Mechano-optical analysis system, manufacturing method, use for identification and classification of

专利摘要:
Mechano-optical analysis system, manufacturing method, use for identification and classification of cell populations, and mechano-optical analysis procedure. Analysis system comprising: a reflective substrate; a hollow elongated structure with two ends; two polymeric supports, coupled to the ends and attached to the substrate; a piezoelectric device, coupled to the substrate and adapted to produce vibrations in the elongated structure; a laser, to emit a beam, a beam splitter, a photoreceptor, an amplification module, and a processor, where the laser beam passes through the cavity, and is absorbed by the photoreceptor, which generates a signal (S1) that transmits to the amplification module, which separates it into a modulated (AC) and unmodulated (DC) component, and transmits it to the processor to obtain the resonance frequency and reflectivity and to provide the piezoelectric device with an excitation signal (S2) at the resonant frequency. (Machine-translation by Google Translate, not legally binding)
公开号:ES2786848A1
申请号:ES201930327
申请日:2019-04-10
公开日:2020-10-13
发明作者:Pérez Alberto Martín；Vega Daniel Ramos；Gómez Montserrat Calleja；De Miguel Francisco Javier Tamayo；Vidal Oscar Malvar
申请人:Consejo Superior de Investigaciones Cientificas CSIC；
IPC主号:

专利说明:

