Optical method for detecting target molecule through amplification of interference response caused by refractive index and dispersion rate

Abstract

To solve the problem related to detection limit, specificity and the like of an interference-based optical biosensor.SOLUTION: An optical method for detecting a target molecule (MO) includes: a) forming a sample including MO, in a liquid, into (NP-BR), NP-BR-MO in which the surface of nano particles (NPs) is functionalized with a specific bioreceptor (BR) of the target molecule; b) separating NP-BR-MO and / or NP-BR; c) bringing NP-BR-MO and / or NP-BR into contact with a sensor surface of an optical transducer through reflection and / or transmission, wherein the sensor surface is immobilized and functionalized on (i) MO or (ii) BR or BR1; and d) determining an optical reading value from a change in the interference response of the optical transducer caused by a change of a real portion of the refractive index of the NP conjugate and / or a change in the interference response caused by a result of dispersion or a variation in the complex portion of the refractive index, or both effects.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The invention is located in the technical field of immunoanalysis and / or diagnostics by the use of optical biosensors that function by the optical interference of light, such as those described in the invention of an Optical detection system for labelling-free high-sensitivity bioassays (Spanish Patent No. ES2574138 (T3) - 15 June 2016) or in the publication of the scientific review: Emerging applications of label-free optical biosensors (G. Zanchetta, et al., Nanophotonics, 6, 1-18). (2017). More specifically, the invention described therein refers to a method for detecting and / or quantifying target molecules (MOs), particularly those with immunoreactivity or affinity for biological receptors or antigens, which involves concentrating the target molecules (also called target analytes) in a sample using functionalized nanoparticles and subsequent analysis by direct optical reading of the reflection and / or transmission of the system. The interference response is affected by the conjugates of the nanoparticles recognized on their surface due to variations in the real part of the refractive index, and by possible loss of intensity due to variations in the complex part of the refractive index and / or dispersion of the conjugates in the directly reflected or transmitted light, without the need for additional detection methods.

[0002] Additionally, the present invention refers to a method that can be applied to a number of optical reading systems or devices, preferably portable, such as the one described in the invention INTERFEROMETRIC DETECTION METHOD (EP 2880396 (A1) - June 10, 2015) and later reported in scientific publications, for example, "Towards reliable optical label-free point-of-care (PoC) biosensing devices" (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 for the diagnosis of immunologically based diseases within a reasonable time frame. [Background technology]

[0003] To date, the most common highly sensitive in vitro detection systems available on the market for analyzing biomolecules are based on chemical development and amplification, with ELISA (Enzyme-Linked Immunosorbent Assay) technology being the most common and established on the market. There is a wide variety of devices available. In the allergy sector, IMMUNOCAP and IMMUNOCAP ISAC stand out as being able to provide quantitative information and primarily qualitative measurements (yes / no answers) regarding the presence of allergy-specific IgE antibodies. This precludes the definition of a component-resolved sensitization profile for each patient, which is necessary for complex cases. Furthermore, these devices only offer a limited range of food allergens, which can be tested using crude allergen extracts rather than individual allergens, potentially leading to misleading information. Additionally, these types of devices require highly skilled installation and staff.

[0004] The number of bibliographical documents related to the field of optical biosensors is enormous, both in scientific and patent literature. In many of these documents, the physical detection principle of optical biosensors is based on the phenomenon of optical interference due to changes in refractive index when biological materials accumulate on the sensor surface. However, because these devices are not yet widely available on the market, traditional methods based on chemical amplification or development remain the most commonly available commercially. Both, like the aforementioned ELISA, are based on nitrocellulose strips that function by colorimetry (lateral flow). One possible reason for this is the high sensitivity required for the various applications of these sensors and the difficulty of working with real biological samples using complex matrices. Among other factors, the concentrations of biomolecules to be recognized are generally very low, and the complexity of working with real samples is high due to the large number of other existing agents (biological or nonbiological) that can generate high nonspecific adsorption and the lack of specificity. This problem is further amplified by the limited ability to detect very low molecular weight molecules, whose effect on the increase in refractive index of the detection surface is much smaller. Therefore, the sensitivity factor of the transducer is much more demanding. At the same time, the requirements for the measurement error or uncertainty in the reading of the device become more crucial in order to reach the required detection limit, which can be estimated as the ratio of the measurement uncertainty of the reading to the sensitivity of the transducer. In short, these requirements mean that in order to reach a competitive detection limit for the required needs, the system should be very competitive, the sensitivity of the transducer should be very high, and the uncertainty of the reading should be very small. In addition, the system must be able to specifically recognize target biomolecules, including those with very low molecular weights, in real samples containing a large number of components or drugs in a very specific way, and must avoid as much as possible non-specific detection that may invalidate the analysis.

[0005] In summary, these very stringent requirements for specificity, sensitivity and detection limits represent a major difficulty in developing a method and / or system for analyzing molecules of immunological nature through the use of optical biosensors based on interference changes due to refractive index variations at the sensing surface when acting on real samples, especially when the matrix effects of the real samples are excluded, and with the very significant additional difficulty of being able to compete with traditional chemical amplification and development systems, such as those based on ELISA, due to the large number of other existing agents that may generate high non-specific adsorption.

[0006] In this field of optical detection of biomolecules, particularly using the principle of optical interference, the transduction process is carried out on a functionalized surface that incorporates specific molecular receptors to exclusively recognize target molecules and blocking agents to prevent nonspecific adsorption of other components found in liquid biological samples (G. Zanchetta et al., Emerging applications of label-free optical biosensors, Nanophotonics, 2017; pp. 1-19). The specific surface is part of the transducer, which, once functionalized, increases its volume and / or density due to the presence of the aforementioned specific molecular receptors and blocking agents, transforming the sensor into a biosensor. This increase in the amount of material produces a change in the interference response, called the reference signal (see Figure 1A). When these molecular receptors recognize the target biomolecule, the amount of material increases again (see Figure 1C), in this case due to an increase in the specific biomaterial that the surface was able to recognize. These molecules recognized by the transducer form a biological layer on the surface of the transducer or biosensor, with a thickness of a few nanometers depending on the type of target molecule.

