Patent Application
20230003739
Optical method for the detection of a target molecule by means of amplification in the interference response by refractive index and scattering.
The present invention belongs to the technical sector of immunological analysis and/or diagnosis using optical biosensors which work by optical interference of light, such as those described in the invention Optical detection system for labelling-free high-sensitivity bioassays (ES2574138 (T3)—2016 Jun. 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 herein relates to a method for the detection and/or quantification of a target molecule (TM), in particular an immunologically reactive molecule or one with bioreceptor-antigen affinity, wherein the method comprises enriching the target molecule (also referred to as target analyte) in a sample by using functionalized nanoparticles, and subsequently analyzing it by direct optical reading of the reflection and/or transmission of the system, the interference response of which will be affected by the conjugates of the nanoparticles specifically recognized on its surface due to variation of the real part of the refractive index and possible loss of intensity due to variation of the complex part of the refractive index and/or scattering of the aforementioned complexes in the reflected or directly transmitted light, without the need to use additional detection methods.
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 invention INTERFEROMETRIC DETECTION METHOD (EP2880396 (A1)—2015 Jun. 10) and subsequently reported for example in the scientific publications: 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 the diagnosis of diseases with an immunological basis in a reasonable time.
So far the most common high-sensitivity in-vitro detection systems available on the market for biomolecular analysis are based on chemical development and amplification, with the ELISA technique (Enzyme-Linked Immunosorbent Assay) being the most common and established on the market. There exist a wide variety of devices; to refer to a specific case, notable in the allergy sector are IMMUNOCAP, IMMUNOCAP ISAC, which can offer quantitative information and particularly a qualitative measurement (yes/no response) with respect to the presence of specific IgE allergy antibodies, which does not allow to define the sensitization profile resolved by components for each patient, necessary for complex cases. In addition, these devices offer only a limited range of food allergens that can be tested, using raw allergen extracts instead of individual allergens, which can lead to misleading information. Additionally, this type of devices require highly qualified facilities and personnel.
The number of bibliographic references relating 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 the change in the refractive index when biological material accumulates on a sensor surface. However, these devices have not yet been marketed in mass and, therefore, traditional methods based on chemical amplification or development remain the usual and commercially available ones, including both nitrocellulose strips based on lateral flow that work by colorimetry, as well as the aforementioned 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 with 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) present that can generate a high non-specific adsorption and to the lack of specificity. In addition, this problem is amplified and there is a limitation in the ability to detect molecules of very low molecular mass, whose impact on the increase in the refractive index on the sensor surface is much smaller, such that the sensitivity factor of the transducer must be much greater, while at the same time the requirements of error or measurement uncertainty in the reading of the device are more critical to reach the necessary detection limit, that can be estimated as the ratio between the measurement uncertainty of the reading and the sensitivity of the transducer. In short, these requirements mean that, in order to reach a detection limit in accordance with the needs, the system must be very competitive, the sensitivity of the transducer must be very high, the reading uncertainty very small and, additionally, the system must be able to specifically recognize the target biomolecules, including those of 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 non-specific detection that could invalidate the analysis.
In summary, these very strict requirements of specificity, sensitivity and detection limit pose a great difficulty when developing a method and/or system for analyzing molecules of an immunological nature, using optical biosensors based on the changes in interference due to the variation in the refractive index on the sensitive surface, in particular 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 a high non-specific adsorption, entails a very significant additional difficulty for competing with conventional chemical amplification and development systems such as those based on ELISA.
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 non-specific adsorption 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). Said specific surface is part of the transducer which, once functionalized, increases its volume and/or density due to the presence of the specific molecular receptors and blocking agents mentioned, turning the sensor into a biosensor. This increase in the amount of matter generates a change in the interference response that we call the reference signal (see
A technical problem of great difficulty is the specific recognition or detection of molecules in a real sample, especially when these target molecules have a small size. Likewise, the processes of receptor functionalization (biofunctionalization) are also highly difficult. However, despite the difficulties, a high number of examples for the anchoring of specific bioreceptors are reported in the literature, for example by direct adsorption using polymer base materials by direct anchoring of antibodies and peptides, or by using reactions such as streptavidin and biotin, or by using protein A and protein G to orient antibodies in the biofunctionalization process. Other alternatives for making covalent surface anchors 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.