[0005] OBJECT OF THE INVENTION
[0007] The invention relates to a mechanical and optical sample analysis system, more specifically, a system capable of identifying biological particles and / or cells in aqueous suspension or in physiological media.
[0009] The system of the invention allows obtaining mechanical and optical data at the same time, and combining them in such a way that it is possible to make an unambiguous distinction between particles, even if they have the same buoyant mass.
[0011] BACKGROUND OF THE INVENTION
[0013] The detection of particles in liquid is of vital importance today for numerous applications, both in the field of health and in the environment or security, so there are different methods to detect them. In addition to methods based on filtering and subsequent analysis, some of these methods are based on the use of resonators, which can use cantilevered elements that perform mechanical measurements when in resonance. The measurement method is based on the change in resonance frequency of the resonator due to the change in mass that it experiences when particles are added to its surface. These methods allow high precision and adequate resolution for these types of applications. Being used as mass sensors in the laboratory, they have even reached a sufficient resolution to carry out measurements of atomic sizes in controlled environments of ultra-high vacuum and low temperature.
[0015] However, the measurements are greatly deteriorated when we try to apply these measurement techniques based on mechanical resonators in other types of environmental conditions, that is, it is not possible to have strict control conditions, for example, in aqueous solutions such as physiological environments for biological applications. This is due to the interaction with the environment, where the Viscous friction with a fluid surrounding the resonator dampens the movement. That is why hollow resonators have been developed that transport the liquid inside, instead of being surrounded by it.
[0017] This type of system, despite being highly sensitive, presents an added problem: the measurement of mass in a sample that is immersed in a liquid is not direct. The change in frequency of the resonator is influenced by the difference in the mass of the particle and the volume of liquid that it displaces, which is known as buoyant mass. A known method of overcoming this limitation is to perform measurements using several different carrier liquids with known mass densities. In this way, it is possible to determine the influence of the density of the carrier liquid in taking measurements, and to differentiate particles with the same buoyant mass. This differentiation is necessary, as the buoyant mass of a particle depends on its density and its volume, it may be the case that two particles with different density and volume have the same buoyant mass and, therefore, give the same change. resonant frequency in the resonator. However, this implies the realization of multiple measurements in order to distinguish between different populations of analytes, with the consequent cost and with a high complexity, which in many cases implies the impossibility of carrying out these measurements, since in biological analyzes the carrier liquid many times it must be a certain one.
[0019] Other proposals are based on the measurement of other parameters of the particles, such as their compressibility. To do this, the resonance frequency of radial extensional modes of the resonator is measured, which have a higher frequency and a lower amplitude, and which can only be measured with the coupling of optical modes confined to the surface of the resonator. In order to excite such optical modes, the evanescent field of an optical fiber that approaches a distance of nanometers from the surface is often used. This process involves a great technical complexity, with a very high cost in elements to be able to measure mechanical modes that are difficult to measure.
[0021] DESCRIPTION OF THE INVENTION
[0022] The mechano-optical system for analyzing samples of the invention allows the unequivocal distinction of particles by determining their buoyant mass and reflectivity, the system comprising:
[0023] - a reflective substrate;
[0024] - an elongated hollow structure, having two ends;
[0025] - two polymeric supports, attached to the ends of the hollow structure and attached to the reflective substrate;
[0026] - at least one piezoelectric device, coupled to the reflective substrate and in contact with the hollow structure, and adapted to produce vibrations in said structure;
[0027] - at least one laser, configured to emit a laser beam,
[0028] - at least one photoreceptor, adapted to absorb the laser beam and produce an electrical signal (S1) associated with the intensity and frequency of the received laser beam,
[0029] - at least one amplification module, coupled to the piezoelectric device and to the photoreceptor, and configured to amplify and filter the electrical signal (S1) provided by the photoreceptor; Y
[0030] - a processor, coupled to the amplification module, and configured to process the amplified and filtered electrical signal (S1) in order to provide a measure of the cavity's resonance frequency at each instant of time, which varies as a function of the variation of the buoyant mass of the sample;
[0032] where the laser beam passes through the cavity before reaching the photoreceptor, the photoreceptor generates a signal (S1) that transmits to the amplification module, said amplification module being adapted to separate the modulated (AC), and unmodulated (DC) component from the signal (S1), and the processor is configured to obtain a measure of the resonance frequency at each instant of time, from the modulated component (AC) of the signal (S1), and the reflectivity inside the hollow structure at each instant of time, starting from the unmodulated component (DC) of the signal (S1) and to provide the piezoelectric device, through the amplification module, an excitation signal (S2) at the resonance frequency , in order for said piezoelectric device to produce vibrations in the cavity at the resonance frequency at each instant of time.
