WO2014198813A1

WO2014198813A1 - Method and device for determining undissolved particles in a fluid

Info

Publication number: WO2014198813A1
Application number: PCT/EP2014/062200
Authority: WO
Inventors: Michael Bassler; Christian Sommer; Stephan Quint; Peter Spang
Original assignee: Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V.
Priority date: 2013-06-12
Filing date: 2014-06-12
Publication date: 2014-12-18
Also published as: DE102013210952A1; DE102013210952B4

Abstract

The invention relates to a method and device for differentiating, classifying or for determining undissolved particles located in a flowing fluid, which fluid has the density p and the viscosity µ, in which the fluid comprising the particles is conveyed through a measurement channel (30, 40, 60), wherein the fluid comprising the particles flows through a measurement section (42, 80). The fluid is conveyed through the entire measurement channel (30, 40, 60) at an average flow speed (I), at which the Reynolds number RK (II) of the channel assumes values between 0.1 and 500, wherein d_hyd designates the hydraulic diameter of the measurement channel (30, 40, 60). The migration speed of the particles is determined along the measurement section (42, 80), and the flow behaviour of the particles is analysed with the aid of the determined migration speed and is used for differentiating, classifying or determining the particles.

Description

Method and device for determining undissolved particles in a fluid

description

The invention relates to a method and a device for differentiation, for the classification or for the determination of undissolved particles present in a flowing fluid, which fluid has the density p and the viscosity μ. The method provides that the fluid is conveyed with the particles through a measuring channel, wherein the fluid flows through a measuring path with the particles. Accordingly, the device has a measuring channel, through which the fluid can be conveyed with the particles and which comprises a measuring section, and a pumping device for conveying the fluid with the particles through the measuring channel.

The invention has its origin in the field of flow cytometry. In simple terms, this is a process in which the fluid with the particles, in this case biological cells, is pumped through a flow cell in which they are excited to emit fluorescence by means of incident light. The cells are therefore usually previously labeled with fluorescent substances. The emitted light is picked up by a detector as the particle passes through the flow cell. Between the measuring channel of the flow cell and the detector is a mask, also shadow mask, with which the emitted light is modulated in time due to the relative movement of the particles to the mask. Such a signal is recorded at a certain sampling rate. The result is called sampling.

The correspondingly temporally modulated detection signal is then subjected to a correlation analysis in which a reference sequence is mathematically formed from the known pattern of the mask, this in its absolute length set (scaled) and compared or folded with the sampling. The comparison is done by shifting the scaled reference sequence across the signal. The process is iteratively repeated at other scales until a match is found. The correspondence is shown for each particle passing through the detection cell in the form of a correlation peak, by means of which the particles can be counted, for example.

Moreover, it is known to use this arrangement to measure the intensity of the emitted light and / or additionally a scattering signal emanating from the particle, from which further information about the particle can be obtained. For example, such a statement about its size can be made. Reference is made by way of example to the documents US 2010/0016335 A1, US Pat. No. 8,373,860 B2, US 2008/0181827 A1, US 2008/0183418 A1 and US Pat. No. 7,763,856 B2, which deal with this technology.

The present invention proposes an alternative or additional method with which particle properties in a flowing fluid can be determined in order, for example, to differentiate different particles, to be able to classify the particles or even to determine the particles in the fluid. The inventive method for differentiation, for classification or for determining in a flowing fluid undissolved particles of the type described above provides that the fluid is conveyed through the entire measuring channel with a mean flow velocity v, in which the channel Reynolds number _ p 'v ^■ d _hyd

_Takes values between 0.1 and 500, where _{dhyd denotes} the hydraulic diameter of the measuring channel. The migration speed of the particles or their equivalent is determined over the measuring section and the flow behavior of the particles is analyzed on the basis of the determined migration rate or its equivalent and used for differentiation, classification or determination of the particles.