In the case of the optical interference transducers object of this invention, the materials employed form a micro/nano photonic 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 anchoring and molecular recognition reactions, which makes the change the basis of the reading in the transduction of biochemical sensing (W. 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 used for this type of sensor are those based on Match Zehnder interferometers, resonator rings and discs, Fabry-Perot interferometers, bimodal guides, nano-resonator networks or resonant nano-pillar networks, etc., such as those available in G. Zanchetta et al. “Emerging applications of label-free optical biosensors”. Nanophotonics 2017; pages 1-19.
As mentioned above, the operating principle of an interference-based biosensor is the fact that when the bioreceptors are anchored to the surface of the interferometer, the interference signal changes, for example generating a change in the Interference signal caused by the sensor (
However, these changes in wavelength, frequency or wavenumber related to the concentration of the biomolecule or molecule to be detected are limited by the response of the photon structure used to manufacture the sensor producing the optical interference. In addition, the dynamic range of concentrations that the sensor can measure depends on the construction thereof, where the detection area of the sensor plays a fundamental role. It is an unquestionable fact that non-specific adsorption on the surface of the sensor of the multiple and diverse components found in a real sample represents a very relevant difficulty that sometimes requires the use of blocking agents, and is a fundamental aspect when working with real biological samples of various nature, such as: blood, serum, saliva, tears, urine, among others, given the high number of different biological components or agents that are found together with the specific molecule to be specifically detected. For this reason, when developing a biosensor specificity is one of the most critical aspects, the elimination of this specific adsorption or the aforementioned matrix effect being a key factor to allow working with real samples. In this sense, a good and efficient functionalization or upholstery of the surface of the sensor can reduce the aforementioned matrix effect, although in many cases it is necessary to use blocking agents of various types such as serum albumin, ethanolamine, among others.
The invention described herein resolves 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, an optical method is described herein of detecting a target molecule by amplifying the interference response by refractive index and variation of the light intensity of this response as a result of the change of the refractive index itself and by the possible scattering produced by the type and size of the conjugates based on the NPs used for the performance of the assay. This method can be used for the measurement of target molecules of very low molecular mass, allows working with real samples, that is, samples that have not been subjected to a pre-treatment, as the specificity is increased and, above all, allows modifying the dynamic range of response and the sensitivity to demand of the bio-amplification to be used.
The present invention provides an optical method for the detection of at least one target molecule based on biological interactions of said molecule. In particular, the method is based on the amplification in the interference response due to the variation in the refractive index generated by the NP conjugates used in the biological recognition process added to the possible further amplification caused by the phenomenon of scattering of said NP conjugates whose number on the sensor surface depends on the biological interactions of the target molecule in the different stages of the method. Accordingly, the method described herein allows qualitative and/or quantitative analysis of a target molecule in a real sample, in particular in a complex-matrix biological sample, improving the limit of detection 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 separation of the NPs conjugates from the real sample to avoid the multiple components of the biological sample at their origin.
The method of the present invention makes it possible to improve optical interference sensing compared to the usual methods reported in the literature for interference-based optical sensors or transducers, which herein is also referred to as 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, resonator rings and disks, Match Zehnder Interferometers, BICELLs or bimodal guides, among many others. Thus, these devices will be able to compete and improve the performance of classic detection systems by chemical amplification or development, generally based on fluorescence or colorimetry (eg. ELISA).
In particular, the present invention provides an optical method for the detection of at least one target molecule (TM, also referred to as analyte) in a sample, characterized in that the method comprises the following steps:
The aforementioned NP-based conjugates that are recognized on the sensor surface of the transducer will cause a change in the refractive index, mainly in its real part, which causes a change in the interference of the reflected or transmitted light. However, it may also occur 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 the reflected or transmitted light. In addition to this effect the scattering of the cited complexes of NPs can cause scattering of the light to a greater or lesser degree depending on the type and size of the NPs, and therefore, another change of the intensity of the received light, both in transmission and in reflection, which are added to the aforementioned effects.
