• 专利附录
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    • 专利摘要:
    Optical method of detecting a target molecule by amplifying the interference response by refractive index and dispersion. Optical method of detecting at least one target molecule (OM) contained in a sample at a certain concentration, comprising: a) contacting, in a liquid medium, the sample containing the MO with a solution containing nanoparticles (NPs)) whose surface has been upholstered or functionalized with at least one type of bioreceptor (BR) specific to the target molecule to be detected (NP-BR), so that these BRs specifically recognize said OM, and therefore conjugates of the NP are formed -BRs with MOs (NP-BR-MOs); b) separating the nanoparticle conjugates (NP-BR-MOs and/or NP-BRs) formed in the previous step; c) contacting the conjugates of said nanoparticles (NP-BR-MOs and/or NP-BRs) with a sensor surface of an optical transducer that works by reflection and/or transmission, whose response is based on optical interference and where This sensor surface will be functionalized by immobilizing on its surface: i) the target molecule (MO), or ii) at least one specific bioreceptor, which may be of the same type (BR) or of another type (BR1) of the target molecule; and c) determining the optical reading on the sensor surface by the change in the interference response of the optical transducer caused by the change in the real part of the refractive index caused by said NP conjugates recognized on the sensor surface, and/or by the intensity change in said interference response caused by the intensity variation caused either by the dispersion or by the variation in the complex part of the refractive index of the mentioned NPs conjugates, or by a combination of both effects. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2750374A1
    申请号:ES201931066
    申请日:2019-12-02
    公开日:2020-03-25
    发明作者:Bolanos Miguel Holgado；Perales Araceli Diaz；Arandia María Garrido；Espinosa Rocío Lopez；Sahagun Alejandro Romero；Heras María Fe Lagunas；Pacios Luis Fernandez；Fernandez Beatriz Santamaria；Alonso Yolanda Ramirez；Gutierrez Francisco Javier Sanza
    申请人:Bio Optical Detection S L；Universidad Politecnica de Madrid；
    IPC主号:
    专利说明:

    [0001]
    [0002]
    [0003]
    [0004] TECHNICAL SECTOR
    [0005]
    [0006] The present invention is located in the technical sector of immunological analysis and / or diagnosis by using optical biosensors that operate by optical interference of light, such as those described in the invention Optical detection system for labeling-free high-sensitivity bioassays ( ES2574138 (T3) - 2016-06-15) or in the scientific review publication: Emerging applications of label-free optical biosensors (G. Zanchetta, et al. Nanophotonics, 6, 1-18 (2017). More specifically , the invention described in this document refers to a method of detection and / or quantification of a target molecule (OM), in particular a molecule with immunological reactivity or that has bioreceptor-antigen affinity, where the method comprises the enrichment of the target molecule (also called target analyte) in a sample by using functionalized nanoparticles, and its subsequent analysis by direct optical reading of the reflection and / or transmission of the system, whose interference response will be affected by the nanoparticle conjugates specifically recognized on its surface due to the variation of the real part of the refractive index and the possible loss of intensity by the variation of the complex part of the refractive index and / or the scattering of the aforementioned complexes in the reflected or directly transmitted light, without the need to use additional detection methods.
    [0007]
    [0008] Additionally, the present invention relates to a method that can be applied to multiple optical reading systems or devices, preferably portable, such as that described in the INTERFEROMETRIC DETECTION METHOD (EP2880396 (A1) - 2015-06-10) and reported later for example in scientific publications: Towards reliable optical label-free point-of-care ( PoC) biosensingdevices (Sensors and Actuators B 236 (2016) 765-772) or Description of an Advantageous Optical Label-Free Biosensing Interferometric Read-Out Method to Measure Biological Species (Sensors 2014, 14, 3675-3689), which allows the diagnosis of immunologically based diseases in a reasonable time.
    [0009] STATE OF THE ART
    [0010]
    [0011] Until now, the most common in -Vitro detection systems with high sensitivity available on the market for the analysis of biomolecules are based on chemical amplification and development, with the ELISA ( Enzyme-Linked Immunosorbent Assay) technique being the most common and established in the market. There are a wide variety of devices, and by referring to a specific case, in the allergy sector the IMMUNOCAP, IMMUNOCAP ISAC can be highlighted, which can offer quantitative information and mainly a qualitative measurement (yes / no response) regarding the presence of IgE specific allergy antibodies, which does not allow defining the component-resolved sensitization profile for each patient necessary for complex cases. Furthermore, these devices offer only a limited range of food allergens that can be analyzed, using extracts of crude allergens instead of individual allergens, which can lead to misleading information. Additionally, these types of devices require highly qualified personnel and facilities.
    [0012]
    [0013] The number of bibliographic references related to the field of optical biosensors is very high, both in the scientific literature and in patent documents. In many of these documents, the physical principle of detection of optical biosensors is based on light interference phenomena, by changing the index of refraction when biological material accumulates on a sensor surface. However, these devices have not been commercialized massively yet and, therefore, remain the traditional methods based on amplification or chemical development of conventional and commercially available, both the nitrocellulose - based lateral flow (lateral flow ) that works by colorimetry, such as those already mentioned ELISA. One of the possible causes may be the requirement of the high sensitivity required for the different applications of these sensors, as well as the difficulty in working with real biological samples of a complex matrix. Among other factors, the concentration of the biomolecules to be recognized is generally very small, the complexity when working with real samples is high due to the high number of other agents (biological or not) that can generate high non-specific adsorption and the lack of specificity, in addition this problem is amplified and there is a limitation to be able to detect molecules of very low molecular mass, whose impact on the increase of the refractive index on the sensor surface is much smaller, therefore, the sensitivity factor of the transducer, as well as the error or uncertainty requirements of Measured in the reading of the device are more critical to reach the necessary detection limit that can be estimated as the quotient between the measurement uncertainty of the reading and the sensitivity of the transducer. In short, these requirements mean that, in order to reach a competitive detection limit to the needs demanded, the system must be very competitive, the sensitivity of the transducer must be very high, the reading uncertainty very small and, additionally, the system it is necessary to be able to specifically recognize the target biomolecules, including those with very low molecular mass, in real samples with a multitude of components or agents in a highly specific way, avoiding as far as possible any nonspecific detection that could invalidate the analysis.
    [0014]
    [0015] In summary, these strict requirements of specificity, sensitivity and limit of detection pose great difficulty when developing a method and / or system to analyze molecules of an immunological nature, using optical biosensors based on changes in interference. due to the variation of the refractive index on the sensitive surface, particularly when working with real samples where the elimination of the matrix effect of the real sample, due to the high number of other existing agents that can generate high nonspecific adsorption, implies a very significant added difficulty in being able to compete with conventional chemical development and amplification systems, such as those based on ELISA.
    [0016]
    [0017] In this field of optical detection of biomolecules, in particular using the principle of optical interference, the transduction process is carried out on a functionalized surface that incorporates specific molecular receptors to exclusively recognize the target molecule, and blocking agents to prevent adsorption. nonspecific of other components found in the liquid biological sample ( G. Zanchetta et al. "Emerging applications of label-free optical biosensors". Nanophotonics 2017; pp. 1-19). This specific surface is part of the transducer that, a Once functionalized, it increases its volume and / or density due to the presence of the specific molecular receptors and blocking agents mentioned, making the sensor a biosensor.This increase in the amount of matter generates a change in the interference response that we call the signal of reference (see figure 1A) .When these molecular receptors recognize the target biomolecule, the amount of matter increases again (see figure 1C), in this case due to the increase in the specific biological material that this surface has been able to recognize. These molecules recognized in the transducer form a biological layer on the surface of the transducer or biosensor, several nanometers thick depending on the type of target molecules.
