OPTICAL METHOD FOR DETECTING A TARGET MOLECULE BY MEANS OF THE AMPLIFICATION IN THE INTERFERENCE RESPONSE, RESULTING FROM THE REFRACTIVE INDEX AND DISPERSION

Abstract

The invention relates to an optical method for detecting at least one target molecule (TM) contained in a sample at a determined concentration, which comprises: (a) bringing a sample containing the TM into contact, in a liquid medium, with a solution containing nanoparticles (NPs), the surface of the NPs having been coated or functionalised with at least one type of specific bioreceptor (BR) of the target molecule to be detected (NP-BR), such that the BRs specifically recognise the TM, thus forming conjugates of the NP-BRs with the TMs (NP-BR-TMs); (b) separating the nanoparticles conjugates (NP-BR-TMs and/or NP-BRs) formed in the previous step; (c) bringing the nanoparticles conjugates (NP-BR-TMs and/or NP-BRs) into contact with a sensor surface of an optical transducer that operates by means of reflection and/or transmission, the response of which is based on optical interference, the sensor surface being functionalised by immobilising thereon: (i) the target molecule (TM) or (ii) at least one specific bioreceptor of the target molecule, which may be of the same type (BR) or of another type (BR1); and (d) determining the optical reading on the sensor surface by means of change in the interference response of the optical transducer, caused by change in the real part of the refractive index as a result of the NP conjugates recognised on the sensor surface, and/or by means of change in intensity in the interference response, caused by variation in intensity as a result of dispersion or as a result of variation in the complex part of the refractive index of the NP conjugates, or by means of a combination of both effects amplification in the interference response by refractive index and scattering.

Claims

1. An optical method for the detection of at least one target molecule in a sample, the method comprising the steps of: a. measuring the interference response of an optical sensor or interferometric transducer with its biofunctionalized sensor surface with: i. the target molecule (TM) to be analyzed comes from a sample selected from a group consisting of a biological sample, a clinical sample, an agri-food sample and water ii. or at least one specific bioreceptor (BR or BR1) of the target molecule; b. putting in contact, in a liquid medium, a sample to be analyzed with functionalized nanoparticles (NPs) with at least one specific bioreceptor (NP-BR) of the target molecule (TM), forming a conjugate (NP-BR-TM) with the functionalized nanoparticles and the target molecules present in the sample; c. separating NP-BR conjugates from the sample and, provided the sample comprises the target molecule, NP-BR-TM conjugates formed in the mixture obtained after step b); d. putting in contact said NP-BR, and if applicable, the NP-BR-TM conjugates obtained in step b) with said biofunctionalized sensor surface of the interferometric transducer; and e. determining the optical reading, measuring the variation of the actual part of the refractive index and/or the variation of intensity caused by the scattering or variation in the complex part of the refractive index of the NP-BR conjugates, and if applicable, the NP-BR-TM conjugates, or a combination of the foregoing, on the sensor surface of the interferometric transducer.

2. The optical detection method according to claim 1, wherein the target molecule is an IgE allergy specific antibody specific to an allergenic molecule (AM).

3. The optical detection method according to claim 2, wherein the BR1 is the allergenic molecule (AM) specific for an allergy specific antibody and the sensor surface is functionalized with said allergenic molecule (AM).

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 claim 1, wherein the sample to be analyzed is a clinical sample and the target molecule is a biomarker for in vitro diagnosis.

6. The optical detection method of claim 5, wherein the biomarker is selected from the group consisting of proteins, hormones, immunoglobulins, toxins, or any molecule that is recognized by immunological processes.

7. The optical detection method according to claim 1, wherein steps a) and c) take place at a temperature between 0 and 40° C., the temperature conditions of these steps being equal or different from each other.

8. The optical detection method according to claim 1, wherein step b) of separation takes place by a technique selected from the group consisting of centrifuging, electric field separation, magnetic field separation, and a combination of the foregoing.

9. The optical detection method according to claim 1, 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. The optical detection method according to claim 1, 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 optical detection method according to claim 10, wherein the concentration of NPs is between 10.sup.8 to 10.sup.1 NPs/μL.

12. The optical detection method according to claim 1, wherein 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.

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

Description

BRIEF DESCRIPTION OF THE FIGURES

[0071] FIGS. 1A-1C: FIG. 1A illustrates an optical signal from an interferometric transducer (e.g. Fabry-Perot type with Vertical Optical Interrogation) in which specific bioreceptors have been immobilized. FIGS. B1a-B3a are a schematic representation of the traditional operation process, while figures B1b-B3b refer to the process described in the present invention in which NPs conjugates are used. FIG. C1 shows schematically the detection of a target molecule in an interference sensor, where the profile of the spectral response is modified by the recognition of a target molecule, while FIG. C2 shows how the detection would be when using what is described in the present invention, where the change of the spectral optical signal is significantly amplified, and also can lead to a modification in the aforementioned amplitude of the said optical signal, so that both effects significantly improve the detection capacity of the system.