[0033] The use of optical measurement methods allows a correct determination of the buoyant mass of the sample particles, providing a differentiation parameter between particles. In this way, it is possible to distinguish between particles with the same buoyant mass but which are actually different.
[0035] Two particles with different density and different volume can have the same buoyant mass, therefore, the use of a laser beam and a photoreceptor to obtain a measure of the reflectivity of the particles that circulate through the cavity, allows to distinguish between these particles in real time, providing measurements of their buoyant mass and scattered light, performing a reliable analysis and reducing costs associated with carrying out more measurements in order to differentiate these particles.
[0037] These advantages are due to the simultaneous acquisition of the buoyant mass and the reflectivity of each particle as it passes through the hollow structure, therefore, even if the measurement of buoyant mass and reflectivity were performed in consecutive, but not simultaneous, processes, The desired objective would not be achieved, since the measurement obtained would be an average of the buoyant mass of the sample and an average of the reflectivity of the sample, thus preventing the differentiation between the particles that make up said sample.
[0039] The analysis system of the invention may also comprise optical elements that allow better performance, among which an optical isolator can be found, to prevent the laser beam from returning to the inside of the laser itself, causing it to malfunction. .
[0041] Additionally, the system can comprise a neutral density filter to allow the passage of light at a determined intensity and a non-polarized beam splitter, designed to divide the light beam and deflect part of it in a determined direction, more specifically, the Beam splitter can be positioned at 45 °, so that it deflects part of the beam in a direction perpendicular to the original beam.
[0043] Preferably, the amplifier is of the synchronous type, which makes it possible to extract the signal (S1) even in the presence of high noise in the signal.
[0045] Furthermore, preferably, the drive signal (S2), which is sent to the piezoelectric device, is a sinusoidal signal at the resonant frequency.
[0046] The piezoelectric device of the system can be made of ceramic, which provides greater flexibility in geometry and dimensions and improved properties over other piezoelectric crystals.
[0048] Preferably, the hollow elongated structure is made of fused silica and has a diameter of approximately 50pm, but can be up to 1pm in diameter. The use of fused silica, which is a transparent material, allows the laser beam to pass through it, so that the beam passes through the sample and reaches the photoreceptor. The reduced diameter of the cavity also makes the flow of particles orderly and not random.
[0050] The analysis system according to claim 1, where the polymeric supports are made of SU8 photoresin, allowing better control of their adherence to the ends of the cavity.
[0052] The laser used to produce the beam can be of the Helium-Neon type, as it is one of the most common and easily accessible.
[0054] Preferably, the system also comprises a camera with an infinity lens that has a sensor with photoelectric cells, a white light source and a monitor, which allows the interior of the cavity to be seen.
[0056] The camera makes it possible to check whether the laser is correctly positioned to precisely point to the center of the elongated hollow structure and, furthermore, that there is no jamming inside the cavity.
[0058] The system can also comprise a pump designed to control the flow of the liquid inside the hollow structure. The system pump can be a syringe pump, or preferably, it can be a pressurized gas pump, and more specifically, a pressurized carbon dioxide and / or nitrogen pump, configured to control the flow within the elongated hollow structure, while avoiding the appearance of noise in the measurement of the resonance frequency made by the system. Thus, the pump makes it possible to provide a constant flow, without abrupt sudden variations that could affect said measurement.
[0059] The invention also relates to a method of manufacturing the hollow elongated cavity suspended on the reflective substrate, comprising the steps of:
[0061] - provide an elongated hollow structure, with two ends, having a protective polyimide layer,
[0062] - remove the protective layer in the central area of the elongated hollow structure, heating said area,
[0063] - apply an axial tension to the elongated structure without a protective layer, to plastically deform its central area, until a specific diameter is achieved,
[0064] - Adhering two or more polymeric supports (5), manufactured on a reflective substrate (2), to each of the ends (4) of the deformed hollow structure (3), leaving the structure (3) suspended on the reflective substrate by means of the polymeric supports (5).
[0066] The proposed manufacturing method allows effective control of the diameter of the resulting elongated hollow structure, which determines the ability of the system to allow an ordered flow of particles without impeding the passage of the particles to be analyzed, and the suspended length, which which controls the mechanical resonance frequency of the structure.
[0068] Preferably, the step of adhering polymeric supports to each of the ends of the cavity is performed by means of a centrifugal coating and using optical lithography, which applies a defined pattern of polymer to the ends of the elongated hollow structure.