    [0018]
    [0019] A technical problem of great difficulty is the specific recognition or detection of molecules in a real sample, especially when these target molecules are small. Likewise, the processes of receptor functionalization (biofunctionalization) are also highly difficult. However, despite the difficulties, a large number of examples are reported in the literature for the anchoring of specific bioreceptors, for example by direct adsorption using polymer-based materials by direct anchoring of antibodies and peptides, or using reactions such as streptavidin and biotin, or using protein A and protein G to target antibodies in the biofunctionalization process. Other alternatives for making covalent anchors of the surfaces may be based on silanization processes, avidin-biotin reactions, among others that may be applicable to oxides, nitride, silicon, etc. Multiple biofunctionalization pathways can be found in Bioconjugate Techniques, Greg T. Hermanson, Second Edition, 2008, ISBN 978-0-12-370501-3.
    [0020]
    [0021] In the case of the interference optical transducers object of this invention, the materials used make up a photonic micro / nano structure that generates an interference or resonance pattern. This interference or resonance pattern will be modified both in wavelength, frequency or wave number, as well as in intensity or amplitude of the signal by the anchorage and molecular recognition reactions, which makes the change the basis of the reading in the biochemical sensing transduction ( M. Holgado et al. “Description of an Advantageous Optical Label-Free Biosensing Interferometric Read-Out Method to Measure Biological Species. Sensors 2014, 14, 3675-3689). The most used photonic structures for this type of sensor are those based on Match Zehnder interferometers, rings and resonator discs, Fabry-Perot interferometers, bimodal guides, nano-resonator networks or resonant nano-pillar networks, etc., such as those can be found in G. Zanchetta and coi "Emerging applications of label-free optical biosensors". Nanophotonics 2017; pp. 1-19.
    [0022]
    [0023] As mentioned above, the principle of operation of a biosensor that works by interference is based on the fact that when the bioreceptors anchor on the surface of the interferometer, the interference signal changes, for example, generating a change in the interference signal caused by the sensor (figure 1A). When this biosensor undergoes a recognition process, the bioreceptors specifically capture the target molecules causing said interference profile to change again, for example, a resonance or a minimum of interference can shift and change its position in wavelength (see figure 1 C). One way to measure sensitivity can be by evaluating the wavelength shift of a minimum of interference as a function of the number of target molecules specifically detected or recognized on the sensor surface. These relative variations of the resonance mode with respect to its initial reference (see Figure 1C) determine the target molecules recognized on the surface, and therefore, allow the concentration to be easily established quantitatively in an analyzed sample, from the performance of a calibration curve with known samples from which the source concentration is known. This method of proceeding to obtain quantitative information is widely reported, for example in RL Espinosa et al., “A Proof-of-Concept of Label-Free Biosensing System for Food Allergy Diagnostics in Biophotonic Sensing Cells: Performance Comparison with ImmunoCAP. Sensors 2018, 18, 2686.
    [0024]
    [0025] However, these changes in wavelength, frequency or wave number related to the concentration of the biomolecules or molecules to be detected are limited by the response of the photonic structure used to manufacture the sensor that produces the optical interference. Furthermore, the dynamic range of concentrations that the sensor can measure depends on the construction of the sensor, where the detection area of the sensor plays a fundamental role. It is an indisputable fact that the nonspecific adsorption on the sensor surface of the multiple and diverse components found in a real sample represents a very significant difficulty that sometimes requires the use of blocking agents, and is a fundamental aspect when working with biological samples. real of diverse nature, such as: blood, serum, saliva, tears, urine, among others, given the high number of components or different biological agents that are found together with the specific molecule that you want to detect specifically. For this reason, the specificity when developing a biosensor is one of the most critical aspects, being the elimination of this nonspecific adsorption, or the previously mentioned matrix effect, a key factor to be able to work with real samples. In this sense, a good and efficient functionalization or upholstery of the sensor surface can reduce the mentioned matrix effect, although in many cases it is necessary to use blocking agents of various types such as albumin, ethanolamine, among others.
    [0026] The invention described herein solves the limitations of interference-based optical biosensors known to date in terms of their limited sensitivity, insufficient detection limit, lack of specificity and difficulty in working with real samples due to the matrix effect. Thus, this document describes an optical method of detecting a target molecule by amplifying the interference response by refractive index and variation in light intensity of this response as a consequence of changing the refractive index itself and by the possible dispersion produced by the type and size of the conjugates based on the NPs used to carry out the assay. This method can be used to measure target molecules with a very low molecular mass, it allows working with real samples, that is, samples that have not been subjected to a previous treatment, by increasing specificity and, above all, it allows modifying the dynamic range of the response and sensitivity to demand of the bio-enlargement to use.
    [0027]
    [0028] DESCRIPTION OF THE INVENTION
    [0029]
    [0030] The present invention provides an optical method of detecting at least one target molecule based on the biological interactions of said molecule. In particular, the method is based on the amplification in the interference response by the variation of the refractive index generated by the NPs conjugates used in the biological recognition process, added to the possible additional amplification caused by the dispersion phenomenon of said conjugates. of NPs whose number on the sensor surface depends on the biological interactions of the target molecule at the different stages of the method. Consequently, the method described here allows qualitative and / or quantitative analysis of a target molecule in a real sample, in particular in a complex matrix biological sample, improving the detection limit by signal amplification caused by the variation of the interference signal and the specificity of the interference-based optical biosensors due to the possibility of separating the NPs conjugates from the real sample to avoid the multiple components of the biological sample in their origin.
    [0031]
    [0032] The method of the present invention makes it possible to improve optical detection by interference compared to the usual methods reported in the literature for optical transducers or sensors based on interference, which in this document is also called an interferometric sensor or transducer. In turn, this method could be used totally or partially in most of the optical biosensors based on interference or optical resonance reported in the literature, such as Fabry-Perot interferometers, rings and resonator discs, Match Zehnder interferometers, BICELLs or bimodal guides, among many others. Thus, these devices can compete and improve the performance of classic detection systems by chemical amplification or development, generally based on fluorescence or colorimetry (eg ELISA).
    [0033]
    [0034] In particular, the present invention provides an optical method of detecting at least one target molecule (MO, also called analyte) in a sample, characterized in that the method comprises the following steps:
    [0035] a) Measure the interference response or reference signal of an optical sensor or interferometric transducer with its biofunctional sensing surface (Figure 1A) with:
    [0036] i. the target molecule (MO),
    [0037] ii. or at least one specific bioreceptor (BR or BR1) of the target molecule. b) Contact, in a liquid medium, a sample to be analyzed with nanoparticles functionalized with at least one specific bioreceptor (NP-BR) of the OM (Figure 1B1b), preferably at a temperature between 2 and 37 ° C, forming a conjugate (NP-BR-MO) with the functionalized nanoparticles and those target molecules present in the sample;
    [0038] c) Separate the NP-BR conjugates from the sample and, provided that the sample includes the target molecule, the NP-BR-MO conjugates, since both conjugates will be together in the mixture obtained after step b (Figure 1B3b);
    [0039] d) Contacting said NP-BR conjugates, and if applicable, the NP-BR-MO conjugates obtained in step b) with said biofunctionalized sensor surface of the interferometric transducer (Figure 1C2);
    [0040] e) Determine the optical reading, in particular the interference response by reflection and / or transmission, by the variation of said interference profile caused by the variation of the real part of the refractive index and / or the variation of intensity caused by either the dispersion or variation in the complex part of the refractive index of the mentioned NPs conjugates, or a combination of the above, on the sensor surface of the optical transducer.