The invention further provides that the device of the type mentioned has a control device associated with the pump device, which is set to set a flow velocity v for the fluid through the entire measuring channel, wherein the channel Reynolds number

_ p - v ^■ d _hyd

_Takes values between 0.1 and 500, where _{dhyd denotes} the hydraulic diameter of the measuring channel. Furthermore, the device according to the invention has a measuring device arranged along the measuring section, which is set up to ascertain the migration speed of the particles or their equivalent over the measuring section, and an analysis device which is set up, to determine the flow behavior of the particles on the basis of the determined migration speed or its equivalent analyze and output for differentiation, classification or determination of the particles.

The finding underlying the invention is that identical particles have a significant flow behavior in the fluid only under the aforementioned conditions, as explained in more detail below with reference to FIGS will be. Namely, by observing the above experimental conditions, the particles arrange in the flowing fluid due to an interaction with the fluid on certain flow paths in the measurement channel, for example, entraining them with a significantly narrower velocity distribution in the fluid than outside said conditions Case is. This is attributed to the hydrodynamic properties of the particles, the term "hydrodynamic particle property" in the sense of this document being understood in particular to mean the particle size, the elasticity, the shape and the density of the particles.

The effect occurs depending on the hydrodynamic properties at just that channel Reynolds number between 0.1 and 500, preferably between 0.5 and 200 and more preferably between 1, 0 and 50, which at given fluid and hydraulic diameter by the flow rate v is set. Below R _K = 0.1, the flow profile of the fluid is too shallow, so that the forces acting on the particles in the fluid flow are insufficient to arrange them within an acceptable period of time. In cases of some particles with particular hydrodynamic properties, a lower limit of R _K = 0.5 or even R _K = 1.0 may be preferred. On the other hand, above the limit of R _K = 500, turbulence is expected on the particle, so that an uncontrollable behavior of the particles is to be expected. Depending on the shape of the measuring channel, more precisely on its cross-sectional dimension, cross-sectional shape, and length, already a channel Reynolds number of more than 200 or even more than 50 to a very high pressure in the measuring channel or the entire line system of the device not every basically suitable material (eg plastic, glass or steel) can withstand. In addition, also depending on the channel shape, the flow velocity in the measuring channel increases above values, which involve a considerable higher technical outlay for the detection of the particles or even a measurement of their velocity in the measuring channel make necessary. For these reasons, it is advantageous in some cases to limit the channel Reynolds number to 200 or even 50.

The "flow behavior" can as a generic term of at least one of the parameters

- mean migration speed of a particle population

- Velocity distribution of a particle population

Change in migration rate depending on a change in flow conditions, i. in particular the flow velocity and thus the channel Reynolds number

Change in the distribution of the migration speed of the particles is understood as a function of the change in the flow conditions, of which not only the velocity distribution used so far, but typically each parameter behaves in a significant manner as a function of the hydrodynamic particle properties while maintaining the flow conditions. Due to the significance of the flow behavior, it is possible to differentiate, classify or determine individual particles.

It may happen that different hydrodynamic particle properties lead quantitatively to the same effect, ie to the same significant flow behavior and therefore an unambiguous determination of the particles is not possible. In this case, only one classification can be made.

For the purposes of this document, the term "particles" includes extended solids as well as droplets of a liquid, meaning that the fluid with particles may be a suspension or also an emulsion flowable medium are understood and in particular rewrite gases and liquids. By "determining" the migration speed in the sense of the invention, it is meant neither that the migration rate is measured directly nor that it must be determined as an absolute value at all Particles in the measuring channel are made over the distance of the measuring section as their equivalent, whereby the dwell time as such, ie without conversion into a speed, can be fed to further evaluation described by sampling. The physical quantity of interest is determined in a manner known per se, usually at the end of the digital evaluation from the sampling data by calibration, wherein the speed need not have been determined at any point as an absolute (intermediate) value.

In many cases, it is not sufficient to determine the migration speed of one particle or one particle population absolutely, but relative to that of another particle or another particle population, especially if the goal is not to determine the particles, but only to differentiate them ,

Extracting the parameter (s) relevant for the differentiation, classification or determination of the particles from the data of the determined migration speed is part of the "analysis" in the sense of the invention and is carried out by the analysis device. The analysis may include an evaluation of individual or a plurality of events or individual measurements, for example to determine a distribution or a mean value .At the end of the analysis, the analysis device outputs the result of the analysis, that is, the flow behavior characterizing one or more Parameter according to user request for image display, print out, in tabular form, as graphic or as electronic data record.