[0070] Optical lithography is an extended, cheap and simple technique, as it allows a centrifugal coating to be carried out and a pattern to be drawn, leaving part of the hollow structure embedded within the solid material of the coating while other parts remain free of said coating. This method allows to achieve a high resolution and speed in the realization of the coating, at the same time that it avoids being limited to structures of very low thickness.
[0072] The invention also relates to the use of the analysis system of the invention for the identification and classification of different cell populations present in an isolated biological sample. That is, according to this application, the described system makes it possible to detect the existence and differentiate at least two different cell lines or populations in the biological sample analyzed and classify said cells.
[0074] Preferably, this aspect of the invention relates to the identification and classification of tumor cells and non-tumor cells. The system therefore makes it possible to detect the presence of different cell populations in the same sample analyzed and classify said cells as tumorous or non-tumorous, which enables their clinical application, for example, for the in vitro diagnosis of tumors, preferably of cancer, from biological samples isolated from the patient. More preferably the cells are epithelial cells, even more preferably from breast tissue.
[0076] DESCRIPTION OF THE DRAWINGS
[0078] To complement the description that is being made and in order to help a better understanding of the characteristics of the invention, according to a preferred example of a practical embodiment thereof, a set of drawings is attached as an integral part of said description. where, for illustrative and non-limiting purposes, the following has been represented:
[0080] Figure 1.- Shows a schematic view of a preferred embodiment of the system of the invention.
[0082] Figure 2.- Shows a schematic view of the manufacturing process of the system of the invention.
[0084] Figure 3.- Shows a graph that compares the measurement of the buoyant mass obtained for two different particles, by means of the system of the invention.
[0086] Figure 4.- Shows a graph that compares the measure of reflectivity obtained for the two particles of Figure 3, by means of the system of the invention.
[0088] Figure 5.- Shows a graph that combines the measurements of the buoyant mass and the reflectivity obtained for the two particles of Figure 3, by means of the system of the invention.
[0089] Figure 6.- Shows a graph that compares the measurement of the variation of the resonance frequency and the reflectivity obtained when the cavity is traversed by a particle, two particles and a particle and a dimer, respectively, by means of the system of the invention.
[0091] PREFERRED EMBODIMENT OF THE INVENTION
[0093] The mechano-optical system (1) for analyzing samples of the invention allows the unequivocal distinction of particles by simultaneously determining their buoyant mass and reflectivity. For this, the fundamental mode of vibration of an elongated structure (3) is measured interferometrically, which we will call capillary, transparent and hollow while liquid flows inside. The fundamental mode of vibration, in contrast to the radial extensional modes, has a lower resonant frequency, and a lower stiffness, therefore, a greater amplitude. The measurement by means of said fundamental mode allows to carry out the interferometric optical measurement, without the need to excite optical modes.
[0095] On the contrary, to carry out the optical measurement, the system (1) of the invention uses an interference pattern generated with a reflective substrate (2) to measure the oscillation of the fundamental mode of vibration. The use of a transparent capillary (3) allows us to measure the light scattered by the particles flowing inside it, which allows us to measure its reflectivity and thus, based on two parameters measured simultaneously, the resonance frequency and the reflectivity, differentiate between particles with the same buoyant mass.
[0097] Figure 1 shows a complete view of a preferred embodiment of the system (1) of the invention, capable of combining optical and mechanical measurements in order to obtain a correct determination of particles in aqueous suspension or cells in a physiological medium.
[0099] The mechanical measurements are carried out by means of an elongated hollow resonant structure (3) arranged in the form of a suspended channel with the liquid containing the samples to be analyzed flowing inside.
[0101] Figure 2 shows the manufacturing process of the fused silica resonant elongated hollow structure (3), where the process comprises the following steps: - Use is made of a capillary, a hollow and elongated structure (3) with a reduced diameter, which has two ends (4) and which has a polyimide protective layer (16) that gives it flexibility and robustness. The capillary (3) is made of fused silica, as it needs to be transparent. - The protective layer is eliminated in the central area of the capillary (3), heating the capillary (3), by using a controlled flame, which pyrolyses said protective layer (16) and exposes the capillary (3).
[0102] - An axial tension is applied to the capillary (5), while it is heated, and in this way a plastic deformation occurs in its central area, which makes the capillary (3) more elongated and progressively reduces the diameter of said capillary ( 3) until reaching a diameter of 50pm.
[0103] - The capillary (3) is placed on a reflective substrate (2), where liquid resin is poured that covers both the capillary (3) and the substrate (2).
[0104] - A mask is used to expose the resin on the ends (4) of the capillary (3) to ultraviolet light
[0105] - The solid set, capillary (3) and substrate (2) are introduced in a solvent that dissolves the parts of the resin that have not been exposed to ultraviolet light, leaving two polymeric supports (5) on which the capillary is suspended (3).