The method of the present invention allows the detection of any TM, including those of very low molecular mass difficult to detect for optical biosensors, having at least one specific bioreceptor. Thus, this method can be used to detect any target molecule that may result in an immunological reaction or present bioreceptor-antigen affinity. In particular, the TM may 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 TM, in such a way that the NP-antiIgE conjugate (NP-BR in its initial notation in the present invention) that would recognize the IgE present in the biological sample forming the NP-antiIgE-IgE complex (NP-BR-TM in its initial notation in the present invention), and on the surface of the sensor at least one allergenic molecule (BR1) has been immobilized that is recognized by the specific IgE of the aforementioned antibody (BR1 in its initial notation in the present invention).
The optical detection method described herein can be used in any sector where specific detection of an in-vitro molecule is required. Therefore, application sectors of great relevance can be, in addition to the clinical, the agri-food sector, where the detection of diseases in animals is required, or the detection of pathogens in their food 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 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.
Thus, in the method described herein, the sample to be analyzed can be a biological sample such as, for example, blood, serum, urine or tears among others; an agro-food sample such as, that which is obtained directly from food, or from the environment where the animals are located; or a water sample, either for human, animal or any other use. As mentioned above, the method of the invention can be applied to a real sample, i.e. a sample taken directly, without any pre-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, the sample will be subjected to a previous step of dissolution in a liquid medium.
The functionalized NPs used in the method of the present invention are upholstered, i.e., functionalized on their surface, with at least one TM-specific BR, in particular a BR capable of specifically interacting with the TM (or analyte) by an immunological reaction, an antibody-antigen affinity reaction or a BR-TM reaction in general. Thus, on those occasions when the sample, preferably an untreated biological sample, comprises the TM, in step b) of the method of the present invention an affinity reaction takes place between these MOs and the NP-BR conjugates, resulting in the formation of NP-BR-MO conjugates. This step of the method can be performed at a temperature preferably between 2 and 37° C. Within this range, higher temperatures favor the immune reaction between the BR and the TM, requiring a shorter reaction time or incubation. Therefore, this time may vary, depending on the temperature in the incubation process, between minutes and hours, depending on the association constant and the kinetics of the recognition reaction and affinity of the bioreceptor used.
In general, an incubation time of 30 min is usually set, although depending on the biological application, temperature, and kinetics of the particular reaction, this time may typically range from less than 30 min to hours.
In step c) of the method described herein, the separation of the NP-BR and NP-BR-TM (also referred to, respectively, as NP-BR conjugates and NP-BR-TM conjugates herein) generated in the affinity reaction taking place in step a) takes place, provided that the sample analyzed comprises TMs. Both conjugates (NP-BRs and NP-BR-TMs) are present in the resulting mixture to a greater or lesser degree. For such separation, any physical separation mechanism of the aforementioned NPs conjugates from the liquid medium of the sample to be analyzed can be used, such as for example centrifugation, separation by electrical or magnetic field, among other possible mechanisms. In particular, these NP conjugates can be readily separated from the rest of the sample by filtration, by means of one or more centrifugation cycles.
In particular embodiments, the NPs used may be capable of being attracted or repelled in the presence of an electric field due to the surface charge. In this case, step b) of separating the NP-BR and NP-BR-TM conjugates from the rest of the sample can preferably be performed by electric field and electrophoresis.
In other particular embodiments, the NPs used may be attracted or repelled by a magnetic field. In this case step b) of separating the NP-BR and NP-BR-TM conjugates from the rest of the sample can preferably be performed by application of a magnetic field.
Additionally, the NPs used may exhibit the ability to be attracted or repelled in an electrical or magnetic field, e.g., silica NPs coated with a magnetic material. In these cases a mixed separation mechanism based on the application of electric field, magnetic field and, optionally, centrifugation can be used.
