear concentration range of 865-6.8 nM (250-2) Alternatively, a second calibration curve is performed where the SPR response is represented after the end of the injection (during the dissociation stage) as a function of the concentration of benzoylecgonine (Figures 3B and 4B) In this case the calibration curve follows a linear behavior in the range of concentrations 54 - 6.8 nM (15.6 - 2 μg / L) and the LOD values are maintained and LOQ measured during the association stage in both immunosensors. The following describes a series of tests carried out in these examples that show the reproducibility and stability of the immunosensor surfaces as well as their usefulness for detecting benzoylecgonine in oral fluid:
- Se realizan inyecciones de muestras de benzoilecgonina de concentración conocida en fluido oral, siguiendo el procedimiento experimental descrito anteriormente, que se prepararon según la siguiente descripción: 1 mi de fluido oral se diluye en 3 mi de disolución tamponada HEPES*. A continuación, la muestra se filtra en dispositivos Amicon ultra (MWCO 3KDa), centrifugando a 6000 rpm y 25 ° C durante 40 min. En 1 mi de la muestra filtrada se añade un volumen de disolución madre de benzoilecgonina (1 mg/ml en metanol) para obtener una concentración final de benzoilecgonina igual a 3,46 μΜ. A partir de esta disolución se prepararan otras de menor concentración por dilución con los 3 mi restantes de muestra de fluido oral sin dopar. Bajo estas condiciones experimentales ambos inmunosensores detectan una concentración 13,5 nM (4 μg/l) de benzoilecgonina en la muestra de fluido oral. - Injections of benzoylecgonine samples of known concentration in oral fluid are performed, following the experimental procedure described above, which were prepared according to the following description: 1 ml of oral fluid is diluted in 3 ml of HEPES * buffered solution. The sample is then filtered on Amicon ultra devices (MWCO 3KDa), centrifuging at 6000 rpm and 25 ° C for 40 min. In 1 ml of the filtered sample, a volume of benzoylecgonine stock solution (1 mg / ml in methanol) is added to obtain a final concentration of benzoylecgonine equal to 3.46 μΜ. From this solution, others of lower concentration will be prepared by dilution with the remaining 3 ml of oral fluid sample without doping. Under these experimental conditions both immunosensors detect a 13.5 nM concentration (4 μg / l) of benzoylecgonine in the oral fluid sample.
- Se realizan ciclos consecutivos de inyección-regeneración sobre las dos superficies inmunosensoras, realizando inyecciones de benzoilecgonina en HEPES* o fluido oral y manteniendo una temperatura constante de 25 °C. Bajo estas condiciones experimentales el inmunosensor I que comprende la superficie inmunosensora I mantiene su actividad durante 4 días y un número total de 54 ciclos de inyección-regeneración. El inmunosensor II que comprende la superficie inmunosensora II mantiene su actividad durante 10 días y un número total de 70 ciclos de inyección-regeneración. - Consecutive cycles of injection-regeneration are carried out on the two immunosensor surfaces, making injections of benzoylecgonine in HEPES * or oral fluid and maintaining a constant temperature of 25 ° C. Under these experimental conditions the immunosensor I comprising the immunosensor surface I maintains its activity for 4 days and a total number of 54 injection-regeneration cycles. The immunosensor II comprising the immunosensor surface II maintains its activity for 10 days and a total number of 70 injection-regeneration cycles.