[0107] The capillary (3) once in position allows the flow of the sample on a carrier liquid through the channel. The flow presents a practically linear particle arrangement, in which the particles are placed one after the other due to the reduced diameter of the capillary (3).
[0109] The flow control inside the capillary (3) is carried out with a pressurized nitrogen pump (12) that allows to obtain a continuous flow, free of random pulsations, and sets the pressure range from 10mbar to 7bar, as can be seen in Figure 1.
[0111] Because the capillary (3) is in suspension, it defines a series of its own mechanical modes, which present certain resonance frequencies. The eigenmodes of the capillary (3) depend, among other things, on the length of the suspended capillary (3), determined during the optical lithography process.
[0112] The resonant frequency of the capillary (3), however, will vary as a function of the mass of the sample along with the mass of the carrier liquid.
[0114] Figure 1 also shows a piezoelectric ceramic (6) that is responsible for producing vibrations in the suspended capillary (3). A piezoelectric crystal (6) is an element that is capable of converting the voltages applied to it into a potential difference that creates an electrical signal, and in the same way, it deforms under the application of an electrical charge. This operation allows the piezoelectric ceramic (6) to exert a certain vibration in the capillary (3), controlled by the electrical signal that is provided to it.
[0116] The excitation signal (S2) of the piezoelectric ceramic (6) is determined by a synchronous amplifier (10) that receives the signal that is intended to be applied to the capillary (3) of a processor (11). The signal applied in this case will be a sinusoidal signal equal to the fundamental resonance frequency, the lowest, with an amplitude of 1.5V. Alternatively, a higher eigenmode could be used to produce resonance with a higher frequency.
[0118] The system (1) also comprises a second optical type measurement source, shown in Figure 1. The system (1) comprises a Helium-Neon type laser (7) that emits a beam (8) of light, the beam (8) of light passes through an optical isolator (17), which prevents a reflected beam (8) from reaching the laser (7) and causing a malfunction, and then, passes through a neutral density filter (18) that allows regulate the intensity of the beam (8) of the laser (7). The beam (8) of the filtered laser (7) then passes through a non-polarized beam splitter (19), which generates two beams, one of the beams (8) passes through the objective (20) towards the capillary (3) . The beam (8) of light passes through the capillary (3), is reflected on the substrate (2) and goes back through the capillary (3) and the objective (20) until it reaches the beam splitter (19), which it deflects towards a photoreceptor (9) that collects the modified beam (8) due to the passage through the capillary (3) and generates a signal (S1) associated with the properties of the collected beam (8).
[0120] The photoreceptor (9) delivers the signal (S1) generated to the synchronous amplifier (10), which is responsible for dividing the modulated component (AC) of the signal and the unmodulated component (DC).
[0121] The modulated component (AC) makes it possible to obtain the power spectral density as a function of the frequency, so that it is possible to calculate the resonance frequency, using an optical technique, at each instant of time.
[0123] With these data a measurement of the buoyant mass is made with a closed loop configuration (PLL), in which the system feeds back and calculates the resonance frequency, at each instant of time, adapting, within previously set margins, the frequency of the excitation signal (S2) of the piezoelectric ceramic (6), which coincides with the demodulation frequency, at the resonant frequency at the previous instant, so that the phase of the signal (S1 ) sent by the photoreceptor (9). At each instant of time, the flow of particles inside the capillary (3) causes the resonant frequency to vary, so the resonant frequency is continuously recalculated. The PLL configuration is very useful when you want to measure changes in the resonance frequency greater than 1kHz and / or that occur in times of the order of one second.
[0125] The processor (11) therefore receives the modulated signal (AC) in the form of an oscillation spectrum, from which it is able to determine the resonance frequency, which has changed with respect to the initial one, due to the movement of the carrier liquid inside the capillary (3) and the flow of particles through said capillary (3). In this way, it is possible to determine the resonant frequency of the capillary (3) at each instant of time.
[0127] Alternatively, the calculation of the resonance frequency can be performed with an open-loop configuration, where the frequency of the signal (S2) that is sent to the piezoelectric ceramic (6) is fixed, so that by means of the amplifier (10) synchronous, the phase change of the signal (S1) provided by the photoreceptor (9), between the empty capillary (3) and the capillary (3) with a particle inside, is measured with a sampling frequency of up to 2kHz and with a demodulation frequency equal to the resonant frequency of the capillary (3).
[0129] Taking into account that the phase curve as a function of frequency presents a linear area for frequencies similar to the resonance frequency, the measurement of the phase change of the signal (S1) at each instant of time can be converted into a measurement of change of frequency at each instant of time, from which to obtain the resonance frequency at each instant of time.
[0130] The open loop configuration is very useful when you want to measure small changes in the resonance frequency that occur in times of the order of 100ms, being this configuration, in that operating range, faster and also eliminating noise problems.