[0007] A very challenging technical problem is the specific recognition or detection of molecules in real samples, especially when these target molecules are small in size. Similarly, the receptor functionalization process (biofunctionalization) is also very challenging. However, despite the difficulties, numerous cases of immobilization of specific bioreceptors have been reported in the literature, for example, by direct adsorption using polymer-based materials, by direct immobilization of antibodies or peptides, or by utilizing reactions such as streptavidin and biotin, or by using protein A and protein G with target antibodies in biofunctionalization processes. Other alternatives for achieving covalent immobilization of surfaces may be based on silanization processes, avidin-biotin reactions, etc., and may be applicable to oxides, nitrides, and silicon, among others. Several biofunctionalization routes can be found in Bioconjugate Techniques, Greg T. Hermanson, Second Edition, 2008, ISBN978-0-12-370501-3.

[0008] In the case of the optical interference transducer object of the present invention, the materials used constitute photonic micro / nanostructures that generate interference or resonance patterns. This pattern of interference or resonance is altered both in wavelength, frequency, or wavenumber, as well as in the intensity or amplitude of the signal resulting from immobilization and molecular recognition reactions, which alters the basis of the readout in 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 photonic structures most commonly used for this type of sensor are based on Mach-Zehnder interferometers, resonator rings and disks, Fabry-Perot interferometers, bimodal guides, networks of nanocavities, or networks of resonant nanopillars. These can be found in the following literature: G. Zanchetta et al., “Emerging applications of label-free optical biosensors,” Nanophotonics 2017; pp. 1-19.

[0009] As described above, the operating principle of interference-based biosensors is based on the fact that when a bioreceptor is immobilized on the interferometer surface, it changes the interference signal, e.g., the interference signal generated by the sensor (Figure 1A). When this biosensor undergoes a recognition process, the bioreceptor specifically captures the target molecule, again altering the interference profile. For example, the resonance or interference minimum can shift, changing its position within the wavelength range (see Figure 1C). One way to measure sensitivity is to evaluate the wavelength shift of the interference minimum as a function of the number of target molecules specifically detected or recognized on the sensor surface. These relative resonance mode variations relative to the initial reference (see Figure 1C) determine the target molecules recognized on the surface. Therefore, the concentration of the analyzed sample can be easily established by realizing a calibration curve of a known sample with a known concentration of origin. This procedure for obtaining quantitative information has been extensively reported, for example, in RLE Spinosa 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.

[0010] However, these changes in wavelength, frequency, or wavenumber, related to the concentration of the biomolecule or molecules to be detected, are limited by the response of the photonics structure used to manufacture the sensor, which generates optical interference. Additionally, the dynamic range of concentrations that a sensor can measure depends on the sensor's structure, in which the sensor's detection area plays a fundamental role. Nonspecific adsorption of multiple and diverse components found in real samples to the sensor surface represents a very relevant difficulty, sometimes necessitating the use of blocking agents. It is undoubtedly a fundamental aspect of working with real biological samples of various natures, such as blood, serum, saliva, tears, and urine, given the large number of components or various biological agents found along with the specific molecules to be specifically detected. For this reason, specificity is one of the most important aspects when developing biosensors, and elimination of this nonspecific adsorption, or the aforementioned matrix effect, is a key factor for functioning with real samples. In this sense, although successful and efficient functionalization or coating of the sensor surface can reduce the aforementioned matrix effect, it is often necessary to use various types of blocking agents, such as serum albumin and ethanolamine, among others.

[0011] The invention described herein overcomes the limitations of interference-based optical biosensors known to date, including their limited sensitivity, insufficient detection limit, lack of specificity, and difficulty in handling real samples due to matrix effects. This document therefore describes an optical method for detecting target molecules that utilizes the amplification of interference responses due to variations in refractive index and the light intensity of this response as a result of changes in the refractive index itself, as well as dispersion that may be generated by the type and size of NP-based conjugates used to realize the assay. This method can be used to measure very low molecular weight target molecules, and its increased specificity makes it possible to handle real samples, i.e., samples that have not been pretreated, and in particular to modify the dynamic range of response and sensitivity to suit the requirements of the biogrowth used. Summary of the Invention

[0012] The present invention provides a method for optical detection of at least one target molecule based on the biological interaction of that molecule. In particular, this method relies on the amplification of the interference response due to the variation in the refractive index generated by the NP conjugates used in the biological recognition process. This is in addition to the potential amplification caused by the dispersion phenomenon of the NP conjugates, whose number on the detection surface depends on the biological interaction of the target molecule at various steps of the method. Thus, the method described herein enables qualitative and / or quantitative analysis of target molecules in real samples, especially biological samples with complex matrices, improving the detection limit due to the amplification of the signal caused by the specificity of interference-based optical biosensors due to the variation in the interference and the possibility of separation of the NP conjugates from the real sample, avoiding the multiple components of the biological sample at its origin.

[0013] The method of the present invention allows for improved optical detection by interference compared to conventional methods reported in the literature for interference-based optical sensors or transducers, also referred to herein as interferometric sensors or transducers. Consequently, this method may be used in whole or in part with most optical biosensors based on interference or optical resonance reported in the literature, such as Fabry-Perot interferometers, resonator rings and disks, Mach-Zehnder interferometers, BICELLs, or bimodal guides, among others. These devices may therefore be able to compete with and improve the performance of conventional detection systems, typically based on chemical amplification or development based on fluorescence or colorimetry (e.g., ELISA).

[0014] In particular, the present invention provides an optical method for detecting at least one target molecule (MO, also called analyte) in a sample, the method comprising: Step a) measuring the interference response or reference signal of an optical sensor or an interferometric transducer for the following i and ii using a bio-functionalized detection surface (FIG. 1A), wherein i and ii are: i. the target molecule (MO), or ii. measuring at least one specific bioreceptor (BR or BR1) of said target molecule; step b) contacting, in a liquid medium, the sample to be analyzed with nanoparticles (NP) functionalized with at least one specific bioreceptor (NP-BR) of said target molecules (MO) (Fig. 1B1b), to form conjugates (NP-BR-MO) comprising said functionalized nanoparticles and their target molecules present in said sample, preferably at a temperature between 2 ° C and 37 ° C; Step c) separating the NP-BR conjugate from the sample, wherein the sample contains the target molecule, the NP-BR-MO conjugate formed in the mixture obtained after step b) (Figure 1B3b); Step d) contacting the NP-BR and, if applicable, the NP-BR-MO conjugate obtained in step b) with the biofunctionalized detection surface of an interferometric transducer (FIG. 1C2); step e) determining the optical readings, in particular the interference response, comprising the variations in the interference profile caused by variations in the real part of the refractive index due to reflection and / or transmission and / or the variations in intensity caused either by the dispersion or variations in the complex part of the refractive index of a conjugate of said NP, or a combination thereof, at the sensor surface of the optical transducer.