The parameter may be used to differentiate, classify or determine the particles in a variety of ways, and if the method or apparatus is calibrated, the parameter may lead directly to the classification or determination of the particle and result in classification or determination of the particle based on experience, a tabular comparison, or further data processing.

"Classification" means, for example, if only one or more of the hydrodynamic properties of the particles but not the particle itself can be clearly determined, the classification of the particle into a particle class with just those hydrodynamic properties.

In particular, the present invention finds application in so-called microfluidics in which minute volumes of liquid in the range of μΙ are handled and analyzed. In particular, the device according to the invention for miniaturization is suitable for a so-called microfluidic chip or lab-on-a-chip system with which, for example, blood samples or other organic cell material can be analyzed.

The term "measuring path" in the sense of the patent claims is the section of the measuring channel in which the migration speed of the particles is determined In addition, the measuring channel can in special embodiments, which are explained in more detail below, also an "excitation zone" and / or All three terms measuring path, excitation zone and start-up path describe functional sections of the measuring channel, while the start-up section and the measuring section are arranged in this order in Flow direction are arranged spatially one behind the other, but the excitation zone and the measuring section or even the excitation zone and the start-up path but spatially coincide or overlap. It should be noted that the adjustment of the significant flow behavior due to the hydrodynamic properties of the particles does not necessarily require a constant channel cross-section. All that is required is that the Reynolds number R _K lies in the claimed range in all sections, that is to say the start-up section and the measuring section of the measuring channel. Nevertheless, a consistently constant channel cross-section - and thus both the cross-sectional geometry and the cross-sectional area is preferred - for reasons of simplicity and process reliability, because then due to constant flow conditions over a longer channel section, a flow equilibrium can form within the flowing fluid, which increases the measurement precision.

Preferably, the method provides that the particles have a maximum particle diameter a _p and that the fluid with the particles in the measuring channel in the flow direction in front of the measuring section a start-up path with a length L of at least

1 h ⁴

L = 3π- _g

° K, a ^a p flows through, wherein R _{K a} denotes the channel Reynolds number in the start-up section and wherein the measuring channel has a height h at least in the region of the start-up section, but preferably in the entire measuring channel.

In a corresponding manner, the device according to the invention preferably provides that the measuring channel in the flow direction upstream of the measuring section is provided with a start-up has this length L, wherein the measuring channel has a height h at least in the region of the run-up distance, but preferably in the entire measuring channel. The particle diameter a _p is assumed to be the largest extent in the case of non-spherical particles, that is to say the main axis in the case of a rotational ellipsoid.

For a measuring channel with unequal extent of the cross section in two spatial directions, the term "height h" is understood to mean the dimension of the lesser extent, for example, for a channel with a rectangular cross section, the length of the short side of the cross section In the case of a circle, the height h as well as the width w correspond in each case to the diameter of the cross section.Any changes in the transverse section should generally and in particular in the direction of the height h not abruptly but only steadily, so that the flow within In general, however, the height h and the width w are constant throughout the measuring channel in order to avoid accelerating forces which can disturb the arrangement of the particles on certain flow paths in the measuring channel and thus widen their velocity distribution.

Compliance with the above condition ensures that an equilibrium flow has formed at the end of the starting section L and thus at the beginning of the measuring section, in which the particles or individual particle populations with uniform hydrodynamic properties in the flowing fluid are arranged in their equilibrium configuration and therefore the velocity distribution of the Particle has the mentioned significance.

The method and the device preferably further provide that the measuring channel at least in the region of the measuring section, but preferably via the Whole length of the measuring channel, a cross section with a width w and a height h, wherein the measuring channel always the limit for the

Aspect ratio e = ^ <0.1.

In order to be able to enforce a sufficiently high amount of fluid, so that sufficient statistical significance can be achieved for particular cases in a reasonable time frame, the cross section of the measuring channel in the second spatial direction, the width w, a much greater extension than the height h and just preferred at least ten times the extent.