[0132] Then, by means of a calibration process, a linear adjustment can be made of a curve that represents the change in the resonance frequency as a function of the variation in the mass of the capillary (3). In this way, the buoyant mass of the sample flowing inside the capillary (3) can be calculated based on the variations in the resonance frequency, calculated using the closed-loop configuration or, preferably, the open-loop configuration. .
[0134] On the other hand, the unmodulated component of the signal provides information, in this case the reflectivity of the sample that flows inside the capillary (3). At each instant of time, the passage of the sample particles modifies the intensity of the reflected beam, leaving a signal that shows the dimension and refractive index of the particles.
[0136] In this way, the system (1) allows the measurement of the buoyant mass and the light scattering produced by a given sample, so that both measurements allow the unequivocal differentiation of different particles, with different density and volume, even though they have the same buoyant mass.
[0138] The system (1) of the invention also maintains its high precision in the face of changes in the density of the carrier liquid and its refractive index.
[0140] Additionally, the system (1) can be used for the measurement of particles in aqueous suspension, for example, for the analysis of contaminating agents, or cells in a physiological environment, for the diagnosis of diseases.
[0142] Figure 1 also shows a camera (13) with an infinite objective that allows to see in real time the changes that occur inside the capillary (3). In this way, the passage of particles through the capillary channel (3) can be more effectively controlled, and ensure that the laser is well directed.
[0143] The camera (3) comprises a sensor (14) with photoelectric cells known as a charge coupled device (CCD) and a white light source (15).
[0145] Figure 3 shows an example of buoyant mass measurements made on 12.5pm polymethylmethacrylate (PMMA) particles and 8.5pm silica particles. Where the vertical axis represents the amount of particles of the same type in percentage and the horizontal axis, the buoyant mass. The buoyant mass of both types of particles is very similar, which makes it practically impossible to distinguish with this measurement the presence of two different types of particles.
[0147] Figure 4 shows an example of the measurements made of the reflectivity of PMMA and silica particles, analyzed in Figure 4. Where the vertical axis represents the amount of particles of the same type in percentage and the horizontal axis, the normalized reflectivity . The reflectivity of both particles is very different, allowing a clear distinction between both types of particles.
[0149] Figure 5 shows a map representing the buoyant mass versus the change in normalized reflectivity. This graph combines the mechanical and optical data obtained by the system (1) of the invention. Unlike the graph in Figure 3, which is typically obtained from a mechanical analysis device such as those found in the state of the art, in this new graph a clear distinction can be made between different types of particles, such as They are made of PMMA and silica with different sizes, but which have a very similar buoyant mass and are difficult to differentiate without the contribution of optical measurements.
[0151] Figure 6 also shows a graph that represents the capacity of the system (1) to distinguish between the measurements of a particle and several of them, when they pass through the capillary at the same time. Where the vertical axis represents the normalized change in reflectivity, in the case of optical measurements, and the resonant frequency variation, in the case of mechanical measurements, and the horizontal axis represents time. It could be the case that two particles pass very close together through the capillary, so that both are in the suspended area at the same time. This significantly modifies the results, since the measurement of buoyant mass is no longer one, but two particles. To avoid confusing this measurement with the measurement of a single larger buoyant mass particle, use can be made of optical measurements.
[0152] Because the measurement of optical parameters is carried out only in the area where the beam (8) of the laser (7) hits, while the mechanical measurements are affected by particles found at any point along the length of the capillary ( 3) suspended, the optical signal has a shorter time duration than the mechanical signal. This allows, as shown in Figure 6, to use the optical measurements to determine the number of particles that pass through the capillary (3) at the same time.
[0154] In a particular embodiment, the optical measurements are sensitive in a region coinciding with the size of the laser beam, in this case around 20pm, while the mechanical measurements are sensitive in a region of 500pm. The reflectivity signal allows us to know if an event measured in the resonance frequency signal is due to one or more particles and, in the case of several particles, to separate the contribution of each one of them to the measure of change in resonant frequency. If two particles pass through the capillary at the same time separated by a distance less than 20pm, they will not be distinguishable in the reflectivity signal and, in this case, it will be a dimer.
[0156] The analysis system shown is also capable of uniquely classifying and differentiating cell lines. For example, in the case of the MCF-10A, healthy, and MCF-7, cancer cell lines, they have a very similar mean buoyant mass, specifically 110 pg with a standard deviation of 40 pg and 90 pg with a standard deviation of 60 pg, respectively, so they are indistinguishable by using only measurements derived from resonant frequency. However, by simultaneously acquiring the buoyant mass and the reflectivity of each particle measured by the system of the invention, the cells of both cell lines can be unequivocally distinguished.