[0072] FIG. 2: It is a schematic representation of the different types of conjugates formed with the nanoparticles (NP) in the method of the present invention, i.e., functionalized nanoparticles (NP-BR) and conjugate (NP-BR-TM) formed by these functionalized nanoparticles and the target molecule. In the particular embodiments depicted in this figure, both conjugates comprise a blocking agent (BA), however, the method of the present invention could also be selectively performed when the NP-BR and NP-BR-TM do not comprise any blocking agent.

[0073] FIG. 3: This figure shows the detection mechanism when the TM is immobilized on the sensor surface. More specifically, in the left part of FIG. 3A 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 TM and NP-BR-TMs in the case that if there were, the extreme case in which all the NP-BRs present in the reaction medium capture all the TM present in the sample is exemplified here. FIG. 3B depicts the recognition step (step d and step e), in the event that the sample did not contain TMs, NP-BRs conjugates would be recognized on the surface of the sensor and the signal would be maximum (see bottom of FIG. 6C), i.e. the sensor interference signal changes significantly. In the event that the sample did have the TM, then the NP-BR-TM conjugates would not be recognized on the surface of the sensor since it also has the TM immobilized (see the top of FIG. 6C)

[0074] FIG. 4: This figure first depicts the recognition and filtering mechanism (FIG. 4), then the detection mechanism when what is immobilized is a specific receptor (BR) of the target molecule (TM) in the sensor (FIG. 4B). Contrary to what was described in FIG. 3, the sample containing the NP-BR-TMs conjugates 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 upper part of FIG. 4C), while when the sample does not have the TM the NP-BRs conjugates will not be recognized on the surface of the sensor (see lower part of FIG. 4C) and therefore the interference signal will practically not be altered.

[0075] FIG. 5: Example of tuning of NPs on a biosensor system. In this case, a Fabry-Perot type interference sensor has been used as a reading mechanism based on 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 monotonically decreasing and valid to be used as a sensor, while for 200 nm the response curve would not be valid.

[0076] FIG. 6A: In FIG. 6A the change of the signal in a real sample of serum with high concentration of specific IgE to Pru p 3 in 14 wells (P1 to P14) can be seen. Each well contains a Fabry-Perot type sensor. In FIG. 6B, scanning electron microscopy (SEM) images can be seen, where an image is shown without NPs on the surface, and another with NPs on the surface. This is the case where the signal increase is observed and therefore the recognition of the NP-IgG-IgE-Pru p 3 conjugate is certified. It should be noted that in this figure the readings of the signals of a 14-well diagnostic kit (P1 to P14) in serum matrix (in this case expressed in Δl.sub.rop %) obtained in example 1 (see below) are shown. The lowest signal of these readings relates to the immobilization of the bioreceptor (BR1), while the high ones relate to the specific detection of the target molecule (TM), in this case a specific IgE of 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 limunoglobulins G (IgGs) that specifically recognize all the immunoglobulins E (IgEs) present in the biological sample, i.e. 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 the optical reading signal changes.

[0077] FIG. 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 (TM) that is the metalloproteinase MMP9. In this case the non-biological sample did not contain the MMP9 and therefore the NP-antiMMP9 conjugates are recognized in the sensor, significantly increasing their signal (FIG. 7B); in addition it is verified that the high signal is due to the NPs that are observed by microscopy (images 7A-3 and 7A-4 of FIG. 7A). As an additional test, another TM (cystatIn CST4) was immobilized on another sensor. When the sample was contacted with this other sensor, it could be observed that there was practically no recognition of the NP-antiMMP9 conjugates with the CST4, as can be seen by the limited number of NPs observed by microscopy (images 7A-1 and 7A-2 of FIG. 7A) and the smallest measured signal (FIG. 7B).

[0078] FIG. 8: In this figure PMMA NPs are observed on the surface of a sensor by means of which an experimental test is carried out to certify that, depending on the size of the NPs, the scattering of light is also a phenomenon that can be used and/or added to the change of interference. In this case, by means of PMMA NPs, it can be observed how the interference profile is reduced in signal amplitude and used as a detection mechanism.

EXAMPLES

[0079] In order to contribute to a better understanding of the invention, and in accordance with a practical embodiment thereof, several preferred embodiments of the present invention are accompanied forming an integral part of this disclosure.