    [0041]
    [0042] The conjugates mentioned based on NPs that are recognized on the sensor surface of the transducer will cause a change in the refractive index, mainly in its real part, that causes a change in the interference of the reflected or transmitted light, but it can also happen that changes are generated in its complex part (depending on the type of NPs used), thus generating a change in the extinction coefficient and a reduction in the intensity of reflected or transmitted light. It is added to this effect that the dispersion of the aforementioned NPs complexes can cause light scattering to a greater or lesser degree depending on the type and size of NPs, and therefore, another change in the intensity of the light received, both in transmission as in reflection, which add to the aforementioned effects.
    [0043]
    [0044] The method of the present invention allows the detection of any MO, including those of very low molecular mass difficult to detect for optical biosensors, that has at least one specific bioreceptor. Thus, this method can be used to detect any target molecule that may give rise to an immune reaction or present bioreceptor-antigen affinity. In particular, the MO can be an antibody, for example an allergy specific antibody (IgE) to a specific allergen or any other protein, hormone, toxin, among others. As one of the practical examples of this invention, an antibody specific to an allergy molecule (IgE) has been detected as MO, such that the conjugate NP-antiIgE (NP-BR in its initial notation in the present invention) that it would recognize the IgE present in the biological sample by forming the NP-antiIgE-IgE complex (NP-BR-MO in its initial notation in the present invention), and on the surface of the sensor it has been immobilized with at least one allergenic molecule (BR1 ) which is recognized by the specific IgE of the mentioned antibody (BR1 in its initial notation in the present invention).
    [0045]
    [0046] The optical detection method described in this invention can be used for any sector where specific detection of a molecule is required in-vitro. Application sectors of great relevance, therefore, can be, in addition to the clinical one, the agri-food sector, where the detection of diseases in animals is required, or the detection of pathogens in their diet or in their environment. Additionally, it can also be applied in the analysis of water in aquaculture. Other applications of the method are, for example, for the process and quality control of products such as milk, wine, oils, as well as the detection of contaminants in agriculture and water monitoring, among many others.
    [0047]
    [0048] Therefore, in the method described here, the sample to be analyzed can be a biological sample such as, for example, blood, serum, urine or tears, among others; a agro-food sample such as that obtained directly from food or from the environment where the animals are found; or a water sample, whether for human, animal or any other use. As mentioned above, the method of the invention can be applied to a real sample, that is, a sample taken directly, without any prior treatment. Although the application of the method to a liquid sample is preferred, in those embodiments in which the sample to be analyzed is a solid sample, it will be submitted to a previous step of dissolution in a liquid medium.
    [0049]
    [0050] The functionalized NPs that are used in the method of the present invention are upholstered, that is, functionalized on their surface, with at least one MO-specific BR, in particular, a BR capable of specifically interacting with MO (or analyte) by means of an immunological reaction, an antibody-antigen affinity reaction or BR-MO reaction in general. Thus, on those occasions when the sample, preferably a biological sample without prior treatment, comprises the OM, in step b) of the method of the present invention an affinity reaction takes place between these MOs and the NP-BR conjugates, giving rise to the formation of conjugates NP-BR-MOs. This step of the method can be carried out at a temperature preferably between 2 and 37 ° C. Within this range, the higher the temperature, the immunological reaction between BR and OM is favored, requiring a shorter reaction time or incubation. Therefore, this time can vary, depending on the temperature in the incubation process, between several minutes and hours, depending on the association constant and the kinetics of the recognizing reaction and the affinity of the bioreceptor used. In general, an incubation time of 30 min is usually established, although depending on the biological application, temperature and kinetics of the reaction in particular, this time can typically range from less than 30 min to several hours.
    [0051]
    [0052] In step c) of the method described here, the separation of NP-BR and NP-BR-MO (also called, respectively, NP-BR conjugates and NP-BR-MO conjugates herein) generated in the affinity reaction that takes place in step a), provided that the sample analyzed comprises MOs. Both conjugates (NP-BRs and NP-BR-MOs) are present in the resulting mixture to a greater or lesser degree. For said separation, any physical separation mechanism of the mentioned NPs conjugates can be used from the liquid medium of the sample to be analyzed, such as centrifugation, separation by electric or magnetic field, among other possible ones. In In particular, these NPs conjugates can be easily separated from the rest of the sample by filtering, by means of one or more centrifugation cycles.
    [0053]
    [0054] In particular embodiments, the NPs used may be able to attract or repel themselves in the presence of an electric field due to surface charge. In this case, step b) of separating the NP-BR and NP-BR-MO conjugates from the rest of the sample can preferably be carried out by means of an electric field and electrophoresis.
    [0055]
    [0056] In other particular embodiments, the NPs used can be attracted or repelled by a magnetic field. In this case, step b) of separating the NP-BR and NP-BR-MO conjugates from the rest of the sample can preferably be carried out by applying a magnetic field.
    [0057]
    [0058] Additionally, the NPs used can have the ability to be attracted or repelled in an electric or magnetic field, for example, silica NPs coated with a magnetic material. In these cases a mixed separation mechanism can be used based on the application of electric field, magnetic field and, optionally, centrifugation.
    [0059]
    [0060] As mentioned above, in step d) of the optical detection method ( in vitro) of a target molecule in a sample, the NP-BRs and the NP-BR-MOs are contacted, if any, because has produced the recognition of MOs present in the sample, obtained in step c) with a sensor surface of a biofunctionalized optical transducer (upholstered with MO or BR1) whose response, in particular, an optical sensor whose response is based on interference, resonance generated by a certain photonic structure such as those mentioned in the bibliographic references mentioned in this document. In particular, any optical sensor operating by optical interference whose response changes in the presence of the recognized material on its sensing surface (the said NPs conjugates in the present invention) can be used. Likewise, this interference or resonance response as a function of wavelength, wave number or frequency, not only varies due to the variation of the refractive index in its real part or optical thickness (this being referred to the refractive index of NPs conjugates). multiplied by the increase in average thickness of these NPs in the sensitive surface of the transducer), but also their amplitude (usually measured in optical intensity) can change depending on the variation of the complex component of the refractive index and / or the dispersion caused by conjugates of recognized NPs, and sites therefore, on the surface of said sensor.
    [0061]
    [0062] In particular embodiments, the sensor employed is an interferometric sensor that operates by measuring the variation in interference which is monitored by the change in light intensity in a given spectral range. For this, said transducer is tuned so that the measurement is due to loss of light intensity ( M. Holgado, et al. Sensors and Actuators B 236 ( 2016) 765-772).