权利要求:
Claims (19)
[1]
1. A sample analysis system (1) characterized in that it comprises:
- a substrate (2);
- a hollow elongated structure (3), having two ends (4);
- two polymeric supports (5), attached to the ends (4) of the elongated hollow structure (3) and attached to the substrate (2);
- at least one piezoelectric device (6), coupled to the substrate (2) and in contact with the elongated hollow structure (3), and adapted to produce vibrations in said cavity (3);
- at least one laser (7), configured to emit a laser beam (8),
- at least one photoreceptor (9), adapted to absorb the laser beam (8), which passes through the cavity (3) before reaching the photoreceptor (9), and produce an electrical signal (S1) associated with the intensity and frequency of modulation of the received laser beam (8),
- at least one amplification module (10), coupled to the piezoelectric device (6) and to the photoreceptor (9), and configured to amplify and filter the electrical signal (S1) provided by the photoreceptor (9), and to separate the modulated (AC) and unmodulated (DC) component of the signal (S1); Y
- a processor (11), coupled to the amplification module (10), and configured to process the amplified and filtered electrical signal (S1) and obtain, from the modulated component (AC) of the signal (S1), a measurement of the resonance frequency of the cavity (3) at each instant of time, which varies as a function of the variation of the buoyant mass of the sample, and from the unmodulated component (DC) of the signal (S1) the reflectivity inside the cavity (3) at each instant of time;
where the processor is further configured to provide the piezoelectric device (6), through the amplification module (10), an excitation signal (S2) at the resonance frequency, so that said piezoelectric device (6) produces vibrations in the cavity (3) at the resonant frequency at each instant of time.
[2]
2. The analysis system according to claim 1, characterized in that the amplifier (10) is of the synchronous type.
[3]
The analysis system according to claim 1, characterized in that the excitation signal (S2), which is sent to the piezoelectric device (6), is a sinusoidal signal at the resonant frequency.
[4]
4. The analysis system according to claim 1, characterized in that the piezoelectric device (6) is made of ceramic.
[5]
The analysis system according to claim 1, characterized in that the hollow elongated structure (3) is made of fused silica.
[6]
The analysis system according to claim 1, characterized in that the final diameter of the elongated hollow structure (3) is approximately 50pm.
[7]
The analysis system according to claim 1, characterized in that the polymeric supports (5) are made of SU8 photoresin.
[8]
The analysis system according to claim 1, characterized in that the laser (7) used is of the Helium-Neon type or a diode laser.
[9]
The analysis system according to claim 1, characterized in that it further comprises a pump (12), configured to control the flow inside the elongated hollow structure (1).
[10]
The analysis system according to claim 1, characterized in that it further comprises a camera (13) with an infinite objective having a sensor (14) with photoelectric cells, a source of white light (15) and a monitor, which allows you to see inside the cavity (3).
[11]
The analysis system according to claim 1, characterized in that it further comprises at least one beam splitter (20).
[12]
12. The analysis system according to claim 1, characterized in that it also comprises at least one filter (19) of neutral density.
[13]
The analysis system according to claim 1, characterized in that it further comprises at least one optical isolator (18).
[14]
14. Manufacturing method of a hollow elongated structure (3), characterized in that it comprises the steps of:
- providing a hollow elongated structure (3), with two ends (4), having a protective layer (16),
- removing the protective layer (16) in the central area of the elongated hollow structure (3), heating said area (3),
- applying an axial tension to the heated elongated hollow structure (3), to plastically deform its central area, until a determined diameter is achieved,
- Adhering two or more polymeric supports (5), manufactured on a reflective substrate (2), to each of the ends (4) of the deformed hollow structure (3), leaving the structure (3) suspended on the reflective substrate by means of the polymeric supports (5).
[15]
Manufacturing method according to claim 14, characterized in that the step of adhering polymeric supports (5) to each of the ends of the cavity (3) is carried out by means of a centrifugal coating and using optical lithography, which applies a pattern defined polymer in cavity (3).
[16]
16. Sample analysis procedure characterized in that it comprises the steps of:
- providing a system (1) according to any of claims 1 to 13;
- actuating the laser (7), which emits a laser beam (8), which hits the hollow structure (3) containing the sample and is collected in the photoreceptor (9);
- generating a signal (S1), by means of the photoreceptor (9);
- amplifying and filtering the signal (S1) generated, by means of the amplification module (10);
- dividing the amplified and filtered signal (S1), by means of the amplification module (10), into a modulated component (AC) and an unmodulated component (DC);
- processing the modulated component (AC) of the signal (S1), by means of a processor to obtain a measure of the resonance frequency of the cavity (3) at an instant of time;
- calculating from the measured resonance frequency a buoyant mass value at an instant of time;
- processing the unmodulated component (DC) of the signal (S1), by means of a processor to obtain a measure of the reflectivity of the sample inside the cavity (3) in an instant of time;
- generating an excitation signal (S2) at the calculated resonance frequency, by means of the processor; Y
- transmitting the excitation signal (S2) generated to the piezoelectric device by means of the amplification module (10).
[17]
17. Use of the analysis system according to claim 1 for the identification and classification of different cell populations present in an isolated biological sample.
[18]
18. Use according to claim 16, for the identification and classification of tumor cells and non-tumor cells.
[19]
19. Use according to any of claims 16 or 17, wherein the cells are epithelial cells of breast tissue.

