Patent Application
20210333296
The present invention relates to a method for substance (organic, inorganic or biological) detection. In particular, the invention uses a novel magnetophoresis technique wherein materials in a sample are mixed with functionalized magnetic particles, such that the variation of the optical properties versus time are monitored and compared with the time-variation in absence of the targeted biological or chemical substance.
Recent years have witnessed an increase in the use of clinical diagnostic methods involving immunological procedures because they are specific and have high sensitivity. Of the many heterogeneous and homogeneous immunological assay methods available, the homogeneous assays based on the agglutination of latex particles continue to be widely used in biology and medicine for the detection of small quantities of an antibody or antigen in a fluid test sample. Some advantages of these assays are that the procedures are simple, widely applicable, nonhazardous, and test results are obtained in a very short time. The agglutination reaction involves in vitro aggregation of microscopic carrier particles (usually of polymeric nature, referred to as latex). This aggregation is mediated by the specific reaction between two molecules, such us antibodies and antigens, one of which is immobilized on the surface of the latex particles to enhance the sensitivity and extend the point of equivalence. A fluid containing the ligand of interest is introduced into a suspension of the sensitized carrier particles, and the agglutination is noted as indicative of the ligand. The agglutination reaction may be used in several different modes to detect the ligand of interest, and each has its own limitations and applications. There are a number of techniques based on light scattering phenomena to detect latex particles' agglutination: turbidimetry, nephelometry, angular anisotropy, and photon correlation spectroscopy. Turbidimetry involves measurement of the intensity of the incident beam as it passes through the sample. The light beam may pass through a suspension or be absorbed, reflected, or scattered by the particles. As a consequence, the intensity of light decreases as it propagates through the suspension. For nonabsorbing particles the decrease in light intensity due to scattering is expressed as turbidity. This technique is rapid and easy to use. In fact turbidimetry does not require any special equipment other than a spectrophotometer, which is generally available in clinical laboratories.
There are fully automatic spectrophotometers that not only measure transmitted light automatically at a desired time but also dilute, pipette, and transfer to the cuvette the convenient volumes of reagents buffers and samples, incubate at a programmed temperature and make the necessary calculations using the selected algorithms and calibration curves. The possibility of running latex agglutination tests into these automatic analyzers allows the processing of hundreds of samples in a short time without investment in new instrumentation or personnel.
However, to optimize the turbitidy change, which occurs during agglutination, it is important to select the appropriate particle size. For example, the number of antigen-antibody bridges between pairs of particles during the immuno agglutination is about 2-10. With larger particles, the shear forces across these bridges may result in disruption of agglutinates when pumped at high speed in automatic machines. Thus, particles of smaller diameter may yield more robust assays. For particles to agglutinate they must first collide so that antigen-antibody bridges can form. For molecules and small particles diffusion is fast enough to produce the initial collisions necessary for aggregate formation. If the particles are large diffusion is reduced (i.e., the agglutination kinetics) because the diffusion coefficient is inversely proportional to particle size. Small particles are desirable because of the requirement for increasing the collision frequency between particles or aggregates to enhance the rate of immunoaggregate production.
The turbidimetric detection depends strongly on the particle size and the incident light wavelength. It is important to select carefully the suitable particles (size) for a suitable wavelength, since the turbidity reaches a maximum with time. This maximum occurs when the signal change exceeds the optical limits of the measuring system. It has been observed by photon correlation spectroscopy that changes in aggregates size continue beyond the plateau observed in turbidimetric assays. The optimal performance may be a function of the ratio of the particle diameter to the incident wavelength, and the refractive index of particle. Thus, the selection of particle material, particle size, and wavelength of detection of the agglutination reaction are all important factors in optimizing assay sensitivity. For particles that are small in comparison with the wavelength of light, the scattering increases with the inverse fourth power of the wavelength. Shorter wavelengths, such as 340 nm, give larger signal differences during agglutination than longer wavelengths, such as 450 nm. On the other hand, the higher the refractive index of the particles at the wavelength of choice, the higher the light scattering signal.
In general, the refractive index of a material is greater at shorter wavelengths. Particles with a polyvinylnaphthalene core have been proposed to enhance sensitivity of latex immunoagglutination assays. Galvin et al. claimed that for the lowest detection limits particles should be in the size range 40-70 nm, with a high refractive index but low absorbance at the wavelength of light used.
