Method for hydrodynamically assisted multi-parametric analyte spectroscopy determination

Abstract

The method uses a suspended resonant microcapillary device and simultaneously obtains three parameters of the analyte: mass, size and refractive index, enabling real-time unambiguous classification of the flowing analyte based on the resonant frequency shift and changes in the transparency microcapillary reflectance. The method comprises the following stages: obtaining a measurement of the reflectance of the sample analyte within the capillary at each moment in time; obtaining a mechanical reference signal (T t ) of the resonant frequency of the microcapillary as a function of time caused by the sample analyte; and detecting the region of passage of the particle through the capillary and obtaining the point of the center of the region illuminated by the end of the analyte, thus obtaining the optical signal ΔT.

Description

[0001] Purpose of the invention

[0002] The purpose of this invention is to provide a method for hydrodynamic-assisted multi-parameter analyte spectroscopy, which utilizes a suspended resonant microcapillary device and allows for the simultaneous acquisition of three parameters of the analyte: mass, size, and refractive index. This enables the precise classification of real-time flowing analytes based on the resonant frequency displacement of the resonant microcapillary and changes in its reflectivity. Background Technology

[0003] High-throughput classification and characterization of individual micron and nanoparticles in the physiological environment is of particular interest in many different fields such as environmental monitoring, clinical trials, nanotoxicology, and cell biology. For these purposes, the use of microfluidic devices has been shown to be a robust technique for passive particle classification, achieving throughputs up to 10-10. 7 Flux operation of particles per minute simply utilizes the force exerted on the particles by the moving fluid.

[0004] These hydrodynamics depend on the shape and size of the particles, ultimately causing them to move with the fluid. While these devices allow for particle analysis based on geometric properties, static microfluidic devices cannot distinguish particles with the same shape but composed of different materials. Therefore, it must be combined with other techniques (e.g., optical methods for measuring refractive index) to distinguish between particles of the same shape.

[0005] Furthermore, nanomechanical resonators have proven to be a powerful analytical tool because they can measure a variety of physical properties (force, mass, stiffness, etc.) with extremely high sensitivity by recording changes in the mechanical resonant frequency of a vibrating structure.

[0006] However, when the device is immersed in a liquid, viscous friction is detrimental to this high sensitivity because the liquid mass is displaced during the oscillation cycle. Suspended microchannel resonators (SMRs) have been developed for analyzing particles in liquid environments, overcoming the aforementioned problems by incorporating microfluidics and nanomechanics.

[0007] The SMR method consists of a nanomechanical resonator with integrated internal microchannels, which allows the resonator to vibrate in a gaseous or vacuum environment while particles are characterized in a liquid flowing through the microchannels. Because viscous friction is significantly reduced by placing the liquid inside the resonator, the resolution of buoyancy mass is improved, reaching up to 10 atks in the latest generation of devices operating in a vacuum.

[0008] In SMR devices, transparent microcapillary resonators (TMRs) not only combine nanomechanical resonators with microfluidic channels but also allow for the measurement of the optical properties of particles flowing within these channels. These devices have proven to be a promising alternative to SMR for mass detection, with particle-based optical characterization introducing a new source of information about the particles. This makes this mechatronic particle detection technique a highly reliable method for particle classification, even when particles have similar masses.

[0009] Specifically, document US2012 / 0118063A1 is known, which describes a method for determining the buoyant mass and deformability of a cell. The method includes introducing a cell into a microchannel resonator, the microchannel resonator including a contraction near a distal position in the resonator. A first frequency change in the resonator, which is related to the buoyant mass of the cell, is monitored. The transit time of the cell through the contraction is measured by monitoring a second frequency change, which is a result of the cell's positional change as it passes through the contraction, thereby determining deformability based on the measured buoyant mass and transit time.

[0010] In existing work, prior information about particle size still needs to be obtained using other techniques to calculate multiple particle parameters, such as refractive index, or each particle needs to be measured twice in liquids with different properties to obtain parameters such as mass density. In particular, due to the relationship between cell density and activity, density has been shown to be a parameter of interest for characterizing cell life cycle in previous work using SMR.