[0015] The aforementioned conjugates based on NPs recognized at the detection surface of the transducer cause a change in the refractive index, primarily in its real part, which may cause a change in the interference of reflected or transmitted light. They may also cause a change in its complex part (depending on the type of NP used), thus resulting in a change in the extinction coefficient and a decrease in the intensity of the reflected or transmitted light. In addition to this effect, the scattering of the aforementioned NP conjugates may cause more or less light scattering, depending on the type and size of the NP, and therefore cause another change in the intensity of the received light, both in transmission and reflection, which is in addition to the effects mentioned above.

[0016] The method of the present invention allows the detection of any MO, including those with very low molecular weights that are difficult to detect with optical biosensors, that have at least one specific bioreceptor. Therefore, this method can be used to detect any target molecule that may provoke an immune response or exhibit bioreceptor-antigen affinity. In particular, the MO can be an antibody, such as an allergy-specific antibody (IgE) against a specific allergen or any other protein, hormone, toxin, among others. In one practical example of the present invention, a specific antibody against an allergen molecule (IgE) was detected as an MO in such a way that a NP-anti-IgE conjugate (NP-BR in the first description of the present invention) that recognizes IgE present in a biological sample forms a NP-anti-IgE-IgE complex (NP-BR-MO in the first description of the present invention) and is immobilized on the sensor surface with at least one allergen molecule (BR1) that is recognized by the specific IgE of the aforementioned antibody (BR1 in the first description of the present invention).

[0017] The optical detection method described in the present invention can be used in any field sector where specific in vitro detection of molecules is required. Therefore, highly relevant fields of application include the clinical as well as the agri-food sector, where the detection of animal diseases or pathogens in food or the environment is required. In addition, it can also be applied to the analysis of water in aquaculture. Other applications of this method include, for example, process and quality control of products such as milk, wine, and oil, as well as the detection of pollutants in agriculture and water monitoring.

[0018] Thus, in the methods described herein, the sample to be analyzed can be, for example, a biological sample such as blood, serum, urine, or tears, among others; an agricultural food sample, such as one obtained directly from food or from the environment in which animals are found; or a water sample intended for human consumption, for animals, or for other uses. As noted above, the method of the present invention can be applied to real samples, i.e., samples collected directly without pretreatment. While it is preferred to apply the method to liquid samples, in embodiments in which the sample to be analyzed is a solid sample, it will be subjected to a prior step of dissolution in a liquid medium.

[0019] The functionalized NPs used in the methods of the present invention are coated with at least one MO-specific BR, particularly a BR capable of specifically interacting with MOs (or test substances) through an immune reaction, an antibody-antigen affinity reaction, or a general BR-MO reaction, i.e., their surfaces are functionalized. Therefore, if a sample, preferably a biological sample without pretreatment, contains MOs, an affinity reaction occurs between these MOs and NP-BR conjugates in step b) of the method of the present invention, leading to the formation of the NP-BR-MO conjugate. This step of the method can be preferably carried out at temperatures between 2°C and 37°C. Within this range, higher temperatures promote the immune reaction between BRs and MOs and shorten the required reaction or incubation time. Therefore, depending on the temperature of the incubation process, this time can vary from several minutes to several hours, depending on the binding constant, the kinetics of the recognition reaction, and the affinity of the bioreceptor used. Generally, an incubation time of 30 minutes is usually established, but depending on the biological application, temperature, and the kinetics of the specific reaction, this time usually ranges from less than 30 minutes to several hours.

[0020] In step c) of the method described herein, separation of the NP-BR and NP-BR-MO (also referred to in this document as NP-BR conjugates and NP-BR-MO conjugates, respectively) generated in the affinity reaction carried out in step a) occurs, provided that the analyzed sample contains MOs. Both conjugates (NP-BR and NP-BR-MO) are present to a greater or lesser extent in the resulting mixture. For this separation, any physical mechanism can be used to separate the aforementioned NP conjugates from the liquid medium of the sample to be analyzed, such as centrifugation, separation by electric or magnetic fields, among others. In particular, these NP conjugates can be easily separated from the rest of the sample by filtration through one or more centrifugation cycles.

[0021] In certain embodiments, the NPs used may be able to attract or repel each other in the presence of an electric field due to their surface charges, in which 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 electric field and electrophoresis.

[0022] In other particular embodiments, the NPs used can be attracted or repelled by a magnetic field, in which 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.

[0023] In addition, the NPs used may have the ability to be attracted or repelled in electric or magnetic fields, such as silica NPs coated with magnetic materials, etc. In these cases, mixing-separation mechanisms can be used based on the application of electric or magnetic fields, and optionally centrifugation.

[0024] As described above, in step d) of the method for optical detection of target molecules in a sample (in vitro), NP-BR and NP-BR-MO, if present, are brought into contact. This allows recognition of the MO present in the sample, which is acquired in step c) using the detection surface of a biofunctionalized optical transducer (coated with MO or BR1), whose response is based in particular on interference, resonance, generated by certain photonic structures, such as those cited in the bibliographic references in this document. In particular, any optical sensor that functions by optical interference and whose response changes in the presence of a material to be recognized on its detection surface (a conjugate of the aforementioned NP in the present invention) can be used. Similarly, the response of this interference or resonance as a function of wavelength, wavenumber or frequency not only depends on the variation of its real part of the refractive index or optical thickness (which refers to the refractive index of the conjugates of NPs multiplied by the increase in the average thickness of these NPs on the sensing surface of the transducer), but also its amplitude (usually measured in light intensity) can vary depending on the variation of the complex component of the refractive index and / or scattering caused by the conjugates of the recognized NPs and therefore located on the surface of the sensor in question.

[0025] In a particular embodiment, the sensor used is an interferometric sensor that works by measuring the fluctuations in interference monitored by changes in light intensity in a given spectral range. To do this, the transducer is adjusted so that the measurements are due to losses in light intensity (W. Holgado, et al. Sensors and Actuators B 236 (2016) 765-772).

[0026] In addition, NPs and biological materials accumulated or recognized on their surface increase the refractive index of the transducer, which modifies this response and therefore the intensity. In addition, NPs attached to biological materials can generate scattering losses (from the English term scattering), so that this phenomenon can be added to the intensity losses generated by interference changes.