- La reproducibilidad intra-ensayo se verifica mediante el análisis de muestras por duplicado donde el valor promedio de coeficiente de variación calculado entre valores correspondientes a la misma concentración es de 8,4 % y 10,9 % para las superficies inmunosensoras I y II, respectivamente. Estos valores son satisfactorios y se encuentran dentro del rango de valores de coeficiente de variación calculados en la determinación de cocaína mediante cromatografía líquida en combinación con espectrometría de masas (Concheiro, M., Gray, T.R., Shakleya, D.M., Huestis, M.A., 2010. Anal Bioanal Chem. 398(2), 915-924). - La reproducibilidad inter-ensayo del inmunosensor de mayor durabilidad (inmunosensor II), se verifica mediante el análisis de dos sets de muestras de concentración conocida de benzoilecgonina preparadas. El primer set se analiza y se genera la curva de calibrado correspondiente. El segundo set se analiza 8 días después y los valores obtenidos se comparan con los valores teóricos que se obtienen de la recta de calibrado preparada con el primer set de muestras (Figura 5). La excelente correlación entre estos valores (r ~ 0,99; pendiente ~ 1,00) pone de manifiesto la notable reproducibilidad inter-ensayo. - Intra-assay reproducibility is verified by duplicate sample analysis where the average value of coefficient of variation calculated between values corresponding to the same concentration is 8.4% and 10.9% for immunosensor surfaces I and II, respectively. These values are satisfactory and are within the range of coefficient of variation values calculated in the determination of cocaine by liquid chromatography in combination with mass spectrometry (Concheiro, M., Gray, TR, Shakleya, DM, Huestis, MA, 2010 Bioanal Chem Anal. 398 (2), 915-924). - The inter-assay reproducibility of the longest lasting immunosensor (immunosensor II) is verified by analyzing two sets of samples of known concentration of benzoylecgonine prepared. The first set is analyzed and the corresponding calibration curve is generated. The second set is analyzed 8 days later and the values obtained are compared with the theoretical values obtained from the calibration line prepared with the first set of samples (Figure 5). The excellent correlation between these values (r ~ 0.99; pending ~ 1.00) shows the remarkable inter-assay reproducibility.

Claims

Reivindicaciones Claims
1. Superficie inmunosensora que comprende un soporte recubierto por una capa metálica a la que se une un polímero que inmoviliza al menos un anticuerpo anti-benzoilecgonina. 1. Immunosensor surface comprising a support coated by a metal layer to which a polymer that immobilizes at least one anti-benzoylecgonine antibody binds.
2. Superficie inmunosensora, según la reivindicación 1, que comprende entre dos y seis anticuerpos anti-benzoilecgonina caracterizados por presentar diferente afinidad por benzoilecgonina. 2. Immunosensor surface according to claim 1, comprising between two and six anti-benzoylecgonine antibodies characterized by having different affinity for benzoylecgonine.
3. Superficie inmunosensora, según cualquiera de las reivindicaciones 1 ó 2, donde los anticuerpos anti-benzoilecgonina son anticuerpos monoclonales.  3. Immunosensor surface according to any one of claims 1 or 2, wherein the anti-benzoylecgonine antibodies are monoclonal antibodies.
4. Superficie inmunosensora, según cualquiera de las reivindicaciones anteriores, donde los anticuerpos se unen al polímero mediante enlaces covalentes tipo amida o tipo carbonilo- hidrazina.  4. Immunosensor surface according to any of the preceding claims, wherein the antibodies are bound to the polymer by covalent bonds of the amide or carbonyl hydrazine type.
5. Superficie inmunosensora, según cualquiera de las reivindicaciones anteriores, donde el polímero se selecciona entre un polímero policarboxilado y un polímero policarboxilado modificado.  5. Immunosensor surface according to any of the preceding claims, wherein the polymer is selected from a polycarboxylated polymer and a modified polycarboxylated polymer.
6. Superficie inmunosensora, según cualquiera de las reivindicaciones anteriores, donde el polímero se selecciona entre polímeros con capacidad de inmovilizar una cantidad de anticuerpos anti-benzoilecgonina comprendida entre 730 pg/mm2 y 73000 pg/mm2. 6. Immunosensor surface according to any of the preceding claims, wherein the polymer is selected from polymers capable of immobilizing an amount of anti-benzoylecgonine antibodies between 730 pg / mm 2 and 73000 pg / mm 2 .