[0011] Furthermore, current technology requires measuring the buoyant mass of the same particle in two media with different densities in order to measure its density, which significantly reduces the measurement throughput. Summary of the Invention

[0012] The present invention provides a hydrodynamic-assisted method for the spectroscopic determination of multiple parameters of analytes, utilizing a suspended microcapillary device, particularly a transparent microcapillary resonator (TMR). A fluid and multiple suspended analytes of interest circulate through the interior of the device, and using this method, three parameters can be measured simultaneously: buoyant mass, particle size, and refractive index. These parameters are obtained by measuring the frequency displacement and the change in reflectivity of the TMR device measured at different instants in time. This method allows for the precise classification of analytes flowing in real time.

[0013] The proposed method allows for the measurement of intensive parameters, such as mass density, for the development of real-time analyte spectrometry in liquids. Specifically, a hydrodynamic analyte approach is proposed to obtain ordered particles with known positions as they pass through the free region of the TMR device. Knowing the analyte's position inside the tube beforehand facilitates the analysis of signals caused by changes in frequency mechanical displacement and reflectivity resulting from the passage of particles through the TMR, which ultimately allows for the determination of the analyte's size.

[0014] This method of simultaneously acquiring particle diameter measurements along with buoyancy mass and reflectivity allows for high-throughput three-parameter analyte characterization techniques and the calculation of strength parameters such as mass density.

[0015] As indicated, the method utilizes a sample analysis device of the transparent microcapillary resonator (TMR) type, comprising: a substrate; a capillary-type elongated transparent hollow structure having two ends through which fluid and suspended analyte of interest circulate; two supports attached to the ends of the capillary; a vibration module, preferably a piezoelectric device, coupled to the substrate and adapted to generate vibrations within the substrate; a laser that emits a light beam into the capillary; and a photosensitive sensor adapted to receive the light beam passing through the capillary.

[0016] The method of the present invention utilizing the described apparatus includes the series of stages described below.

[0017] First, the laser is activated, emitting a beam that strikes a capillary containing the sample of interest, which is a fluid containing suspended analytes. The beam passes through the capillary, is reflected by the substrate, and is picked up by a photosensor.

[0018] The next stage involves generating a first signal using a photosensor, and then amplifying and filtering the first signal. The amplified and filtered signal is then separated into modulated and unmodulated components.

[0019] The unmodulated component of the signal is processed to obtain a continuous measurement of the capillary reflectivity over time, which generates a change in reflectivity signal (reflection power) as a particle passes through an area illuminated by a laser beam. Based on this change in reflection power, the time difference (ΔT) between the point where the particle enters and leaves the illuminated area can be obtained.

[0020] Next, the modulation component of the signal is processed to obtain a reference mechanical signal (T) whose resonant frequency varies with time due to the passage of the particle. t The maximum change in frequency depends on the buoyant mass of the particle, and the change in time depends on the velocity (which is obtained by multiplying the reciprocal of the transit time by the length of the capillary).

[0021] As particles pass through the capillary, the resonant frequency decreases due to the added mass. Existing literature only analyzes the maximum changes in resonant frequency and reflected power; in this application, both curves are adjusted to save time during passage: T t And ΔT. The suspension length of the capillary is precisely known, which is understood as the length of the capillary including the supports. By knowing the distance and time taken to travel through said space, the particle velocity can be calculated very accurately.

[0022] Starting with the length of the capillary tube, which lies between the two supports of the device, the mechanical and optical signals, and the particle size can be calculated as follows:

[0023] Attached Figure Description

[0024] To supplement the ongoing description and to aid in a better understanding of the features of the invention according to preferred practical exemplary embodiments, a set of drawings is attached as an integral part of the description, in which the following is described in an illustrative and non-limiting manner:

[0025] Figure 1 shows a diagram of a transparent microcapillary resonator (TMR), which belongs to the prior art of mechatronic measurement.

[0026] Figure 2 A cross-sectional view of a capillary located between two supports and traversed by a laser beam is shown, and a graph of the resonant frequency signal of a particle traversing the capillary is shown in the lower portion.