[0027] It is worth mentioning here that the refractive index plays a key role in the optical interferometric response of photonic sensors, with a variety of sensor types reported in the technical and scientific literature. When NP conjugates specifically attach 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 Fabry-Perot thin-film interferometers, the refractive index product of the NPs with their average thickness changes the interferometric response (e.g., "New Device Based on Interferometric Optics! Detection Method for Label-Free Screening of C-Reactive Protein," IEEE Transactions on Instrumentation and Measurement, 68, 9, 3193-3199, 2019). In the case of sensors based on evanescent field detection, usually via horizontal optical interrogation, and those based on waveguides, the surface concentration of NPs on the surface increases the average refractive index seen by light as it propagates through a given waveguide. In both cases, a change in the interference profile is produced, as previously described, e.g., Label-free optical biosensing with slot-waveguides, Optics Letters, 33, 7, 2008. On the other hand, the imaginary part of the refractive index produces a signal loss that can be explained by the observed loss in amplitude by reducing the signal strength. Finally, and primarily, in the case of perpendicular interrogation sensors, scattering losses also reduce the amplitude of the interference signal, allowing it to be used as a detection mechanism.

[0028] Therefore, the method described in this document can be primarily used in vertical optical interrogation interferometers, such as those based on Fabry-Pérot interferometers, high-sensitivity cells based on nanostructured structures, nanopillar networks, and high-sensitivity cells based on resonant nanopillars, among others (e.g., 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 antibody biosensing, Biosensors and Bioelectronics 26(2011)4842-4847; or Resonant nanopillar arrays for label-free biosensing, Optics Letter, 41, 23, 2016). Similarly, in-plane optical interrogation interferometers can be used, such as Mach-Zehnder interferometers, resonator rings, bimodal guides, grating guides, and resonator disks, as described in the following references: G. Zanchetta et al., 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). Due to its simplicity, the use of a Fabry-Perot interferometer for perpendicular interrogation is preferred.

[0029] Step c) of this method is carried out at a temperature between 0°C and 40°C, preferably between 2°C and 37°C. °The procedure can be carried out at temperatures between 2°C and 37°C, and even more preferably between 18°C ​​and 37°C when working with continuous flow systems without evaporation. Nevertheless, when working with sample droplets, and depending on the volume, it is possible to work at temperatures between 2°C and 37°C (incubation process) using an incubator to avoid evaporation and promote the immunoreaction, shortening the incubation time between the conjugate and the detection surface of the biosensor. On the other hand, lowering the temperature can reduce the evaporation coefficient and eliminate the need for an incubator. Depending on the option chosen, the incubation time can range from a few minutes to several hours, depending on the binding constant, the kinetics of the recognition reaction, and the affinity of the bioreceptor used.

[0030] The sensing surface of a transducer or optical sensor as referred to in the present invention can be functionalized to have an MO or at least one BR1 that recognizes an MO immobilized on the surface of the sensor. In addition, the surface of the sensor may or may not contain one or more blocking agents (AB) on gaps that may not be covered by either the MO or BR1 to prevent non-specific responses from other components that may be present in the sample to be analyzed.

[0031] In embodiments in which the detection surface contains MOs, i.e., these molecules are immobilized or coated on the sensor surface, the maximum variation in the optical read signal is obtained by the mechanism described in the present invention when the analyzed sample does not contain MOs. This is because a large number of NP-BRs interact and are recognized on the sensor surface (see the scheme in the lower right part of Figure 3). Nevertheless, when the analyzed sample contains MOs, if the concentration of this analyte is sufficient to coat all NP-BR conjugates, the variation in the read signal is essentially zero, i.e., it remains unchanged before and after step d). In other words, the response curve of the system changes as follows: The read signal decreases as the concentration of MOs in the sample increases. Therefore, if the amount of MOs in the sample is equal to or greater than the amount required to completely coat the surface of the total amount of NP-BR used in the method described herein, and the resulting filter obtained in step c) contains only NP-BR-MO conjugates, the optical read signal will not be altered (see the diagram on the left of Figure 3). Quantification is performed 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 as a function of concentration in a certain tabular format. It should be noted here that the ratio of NPs to MOs present in the sample can be easily changed to adjust and adapt the dynamic range of the sensor.

[0032] On the other hand, in an embodiment in which the sensor surface is functionalized or coated by immobilizing at least one type of BR1 that specifically recognizes MOs, i.e., a biomolecule (BR1) that can specifically react with MOs, the change in the optical response of the sensor increases as the MO concentration increases in the analyzed sample (see the upper left part of Figure 4 ). However, if the sample does not contain the analyte or MO to be detected, the readout signal remains unchanged. This is because the NP-BR-MO conjugate is not formed (see the lower right part of Figure 4 ) and is not recognized by the BR1-coated surface. As in the previous case, quantification of MOs is performed by calibrating the sensor between the two situations illustrated in Figure 4 . For this purpose, a calibration curve is obtained in which the sensor's response signal is obtained as a function of concentration in a certain tabular format.

[0033] The specific bioreceptor (BR1) contained in the sensing surface of the interferometric optical sensor can be the same as or different from the bioreceptor (BR) contained in the functionalized nanoparticle (NP-BR).

[0034] Additionally, in other specific embodiments of the present invention, the described methods can be used to detect allergy-specific antibodies (IgE), denoted as MO in the present invention, of specific allergens in biological samples. In these embodiments, the BR immobilized on the surface of the NP (NP-BR) is a specific biological receptor (anti-IgE) that recognizes the allergy-specific antibody (i.e., the test substance or MO, in this case IgE) on the biological sample to be analyzed. In this complex case, the NP-BR conjugate (NP-anti-IgE) captures IgE in the patient's sample to form a NP-anti-IgE-IgE conjugate (denoted NP-BR-MO). In one embodiment of the present invention, the specific receptor (BR1) is an allergenic molecule (MA), and in the particular case of Prup3 in the assay shown in Figure 6 of this document, the detection surface is covered with MA, so that when an NP-BR-MO conjugate (i.e., NP-anti-IgE-IgE) is recognized on the sensor surface, the signal is zero if the patient does not have MA-specific IgE immobilized on the sensor. If the patient has allergy-specific antibodies, the NP-BR-MO conjugate (i.e., NP-anti-IgE-IgE) is recognized on the sensor surface, changing the optical readout signal as described in this invention. In summary, in these embodiments, if the patient from whom the analyzed sample was obtained has an allergy, the interference signal is modified by the presence of an NP-BR-MO conjugate, in which the MO is an allergy-specific antibody (IgE) against the allergenic molecule (MA). Thus, the method of the present invention represents a major advantage, as it allows for the in vitro detection of possible allergies in patients. This determination is currently carried out in vivo via a "prick test," in which an allergen molecule or extract is introduced into a patient's arm and it is observed whether the body reacts.