    [0063]
    [0064] Additionally, the NPs plus the biological material accumulated or recognized on its surface, increases the refractive index of the transducer causing this response to be modified and, therefore, the intensity to change. In addition, the NPS attached to the biological material, can generate losses by dispersion (of the English term scattering ) in such a way that this phenomenon can be added to the loss of intensity generated by the change in interference.
    [0065]
    [0066] It should be mentioned here that in the optical response by interference of photonic sensors, the refractive index plays an important role in the diversity of sensor types reported in the technical and scientific literature. When a conjugate of NPs is specifically adhered to the sensing surface of a photonic transducer, and due to this recognition phenomenon, the average refractive index of the transducer increases. In the case of sensors based on interferometers based on thin Fabry-Perot type sheets, the product refractive index by the average thickness of NPs causes the response to be changed by interference ( eg New Device Based on Interferometric Optical Detection Method for Label-Free Screening of C-Reactive Protein, IEEE Transactions on Instrumentation and Measurement, 68, 9, 3193 - 3199, 2019), and in the case of those based on evanescent field detection, usually of horizontal optical interrogation and based on waveguides, the surface concentration of NPs on the surface increases the average refractive index that light sees when it propagates through a certain waveguide. In both cases, as mentioned, a change in the interference profile is generated, such as Label-free optical biosensing with slot-waveguides, Optics Letters, 33, 7, 2008. On the other hand, the imaginary part of the index Refraction generates signal losses that can be accounted for in a loss of amplitude that will be observed by reducing signal intensity. Finally, and mainly, for vertical interrogation sensors, the losses due to dispersion will also reduce the amplitude of the interference signal and, for Therefore, it should be used as a detection mechanism.
    [0067]
    [0068] Thus, the method described in this document can be used mainly in vertical optical interrogation interferometers, such as those based on Fabry-Perot interferometers, Sensitive cells based on nanostructured structures, Sensitive cells based on Nano-Pillar networks and Resonant Nano Pillars, among others ( eg Label-free biosensing by means of periodic lattices of high aspect ratio SU-8 nanopillars, Biosensors and Bioelectronics 25 ( 2010) 2553-2558; Bio-Photonic Sensing Cells over transparent substrates for anti-gestrinone antibodies biosensing, Biosensors and Bioelectronics 26 ( 2011) 4842-4847; o Resonant nanopillars arrays for label-free biosensing, Optics Letter, 41, 23, 2016). Likewise, in-plane optical interrogation interferometers can also be used, such as Match-Zehnder interferometers, resonator rings, bimodal guides, grid guides, resonator discs, etc. such as those described in the aforementioned reference G. Zanchetta and coi “Emerging applications of label-free optical biosensors. "Nanophotonics Nanophotonics 2017; 6 ( 4): 627-645. DOI 10.1515 / nanoph-2016-0158; or MC Estevez, M. Alvarez, and LM Lechuga, Laser Photon. Rev. 6, 463 ( 2012). Because of its simplicity, the use of a Fabry-Perot Interferometer vertical interrogation is preferred.
    [0069]
    [0070] This step c) of the method can be carried out at a temperature between 0 and 40 ° C, preferably at a temperature between 2 and 37 ° C, and even more preferably, between 18 and 37 ° C when working in continuous flow systems where there is no evaporation. However, when working with sample drops and, depending on the volume, it is possible to work at temperatures between 2 to 37 ° C (incubation process) using incubators to avoid evaporation and to promote the immune reaction by reducing said incubation time. between the conjugates and the sensor surface of the biosensor. On the other hand, the temperature can be reduced to reduce the evaporation factor, avoiding the need to use incubators. Depending on the option chosen, the incubation time can range from several minutes to hours, depending on the association constant and the kinetics of the recognizing reaction and the affinity of the bioreceptor used.
    [0071]
    [0072] The sensor surface of the transducer or optical sensor as mentioned in the present invention can be functionalized to have, immobilized on the sensor surface, the MO or, alternatively, at least one BR1 that also recognizes the MO. Additionally, the surface of said sensor may or may not comprise one or more agents. blocking agents (AB) on the gaps that would not have been covered, either by the MO or by the BR1, in order to prevent a non-specific response from other components that may be present in the sample to be analyzed.
    [0073]
    [0074] In those embodiments in which the sensor surface comprises the MO, that is, this molecule is immobilized or upholstered on the sensor surface, the maximum variation of the optical reading signal is obtained by the mechanisms described in the present invention when the sample analyzed does not contain OM, since in this case the number of NP-BRs that interact and are recognized on the surface of the sensor is greater (see diagram in the lower right of Figure 3). However, when the analyzed sample comprises OM, if this analyte is in a sufficient concentration to cover all NP-BR conjugates, the variation of the reading signal will be practically nil, that is, it will not be altered before and after the stage. d). In short, the response curve of the system will evolve as follows, the reading signal will decrease as the concentration of OM in said sample increases. Therefore, in the event that the amount of OM in the sample is equal to or greater than the amount necessary to completely cover the surface of the total amount of NP-BRs used in the method described here and, consequently, filtering obtained in step c) only includes the NP-BR-MO conjugate, the optical reading signal will not be modified (see diagram in the left part of figure 3). Quantification is carried out by calibrating the sensor between these two states, for which a calibration curve is obtained in which the response signal of the sensor is obtained based on certain tabulated concentrations. It should be noted here that the ratio of NPs and MO present in the sample can now be easily modified to tune and adapt the dynamic range of the sensor.
    [0075]
    [0076] On the other hand, in those embodiments in which the sensor surface is functionalized or upholstered by immobilizing at least one type of BR1 that specifically recognizes OM, that is, a biomolecule (BR1) capable of reacting specifically with OM, The change in the optical response of the sensor will be increased as the concentration of MO in the analyzed sample increases (see top left of Figure 4), while said reading signal will not change when the sample does not contain the analyte. or MO to detect, since in this case the NP-BR-MO conjugate is not formed (see bottom right of figure 4) and is not recognized by the surface covered by said BR1. As in the previous case, the MO quantification is performed by calibrating the sensor between the two situations exemplified in figure 4. To do this, a calibration curve is obtained in which the response signal of the sensor is obtained as a function of certain tabulated concentrations.
    [0077]
    [0078] The specific bioreceptors (BR1) comprised on the sensor surface of the optical interference sensor may be the same or different from the bioreceptors (BR) comprised in the functionalized nanoparticles (NP-BR).
    [0079]
    [0080] Additionally, in other particular embodiments of the invention, the described method can be used to detect allergy specific antibodies (IgE), noted as MO in the present invention of a specific allergen in a biological sample. In these embodiments, the BR immobilized on the surface of the NPs (NP-BR) is a specific bioreceptor (anti-IgE) that recognizes allergy-specific antibodies (i.e., the analyte or OM, in this case IgE) in the biological sample to be analyzed. In this complex case, the NP-BR (NP-antiIgE) conjugate captures IgEs in the patient's sample forming the NP-antiIgE-IgE conjugate (noted as NP-BR-MO). In one embodiment of the present invention, when the specific receptor (BR1) is an allergenic molecule (MA), and in a particular case, Prup3 in the assay depicted in Figure 6 herein, the sensor surface is covered with MA, such that if the NP-BR-MO conjugate (i.e. NP-antiIgE-IgE) is recognized on the sensor surface, the signal will be zero when the patient does not have IgEs specific to the MA immobilized on the sensor , while in the case that the patient has allergy-specific antibodies, the NP-BR-MO conjugate (that is, NP-antiIgE-IgE) is recognized on the sensor surface, altering the optical reading signal such and as described in this invention. In short, in these embodiments, if the patient from whom the analyzed sample was obtained has an allergy, the interference signal will be modified by the presence of the NP-BR-MO conjugate, where the OM are the allergy-specific antibodies (IgE ) to the allergenic molecule (MA). In this way, the method of the present invention supposes a great advantage, since it allows the in vitro detection of the possible allergies that a patient may have. This determination is currently carried out in vivo by means of the "prick tesf", which consists of introducing allergenic molecules or extracts into the patient's arm and observing if the body reacts.