[0027] Figure 3 The graphs showing the optical and mechanical signals of the particles are shown.

[0028] Figure 4 Experimental measurements of the mass density of individual MCF-7 cells (squares) corresponding to human mammary epithelial cells are shown.

[0029] Figure 5 The particle size distribution of a sample consisting of 67 PMMA particles and 192 silica particles is shown. Detailed Implementation

[0030] The following is based on Figure 1 to... Figure 5 Preferred embodiments of a method for hydrodynamic-assisted multiparameter analyte spectroscopic analysis are described.

[0031] The method of the present invention utilizes a transparent microcapillary resonator (TMR) device (12), as shown in the overall view of FIG1 and Figure 2As shown in the detailed view, and includes: a substrate; an elongated transparent hollow structure in the form of a capillary having two ends (3, 4) through which fluid and suspended analytes of interest circulate; a support (2) coupled to the ends of the capillary; a piezoelectric device coupled to the substrate and in contact with the capillary, adapted to generate vibrations in the capillary; a laser (1) emitting a light beam; and a photosensitive sensor (6) adapted to receive the light beam passing through the capillary before reaching the photosensitive sensor (6).

[0032] The analyte can be the biological entity of interest, such as cells or bacteria, or inorganic particles.

[0033] As shown in Figure 1, the light beam from the laser (1) passes through the beam splitter (5). After the beam splitter (5), the light is focused into the capillary (12) by the microscope objective (11). The light beam reflected in the substrate returns through the capillary and the microscope objective (11) until it reaches the beam splitter (5), which directs the beam towards the photosensitive sensor (6). The photosensitive sensor picks up the beam beam altered by passing through the capillary and generates a signal associated with both the motion of the capillary and the properties of the transparent capillary, as well as any material passing through the capillary at that moment.

[0034] The capillary has an outer diameter of 44 μm and an inner diameter of 34 μm, and is obtained by locally elongating a fused silica capillary with an outer diameter of 350 μm and a wall thickness of 50 μm. The substrate is made of silicon, and the support (2) is a photolithographed polymer pad to obtain a 500 μm long suspension region for the capillary, in which the capillary can mechanically oscillate in a manner similar to a guitar string, like a resonator.

[0035] The mechanical modes of the capillaries are excited by means of piezoelectric actuators, and their resonant frequencies are monitored in real time by means of an interferometric reading system and a lock-in amplifier (9), as shown in Figure 1.

[0036] This interferometric measurement system also allows monitoring of the power of light reflected by the capillary to obtain information about the optical properties of the analyte in the flow by analyzing the scattered light in real time.

[0037] In addition, the capillary is pressurized by a first nitrogen pressure pump (10), which allows the flow to be controlled by establishing a controlled pressure difference through a microfluidic resistor (8).

[0038] Finally, the outlet end (4) of the capillary is linked to a second nitrogen pump (7), which allows a minimum pressure difference of 5 mbar to be established between the inlet end (3) of the capillary and the outlet of the microfluidic resistor (8). The microfluidic resistor (8) allows for better control of the flow. This allows for control of the rate at which the particles or analytes of interest pass through the suspended capillary.

[0039] In itself, the method of the present invention utilizing the disclosed apparatus (12) aims to include the following stages:

[0040] An actuated laser (1) emits a beam that strikes a capillary containing the sample and is picked up by a photosensitive sensor (6).

[0041] A first signal is generated by means of a photosensitive element (6), and the first signal is amplified and filtered.

[0042] The amplified and filtered signal is divided into modulated and unmodulated components.

[0043] The unmodulated component (DC) of the signal is processed by a processor to obtain a measurement of the sample reflectance in the capillary at each moment.