As mentioned above, in step d) of the optical method of detection (in vitro) of a target molecule in a sample, the NP-BRs and the NP-BR-TMs, if any, are put in contact because the recognition of TMs present in the sample has occurred, obtained in step c) with a sensor surface of a biofunctionalized optical transducer (upholstered with TM or BR1) whose response, in particular, an optical sensor whose response is based on interference, resonance that generates a certain photonic structure such as those cited in the bibliographic references mentioned herein. In particular, any optical sensor operating by optical interference whose response changes in the presence of the material recognized on its sensor surface (the aforementioned NP 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 changes due to the variation of the real part of the refractive index or optical thickness (referred to the refractive index of the NP conjugates multiplied by the average thickness increase of these NPs on the sensitive surface of the transducer), but also its amplitude (usually measured in optical intensity) can change as a function of the variation of the complex component of the refractive index and/or of the scattering caused by the NP conjugates recognized, and therefore located, on the surface of said sensor.
In particular embodiments, the sensor employed is an interferometric sensor that operates by measuring the variation of the interference, which is monitored by the change of light intensity in a given spectral range. To do this, said transducer is tuned so that the measurement is due to loss of light intensity (W. Holgado, et al. Sensors and Actuators B 236 (2016) 765-772).
Additionally, the NPs and the biological material accumulated or recognized on its surface increase the refractive index of the transducer, causing this response to be modified and, therefore, the intensity to change. In addition, the NPs linked to the biological material, can generate scattering losses in such a way that this phenomenon can be added to the loss of intensity generated by the change in interference.
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 types of sensors reported in the technical and scientific literature. When on the sensor surface of a photonic transducer a conjugate of NPs are recognized and, due to this recognition phenomenon, specifically adhere, the average refractive index of the transducer is increased. In the case of sensors based on interferometers based on thin sheets of the Fabry-Perot type, the refractive index product by the average thickness of NPs causes the interference response to change (e.g.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 the light sees when propagating in a certain waveguide. In both cases, as discussed, a change in the interference profile is generated, such as Label-free optical biosensing with slot-waveguides, Optics Letters, 33, 7, 2008. Moreover, the imaginary part of the refractive index generates signal losses that can be identified as a loss of amplitude observed by signal intensity reduction. Finally, and mainly, for vertical interrogation sensors, the scattering losses will also reduce the amplitude of the interference signal and, therefore, can be used as a detection mechanism.
Thus, the method described herein can be used primarily in vertical optical interrogation interferometers, such as those based on Fabry-Perot interferometers, sensitive cells based on nano-structured structures, sensitive cells based on nano-pillar networks, and resonant nano-pillars, among others (e.g. Label-free biosensing by means of periodic lattices of high aspect ratio SU-8 nano-pillars, 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; or Resonant nanopillars arrays for label-free biosensing, Optics Letter, 41, 23,2016). Likewise, optical interrogation in plane interferometers can 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 et al. “Emerging applications of label-free optical biosensors”. Nanophotonics Nanophotonics 2017; 6(4): 627-645. DOI 10.1515/nanoph-2016-0158; or M. C. Estevez, M. Alvarez, and L. M. Lechuga, Laser Photon. Rev. 6, 463 (2012). Due to its simplicity, the use of a Fabry-Perot vertical interrogation interferometer is preferred.
This step c) of the method can be performed 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 droplets and, depending on the volume, the work can be performed at temperatures between 2 to 37° C. (incubation process) using incubators to avoid evaporation and to favor 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 alleviate the evaporation factor, avoiding the need to use incubators. Depending on the option chosen, the incubation time may range from several minutes to hours, depending on the association constant and the kinetics of the recognition reaction and affinity of the bioreceptor used
The sensor surface of the optical transducer or sensor as cited in the present invention may be functionalized to have, immobilized on the surface of the sensor, the TM or, alternatively, at least one BR1 that also recognizes the TM. Additionally, the surface of said sensor may or may not comprise one or more blocking agents (BA) on the voids that would not have been covered, either by the TM or by the BR1, in order to prevent a non-specific response of other components that may be present in the sample to be analyzed.