7. Inmunosensor que comprende:  7. Immunosensor comprising:
a. una superficie inmunosensora como se describió en cualquiera de las reivindicaciones de 1 a 6, y  to. an immunosensor surface as described in any one of claims 1 to 6, and
b. un transductor basado en el fenómeno óptico de la resonancia de plasmón superficial (SPR).  b. a transducer based on the optical phenomenon of surface plasmon resonance (SPR).
8. Aparato para la detección y cuantificación directa de benzoilecgonina que comprende un inmunosensor como se describió en la reivindicación 7 y un dispositivo o instrumento para procesar o acondicionar la señal recibida durante la etapa de asociación o de disociación.  8. Apparatus for the direct detection and quantification of benzoylecgonine comprising an immunosensor as described in claim 7 and a device or instrument for processing or conditioning the signal received during the association or dissociation stage.
9. Aparato, según la reivindicación 9, donde además comprende un dispositivo para realizar un cálculo.  9. Apparatus according to claim 9, further comprising a device for performing a calculation.
10. Aparato, según cualquiera de las reivindicaciones de 8 a 9, donde el inmunosensor está miniaturizado y el aparato es portátil. 10. Apparatus according to any of claims 8 to 9, wherein the immunosensor is miniaturized and the apparatus is portable.
11. Kit que comprende una superficie inmunosensora como se describió en cualquiera de las reivindicaciones de 1 a 6. 11. Kit comprising an immunosensor surface as described in any one of claims 1 to 6.
12. Aparato portátil que comprende una superficie inmunosensora como se describió en cualquiera de las reivindicaciones de 1 a 6.  12. Portable apparatus comprising an immunosensor surface as described in any of claims 1 to 6.
13. Método de detección de benzoilecgonina que comprende:  13. Method of detecting benzoylecgonine comprising:
i. poner en contacto la muestra problema con la superficie inmunosensora como se describió en cualquiera de las reivindicaciones de 1 a 6,  i. contacting the test sample with the immunosensor surface as described in any one of claims 1 to 6,
ii. detectar la señal de SPR durante la etapa de asociación y la etapa de disociación.  ii. Detect the SPR signal during the association stage and the dissociation stage.
14. Método de cuantificación de benzoilecgonina que comprende:  14. Method of quantification of benzoylecgonine comprising:
iii. detección de benzoilecgonina durante la etapa de asociación, según la reivindicación 13,  iii. detection of benzoylecgonine during the association step, according to claim 13,
iv. interpolación de la pendiente de la señal (dR/dt) obtenida en la etapa iii) en la recta de calibrado (dR/dt frente a concentración) para obtener el valor de concentración de benzoilecgonina.  iv. interpolation of the slope of the signal (dR / dt) obtained in step iii) in the calibration line (dR / dt versus concentration) to obtain the concentration value of benzoylecgonine.
15. Método de cuantificación de benzoilecgonina que comprende:  15. Method of quantification of benzoylecgonine comprising:
v. detección de benzoilecgonina durante la etapa de disociación, según la reivindicación 13,  v. detection of benzoylecgonine during the dissociation step according to claim 13,
vi. interpolación de la señal (R) obtenida en la etapa v) en la recta de calibrado (R frente a concentración) para obtener el valor de concentración de benzoilecgonina.  saw. interpolation of the signal (R) obtained in step v) in the calibration line (R versus concentration) to obtain the concentration value of benzoylecgonine.
16. Uso de una superficie inmunosensora, según se definió en la cualquiera de las reivindicaciones de 1 a 6, para la detección directa de benzoilecgonina.  16. Use of an immunosensor surface, as defined in any one of claims 1 to 6, for the direct detection of benzoylecgonine.
17. Uso, según la reivindicación 16, donde la detección tiene lugar en tiempo real.  17. Use according to claim 16, wherein the detection takes place in real time.
18. Uso, según las reivindicaciones 16 y 17, donde la detección se realiza en disoluciones acuosas reguladoras, en fluido oral o en mezclas de las mismas.  18. Use according to claims 16 and 17, wherein the detection is carried out in aqueous regulatory solutions, in oral fluid or in mixtures thereof.
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