[0044] Particles were detected passing through the area illuminated by the laser (1) and adjusted to a double Gaussian. The two Gaussian centers ( Figure 3 Points 1 and 3 on the dashed curve mark the center of the illuminated area through which each end of the particle passes; the difference between these points is the value ΔT. Similarly, there is a maximum value between points 1 and 3 on the dashed curve (…). Figure 3 Point 2 on the dashed curve, the maximum value corresponds to the center of the particle through the center of the illuminated area, from which the value of the maximum change in reflectivity can be obtained;

[0045] The modulation component is processed to obtain a mechanical reference signal (Tt) that describes the resonant frequency as a function of time. The change in resonant frequency over time is measured. As the particle passes through the capillary, the resonant frequency decreases due to the added mass. Adjusting the curve saves time. Figure 3 The lower curve (mechanical signal). The precise suspension length of the capillary (500 μm) is known because it was manufactured using photolithography. By knowing the distance and time traveled, the particle velocity can be calculated very accurately;

[0046] Particle size is calculated as follows:

[0047]

[0048] Figure 2 A detailed view of the device (12) is shown, in which particles flow through a free region between supports (2) of the capillary, causing a shift in the resonant frequency signal. The amplitude (Δf) of this decrease depends on the buoyant mass of the particle, while its width (transit time, T) t It depends on the particle's velocity.

[0049] Assuming a constant velocity, the flowing analyte (whose diameter is very small compared to the length of the capillary's suspension region) tests the mechanical modes by following its contour. Therefore, for the umpteenth mechanical mode, the frequency displacement signal tracked over time in the lock-in amplifier (9) can be written as:

[0050]

[0051] (Equation 1)

[0052] f n (t) is the frequency of the infiniteth bending mode over time, f n0 It is the natural resonant frequency of the mechanical mode of the resonator, ψ n，max It is the value of the normalized mechanical mode shape at its maximum amplitude, the time t0 particle is at the center of the suspension region, and β. n These are the feature values ​​of the pattern (the first four feature values ​​are β). n =4.7300, 7.8532, 10.9956, 14.1372).

[0053] Figure 2 The dashed lines in the diagram show the frequency displacement caused by the particles in the first four bending modes of the device (12). Without limiting the foregoing generality, the basic mode is analyzed. Adjusting the time-frequency displacement according to Equation 1 allows the buoyant mass or velocity of the particles to be obtained, which is directly obtained as the reciprocal of the transit time multiplied by the length of the capillary's suspension region.

[0054] The motion of particles is a result of the forces exerted by the liquid on the particle surface. Therefore, the following analysis examines the dependence of hydrodynamics on particle size, which will allow for classification based on the radius of the analyte.

[0055] To verify this experimentally, a uniform aqueous suspension of micron-sized particles was introduced into the device (12). If the particles were only subjected to the tension of the fluid in the flow direction, they would follow a random distribution in the radial direction of the tube. However, in laminar flow, each particle also experiences a displacement orthogonal to the flow direction.

[0056] This is the result of the balance between two lift forces exerted on the moving particle by the fluid: shear gradient (centrifugal) and wall-induced lift (centripetal). To calculate the equilibrium position of the particle, finite element simulations were performed to calculate the net force at the top and direction of spherical particles with nominal diameters of 6.8 μm and 12.4 μm.

[0057] A cylindrical tube was simulated, its dimensions mimicking the experimental suspension region of a capillary, with a pressure difference established between its ends (3, 4). Simulations showed that the dominant force is always centrifugal, making the equilibrium position in this configuration contact with the capillary wall. Therefore, randomly distributed particles introduced into the capillary are subjected to lift, causing all particles to settle onto the capillary wall. Once settled, the thrust and pull of the particles reach a steady state. This balance between hydrodynamic forces causes the particles to pass through the suspension region at a constant velocity, allowing adjustment of the frequency variation according to Equation 1. This particle position was also confirmed by optical inspection using a CCD (charge-coupled device) camera.

[0058] The device (12) and the measurement system (9) also enable the monitoring of the power of the light (optical signal) reflected by the device (12) over time. Therefore, when a particle passes under the area illuminated by the laser (1), it produces a reduction in this optical signal due to the scattered light, which can be used to obtain additional information about the particle.