[0035] Thus, in the method of the invention, two mechanisms are involved: on the one hand, the change in wavelength, frequency or wavenumber interference of the interference profile when the optical path (refractive index in its real part per length that the light must travel) increases, and on the other hand, the possible decrease in amplitude due to light scattering of the corresponding NP complexes on the sensor surface due to the aforementioned scattering mechanisms and / or variations in the complex part of the refractive index.

[0036] The method of the present invention allows for quantitative analysis, ie the determination of the presence or absence of a target molecule (MO) as well as its quantification depending on the test carried out on the sample.

[0037] The nanoparticles used in the method of the present invention can be formed by any type of inert dielectric material, such as oxide or silicon, or by optically transparent materials, such as silica, titania, alumina, silicon, or nanoparticles of another nature, such as gold, aluminum, or silver, among others. In the latter case, the NPs may be transparent to light at certain wavelengths and / or may cause more or less dispersion or variation in the extinction coefficient or the complex part of the refractive index. Thus, the 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).

[0038] 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.

[0039] Therefore, in a preferred embodiment of the method of the invention, when the optical sensor used is a Fabry-Perot interferometer, it is preferred to use a Fabry-Perot interferometer with a diameter between 50 nm and 100 nm or another form with a diameter of the same order of magnitude as the previous one, preferably per microliter (10 8 ~10 12The use of spherical silica NPs (SiO2) with a nanoparticle concentration of (1E8-1E12) is preferred. This concentration can vary mainly depending on the sensing area of ​​the sensor, incubation time, temperature, and volume, but the reaction kinetics determine not only the binding time but also their diffusion and sedimentation, in which the NPs play a relevant role. The higher the concentration of NPs, the shorter the time required for them to reach the surface and cover the sensing area of ​​the sensor. As shown in Figure 5, when the NP diameter is 200 nm, the response (□ of the Fabry-Perot sensor used) is 100 s. IROP The signal) does not decrease monotonically and can only be used from a coverage of 50% or more of the sensor's response. In this case, the NPs are considered untuned and the response of the biosensor system is not improved. However, if the NPs are spherical with a diameter of 50 nm or 100 nm, the response signal decreases monotonically and the sensor signal (□ of this used sensor) can be used. IROP It is observed that the signal decreases approximately linearly with the area covered in the sensor. In this case, thanks to the method of the present invention, the biosensor system was tuned and the sensitivity improved, since the slope of the response curve is higher than that reported in previous studies.

[0040] In another preferred embodiment of the method of the invention, when the optical sensor used is a Fabry-Perot interferometer, preference is given to using gold (Au) NPs with a diameter of 20 nm to 70 nm, preferably 45 nm to 55 nm, which size makes the NPs transparent to the wavelength or optical interrogation spectral range (Modelling the optical response of gold nanoparticles, Chem. Soc. Rev., 2008, 37, 1792-1805) and preferably 10 8 or 10 11 The concentration of NPs / mL. This concentration can vary mainly depending on the sensor's sensing area, incubation time, temperature, and volume, but the reaction kinetics determine not only the binding time but also the diffusion and sedimentation of NPs, which play a relevant role. The higher the concentration of NPs, the shorter the time required for them to reach the surface and be able to cover the sensor's sensing area.

[0041] The relevance of the present invention is that the change in refractive index is generated by biological material immobilized or recognized on the NPs, since it acts as a vehicle and is said to change the refractive index of the transducer and therefore the interference response. This interference response is read by an intensity change in a wavelength or spectral range, with the additional effect of dispersion depending on the size of the NPs containing the corresponding biological material.

[0042] The method of the present invention allows for great versatility, as it allows for the selection of nanoparticle (NP) size and material, which allows for tuning with the transducer's interference response. Through this tuning process, the interference response of the selected transducer, preferably a Fabry-Perot interferometer, can be amplified, while taking into account the reduction in signal amplitude due to the dispersion of the NPs recognized in the sensor. In addition, the method of the present invention also allows for the application of design criteria to determine amplification factors, establish quantitative detection ranges, significantly improve sensitivity, reduce measurement uncertainty, and dramatically improve detection limits. In addition, this method also allows for the modification of the detection signal readout system, thereby reducing measurement uncertainty.

[0043] The detection method described in this patent application therefore achieves the following advantages:

[0044] The optical method described herein detects both low- and high-molecular-weight molecules because it is independent of the molecular weight of the target molecule (MO) being detected, providing an unresolved technical solution to a common problem. More specifically, in the optical method described herein, detection relies on the conjugate (NP-BR or NP-BR-MO) recognized on the sensor surface. Therefore, unlike conventional methods in which the mass on the detection surface changes little when the MO has a very low molecular weight, in the method of the present invention, the mass on the detection surface depends primarily on the nanoparticles (NPs) and bioreceptors (BRs) and is less dependent on the molecular weight of the target molecule (MO). This is a significant advantage over other methods using interferometric sensors, in which the signal and sensitivity are highly dependent on the molecular weight of the target molecule.

[0045] This allows working with real samples, i.e., samples that have not been previously processed (especially biological samples). The problems of matrix effects and nonspecific adsorption are significantly reduced or eliminated. This is because the method involves separating the analyte from the rest of the sample, which advantageously occurs outside the sensor, so that a clean sample is deposited on the detection surface of the biosensor. In this way, in addition to avoiding complex cleaning processes directly on the sensor to eliminate matrix effects or nonspecific adsorption, the need for complex blocking systems on the sensor surface itself is also eliminated. It should be noted here that the blocking (adhesion) and marking processes in optical systems that function without optical marking are one of the main reasons why sensors or diagnostic systems have not been widely commercialized in medical or intensive care settings, a term often used in English to refer to "point-of-care" (i.e., testing at the patient's door).

[0046] By selecting the amount, size, and material of the NPs, it is possible to optimize the response curve, expressed as the measured signal as a function of contraction, to adapt the response to the range of concentrations to be detected, i.e., the dynamic range. In this way, it is possible to optimize the desired concentration range for each type of transducer that functions with the selected interference, based on the selected biological application.

[0047] This method dramatically improves the sensitivity and source detection limit of the interferometric transducer used. Therefore, this method allows for the detection of even the smallest concentrations of target molecules (analyte) due to the increased sensitivity of the transduction process. Additionally, by selecting the amount, size, and material of the NPs, the detection limit can be significantly improved. This is because while signal amplification depends on the NPs used, the uncertainty of the system reading does not. Consequently, by increasing the aforementioned sensitivity, the sensitivity / uncertainty ratio of the reading corresponding to the detection limit can be improved for any reading system.