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US6454710B1|2002-09-24|Devices and methods for monitoring an analyte

---

US8088595B2|2012-01-03|Method for glucose monitoring using fluorescence quenching

---

US6256522B1|2001-07-03|Sensors for continuous monitoring of biochemicals and related method

---

ES2891558T3|2022-01-28|Deep tissue flowmetry using diffuse speckle contrast analysis

---

ES2335293T3|2010-03-24|COMBINED DEVICE FOR MEASURING THE LEVEL OF GLUCOSE IN BLOOD FROM AN ACULAR FLUID.

---

ES2841404T3|2021-07-08|Respiratory gas analysis apparatus for NMR analysis of substances in a sample and procedure for respiratory gas analysis

---

US20050221277A1|2005-10-06|Intracorporeal substance measuring assembly

---

ES2701475T3|2019-02-22|Method and apparatus for in vivo transdermal measurement by Raman spectroscopy

---

ES2879289T3|2021-11-22|Microscope three-dimensional focusing device and method

---

Li et al.2018|Perspective: Biomedical sensing and imaging with optical fibers—Innovation through convergence of science disciplines

---

ES2786848B2|2021-04-22|Mechano-optical analysis system, manufacturing method, use for identification and classification of cell populations, and mechano-optical analysis procedure

---

KR100937219B1|2010-01-20|Excitation Induced Optical Molecular Imaging System

---

EP3218717B1|2019-02-27|Dynamic switching biosensor

---

Wang et al.2005|Dual-capillary backscatter interferometry for high-sensitivity nanoliter-volume refractive index detection with density gradient compensation

---

Dinish et al.2019|Surface‐enhanced Raman scattering‐active photonic crystal fiber probe: Towards next generation liquid biopsy sensor with ultra high sensitivity

---

EP2175258B1|2017-04-12|Measurement instrument of optical characteristics for sample flowing in passage

---

ES2803216T3|2021-01-25|Characterization of particles in open optical resonator cavity

---

JPWO2012046423A1|2014-02-24|Method for measuring the concentration of biological components contained in a living body

---

Tcherniavskaia et al.2010|Detection and identification of microparticles/nanoparticles and blood components using optical resonance of whispering-gallery modes in microspheres

---

Ben Mohammadi et al.2015|Clinical performance of a low cost near infrared sensor for continuous glucose monitoring applied with subcutaneous microdialysis

---

ES2697131T3|2019-01-22|Integrated optical sensor circuit

---

Campagnolo2013|Optical feedback interferometry sensing technique for flow measurements in microchannels

---

US20160081605A1|2016-03-24|Use of a barrier contact medium for chemo-chemo-optical sensors in transcutaneous applications

---

ES2603603B1|2017-12-12|Interfacial rheometer for magnetic needle shearing and its system and method of operation

同族专利:

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公开号 | 公开日

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ES2786848B2|2021-04-22|

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WO2020208285A1|2020-10-15|

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EP3954982A1|2022-02-16|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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WO2016055683A1|2014-10-10|2016-04-14|Consejo Superior De Investigaciones Cientificas |Spectrophotometer|

---

US20170089881A1|2015-09-29|2017-03-30|The Board Of Trustees Of The University Of Illinois|System and method for high-throughput, optomechanical flow cytometry|

---

CN207540944U|2017-08-23|2018-06-26|中国科学院苏州生物医学工程技术研究所|For the micro-fluidic chip system of rare cell screening|

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ES201930327A|ES2786848B2|2019-04-10|2019-04-10|Mechano-optical analysis system, manufacturing method, use for identification and classification of cell populations, and mechano-optical analysis procedure|ES201930327A| ES2786848B2|2019-04-10|2019-04-10|Mechano-optical analysis system, manufacturing method, use for identification and classification of cell populations, and mechano-optical analysis procedure|

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EP20787808.3A| EP3954982A1|2019-04-10|2020-04-13|Mechano-optical analysis system, method for producing same, use for identifying and classifying cell populations and mechano-optical analysis method|

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PCT/ES2020/070236| WO2020208285A1|2019-04-10|2020-04-13|Mechano-optical analysis system, method for producing same, use for identifying and classifying cell populations and mechano-optical analysis method|