    [0081]
    [0082] Thus, in the present method two mechanisms intervene, on the one hand, the change of the interference in wavelength, frequency or wave number of the interference profile to the optical path is increased (refractive index in its real part by length that the light must pass), and on the other hand, the possible reduction in amplitude due to the scattering of light from the corresponding NPs complexes on the sensor surface due to the mentioned mechanisms of dispersion and / or variation of the complex part of the refractive index.
    [0083]
    [0084] The method of the present invention allows quantitative analysis, that is, determining the presence or absence of a target molecule (MO), as well as its quantification depending on the test carried out on a sample.
    [0085]
    [0086] The nanoparticles used in the method of the present invention can be formed by any type of dielectric inert material such as, oxides or silicon; any light-transparent material, for example, silica, titania, alumina or silicon; as well as nanoparticles of another nature such as gold, aluminum, silver, among others. In the latter case, it happens that at certain wavelengths the NPs can be transparent to light and / or generate more or less dispersion or variation in the extinction coefficient or a complex part of the refractive index. Thus, nanoparticles can be selected from the group consisting of silica, gold, aluminum, silver, silicon, and metal oxides, in particular titanium oxide (titania) or aluminum oxide (alumina).
    [0087]
    [0088] It should be noted that the size and concentration of NPs are calculated based on the sensor surface, the type of interference biosensor used and the dynamic range of the concentration to be detected.
    [0089]
    [0090] Therefore, in preferred embodiments of the method of the present invention, when the optical sensor used is a Fabry-Perot interferometer, the use of spherical silica NPs (SiO2) with a diameter between 50 and 100 nm, or of another morphology, is preferred. with diameters of the same magnitude as the previous ones, preferably with a concentration of 108 to 10121 (1E8 to 1E12) nanoparticles per microliter. Although this concentration can vary mainly depending on the sensitive area of the sensor, incubation time, temperature and volume, since not only the kinetics of the reaction determines the association time, but the diffusion and sedimentation of the NPs they play a relevant role. The higher the concentration of NPs, the less time it takes for them to reach the surface and can cover the sensitive area of the sensor. As shown in Figure 5, when the NPs have a diameter of 200 nm, the response (Signal of A | RO p of the Fabry-Perot sensor used) is not monotonous decreasing and only from an upholstery equal to or greater than 50% the response of the sensor could be used. In this case, it is considered that the NPs are not tuned and the response of the biosensor system does not improve. However, when the NPs are spherical with a diameter of 50 nm or 100 nm, it is observed how the response signal is monotonously decreasing and the sensor signal (A | ROp signal of this used sensor) decreases almost linearly with the area upholstered in the sensor. In this case, the biosensor system is tuned and the sensitivity has been improved because the slope of the response curve is higher than that reported in previous works, thanks to the method of the present invention.
    [0091]
    [0092] In other preferred embodiments of the method of the present invention, when the optical sensor used is a Fabry-Perot interferometer, the use of gold (Au) NPs with a diameter of 20 to 70 nm and preferably 45-55, sizes that are preferred, is preferred. make the NPs transparent to the optical interrogation wavelength or spectral range ( .Modeling the optical response of gold nanoparticlesw, Chem. Soc. Rev., 2008, 37, 1792 1805) and preferably a concentration of 108 o 1011 NPs / mL. Although this concentration can vary mainly depending on the sensitive area of the sensor, incubation time, temperature and volume, since not only the kinetics of the reaction determines the association time, but the diffusion and sedimentation of the NPs they play a relevant role. The higher the concentration of NPs, the less time it takes for them to reach the surface and can cover the sensitive area of the sensor.
    [0093]
    [0094] What is relevant to the invention is that the modification of the refractive index is produced by the biological material immobilized or recognized in the NP, since it acts as a vehicle and it is said biological material that changes the refractive index of the transducer, and therefore , your interference response. This interference response is read by the intensity variation of a wavelength or a spectral range and depending on the size of the NP with its corresponding biological material, the effect of the dispersion is added.
    [0095]
    [0096] The method of the present invention allows great versatility, since it is possible to select the size and material of the nanoparticles (NPs) that allow them to be tuned to the interference response of the transducer. By means of this tuning process, the response by interference of the chosen transducer, preferably the Fabry-Perot Interferometer, can be amplified, as well as taking into account the reduction of the signal amplitude due to the dispersion of the NPs that are recognized in the sensor. Additionally, the method The present invention also allows design criteria to be applied to determine the amplification constant, establish the quantitative detection range, significantly improve sensitivity, reduce measurement uncertainty and dramatically improve the detection limit. Additionally, this method also allows modifying the detection signal reading system, which allows reducing the measurement uncertainty.
    [0097]
    [0098] Thus, with the detection method described in this patent application, the following advantages are achieved:
    [0099]
    [0100] - Detection of both low and high molecular mass molecules, since the method is independent of the molecular mass of the target molecule (MO) to be detected and an unsolved technical solution is given to a common problem. More specifically, in the optical method described here, detection depends on the conjugates (NP-BR or NP-BR-MO) recognized on the surface of the sensor. Therefore, unlike conventional methods where the mass on the sensing surface changes very little, if the MO is of very low molecular mass; in the method of the invention the mass on the sensor surface depends mainly on the nanoparticle (NP) and the bioreceptor (BR), not so much on the molecular mass of the target molecule (MO). This is a great advantage over other methods that use interference sensors, where the signal and the sensitivity depend a lot on the molecular mass of the target molecule.
    [0101]
    [0102] - It allows working with real samples, that is, samples (in particular, biological samples) that have not undergone any previous treatment. The problem of matrix effect, nonspecific adsorption, is dramatically reduced or eliminated, since the method comprises the separation of the analyte from the rest of the sample and, advantageously, this separation is done outside the sensor, so that it only deposits a clean sample on the sensing surface of the biosensor. This eliminates the need to use complex blocking systems on the surface of the sensor itself, in addition to avoiding complex washing processes directly on the sensor to eliminate the matrix effect or nonspecific adsorption. It should be noted here that the blocking and marking process in optical systems that operate without optical marking is perhaps one of the main reasons why they have not been massively marketed as a sensor or diagnostic system at the site of care or need " point of care ” for its terminology in the English language.
    [0103] - By selecting the quantity, size and material of the NPs, it is possible to optimize the response curve represented as the measurement signal as a function of the conctration in order to adapt the response to the range of concentrations to be detected, that is, the Dynamic range. In this way, it is possible to optimize the concentration range in which you want to work depending on the selected biological application and for each type of transducer that works by chosen interference.