In those embodiments in which the sensor surface comprises the TM, i.e., 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 analyzed sample does not contain the TM, since in this case the number of NP-BRs that interact and are recognized on the sensor surface is greater (see diagram in the lower right part of
On the other hand, in those embodiments in which the sensor surface is functionalized or upholstered by the immobilization of at least one type of BR1 that specifically recognizes the TM, that is, a biomolecule (BR1) capable of specifically reacting with the TM, the change of the optical response of the sensor will increase as the concentration of TM in the analyzed sample increases (see upper left of
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 on the functionalised nanoparticles (NP-BR).
Additionally, in other particular embodiments of the invention, the disclosed method can be used to detect allergy specific antibodies (Ig), noted as TMs 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 the allergy-specific antibodies (i.e., the analyte or TM, in this case the IgE) in the biological sample to be analyzed. In this complex case, the NP-BR conjugate (NP-antiIgE) captures the IgEs in the patient sample forming the NP-antiIgE-IgE conjugate (noted as NP-BR-TM). In one embodiment of the present invention, when the specific receptor (BR1) is an allergenic molecule (AM), and in a particular case, Pru p 3 in the assay depicted in
Thus, in the present method two mechanisms are involved. On the one hand, the change of the interference in wavelength, frequency or wave number of the interference profile when the optical path is increased (refractive index in its real part times the length that the light must pass through), and on the other hand the possible reduction of the amplitude by the scattering of the light of the corresponding NP complexes on the surface of the sensor due to the aforementioned mechanisms of scattering and/or variation of the complex part of the refractive index.
The method of the present invention allows quantitative analysis, i.e. determining the presence or not of a target molecule (TM), as well as its quantification depending on the assay to be performed on a sample.
The nanoparticles used in the method of the present invention may be formed of any type of dielectric inert material, such as oxides or silicon; any material transparent to light, for example, silica, titania, alumina or silicon; as well as nanoparticles of other nature such as gold, aluminum, silver, among others. In the latter case it occurs that at certain wavelengths the NPs can be transparent to light and/or generate more or less scattering or variation in the extinction coefficient or complex part of the refractive index. Thus, the nanoparticles may be selected from the group consisting of silica, gold, aluminum, silver, silicon, and metal oxides, in particular titanium oxide (titania) or aluminum oxide (alumina).
It should be noted that the size and concentration of NPs are calculated based on the surface of the sensor, the type of interference biosensor used and the dynamic range of the concentration to be detected.
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 shape with diameters of the same magnitude as the previous ones, preferably with a concentration of 108 to 1012 (1E8 to 1E12) nanoparticles per micron is preferred. This concentration can vary, mainly depending on the sensitive area of the sensor, incubation time, temperature and volume, since it is not only the kinetics of the reaction that determines the association time, as the diffusion and sedimentation of the NPs also play an important role. The higher the concentration of NPs, the less time it takes for them to reach the surface and be able to upholster the sensitive area of the sensor. As shown in
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 nm is preferred, sizes that make the NPs transparent to the wavelength or spectral range of optical interrogation (Modelling the optical response of gold nanoparticles, Chem. Soc. Rev., 2008, 37, 1792-1805) and, preferably, a concentration of 108 or 1011 NPs/mL. This concentration can vary, mainly depending on the sensitive area of the sensor, incubation time, temperature and volume, since it is not only the kinetics of the reaction that determines the association time, as the diffusion and sedimentation of the NPs also play an important role. The higher the concentration of NPs, the less time it takes for them to reach the surface and be able to upholster the sensitive area of the sensor.
The relevance of 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, its interference response. This interference response is read by the variation of intensity of a wavelength or a spectral range and, depending on the size of the NP with its corresponding biological material, the effect of scattering is added
The method of the present invention allows great versatility, since it is possible to select the size and material of the nanoparticles (NPs) that allows tuning with the interference response of the transducer. Through this tuning process, the interference response can be amplified in the chosen transducer, preferably a Fabry-Perot interferometer, as well as taking into account the reduction of the signal amplitude due to the scattering of the NPs that are recognized in the sensor. Additionally, the method of the present invention also makes it possible to apply design criteria to determine the amplification constant, establish the quantitative detection range, significantly improve sensitivity, reduce measurement uncertainty, and drastically improve the detection limit. Additionally, this method also makes it possible to modify the reading system of the detection signal, which makes it possible to reduce the uncertainty of the measurement.