[0048] In conclusion, the present invention solves a key problem of optical biosensors that function by interference. I. With the present invention, the sensitivity of the detection system is no longer dependent on the molecular weight of the target molecule to be detected or recognized. II. Its sensitivity and therefore detection limit are significantly improved, allowing molecules with very small molecular weights to be detected at trace concentrations. III. By reducing or eliminating non-specific signals (also called background signals in English), real samples (e.g., biological samples with complex matrices) can be effectively measured, and therefore can be measured. IV. Additionally, the response curve can be fitted to the range of concentrations to be measured.

[0049] Therefore, the method of the present invention can be used to develop non-invasive on-site diagnostic systems by capturing biomarkers present in different samples, preferably biological samples. For clinical diagnosis, biological liquid samples in which these biomarkers can be found may include, inter alia, blood, serum, urine, tears, especially cerebrospinal fluid, water, saliva, etc. Similarly, the diagnostic method can be used in multiple fields where in vitro (e.g., antibody-antigen affinity reaction) bioreceptor-analyte recognition detection is performed.

[0050] Therefore, the present invention also provides an immunologically based in vitro diagnostic method by using photonic transducers based on optical interference (interferometric transducers), which contain on their detection surface: i) the target molecule, or ii) its minus one specific bioreceptor (BR1), and a readout system capable of monitoring the relative fluctuations of the immobilization and / or recognition events, which are greatly amplified by using the above-mentioned NP-BR and NP-BR-MO conjugates.

[0051] In a particular embodiment of the invention, the sample to be analyzed is a biological sample, whether human or animal, and the target molecule to be detected is, for example, a biomarker allowing the in vitro diagnosis of a disease.

[0052] In one preferred embodiment, the optical detection method of the present invention can be used for the in vitro diagnosis of food allergies. The method would consist of detecting allergy-specific immunoglobulins (IgE) in actual patient samples. In this case, the target molecules (MOs) to be detected are IgE specific to the allergen to which the patient is allergic. For this purpose, several food allergen molecules are immobilized in different wells of a bio-kit, for example, using several Fabry-Perot interferometers (e.g., Towards reliable optical label-free point-of-care (PoC) biosensing devices, Sensors and Actuators B 236 (2016) 765-772). NPs are biofunctionalized with bioreceptors (BRs) consisting of IgG immunoglobulins that specifically recognize all existing IgG in the patient sample (serum from a patient with a food allergy). To identify which IgE molecules a patient is allergic to, samples containing various IgE-containing NP conjugates are obtained after incubation and filtering of the patient's sample. The samples are deposited and incubated in various wells containing the allergen molecules to be diagnosed. As a result, specific IgE-containing NP conjugates bind to the corresponding allergens and are then detected by reading the optical signal due to the change in interference generated.

[0053] Therefore, the detection method described herein must additionally include a preliminary step of functionalizing the nanoparticles with at least one specific bioreceptor, resulting in the formation of functionalized nanoparticles (NP-BR). To perform this functionalization, any anchoring method known to those skilled in the art (covalent bonding, direct adsorption or any other biofunctionalization route) can be used.

[0054] In these embodiments where the NPs are silica, the preferred functionalization method is silanization to attach anchor molecules that target antibodies for their nonspecific moieties. On the other hand, when this method is used with gold NPs, it is preferable to use thiols as anchor elements. The mechanisms of biofunctionalization of surfaces have been widely reported in the scientific literature (e.g., Bioconjugate Techniques, Greg T. Hermanson, Second Edition, 2008, ISBN 978-0-12-370501-3).

[0055] As a specific example, biofunctionalization of silica NPs with specific bioreceptors of target molecules can be carried out from a monodisperse solution of nanospheres bearing amino groups on their surface. The carboxyl groups present on the antibody are activated and stabilized by the addition of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbomidine hydrochloride) and NHS (N-hydroxysuccinimide), which cause spontaneous reaction with the primary amines present on the surface of the nanospheres, resulting in the formation of stable NP-BR conjugates by the formation of covalent amide bonds.

[0056] Similarly, the optical detection method of the present invention also requires biofunctionalization of the optical sensor surface, as described in step d) of the recognition step, where NP-BR-MO and / or NP-BR are in contact with the detection surface. Biofunctionalization of the detection surface of the transducer has been widely described in the literature by various routes, including covalent or adsorptive anchors, with or without orientation, and using various blocking agents. Throughout this invention, several references are reported in which these biofunctionalization routes are described, but these are not the subject of this invention. [Brief explanation of the drawings]