    [0104]
    [0105] - This method dramatically improves the sensitivity and limit of origin detection of the transducer by interference used. Thus, the method allows detecting minimum concentrations of the target molecule (analyte) due to the increased sensitivity in the transduction process. Additionally, by selecting the quantity, size and material of the NPs it is also possible to significantly improve the detection limit, since the amplification of the signal depends on the NPs used, while the uncertainty of reading of the system does not depend on the NPs. Consequently, the ratio Sensitivity / Uncertainty of reading corresponding to the detection limit can be improved for any reading system due to the increased sensitivity mentioned.
    [0106]
    [0107] In conclusion, the present invention solves important problems of interference-operated optical biosensors:
    [0108]
    [0109] I. That with this invention the sensitivity of the detection system no longer depends on the molecular mass of the target molecule to be detected or recognized.
    [0110]
    [0111] II. That the sensitivity is drastically increased, therefore improving the detection limit in a very significant way and thus detect molecules of very low molecular mass in minute concentrations.
    [0112]
    [0113] III. What can be measured effectively real samples (for example biological samples of complex matrix) reducing or eliminating the non-specific signal (also called background or background signal in the English language) and therefore be able to measure
    [0114]
    [0115] IV. Furthermore, the response curve can be adapted to the concentration range to be measured.
    [0116] Therefore, the method of the present invention can be used to develop in-situ and non-invasive diagnostic systems by capturing biomarkers present in different samples, preferably biological. In the case of clinical diagnosis, the biological liquid samples where these biomarkers can be found can be, among others, blood, serum, urine, tears, among others, such as spinal fluid, water, salve, etc. Similarly, the diagnostic method can be used for multiple sectors where in-vitro bioreceptor-analyte recognition detection (eg antibody-antigen affinity reaction) takes place.
    [0117]
    [0118] Thus, the invention also provides an immunologically based in-vitro diagnostic method using optical interference based photonic transducers (interferometric transducers), where these transducers comprise on their sensing surface: i) the target molecule, or ii) the less a specific bioreceptor (BR1) of this; as well as a reading system capable of monitoring the relative variations of the immobilization and / or recognition events that are drastically amplified by the use of the NP-BR and NP-BR-MO conjugates described above.
    [0119]
    [0120] In particular embodiments of the present invention, the sample to be analyzed is a biological sample, either human or animal, and the target molecule to be detected is a biomarker that allows in vitro diagnosis of, for example, a disease.
    [0121]
    [0122] In one of the preferred embodiments, the optical detection method of the present invention can be used for in vitro diagnosis of food allergy. This method would consist of detecting allergy specific immunoglobulins (IgE) contained in real patient samples. In this case, the Target Molecules (MOs) to detect are IgEs specific to the allergens to which the patient is allergic. To do this, several food allergen molecules are immobilized in different wells of a biokit, for example, several Fabry-Perot-type interferometers (eg Towards reliable optical label-free point-of-care ( PoC) biosensing devices, Sensors and Actuators B 236 ( 2016) 765-772). The NPs are biofunctionalized with a bioreceptor (BR) consisting of an IgG immunoglobulin that specifically recognizes all the IgEs existing in the patient's sample (sera from patients with food allergy). In order to distinguish which IgE molecules the patient is allergic to, after incubation and filtering of the patient a sample is obtained that will contain the conjugates of NPs with the different IgEs, this sample is deposited and incubated in the different wells that contain the allergenic molecules against which the diagnosis is to be made, so that the conjugates of NPs with the Specific IgEs bind to the corresponding allergen, being then detected when reading the optical signal due to the change in interference that is generated.
    [0123]
    [0124] The detection method described here, therefore, must additionally comprise a previous stage of functionalization of nanoparticles with at least one specific bioreceptor, resulting in the formation of functionalized nanoparticles (NP-BR). To carry out this functionalization, any anchoring method (covalent bond, direct adsorption or any other biofunctionalization route) known to the person skilled in the art can be used.
    [0125]
    [0126] In those embodiments in which the NPs are silica, the preferred functionalization method is silanization, to couple an anchor molecule that orients the antibody for its non-specific part. On the other hand, when the method uses gold NPs, it is preferable to use thiol as an anchoring element. The biofunctionalization mechanisms of surfaces are widely reported in the scientific literature (eg Bioconjugate Techniques, Greg T. Hermanson, Second Edition, 2008, ISBN 978-0-12-370501-3).
    [0127]
    [0128] As a particular example, the biofunctionalization of silica NPs with specific bioreceptors of the target molecule can be carried out from a monodispersed solution of nanospheres with amino groups on their surface. The carboxyl groups present in the antibodies can be activated and stabilized by the addition of EDC (1-ethyl-3- (3-dimethylaminopropyl) carbomidine hydrochloride) and NHS (N-hydroxysuccinimide), which causes a spontaneous reaction with the primary amines present on the surface of the nanospheres, resulting in the formation of a stable NP-BR conjugate by the formation of a covalent amide.
    [0129]
    [0130] Likewise, the optical detection method of the present invention also requires biofunctionalization of the surface of the optical sensor, as described in recognition step d), in which the NP-BR-conjugates are contacted. MO and / or NP-BR with the sensing surface. The biofunctionalization of the sensor surface of the transducers has also been widely described in the literature, different routes, with covalent or adsorptive anchorages, with or without orientation and using different agents. blockers. Throughout this invention, several references have been reported describing these biofunctionalization routes that are not the object of the present invention.
    [0131]
    [0132] BRIEF DESCRIPTION OF THE FIGURES
    [0133]
    [0134] Figures 1A-1C: Figure 1A illustrates an optical signal from an interferometric transducer (eg Fabry-Perot type of vertical optical interrogation) in which specific bioreceptors have been immobilized. Figures B1a-B3a are a schematic representation of the traditional operating process, while Figures B1b-B3b refer to the process described in the present invention in which the NPs conjugates are used. Figure C1 shows schematically what is the detection of a target molecule in a sensor by interference, where the profile of the spectral response is modified by the recognition of a target molecule, while Figure C2 shows what detection would be like in the case of using what is described in the present invention, where the change in the spectral optical signal is significantly amplified, and may also entail a modification in said amplitude of said optical signal, and therefore both effects greatly improve the ability to system detection.
    [0135]
    [0136] Figure 2: It is a schematic representation of the different types of conjugates formed with nanoparticles (NP) in the method of the present invention, that is, functionalized nanoparticles (NP-BR) and conjugate (NP-BR-MO) formed by these functionalized nanoparticles and the target molecule. In the particular embodiments depicted in this figure, both conjugates comprise a blocking agent (AB), however, the method of the present invention could also be selectively performed when the NP-BR and NP-BR-MO do not comprise any blocking agent.