[0059] like Figure 3 As shown, this drop in the optical signal consists of two peaks, with a local maximum corresponding to the particle located at the center of the laser beam (1), and a local minimum corresponding to the light scattered by the edge of the particle: when each of them is aligned with the center of the illuminated area. Therefore, given the particle velocity obtained by adjusting the mechanical signal, the particle diameter can be calculated by measuring the time difference between the two local minimums of the optical signal.

[0060] like Figure 5 As shown, a size distribution of 11 ± 2 μm was obtained for PMMA particles and 7 ± 2 μm for silica particles. These values ​​reproduce the results obtained by analyzing scanning electron microscopy (SEM) images of the same population, which were 12.4 ± 0.2 μm for PMMA particles and 6.8 ± 0.4 μm for silica particles.

[0061] Therefore, the objective of this method is to simultaneously measure three distinct and independent parameters of each individual particle (the buoyant mass, velocity, and reflectivity of the analyte) at extremely high throughput (up to 300 analytes per minute). When plotted on a three-dimensional scatter plot, the mixture of the above analytes can be clearly identified, and highly reliable analyte identification can be performed based on three independent parameters.

[0062] Furthermore, due to the relationship between cell density and its activity, mass density has been shown to be a parameter of interest characterizing cell life cycle. Cell density is known to vary during the cell life cycle. This is particularly important in human cells, where density remains constant throughout the life cycle, except during mitosis, when the cell undergoes a rapid increase in cell volume and a corresponding decrease in its mass density.

[0063] Therefore, there is a dependence on mass density, with larger cells exhibiting lower density. Measuring the density of human epithelial breast cells in MCF-7 human breast adenocarcinoma cells (mean 1.11 ± 0.08 g-ml-1) served as a proof-of-concept for the ability of this mechano-optical technique to characterize pathological cells.

[0064] from Figure 4 The experimental measurements shown indicate an observed dependence of density on its size, consistent with results obtained in previous work. The dashed lines represent adjustments to the experimental values, illustrating the dependence of the measured density on size. This result confirms the capability of the method described in this invention and opens the door to separate studies of the cell cycle.

[0065] In summary, this application discloses a method for multi-parameter analyte spectral analysis using a suspended microcapillary resonator, which simply follows the flexural mechanical resonance mode (preferably the basic mode) and the power reflected by the resonator.

[0066] By monitoring the device's mechanical frequency while simultaneously measuring buoyancy mass and velocity, and by measuring changes in reflectivity via optical signals, differentiation between populations of analytes is possible. Similarly, by combining the results of these measurements, other physical parameters of interest, such as the size or density of the analytes flowing through the device, can be obtained in high throughput. These parameters have proven to be of great interest for the characterization of biological entities such as cells.

Claims

1. A method for hydrodynamic-assisted analyte spectral determination, which utilizes a transparent microcapillary resonator (12), the transparent microcapillary resonator comprising: substrate; A transparent capillary having two ends (3, 4), through which fluid and one or more suspended analytical substances flow; a support (2) connected to the ends (3, 4) of the capillary; a piezoelectric module connected to the substrate and in contact with the capillary, adapted to generate vibration in the capillary; and a laser (1) emitting a beam of light. and a photosensor (6) adapted to receive the light beam, the light beam passing through the capillary before reaching the photosensor (6); wherein the method includes the following steps: The laser (1) is actuated, the laser emits a beam, the beam strikes the capillary and is picked up by the photosensitive sensor (6). A first signal is generated by means of the photosensor (6), and the first signal is amplified and filtered. The amplified and filtered signal is divided into modulated and unmodulated components. The unmodulated component of the signal is processed, and the measured value of the reflected light power at each moment is obtained. The analyte is detected passing through the capillary in the area illuminated by the laser (1), and the time difference between the point where the particle enters the illuminated area and the point where it leaves the illuminated area is obtained by measuring the change in the reflected light power. ,as well as Process the modulation component and obtain a mechanical reference signal showing the resonant frequency of the capillary as a function of time. ), The analyte size is calculated using the following expression: = in It is the free length of the capillary included between the support members (2).

2. The method of claim 1, further comprising calculating the velocity of the particle as... With the mechanical reference signal The stages of the business between them It is the suspension length of the capillary between the support members (2).

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