[0057] [Figure 1]Figure 1A shows the optical signal from an interferometric transducer (e.g., Fabry-Perot vertical interrogation) with immobilized specific bioreceptors. Figures B1a-B3a are schematic diagrams of the conventional operation process. Figures B1b-B3b illustrate the process described in the present invention in which NP conjugates are used. Figure C1 shows a schematic of the detection of a target molecule in a sensor by interference, where the spectral response profile is modified upon recognition of the target molecule. Figure C2 shows what happens when using the method described in the present invention, where the change in the spectral optical signal is significantly amplified and can also lead to a modification of the aforementioned amplitude of the optical signal; therefore, both effects further significantly improve the capacity of the detection system. [Figure 2] 1 is a schematic diagram of different types of conjugates formed with nanoparticles (NP) in the method of the present invention, i.e., functionalized nanoparticles (NP-BR) and conjugates (NP-BR-MO) formed by these functionalized nanoparticles and targeting molecules. In the specific embodiment depicted in this figure, both conjugates contain a blocking agent (AB), although the method of the present invention could also be alternatively carried out in the case where the NP-BR and NP-BR-MO do not contain a blocking agent. [Figure 3]This figure shows the detection mechanism when MOs are immobilized on the sensor surface. More specifically, the left side of Figure 3A shows the results of incubating NP-BR (step b) with a sample and filtering (step c) to obtain NP-BR conjugates when the biological sample did not contain MOs, and NP-BR-MO when the biological sample contained MOs. This illustrates the extreme case in which all NP-BR present in the reaction medium captures all MOs present in the sample. Figure 3B shows the recognition steps (steps d and e). When the sample does not contain MOs, the NP-BR conjugates are recognized on the sensor surface, resulting in a maximum signal (see the bottom of Figure 6C), i.e., the sensor's interference signal changes significantly. When the sample contains MOs, the NP-BR-MO conjugates are not recognized on the sensor surface because the MOs are also immobilized (see the top of Figure 6C). [Figure 4] This diagram first illustrates the recognition and filtering mechanism (Figure 4), and then the detection mechanism (Figure 4B) when the immobilized substance is a specific receptor (BR) for the target molecule (MO) in the sensor. Unlike the method described in Figure 3, a sample containing NP-BR-MO conjugates is recognized on the sensor surface, resulting in a significant change in the signal due to optical interference (see the top of Figure 4C). However, if the sample does not contain MO, the NP-BR conjugates are not recognized on the sensor surface (see the bottom of Figure 4C), resulting in virtually no change in the interference signal. [Figure 5]Figure 1 shows an example of NP tuning in a biosensor system. In this case, a Fabry-Perot interferometric sensor was used as the readout mechanism based on the Increased Relative Optical Power (IROP (%)) as described in Towards reliable optical label-free point-of-care (PoC) biosensing devices, Sensors and Actuators B 236 (2016) 765-772. The response curve simulation shows the readout signal as a function of the percentage of NPs recognized on the surface. For NPs between 50 nm and 100 nm, the response curve decreases monotonically, indicating validity for use as a sensor, whereas for NPs 200 nm, the response curve is invalid. [Figure 6]Figure 6A shows the signal evolution of real serum samples containing high concentrations of IgE specific for Pru ​​p 3 in 14 wells (P1 to P14). Each well contains a Fabry-Perot sensor. Figure 6B shows scanning electron microscope (SEM) images, showing one image without NPs on the surface and another with NPs on the surface. This is the case when an increase in signal is observed, thus proving the recognition of the NP-IgG-IgE-Pru p 3 conjugate. It should be noted that this figure shows the signal readouts of 14 wells (P1 to P14) of the diagnostic kit for the serum matrix (expressed in this case as □IROP%) obtained in Example 1 (see below). The lowest signal of these readouts is associated with the immobilization of the bioreceptor (BR1), whereas the higher signals indicate the specific detection of the target molecule (MO), in this case, specific IgE for the allergen Pru p 3. As can be seen from this graph, the silica NPs used are functionalized with bioreceptors (BR) consisting of immunoglobulin G (IgG) that specifically recognize all immunoglobulin E (IgE) present in the biological sample; that is, the NPs are functionalized with antibodies to IgE (IgG that capture IgE). The sensor surface is functionalized with bioreceptors (BR1). In this case, they are molecules of a specific type of allergen (Pru p 3), and only IgE specific to Pru p 3 (the target molecule in this case) is recognized on the sensor surface. Therefore, when this occurs, the optical readout signal changes. [Figure 7]Figure 7A shows SEM images (Images 7A-3 and 7A-4) of the conjugate recognized on the sensor surface, in contrast to Images 7A-1 and 7A-2, in which a limited number of NPs were recognized. In this case, the method used gold NPs biofunctionalized with a bioreceptor (BR) consisting of immunoglobulin G, which specifically recognizes the target molecule (MO), the metalloproteinase MMP9. In this case, the non-biological sample did not contain MMP9, so the NP-anti-MMP9 conjugate was recognized by the sensor, resulting in a significant increase in signal (Figure 7B). Furthermore, such a high signal is confirmed by microscopy (Images 7A-3 and 7A-4 in Figure 7A). As an additional test, another MO (cystatin CST4) was immobilized on another sensor. When the sample was contacted with this other sensor, virtually no recognition of the NP-anti-MMP9 conjugate containing CST4 was observed, as evidenced by the limited number of NPs observed microscopically (images 7A-1 and 7A-2 in Figure 7A) and the lowest measured signal (Figure 7B). [Figure 8] In this figure, PMMA NPs are observed on the surface of the sensor, through which experimental tests are performed to prove that light scattering is also a phenomenon that can use and / or add interference changes depending on the size of the NPs. In this case, it can be observed how the interference profile is reduced in signal amplitude by the PMMA NPs and how this can be used as a detection mechanism. DETAILED DESCRIPTION OF THE INVENTION

[0058] (Example) In order to contribute to a better understanding of the present invention and in accordance with its practical implementation, a series of examples of preferred embodiments of the present invention are attached as an integral part of this specification.

[0059] Example 1: Detection of food allergy-specific IgE using silica nanoparticles In this case, the NP is functionalized by immobilizing a specific receptor for IgE allergy-specific antibodies (NP-anti-IgE). It should be noted that anti-IgE is an IgG that recognizes and captures all IgE present in the corresponding biological sample and on the surface. One specific allergen molecule is immobilized from the sensor, and we want to know whether the allergy-specific antibody is associated with that molecule, thereby detecting whether the patient has allergy-specific antibodies (IgE) against the Pru p 3 molecule. Measurements are performed on actual serum samples. In this case, an NP-anti-IgE-IgE conjugate is formed, with the specificity that the IgE may or may not be specific for the allergen molecule. The allergen molecule is coated within the sensor (in this assay, Pru p 3) and is recognized as BR1. Therefore, an increase in the sensor signal indicates that the conjugate is recognized on the surface and that the sample is derived from a patient with a specific concentration of allergy-specific antibodies against the Pru p 3 molecule. Conversely, if the sensor signal does not change, the sample does not contain allergy-specific antibodies against Pru p 3.

[0060] Allergen molecules (Pru p3) were immobilized on the sensing surface of the Fabry-Perot interferometer sensor at a concentration of 5 μg / mL.

[0061] On the other hand, antibodies (anti-IgE) that specifically recognize all IgE in NPs were immobilized on silica NPs (NP-anti-IgE conjugates) with diameters of 50 nm to 100 nm.

[0062] A sample was collected from the patient, centrifuged to obtain serum, and then mixed with serum from a patient in which allergy-specific antibodies (IgE) were detected. Functionalized silica NPs were added to the sample at 2.5 x 10 10 The NPs were added at a concentration of 1000 NPs / L and incubated for 30 minutes in an incubator, allowing the NP-anti-IgE conjugate to recognize the expected IgE present in the patient sample. ° It was abandoned at C.

[0063] Once the incubation process was completed, a centrifugation process was performed to precipitate the NP conjugate into the Eppendorf tube. The supernatant was removed to obtain the NP conjugate that recognized the patient's IgE. This centrifugation process was repeated three times.

[0064] Once the sample containing the NP-anti-IgE-IgE (specific) conjugate was filtered, it was introduced to a sensor pre-functionalized with the allergen Pru p 3, 37 ° C for 30 min. After the recognition step, if the NP-anti-IgE-IgE conjugate is specific for the allergen molecule, the conjugate will remain specifically recognized on the sensor surface, resulting in a change in the readout signal.