This compromise between the size and refractive index of the particles (material), and the wavelength of the incident light can be decisive for the sensitivity of the test. As a consequence, more advanced detectors (e.g. high resolution multiwavelength-detectors) may be necessaries to conclude the test. Unfortunately, these detectors are more expensive and then more difficult to implement, especially in developing countries. In addition, the selection of the most suitable particles for each test may requires a high level of optical knowledge and an easy access to a large selection of particles.
Substantial progress has been made in developing technologies in the field of magnetic microspheres and magnetic nanospheres. Those magnetic nanospheres and microspheres containing superparamagnetic cores in a nonmagnetic matrix (hereafter magnetic particles) are used in numerous biological applications. They are used, for example, as carriers, which can be targeted to a particular site using an external magnetic field.
The above mentioned magnetic particles are designed to create magnetic particles with large superparamagnetic response. In fact these magnetic particles are typically made by embedding superparamagnetic nanocrystals in a non-magnetic matrix such as polystyrene and nanoporous silica. The resulting magnetic particles retain the superparamagnetic response of their constituent superparamagnetic nanocrystals and show larger magnetization when an external magnetic field is applied. Further, neither coercivity nor remanence is observed at the working temperature. However, in addition to the intrinsic superparamagnetic behavior of the constituent nanocrystals, one must consider the interactions between the nanocrystals inside the skeleton matrix due to their proximity and surface effects due to the coating; these can lead to changes in the overall magnetic response of the colloidal particle.
Biomagnetic Separation (using the magnetic particles described above) has numerous applications in Life Science. From cell sorting to molecular diagnostics, this technology can be used with volumes ranging from a few nanoliters (lab-on-chip) to tens of liters (production of IVD-reagents). One of the problems of working with small tubes and classical magnetic separators (or simple magnets) is the lack of definition of the magnetic force. As the magnetic field and its gradient change with distance, the force on the magnetic particle in the suspension is not constant and variations in the behavior of the suspension are difficult to control.
EP0339623 discloses a laser magnetic immunoassay method and system based on antigen-antibody reactions. For this method, nano beads are needed and electromagnets are used to generate the magnetic force. The use of magnetic nanoparticles (D<<100 nm) implies that its magnetic moment is very small, thus each particle moves without interacting magnetically with its neighbors. The separation speed is then v=(D2/18n)*μ0*M(H)*∇(H)*ρ, where D is the particle diameter, n the viscosity of the buffer and ρ the density of the particle. This individual magnetophoretic behavior implies a slow separation speed and a strong dependence on the individual nanobeads characteristics. That implies a large variability due the inherent dispersion of size of a set of real magnetic particles. Moreover, the speed would also be dependent on the magnetic field profile. The use of electromagnets, with soft ferromagnetic pole pieces, makes very difficult to generate well controlled magnetic field variations, thus the attainable range of magnetic field gradients is limited, thus the attainable magnetic force. As the permeability of the pole pieces would also strongly depend on the local value of the field (including the inherent hysteresis of ferromagnetic materials), the magnetic flux path would change smoothly from point to point. The local value of the magnetic field on the pole pieces —and by consequence in the gap—will also vary with the applied magnetomotive force (Ampere turns) and its previous values. The value of the magnetic force over an individual magnetic nanobead would depend on the value of the applied intensity on electromagnet, but would be different if the current is increasing or decreasing, and if the pole pieces have been saturated or not (i.e. the applied electrical current have reach a value high enough to reach the maximal magnetization of the pole pieces' material). All these effects imply that for a given set of individual beads, its a large variation on the time needed for reaching the final position (or any specific point), and even for exactly the same distribution of physical characteristics (size, density, magnetic properties), reproducibility would require a precise electrical current control and detailed control on how it is applied to the electromagnet.
All those variations would, partially or totally, mask the magnetophoretical changes induced on the beads due to the immunocaptured target,
Further, the use of a laser makes the optical system costly and complex, as the need to focus the laser at a single point and measure the scattered backlight requires a well-controlled optical environment. Also, the bulky electromagnets used to generate the magnetic force needed require high power consumption that generates heat that is difficult to manage.