    [0137]
    [0138] Figure 3: This figure represents the detection mechanism when the MO is immobilized on the sensor surface. More specifically, the result of having incubated the NP-BRs (step b) with the sample and filtered (step c) to obtain the NP-BRs conjugates in the case that the Biological sample did not contain the OM and NP-BR-MOs in the event that they did, this is exemplified by the extreme case in which all the NP-BR present in the reaction medium capture all the OM present in the sample. Figure 3B represents the recognition stage (stage d and stage e), in the event that the sample did not contain MOs, the NP-BRs conjugates would be recognized on the sensor surface and the signal would be maximum (see bottom of Figure 6C), ie the sensor interference signal changes significantly. In the event that the sample did have OM, then the NP-BR-MO conjugates would not be recognized on the surface of the sensor since the sensor also has the MO immobilized (see the upper part of Figure 6C)
    [0139]
    [0140] Figure 4: This figure represents the recognition and filtering mechanism first (Figure 4), then the detection mechanism when what is immobilized is a specific receptor (BR) of the target molecule (MO) in the sensor (Figure 4B ). Contrary to how it was described in Figure 3, the sample containing the conjugates of NP-BR-MOs will now be recognized on the surface of the sensor and therefore in this case the change of the signal by interference will be very significant (see part upper part of figure 4C), whereas when the sample does not have OM, the NP-BRs conjugates will not be recognized on the surface of the sensor (see lower part of figure 4C) and therefore the interference signal will practically not be altered .
    [0141]
    [0142] Figure 5: Example of tuning NPs on a biosensor system. In this case, a Fabry-Perot type interference sensor was used, used as a reading mechanism based on the Increased Relative Optical Power (IROP (%)) described in Towards reliable optical label-free point-of-care ( PoC) biosensing devices , Sensors and Actuators B 236 ( 2016) 765 772. The simulation of the response curve represents the reading signal as a function of the percentage of NPs recognized on the surface. It can be seen that for NPs from 50 to 100 nm, the response curve is monotonic decreasing and valid to be used as a sensor, while for 200 nm the response curve would not be valid.
    [0143]
    [0144] Figure 6A: Figure 6A shows the change of the signal in a real serum sample with high concentration of IgE specific to Pru p 3 in 14 wells (P1 to P14). Each Well contains a Fabry-Perot sensor. Figure 6B shows the Scanning Electron Microscopy (SEM) images, where one image is observed without NPs on the surface, and another with NPs on the surface. It happens that this is the case where the signal increase is observed and therefore the recognition of the NP-IgG-IgE-Pru p3 conjugate is certified. It should be noted that this figure shows the readings of the signals of a 14-well diagnostic kit (P1 to P14) in serum matrix (in this case expressed in A | RO p %) obtained in Example 1 (see more down). The lowest signal of these readings is that related to the immobilization of the bioreceptor (BR1), while the highest already refer to the specific detection of the target molecule (MO), in this In the case of an IgE specific to the allergen Pru p 3. As can be seen in this graph, the silica NPs used have been functionalized with a bioreceptor (BR) consisting of immunoglobulins G (IgGs) that specifically recognize all the immunoglobulins E (IgEs) present in the biological sample, that is, the NPs have been functionalized with antiIgE (IgGs that capture IgEs). The sensor surface is functionalized with a bioreceptor (BR1). In this case they are molecules of a specific type of allergen (Pru p 3), in such a way that only those IgEs specific to Pru p 3 (in this case the target molecule) will be recognized on the surface of the sensor, and therefore when this occurs changes the optical reading signal.
    [0145]
    [0146] Figure 7A: SEM images of the conjugates recognized on the sensor surface (images 7A-3 and 7A-4) in contrast to images 7A-1 and 7A-2 where a limited number of NPs have been recognized. In this case, the method has consisted of using gold NPs that have been biofunctionalized with a bioreceptor (BR) consisting of an immunoglobulin G that specifically recognizes the target Molecule (MO) that is the metalloproteinase MMP9. In this case, the non-biological sample did not contain MMP9 and therefore the NP-antiMMP9 conjugates are recognized in the sensor, significantly increasing its signal (Figure 7B), and it is also verified that the high signal is due to the NPs observed by microscopy (images 7A-3 and 7A-4 in figure 7A). As additional test, another MO (Cystatin CST4) was immobilized on another sensor. When the sample was put in contact with this other sensor, it was observed that there was practically no recognition of the NP-antiMMP9 conjugates with CST4, as can be seen by the limited number of NPs observed by microscopy (images 7A-1 and 7A- 2 of figure 7A) and the smallest measured signal (figure 7B).
    [0147]
    [0148] Figure 8: In this figure, NPMAs of PMMA are observed on the surface of a sensor, through which an experimental test is carried out to certify that, depending on the size of the NPs, light scattering is also a phenomenon that can be used and / or or add to the change in interference. In this case, using PMMA NPs, it can be seen how the interference profile is reduced in signal amplitude and is used as a detection mechanism.
    [0149] EXAMPLES
    [0150] In order to contribute to a better understanding of the invention, and according to a practical embodiment thereof, a series of examples of preferred embodiments of the present invention are attached as an integral part of this description.
    [0151]
    [0152] Example 1: Detection of food allergy specific IgE using silica nanoparticles
    [0153]
    [0154] In this case the NPs are functionalized by immobilizing a specific receptor for specific allergy IgE antibodies (NP-antiIgE), it should be noted that the anti-IgE are IgGs that recognize and capture all the IgEs existing in the corresponding biological sample, and on the surface One of the specific allergenic molecules for which you want to know whether the allergy-specific antibody that is related to that molecule is immobilized from the sensor, and therefore detect if the patient has allergy-specific antibodies (IgEs) to the molecule Pru p 3 The measurements are made on a real serum sample. In this case, the NP-antiIgE-IgE conjugate is formed, but with the peculiarity that IgE may or may not be specific to the allergenic molecule that has been upholstered in the sensor (Pru p 3, in this test) and that we have noted like BR1. Thus, if the signal from the sensor increases, it means that the conjugate is recognized on the surface and the sample comes from a patient who has a specific concentration of specific allergy antibodies to the Pru p 3 molecule. On the contrary, if the sensor signal does not change, the sample does not contain specific allergies to Pru p 3.
    [0155]
    [0156] An allergenic molecule (Pru p3) was immobilized on the sensor surface of the Fabry-Perot Interferometer Sensor at a concentration of 5 ^ grams / mL.
    [0157]
    [0158] On the other hand, antibody (anti-IgE) that specifically recognizes all IgEs in silica NPs (NP-antiIgE conjugate) with a diameter between 50 nm and 100 nm was immobilized.
    [0159]
    [0160] A sample was withdrawn from the patient, which was centrifuged to obtain the serum. Subsequently, this sample was mixed with the patient's serum, where the allergy specific antibodies (IgEs) were found. The functionalized silica NPs were added, at a concentration of 2.5 x 1010 NPs / | a, L and incubated for 30 min, that is, left at 37 ° C in an incubator for the NP-conjugate. anti-IgE will recognize the possible IgEs present in the patient's sample.
    [0161] Once the incubation process was completed, the centrifugation process was carried out so that the NPs conjugates settled in the eppendorf. The supernatant was removed, thus obtaining the NPs conjugates that have recognized the patient's IgEs. This spinning process was repeated 3 times.
    [0162]
    [0163] Once the sample containing the NP-antiIgE-IgE (specific) conjugates was filtered, it was introduced into the sensor previously functionalized with the allergic molecule Pru p 3 and incubated for 30 min at 37 ° C. After the recognition stage, if the NP-antilgE-IgE conjugate is specific to the allergenic molecule, the conjugate remains specifically recognized on the surface of the sensor and the reading signal changes.