[0065] Figures 6A and 6B show the results obtained in this test. In particular, Figure 6A shows the immobilization bar of the Pru p 3 allergen and the signal increase in well 14 (□ when the NP-anti-IgE-IgE conjugate specific for Pru ​​p 3 in the biological sample was recognized on the sensor surface). IROP In this case, the initial signal corresponding to the immobilization of Pru p 3, IROP In contrast to the signal of 200 (%), IROP This signal is supported by Figure 6B, where the NP conjugate-anti-IgE-IgE Pru p3 A scanning electron microscope (English acronym SEM) image is shown demonstrating the recognition of

[0066] It is important to note here that this sensor works by the light intensity 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 on the signal deviation due to both the interference amplified by the increase in material in the sensor, as well as the loss of light intensity due to the optical scattering of the recognized NPs on the surface.

[0067] Example 2: Detection of metalloproteinase MMP9 (a marker of inflammation) To carry out this experimental test, an MMP9-specific antibody (anti-MMP9) was immobilized on the surface of gold NPs (NP-anti-MMP9 conjugates). In this case, 50 nm gold NPs were chosen because they are transparent to the selected wavelength range around 850 nm (Modeling the optical response of gold nanoparticles, Chem. Soc. Rev., 2008, 37, 1792-1805).

[0068] On the other hand, the Fabry-Perot interferometer type sensor was coated with MMP9 and CST4 at sufficient concentrations on the corresponding sensor surface, resulting in a high coverage of the sensor surface. Specifically, both MMP9 protein (an inflammation marker) and CST4 protein were coated on the surface of the Fabry-Perot type sensor at 10 μg / ml. -1 Thus, two biosensors were obtained: one immobilized a protein related to the anti-MMP9 (target molecule) and the other immobilized a protein unrelated to the MMP9 antibody.

[0069] 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; doi:10.3390 / s17051158) was used to fabricate the interferometer, except that in this case, nitrocellulose was not used for anchoring.

[0070] This analysis was performed directly using the NP-anti-MMP9 conjugate, which must recognize MMP9 as a bioreceptor and must not recognize CST4 as a receptor. To do this, a sample containing the NP-anti-MMP9 conjugate was incubated on the aforementioned detection surface at 37°C for 2 hours. Therefore, if the signal is high, the NP-anti-MMP9 conjugate is recognized by the surface, whereas if the signal is low, the NP-anti-MMP9 conjugate is not recognized by the detection surface and the signal should be low.

[0071] Figure 7 shows the results obtained in this study. In the electron microscope image (see Figure 7A), it is possible to see how the NP-anti-MMP9 conjugate is recognized by the surface (images 7A-3 and 7A-4, thus recognizing the target molecule (MO)). This is further supported by the signals detected by the sensor in Figure 7B (signals 7A-3 and 7A-4). These signals are very high when the aforementioned NP-anti-MMP9 conjugate is recognized. However, results obtained when an unrelated protein (in this case, the aforementioned CST4) is immobilized show that the signal is much lower due to the limited number of NP-anti-MMP9 conjugates. See Figure 7A, images 7A-1 and 7A-2, and signals 7A-1 and 7A-2 in Figure 7B, respectively.

Claims

1. 1. An optical method for detecting at least one target molecule in a sample, comprising: Step a) measuring the interferometric response of an optical sensor or interferometric transducer with a bio-functionalized detection surface (FIG. 1A) for the following i and ii: i. the target molecule (MO) of interest, derived from a sample selected from the group consisting of a biological, clinical, agricultural food sample, and water; or ii. Measuring at least one specific bioreceptor (BR or BR1) of the target molecule; Step b) contacting, in a liquid medium, the sample to be analyzed with nanoparticles (NP) functionalized with at least one specific bioreceptor (NP-BR) of said target molecules (MO) to form conjugates (NP-BR-MO) comprising said functionalized nanoparticles and their target molecules present in said sample; Step c) separating the NP-BR conjugate from the sample, wherein the sample contains the target molecule, the NP-BR-MO conjugate formed in the mixture obtained after step b); step d) contacting the NP-BR and, if applicable, the NP-BR-MO conjugate obtained in step b) with the biofunctionalized sensing surface of an interferometric transducer; step e) measuring intensity variations caused by variations in the real part of the refractive index and / or dispersion or variations in the complex part of the refractive index of the NP-BR conjugate, and, if applicable, the NP-BR-MO conjugate, or a combination thereof, on the detection surface of the interferometric transducer to determine an optical reading.

2. 2. The optical detection method according to claim 1, wherein the target molecule is a specific IgE allergy antibody specific for an allergen molecule (MA).

3. 3. The optical detection method according to claim 2, wherein BR1 is the allergen molecule (MA) specific to an allergy-specific antibody, and the detection surface is functionalized with the allergen molecule (MA).

4. 4. The optical detection method according to claim 1, wherein the clinical sample to be analyzed is selected from the group consisting of blood, serum, plasma, saliva, tears, and urine.

5. The optical detection method according to any one of claims 1 to 4, wherein the sample to be analyzed is a clinical sample and the target molecule is a biomarker for in vitro diagnosis.

6. 6. The optical detection method of claim 5, wherein the biomarker is selected from the group consisting of a protein, a hormone, an immunoglobulin, a toxin, or any molecule recognized by an immunological process.

7. 7. The optical detection method according to any one of claims 1 to 6, wherein steps a) and c) are carried out at a temperature between 0°C and 40°C, and the temperature conditions of these steps are the same or different from each other.

8. 8. The optical detection method according to any one of claims 1 to 7, wherein the separation step b) is carried out by a technique selected from the group consisting of centrifugation, electric field separation, magnetic field separation, and combinations thereof.

9. 9. The optical detection method according to any one of claims 1 to 8, 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.

10. 10. The optical detection method according to any one of claims 1 to 9, 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 100 nm.

11. The concentration of the NP is 10 ８ NP / μL to 10 １２ The optical detection method of claim 10, wherein the concentration is between NP / μL.

12. 10. The optical detection method according to any one of claims 1 to 9, wherein the optical sensor is a Fabry-Perot interferometer and the functionalized nanoparticles (NP-BR) comprise gold nanoparticles having a diameter of 20 nm to 70 nm.

13. The concentration of the NP is 10 ８ NP / mL to 10 １１ The optical detection method according to claim 12, wherein the concentration is NP / mL.

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