The present invention provides a method for detecting the presence or absence of a biological or chemical substance based on a opto-magnetophoresis technique, wherein the materials are reacted with functionalized magnetic particles in the range of 10-1000 nm in the presence of a uniform magnetic field gradient. The method does not depend on the refractive index of the non-magnetic matrix of the magnetic particles, as in known turbidimetric techniques, and the device does not need a laser or electromagnets, the invention solving thus the problems discussed above. The method comprises the steps of providing a light source and detector, providing a magnetic force perpendicular to the light's propagation direction with a constant magnetic field gradient, with an absolute value which is higher than 0.1 T, and measuring the change of the magnetic particle's suspension transparency versus time. Systems for putting the invention into practice comprise a light source and detector, a magnetic force generator made of permanent magnets for generating a magnetic force perpendicular to the light's propagation direction, with a constant magnetic field gradient and with an absolute value which is higher than 0.1 T and processing means for measuring the change of the magnetic particle's suspension transparency versus time. In one embodiment, the magnetic force generator is formed by at least four permanent magnets following the Halbach progression for a quadrupole in a cylindrical support. In another embodiment the magnetic force generator is formed by two permanent magnets with opposite polarities and the light and the detector is an spectrophotometer.
To complete the description and provide for better understanding of the invention, a set of drawings is provided. Said drawings illustrate a preferred embodiment of the invention, which should not be interpreted as restricting the scope of the invention, but just as an example of how the invention can be carried out.
In the present invention, materials in a sample are reacted with magnetic particles (commercially available) that are functionalized to specifically bind to the targeted biological material in solution to produce a magnetic colloidal aggregate. The magnetic particles can be coated with protein antigens and reacted with the corresponding antibody, or with complementary or non-complementary oligonucleotides for genetic and pathogenic disease-detection. Other aggregations are based on electrostatic interaction between the magnetic particles and the biological materials, or by the absorption of the material onto the surface of the magnetic particles. In the case of microorganisms, the magnetic particles are absorbed on the surface of the microorganism. The aggregate is driven by magnetic forces towards the walls of the transparent vessel containing the suspension inducing higher transparency in the suspension. The progressive transparency of the suspension is real-time monitored. The separation speed in the present invention depends on the magnetization and size of the particles. These parameters can be adjusted by adjusting the size and the magnetic content of particles. The most suitable particles for use with the invention are particles of 200-300 nm of diameter with a magnetic content of 20-60%.
When the magnetic particles are functionalized and mixed with the analyte, colloidal aggregates containing the magnetic particles and the analyte are formed. Those aggregates, when brought under a uniform magnetic field gradient, move at a higher velocity than the initial monodispersed magnetic particles (colloids without analyte). As described by Andreu et al (J. S. Andreu et al, PHYSICAL REVIEW E 84, 021402 (2011)), the movement of magnetic particles is a cooperative phenomenon where the individual particle has enough magnetic moment for having a magnetic dipole-dipole interaction with its neighbours, strong enough to overcome thermal agitation.
If that happens, the magnetic particles form chains moving much faster than isolated magnetic particles. The average number of particles forming a chain can be estimated from the formula N=√(ϕ0*.e(T−1)), where ϕ0 is the concentration of particles and Γ is the ratio between magnetic and thermal energy Γ=(μ0πD3Ms2)/(72kBT), where D is the particle diameter and Ms its saturation magnetisation. When N>1 the magnetic particles interact and move much faster than when N<<1 and each particle moves isolated. Thus, selecting the right diameter and saturation magnetisation for the selected magnetic particle, it is possible to have it moving as an isolated particle (slow) when is not agglutinated, but moving cooperatively (fast) when its agglutinated. The particles linked through reaction with the analyte act as particles having a bigger diameter. It should be noticed that the increase in diameter has a larger effect on the value of F (even if the effective Ms would be lower for the agglutinate) and its effect over N is exponential.
This behavior induces an important difference in the magnetic separation time of the formed aggregates and the monodispersed magnetic particles. This difference in magnetic separation time is optically monitored by measuring the real time transparency of the suspension. In the case of the aggregate formation, the suspension reaches higher grades of transparency in shorter times in comparison with the suspension of the monodispersed magnetic particles. This optical monitoring (transparency of the suspension) can be performed in white light (combination of different wavelengths in the electromagnetic spectrum), or at specific wavelengths such as the UV-Visible spectrum (350 nm, 580 nm,). Optical monitoring at a specific wavelength has an additional advantage over the optical monitoring using white light, as it allows the detection of aggregates at concentrations as low as 0.001%. Moreover, as the aggregates concentrate at the walls of the transparent vessel, in the region where the magnetic field is maximum, this behavior allows their collection for further analysis.