Thus, the following advantages are achieved with the detection method described in this patent application:
In conclusion, the present invention solves important problems of optical biosensors operating by interference:
Thus, the method of the present invention can be used to develop in-situ and non-invasive diagnostic systems by capturing biomarkers present in different, preferably biological, samples. In the case of clinical diagnosis, the biological liquid samples where to find these biomarkers may be, among others, blood, serum, urine, tears among others as medullary cerebral fluid, water, saliva, etc. Likewise, the diagnostic method may be used for multiple sectors where a detection of bioreceptor-analyte recognition takes place in-vitro (e.g. antibody-antigen affinity reaction).
Thus, the invention also provides an immunological-based in-vitro diagnostic method using optical interference-based photonic transducers (interferometric transducers), wherein these transducers comprise on their sensor surface: i) the target molecule, or ii) at least one specific bioreceptor (BR1) thereof; 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-TM conjugates described above.
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.
In one of the preferred embodiments, the optical detection method of the present invention may be used for in vitro diagnosis of food allergy. This method would consist in the detection of allergy-specific immunoglobulins (IgE) contained in real samples of patients. In this case, the target molecules (TMs) to be detected are IgEs specific to the allergens to which the patient is allergic. To do this, several allergenic food molecules are immobilized in different wells of a biokit in which, for example, several Fabry-Perot type interferometers are found (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 a bioreceptor (BR) consisting of an IgG Inmonuglobulin that specifically recognizes all existing IgEs in the patient sample (sera from food allergy patients). In order to distinguish which IgE molecules the patient is allergic to, after the incubation and filtering of the patient's sample a sample is obtained that will contain the NPs conjugates with the different IgEs. This sample is deposited and incubated in the different wells containing the allergenic molecules for which the diagnosis is made, so that the NPs conjugates with the specific IgE bind to the corresponding allergen, which are detected when reading the optical signal by the change in the interference that is generated.
The detection method described herein, therefore, has to further comprise a prior step of functionalizing nanoparticles with at least one specific bioreceptor, resulting in the formation of the functionalized nanoparticles (NP-BR). Any anchorage method (covalent bond, direct adsorption or any other biofunctional pathway) known to the skilled person can be used to carry out this functionalization.
In those embodiments in which the NPs are silica, the preferred method of functionalization is silanization, to couple an anchor molecule that orients the antibody in its non-specific part. On the other hand, when the method uses gold NPs, it is preferable to use the thiol as an anchoring element. The mechanisms of surface biofunctionalization are extensively reported in the scientific literature (e.g. Bioconjugate Techniques, Greg T. Hermanson, Second Edition, 2008, ISBN 978-0-12-370501-3).
As a particular example, biofunctionalisation of silica NPs with specific bioreceptors of the target molecule can be performed from a monodispersed solution of nanospheres with amine 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-dimethyl aminopropyl) carbomidine hydrochloride) and NHS (N-hldroxysuccinimide), 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.
Likewise, the optical detection method of the present invention also requires the biofunctionalization of the surface of the optical sensor, as described in step d) of recognition, in which the conjugates NP-BR-TM and/or NP-BR are put in contact with the sensor surface. Different routes have also been widely described in the literature for the biofunctionalization of the sensor surface of the transducers, with covalent or adsorption anchors, with or without orientation and using different blocking agents. Throughout this application several references have been cited in which these biofunctionalization routes that are not the object of the present Invention are described.
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.
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.
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.
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.
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 2.5×1010 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.
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.
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.
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.
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).
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−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.
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.
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.
| Number | Date | Country | Kind |
|---|---|---|---|
| P201931066 | Dec 2019 | ES | national |
This application is a national stage application of PCT/ES2020/070735, filed Nov. 25, 2020, which claims the benefit of and priority to Spanish Patent Application No. P201931066, filed Dec. 2, 2019, the entire contents of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/ES2020/070735 | 11/25/2020 | WO |