Example 1: Detection of Food Allergy Specific IgE Using Silica Nanoparticles

[0080] In this case, the NPs are functionalized by immobilizing a specific receptor of allergy specific antibodies IgE (NP-antiIgE), it is advisable to indicate that the anti-IgE are IgGs that recognize and capture all the existing IgEs in the corresponding biological sample, and on the surface of the sensor one of the specific allergenic molecules is immobilized for which it is desired to know if the specific allergy antibody that is related to said molecule, and therefore detect if the patient has allergy specific antibodies (IgEs) to the Pru p 3 molecule. Measurements are made on a sample of real serum. In this case, the NP-antiigE-IgE conjugate is formed, but with the peculiarity that the 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 noticed as BR1. Thus, if the sensor signal increases, it means that the conjugate is recognized on the surface and the sample comes from a patient who has a specific concentration of allergy-specific antibodies to the Pru p 3 molecule. On the contrary, if the sensor signal does not change, the sample does not contain specific antibodies for allergy to Pru p 3.

[0081] An allergenic molecule (Pru p3) was immobilized on the sensor surface of the Fabry-Perot Interferometer Sensor at a concentration of 5 μg/m L.

[0082] On the other hand, an antibody (anti-IgE) was immobilized that specifically recognizes all IgEs in silica NPs (NP-antiIgE conjugate) with a diameter between 50 nm and 100 nm.

[0083] A sample was taken 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 2.5×10.sup.10 NPs/ΔL and incubated for 30 min, i.e. left at 37° C. in an incubator for the NP-anti-IgE conjugate to recognize the possible IgEs present in the patient sample.

[0084] Once the incubation process was completed, the centrifugation process was carried out so that the NPs conjugates were sedimented in the Eppendorf flask. The supernatant was removed, thus obtaining the NPs conjugates that have recognized the patient's IgEs. This centrifugation process was repeated 3 times.

[0085] Once the sample containing the NP-antiIgE-IgE conjugates (specific) 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 step, if the NP-antiIgE-IgE conjugate is specific to the allergenic molecule, the conjugate is specifically recognized on the surface of the sensor and the reading signal changes.

[0086] FIGS. 6A and 6B show the results obtained in this assay. In particular, in FIG. 6A it is possible to observe the immobilization bars of the Pru p 3 allergen and the increase of signal in 14 wells (signals of 1100 of Δ I.sub.rop (%) when the NP-antiIgE-IgE conjugates specific to Pru p 3 of the biological sample is recognized on the surface of the sensor. In this case, signals of 1200 pp ΔI.sub.rop (%) are obtained, in contrast to the initial signal corresponding to the immobilization of the PRU p 3, signals of 200 Δ I.sub.rop (%)) This signal is corroborated with FIG. 6B, where scanning electron microscopy (SEM) images are shown that demonstrate the recognition of the NP-antiIgE-IgE Pru p 3 conjugate.

[0087] It is important to note here that this sensor works by 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 the light by the optical scattering of the NPs recognized on said surface.

Example 2: Detection of Metalloproteinase MMP9 (Inflammation Marker)

[0088] To perform this experimental test, the MMP9-specific antibody (antiMMP9) was immobilized on the surface of the gold NPs (NP-antiMMP9 conjugate). In this case, 50 nm gold NPs were chosen since they are transparent to the range of wavelengths chosen around 850 nm (Modelling the optical response of gold nanoparticles, Chem. Soc. Rev., 2008, 37, 1792-1805).

[0089] 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 immobilized at a concentration of 10 Δ g mL.sup.−1 on the surface of the Fabry Perot type sensor. Therefore, two biosensors are obtained, one in which a protein related to the antiMMP9 (the target molecule) has been immobilized and in another a protein not related to the MMP9 antibody.

[0090] In this case, to manufacture the interferometer, a pulmérica base 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; DOL: 10.3390/s17051158) was used, except that this case did not use nitrocellulose for anchoring.

[0091] The analysis was performed directly using NP-antiMMP9 conjugates that were meant to recognize MMP9 as bioreceptor and not to recognize CST4 as receptor. To do this, the sample containing the NP-antiMMP9 conjugates was incubated on the aforementioned 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, it is that the NP-antiMMP9 conjugates are not recognized on the sensor surface and the signal must be low.

[0092] FIG. 7 shows the results obtained in this test. It can be seen in the electron microscopy images (see FIG. 7A) how the NP-anti-MMP9 conjugate is recognized on the surface (images 7A-3 and 7A-4, and therefore the target molecule (TM) is recognized. In addition, this is corroborated by the signals detected on the sensors in FIG. 7B (signals 7A-3 and 7A-4) that are very high when the aforementioned NP-antiMMP9 conjugates have been recognized. However, the results found when immobilizing a protein that is not related (in this case the aforementioned CST4) indicate that the number of NP-antiMMP9 conjugates is limited, and therefore, the signal is much lower. See FIGS. 7A, images 7A-1 and 7A-2 and FIG. 7B, signals 7A-1 and 7A-2 respectively.