The inventors have found that a particularly narrow velocity distribution of the particles occurs when its height h or minimum extent does not exceed 200 times, preferably 50 times, the particle diameter.

Accordingly, it is preferred for the method as well as for the device if the height h of the measuring channel at least in the region of the measuring section, but preferably over the entire length of the measuring channel, 200 times, more preferably 50 times, the maximum particle diameter a _p does not exceed.

It is furthermore advantageous if, at least in the region of the measuring path, but preferably over the entire length of the measuring channel, the ratio y = <0.75, particularly preferably γ = <0.5.

This requirement reflects the fact that particles larger than half the minimum channel cross-section no longer have sufficient room to dispose in the direction of the height h, which ultimately leads to ambiguity. - I l

in the speed distribution. Whilst the method yields definite measured values for deformable particles up to a γ of 0.75, this is not ensured for rigid particles. Therefore, the compliance with a γ of the highest 0.5 is to be preferred.

Also, the velocity distribution of the particles can be adjusted if according to another aspect of the invention for the particle Reynolds number

R _P = R _K - 2.5- 10 ^" assumes ⁶ and 0.1, where a _p again indicate the maximum particle diameter and h the height of the measuring channel at least in the region of the measuring section, but preferably over its entire length.

This requirement includes the insight that not only a given range of the channel Reynolds number and a specific ratio of the particle diameter to the height of the measuring channel lead to a significant particle flow but also there is a non-linear relationship between the parameters.

In a simple embodiment, the method provides that the fluid contains at least two populations of particles with at least one different hydrodynamic property and that the particle velocity is used to differentiate the at least two particle populations. According to an advantageous development of the method, the velocity distribution and / or a mean velocity value is obtained from the determined migration speed and used for differentiation, classification or determination of the particles. If, for example, two particle populations are investigated, the particle populations each assuming a different mean equilibrium velocity due to at least one different hydrodynamic particle property, this is shown in the velocity distribution by two maxima, which at least differentiate, classify or in a sufficiently well-determined system (the Dispersion or emulsion consisting of fluid and particles is also referred to as a system) just even a determination of the two particle populations can be based. This presupposes that the velocity distributions of the particles are respectively sufficiently narrow and far enough apart from each other so that the maxima can be clearly identified, which is ensured in particular by compliance with the conditions according to the invention. A distribution according to the experience of the inventors is understood to be sufficiently narrow if the half-width of the velocity distribution of a particle population is less than 4%, preferably less than 2% of the average particle velocity. A preferred form of analysis therefore comprises, in particular, the determination of the average particle velocity and / or the distribution width, particularly preferably the half-width of the determined velocity distribution.

An alternative or additional development of the method provides that the mean flow velocity v is changed in a predetermined manner during the flow through, without the channel Reynolds number the value range between 0.1 and 500, preferably between 0.1 and 200 and more preferably between 0.1 and 50 leaves.

This is useful in connection with a further development of the method in which a change in the measured migration speed and / or a change in the distribution of the migration speed as a result of the changes tion of the flow velocity v is analyzed and used for differentiation, for classification or for the determination of the particles.

The inventors have found that not only the absolute or relative particle velocity or the velocity distribution can be used to classify the particles, but also that the change of one of these two parameters allows a statement about the hydrodynamic properties of the particles. By a combination of both analyzes, under certain circumstances a further degree of freedom or a further hydrodynamic particle property can be determined and thus the classification of the particles can be carried out more accurately or even a determination of the particles becomes possible.

As already discussed, determining the migration rate preferably involves measuring or recording the time that the particles take to pass the measurement path.

Accordingly, the device according to an advantageous embodiment provides that the measuring device comprises a time measuring device with a start sensor at the beginning and a stop sensor at the end of the measuring path, which are each set up to detect the passage of a particle.

Alternatively, the device is designed such that the measuring device comprises a radiation source for electromagnetic radiation, which is aligned with an excitation zone in the measurement channel, a detector aligned with the measurement path, which is set up by the particles as a result of the electromagnetic radiation emission and / or by the particles To detect outgoing scattered light, arranged in the region of the measuring section between the measuring channel and the detector mask for the spatial modulation of the emission and / or the scattered light and an evaluation device for determining the migration speed from the modulated signal.