In the classical turbidimetry technique, as the aggregates remain in the suspension increasing its turbidity (lower transmittance), the particle characteristics and incident light wavelength should be carefully chosen to avoid to excess the optical limits of the measuring system. In comparison, in the present invention, it is not necessary to select particles for a suitable wavelength. As the aggregates are magnetic and are removed by the magnetic force from the suspension, the results of the experiments do not depend on the interaction of the incident light with the aggregate. In addition, many types of magnetic particles can be used (composites of iron oxide nanoparticles embedded in non-magnetic matrices).
To assess the time dependency of the transparency of samples with and without aggregates, a constant magnetic force is generated on the whole sample volume. For obtaining a constant magnetic force it is necessary to fulfil two conditions: a constant magnetic field gradient, and the saturation of the magnetic moment of the magnetic particles. The magnetic force must be perpendicular to the light's propagation direction.
The magnetic force acting over the magnetic particles is the gradient of the product of the magnetic moment by the applied magnetic field. If the magnetic moment of the particles is constant, the force is constant when the magnetic field gradient is also constant. At the same time, the magnetic moment of the particles depends on the applied magnetic field. When the applied field is small, the magnetic moment is proportional to the latter. The resultant magnetic moment is then the product of the volume (or mass) of the particle by the value of the magnetization of the magnetic nanoparticles embedded in the non-magnetic matrix (magnetic particles). The magnetization is the product of the magnetic susceptibility (a constant which is intrinsic to the material) multiplied by the applied field. When the magnetic field reaches a particular value, the magnetization remains almost constant, i.e. becomes saturated, and the magnetic moment of each particle is constant. The value of the applied magnetic field where the magnetic moment changes its response from linear to saturated is known as saturation field (Be).
The magnetic force generator according to the invention is a magnetic field source that generates a magnetic field with a constant gradient and absolute value higher than the saturation field in the region where the samples are placed for measuring the transparency changes. For practical purposes, a 0.1 T field is sufficient to guarantee the saturation of any commercial particles suitable for use with the invention. The speed of the magnetic particles placed at the sample holder is the result of the competition between the magnetic force and the drag force generated by the viscosity of the buffer suspension. The speed resultant of the balance of these force is proportional to the square of the magnetic bead diameter, the gradient of the applied magnetic field, the magnetization and the density of the particle, and it is inversely proportional to the buffer viscosity. For larger beads —keeping constant the other characteristics-, the separation speed is thus faster due the larger diameter. The aggregates act as larger particles and move therefore faster than the single particles
For the suitable range of magnetic beads diameter and magnetic charge selected for this application (i.e. beads that moves as isolated particles when are not linked by the capture of the target, but have a cooperative magnetophoretical movement when are aggregate by the capture of the target substance), magnetic field gradients between 1 T/m and 100 T/m are needed over large volumes if the separation is to be performed in few minutes and not in several hours. These gradients (on relatively large gaps) are very difficult to obtain with electromagnets due to the limitations inherent to the use of soft ferromagnetic pole pieces. However, using modern magneto crystalline anisotropic permanent magnets, like rare-earth based materials, it is possible to add the effect of magnetic field sources with different directions without affecting the magnetisation direction of the neighbour magnets, thus generating high spatial magnetic field variations, including linear constant gradients higher than the ones attainable with larger and bulkier electromagnets and without the need of use of power supplies. Note that for very small magnetic beads as the used for Magnetic Activated Cell Sorting or the patent EP0339623, these magnetic field gradients may not be high enough to perform the separation fast enough for practical applications
When the generated magnetic force is homogenous at the sample's position (i.e. the magnetic gradient is constant and the magnetic field higher than the saturation field), all the beads move at the same speed. Thus, once the farthest beads start moving, their movement marks the border between the transparent buffer and the opaque region. As all the beads move at the same speed, the optical density in the region in front of the border is constant. Under these conditions, changes in the transparency as a function of time are easy to parameterize. This leads to an easier way of correlating the presence/absence of the aggregates and accordingly, the presence of the analyte of interest.
The optical means can be as simple as a LED that generates the light, and a detector such as a photodiode or a light dependent resistor that measures the amount of light transmitted through the sample. This simple arrangement allows to place one sample with its own light source and detector to measure a single analyte, or several samples (each with its own light source and detector) inside a cylindrical cavity, allowing a multiplex or mid-plex assay that can simultaneously measure multiple analytes in a single run of the assay. It is distinguished from procedures that measure one analyte at a time.