    [0164]
    [0165] Figures 6A and 6B show the results obtained in this test. In particular, Figure 6A shows the immobilization bars of the allergen Pru p 3 and the signal increase in 14 wells (1100 signals of A | RO p (%) when the sensor surface is recognized by the NP conjugates. -antilgE-IgE specific to Pru p 3 of the biological sample, in this case 1200 A | RO p (%) signals in contrast to the initial signal corresponding to the immobilization of Pru p 3, 200 A | RO p (%)) This signal is corroborated with figure 6B, where scanning electron microscopy (SEM) images showing recognition of the NP-antilgE-IgE conjugate Pru p3 are shown .
    [0166]
    [0167] It is important to note here that this sensor works due to the intensity of the light generated by the Fabry-Perot interferometer used ( Towards reliable optical label-free point-of-care ( PoC) biosensing devices, Sensors and Actuators B 236 ( 2016) 765- 772) and, in this case, the method described here amplifies the recognition signal, since it depends both on the displacement of the signal by interference, amplified by the increase of matter in the sensor, as well as by the loss of intensity of light by the optical scattering of the NPs recognized on said surface.
    [0168]
    [0169] Example 2: Detection of MMP9 metalloproteinase (inflammation marker)
    [0170]
    [0171] To carry out this experimental test, the MMP9-specific antibody (antiMMP9) was immobilized on the surface of the gold NPs (NP-anti-MMP9 conjugate). In this case 50 nm gold NPs were chosen as they are transparent to the chosen wavelength range around 850 nm ( Modeling the optical response of gold nanoparticlesw, Chem. Soc. Rev., 2008, 37, 1792-1805).
    [0172]
    [0173] On the other hand, Fabry-Perot interferometer-type sensors were upholstered with MMP9 and CST4 on their corresponding sensor surfaces with a concentration sufficient to have a high percentage of upholstery of said sensor surfaces. Specifically, both the MMP9 protein (inflammation marker) and the CST4 protein were mobilized at a concentration of 10 | a, g ml_ "1 on the surface of the Fabry Perot type sensor. Therefore, two biosensors were obtained, one in which it has immobilized a protein related to antiMMP9 (the target molecule) and in another a protein not related to the MMP9 antibody.
    [0174]
    [0175] In this case, a polymer-based material (SU8) similar to that described in Development towards Compact Nitrocellulose-Based Interferometric Biochips for Dry Eye MMP9 Label-Free In-Situ Diagnosis Sensors 2017, 17, 1158 was used to manufacture the interferometer; doi: 10.3390 / s17051158), except that this case did not use nitrocellulose for anchoring.
    [0176]
    [0177] The analysis was carried out directly using NP-antiMMP9 conjugates that must recognize MMP9 as a bioreceptor and must not recognize CST4 as a receptor. For this, the sample containing the NP-antiMMP9 conjugates was incubated on said sensor surfaces for a period of 2 hours, at a temperature of 37 ° C. So if the signal is high, the NP-antiMMP9 conjugates have been recognized on the surface, while if the signal is low, the NP-antiMMP9 conjugates are not recognized on the sensor surface and the signal must be low.
    [0178]
    [0179] Figure 7 shows the results obtained in this test. It can be seen in the electron microscopy images (see figure 7A) how the NP-anti-MMP9 conjugate is recognized on the surface (images 7A-3 and 7A-4, and therefore recognizes the target molecule (MO). this is corroborated with the signals detected in the sensors in Figure 7B (signals 7A-3 and 7A-4) that are very high when those mentioned with NP-antiMMP9 plays have been recognized. However, the results found when immobilizing a protein is not related (in this case the aforementioned CST4) indicate that the number of conjugates two NP-antiMMP9 is limited, and therefore, the signal is much lower. See figures 7A, images 7A-1 and 7A-2 and figure 7B signals 7A-1 and 7A-2 respectively.
    权利要求:
    Claims (14)
    [1]
    1. - An optical method of detecting at least one target molecule in a sample, characterized in that the method comprises:
    to. Measure the interference response of an optical sensor or interferometric transducer with its biofunctional sensing surface (Figure 1A) with:
    i. the target molecule (MO),
    ii. Or at least one specific bioreceptor (BR or BR1) of the target molecule;
    b. Contacting, in liquid medium, a sample to be analyzed with nanoparticles (NPs) functionalized with at least one specific bioreceptor (NP-BR) of the target molecule (MO), forming a conjugate (NP-BR-MO) with the nanoparticles functionalized and those target molecules present in the sample;
    c. Separate the NP-BR conjugates from the sample and, provided that the sample includes the target molecule, the NP-BR-MO conjugates formed in the mixture obtained after step b);
    d. Contacting said NP-BR, and if applicable, the NP-BR-MO conjugates obtained in step b) with said biofunctionalized sensor surface of the interferometric transducer; Y
    and. Determine the optical reading on the sensor surface of the interferometric transducer.
    [2]
    2. - The optical detection method according to claim 1, wherein the target molecule is an IgE allergy specific antibody specific to an allergenic molecule (MA).
    [3]
    3. - The optical detection method according to claim 2, where BR1 is the allergenic molecule (MA) specific for an allergy-specific antibody and the sensor surface is functionalized with said allergenic molecule (MA).
    [4]
    4. - The optical detection method according to any one of claims 1 to 3, wherein the sample with at least one target molecule to be analyzed is selected from the group consisting of a biological, clinical sample, an agro-food sample and water.
    [5]
    5. - The optical detection method according to claim 4, wherein the clinical sample to be analyzed is selected from the group consisting of blood, serum, plasma, saliva, tear and urine.
    [6]
    6. - The optical detection method according to claim 4 or 5, where the sample to be analyzed is a clinical sample and the target molecule is a biomarker for in vitro diagnosis.
    [7]
    7. - The optical detection method according to claim 6, wherein the biomarker is selected from the group consisting of proteins, hormones, immunoglobulins, toxins, or any molecule that is recognized by immunological processes.
    [8]
    8. - The optical detection method according to any one of claims 1 to 7, where steps a) and c) take place at a temperature between 0 and 40 qC, the temperature conditions of these steps being the same or different from each other.
    [9]
    9. - The optical detection method according to any one of claims 1 to 8, wherein the separation step b) takes place by means of a technique selected from the group consisting of centrifugation, separation by electric field, separation by magnetic field and a combination of the previous ones.
    [10]
    10. - The optical detection method according to any one of claims 1 to 9, wherein the nanoparticles are selected from the group consisting of silica, alumina, silicon nitride, silicon, dielectric materials, metal oxides, magnetic materials, gold, aluminum , silver and metallic materials.
    [11]
    11. - The optical detection method according to any one of claims 1 to 10, wherein the optical sensor is a Fabry-Perot Interferometer, and the functionalized nanoparticles (NP-BR) comprise spherical silica nanoparticles with a diameter between 50 nm and 100nm.
    [12]
    12. - The optical detection method according to claim 11, where the NPs concentration is between 108 to 1012 NPs / ^ L.
    [13]
    13. The optical detection method according to any one of claims 1 to 10, where the optical sensor is a Fabry-Perot Interferometer, and the functionalized nanoparticles (NP-BR) comprise gold nanoparticles with a diameter of between 20-70 nm .
    [14]
    14. The optical detection method according to claim 13, where the NPs concentration is between 108 to 1011 NPs / mL.
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