For this purpose, the evaluation device preferably comprises means for correlation analysis.

According to an advantageous development of the device, the detector is set up to quantitatively detect the intensity of the emission emanating from the particles and / or the scattered light emanating from the particles. Correspondingly, the analysis device is set up to output the intensity for differentiation, for classification or for the determination of the particles.

In particular, the method provides that the flow velocity v of the fluid is chosen such that the particles assume a speed distribution whose half-width is less than 4%, preferably less than 2% and particularly preferably less than 1% of the mean velocity of the particles

Further principles, features and advantages of the invention are explained in more detail below with reference to drawings and exemplary embodiments. In the figures show:

Figure 1 is a schematic representation of the particle concentration in a cylindrical channel after Segre and Silberberg;

Figure 2 shows four distributions of the particles in the cylindrical channel in the radial direction at different axial positions;

FIG. 3 shows velocity distributions of the same particles in one

Fluid at different flow rates; Figure 4 shows two velocity distributions of different particles in the same fluid at constant flow rate; Figure 5 is a schematic representation of the grouping different

Particles along the velocity profile of a fluid in a channel;

FIG. 6 a schematic representation of a measuring channel with a simple time measuring device;

FIG. 7 shows a schematic measuring signal of the time measuring device according to FIG. 6; 8 shows a schematic representation of a measuring channel with a time measuring device for a modulated fluorescence or scattered light signal;

FIG. 9 shows the measuring channel according to FIG. 8 in plan view;

FIG. 10 shows a schematic measurement signal of a single particle taken with that of a measuring device according to FIG. 8;

FIG. 11 shows the signal of a particle after correlation analysis has been carried out;

FIG. 12 Derivation of the correlation peak from FIG. 11;

Figure 13 shows a typical signal of several particles taken with a measuring device according to Figure 8; FIG. 14 shows the measurement signal according to FIG. 13 after passing through the correlation analysis and derivation;

Figure 15 is a graph of three velocity distributions of different particles measured at a first flow rate;

Figure 16 is a graph of three velocity distributions of the same three particles measured at a second flow rate;

FIG. 17 shows a two-dimensional intensity-velocity

Diagram of two particle populations;

Figure 18 is a projection of the diagram of Figure 17 on the

Intensity axis; and

Figure 19 is a diagram of two velocity distributions of a spherical and a non-spherical particle.

In a fundamental scientific paper from 1961, Segre and Silberberg have shown that solid particles in a dispersion flowing through a cylindrical tube assume an inhomogeneous distribution after a certain distance, see G. Segre and A. Silberberg, "Radial particle displacement in poiseuille flow of suspensions" , Nature, Vol. 189, 21 January 1961. This behavior is shown schematically in FIGS.

1 shows a longitudinal section through such a cylindrical tube 10 with the central axis 12 and the radius r, in which the fluid with the particles, ie the dispersion, flows in the direction of arrow 14. It is assumed that the particles in the dispersion at the beginning of the tube 10 at position a) are still homogeneous Distributed with the fluid distributed over the entire cross-sectional area. The constant distribution at the position a) is shown schematically in the corresponding diagram a) in FIG. 2, the distribution from the center or symmetry axis 12 to the radius r being plotted there.

As the dwell time, or distance, in the channel increases, interactions between the channel walls, the fluid, and the particles cause a change in the spatial distribution in the fluid. Accordingly, the distribution is radially constricted downstream so that the particle concentration near the peripheral walls rapidly decreases to zero. At the same time, however, forces also occur which displace the particles from the middle of the channel towards the edge. This effect is only slightly weaker, so that the distribution first decreases slightly from the center or symmetry axis "0." As a result of both processes, a distribution maximum tends to form closer to the channel edge, a trend that increases with time or distance in the flow direction until the distribution of the particles is limited to a narrow annular section in the channel This development is shown for three further positions b), c) and d) by the associated diagrams in FIG.

The behavior can also be read in FIG. 1 on the basis of the contour lines 16 and 18. These show that the constriction of the spatial particle distribution with the distance covered in the channel is continuous and parabolic.