In a first embodiment, the device has a cylindrical configuration. In this case, the required magnetic field is quadrupolar. This way, the radial magnetic field gradient is constant, with zero intensity at the cylinder axis. This also has the advantage that the obtained magnetic field profile is large enough to place several samples, where each sample has its light source and optical sensor. For this purpose, several magnets are placed in a ring-like support following the Halbach progression for a quadrupole: the magnetization direction of each magnet should be 3 times the angle respect the angular position of its center in the cylindrical coordinates systems defined by the cavity, as described in
In a preferred embodiment, for a simple and easier manufacturing, square section magnets are used. N identical magnets, with N greater than 4, magnetized along one of their sides are placed at the same radial distance, R, from the center of the cylinder axis and distributed at regular angular intervals, θ, where θ=2π/N. The permanent magnet angular position would be then nθ, for n=1, . . . , N, and the magnetization direction for each magnet is defined by γ(n)=3nθ. (
With this configuration, it is easy to obtain gradients in the range between 1 and 100 T/m. The samples are placed at radial distances where the magnetic field is higher than the saturation field of the magnetic nanoparticle (Bs). When the samples are placed at a distance r>rs (rs=Bs/∇|B|), where ∇|B| is the value of the radial component of the constant magnetic field gradient generated by the permanent magnet assembly (
A cylindrical magnetic force generator according to an embodiment of the invention uses 12 NdFeB permanent magnets of 40 mm height and a square section of 20×20 mm magnetized along one of their sides. The magnets can be placed with their centers along a circumference of 56 mm, with an angular distance of 30° between them. As described, the magnetization should rotate 90° (30°×3) between successive magnets. This magnetic force generator would generate a magnetic gradient of 8 T/m at the central plane of the cylinder defined by the magnets. For an 8 T/m radially constant magnetic field gradient in a cylindrical cavity of 8 cm diameter, the transparent vessel containing the aggregates can be placed at a distance higher than 1.25 cm radius (0.1/8=0.0125 m) from the cylindrical axis of the cavity. As the nanoparticles move in the radial direction, the optical system is aligned perpendicular to it, i.e. tangential to the circumference defined by the sample holder center (
At the end of the separation process, a sigmoidal curve showing the transparency behaviour of the suspension versus the time t is obtained. The difference on the magnetic separation speed due the presence/absence of aggregates is determined measuring the time necessary to reach a certain level of transparency, for example the 50% between the minimal and maximal transparency. The time of reference is obtained using a suspension of magnetic beads without the analyte. When a new sample needs to be tested for the presence of the analyte, the same amount of magnetic beads is added, to keep concentration constant. The sample is incubated and introduced in the magnetic force generator. The change in transparency is monitored and the time necessary to achieve the defined level of transparency determined. This time can be equal to the reference time: that would indicate the absence of aggregates that would not be formed if the analyte is not present in the test sample. The second possible outcome is that the time necessary to reach the defined level of transparency is shorter than the reference time. This second result indicates the presence of aggregates—that move faster during the separation process-, formed because the analyte is present in the tested sample.
To improve the sensitivity, the experimental data can be fitted to a sigmoidal curve using the minimum squares method (or other similar algorithm). The data can then be normalized between its maximal and minimal transparency. The fitting parameters allow to get an analytical curve and determine with more precision the value of the time. For example, the experimental points (transparency vs time) can be fitted by the expression V(t)=V0+(Vf−V0)/(1+(t/t50)p) using the minimum squares method. The fitting parameters would be the V0, the output of the optical sensor at the start of the process (lower value of the transparency); Vf, the output of the optical sensor at t=infinite (maximal transparency); t50, the time when the sensor output is exactly the average of V0 and Vf; and p, an exponent proportional to the slope of the curve at tso. The transparency curve versus time can be normalized (V0=0%, Vf=100%), Vn=100%/(1+(t/t50)p). If the selected level of transparency is 50%, this fitting would directly provide the value of the time (t50).
For experiments where determination of the separation time is done at specific wavelengths, a rectangular magnetic force generator is proposed, such that it can be incorporated into a spectrophotometer. Two sets of permanent magnets comprising two magnets each, is provided, which generates a homogenous magnetic force. The magnets have different thickness embedded in two parallel surfaces and with opposite polarities (
In a particular embodiment, 2 NdFeB magnets of 20×20×40 mm are placed over a 5 mm thick iron plate. Both magnets are magnetized along one of the shorter sides. The magnets are placed such that their longer sides are parallel, separated by 1 mm, aligned in its top, and with the magnetization direction pointing out of the iron plate. A second set of NdFeB magnets, with dimensions 10×10×40 mm, are placed on a second 5 mm thick iron plate. The 4 magnets are magnetized along one of the 10 mm directions, and placed parallel along the 40 mm side (separated by 1 mm) and aligned in the top. The magnetization direction points out of the iron plate. The inner face of the two iron plates (the ones with the magnets attached) should be placed parallel and with the center of the magnets aligned. With this configuration, the magnetic field gradient is constant at the central area, with a value of 7 T/m. The value of the magnetic field is zero at 20 mm from the surface of the 10×10×40 magnets, what is 15 mm from plane equidistant to the inner faces of the iron plates. As consequence, the field at the center (where the sample is placed for optical monitoring) is 0.113 Tesla, high enough to saturate the magnetic beads.
The advantage of this rectangular design over the previous embodiment is the possibility of its easy incorporation into any commercial spectrophotometers, and then the selection of specific wavelengths for measuring the transmittance variation of the suspension as a function of time while the magnetic nanoparticles are moving to the retention position. Using this combination of permanent magnet assembly and spectrophotometer optics, it is possible to distinguish samples with/without aggregates even with magnetic nanoparticle concentration below 0.001% (w/v) just comparing the time dependence of the transmittance.
In a preferred embodiment, for an immunoassay test, the magnetic particles are functionalized with an antibody or antigen, then mixed with the sample to analyse (containing the analyte). After incubation, a shorter separation time indicates the agglutination of the magnetic particles, which can be directly or indirectly proportional to the analyte content present in the sample, depending on whether the format is Immunosandwich or competitive, respectively. The system could also work without the use of antibody or antigen as a molecule capture, but with the use of aptamers (both nucleotide or peptide based), with widely used biological pairs (such as streptavidin and biotin . . . ), with molecules that present a natural capacity for binding to another molecule, and with any other alternative where there is a specific or non-specific binding between functionalized or non-functionalized particles with the analyte present in the sample. The method includes application to microorganisms and bacteria by mixing functionalized magnetic particles that recognize the microorganism in the sample to analyse. The magnetic particles can be functionalized with antibodies (or any molecule capable of recognizing and binding specifically to certain molecules exposed in the microorganism of interest). Then the microorganisms-magnetic particle aggregates are formed and monitored by the magneto-optical system. Moreover, the separated microorganism-aggregates can be collected for their incubation and subsequent analyses.
In another preferred embodiment, for oligonucleotide hybridization test, two populations of magnetic particles are each functionalized with complementary or non-complementary oligonucleotides and mixed with the sample to analyse, which might contain a complementary oligonucleotide to the one or both coated onto the magnetic particles. This sample might be the product of a PCR nucleotide amplification (DNA), the result of the DNA or RNA extraction of a particular cell or microorganism, or any other source of DNA or RNA. After incubation of the mixture, if the separation time is shorter than the separation time of the magnetic particles, this indicates the formation of agglomerates, and then the hybridization of the oligonucleotides from both particles, indicating that the sample does not contain the complementary oligonucleotide. Alternatively, the aggregation of the particles could be due to hybridization or bridging between the DNA/RNA present in the sample and the oligonucleotides conjugated to the particles.
The t50 value for the sample without the presence of CRP (filled symbol) is 14.5 seconds. The open symbol curve has a t50 of 8.7 seconds, shorter than the reference value of 14.5 seconds, thus indicating the presence of magnetic beads aggregates due the presence of CRP in the sample. The method can be applied for detection of proteins, small molecules, microorganisms and DNA between others.
As it is used herein, the term “comprises” and derivations thereof (such as “comprising”, etc.) should not be understood in an excluding sense, that is, these terms should not be interpreted as excluding the possibility that what is described and defined may include further elements, steps, etc.
On the other hand, the invention is obviously not limited to the specific embodiment(s) described herein, but also encompasses any variations that may be considered by any person skilled in the art (for example, as regards the choice of materials, dimensions, components, configuration, etc.) to be within the general scope of the invention as defined in the claims.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/ES2018/070392 | 5/30/2018 | WO | 00 |