In a more recent work, scientists have found that the average radius of maximum particle concentration in a dispersion flowing through a tube depends inter alia on the diameter of the particles, see Bhagat, Kuntaegowdanahalli, Papautsky, "Inertial Microfluidics for Continuous Particle Filtration and Extraction". , Microfluid Nanofluid, Vol. 7, 217-226, 2009. They lead to the parametrization of this behavior the already mentioned particle Reynolds number

a, which indicates the smallest channel diameter in a rectangular channel h. The scientists suggest in their work to take advantage of this effect for particle extraction and filtration. These findings have led the inventors to study the rate at which the particles travel in a fluid in a channel. A first part of their findings is illustrated in FIG. This shows a three-dimensional diagram in which the velocity distribution of the particles measured at six different flow rates in the same channel is plotted. Specifically, the particle velocity in the x-axis direction, the flow rate in the y-axis direction, and the particle number in the z-axis direction are plotted therein. The particle velocity is normalized to the mean flow velocity of the fluid so that the change in flow rate does not result in a shift in the (absolute) particle velocity and the results are easier to compare.

It could be established so that // set at a flow rate of 10 min, a velocity distribution having a maximum which has close to the maximum flow rate of the fluid v _max a steep edge and on the other hand a small spur to a smaller particle out See graph 20. With increasing flow rate and thus flow velocity of the fluid, this maximum decreases and shifts slightly towards a lower particle velocity. At the same time, the initially small spur of slower particles increases until see above curves 21 and 22. As the flow rate continues to increase, the distribution shifts further and further towards slower particles, with a very narrow and high distribution maximum beginning to form as shown in curves 23 and 24 until, finally, at a flow rate of 200 //// min, as can be seen in curve 25, all particles are very much within a very high narrow, dominant velocity distribution peaks are focused, which is at a relative speed just above the average flow velocity v of the fluid. It is noteworthy that, due to this focussing, the distribution width decreases significantly over the initial distribution width at a flow rate of 10 / min. The inventors further made another observation, which is illustrated in FIG. While the diagram in FIG. 3 shows the behavior of a single particle population in a fluid, in a further experiment, whose result is shown in FIG. 4, the inventors have described the behavior of two different particle populations in a fluid under the same experimental conditions (constant channel cross-section and flow rate / Flow velocity).

FIG. 4 shows in a two-dimensional diagram a velocity distribution in which a particle counting rate in the vertical direction against the particle velocity in the horizontal direction is plotted. Two distinct maxima are formed at an average particle velocity of about 432 or 446 mm / sec, the first maximum having a half-width of about 5.1 mm / sec and the second maximum having a half-width of about 1.9 mm / sec , Thus, the two maxima are clearly separated and can each be clearly assigned to one of the two particle populations. This makes it obvious that different particles due to different hydrodynamic particle properties not only in their spatial but also in your velocity distribution in a flowing medium significantly different order and therefore differentiable, classifiable and can also be determined by appropriate behavior with a suitable calibration.

An explanation of the different dynamic behavior of the particles is made with reference to FIG. Shown is a cross section through a flat rectangular channel 30 which is formed symmetrically about the center line 32 and the height h, wherein the height h denotes the smallest extent of the rectangular channel. The channel 30 is traversed by a fluid having two different particle populations in the direction of the arrow 34. In this case, a parabolic-shaped flow profile 36 of the fluid is formed, which has a _maximum velocity v _max in the middle of the channel and decreases symmetrically towards the two edges of the channel 30.

Furthermore, two particle populations are present, of which the particles of a first population are schematized as small circles 37 and those of a second population as large triangles 38. It is assumed that the particles 37 of the first population have a smaller mean particle diameter than the particles 38 of the second population.

The spatial focussing of the particles explained with reference to FIGS. 1 and 2 causes them to be arranged on different equilibrium paths in the channel, the larger particles 38 being oriented slightly closer to the middle 32 of the channel 30 than the smaller particles 37 in that the larger particles 42 occupy a higher equilibrium velocity v ₂ corresponding to the flow profile 36 than the smaller particles 40 whose equilibrium velocity lies at v ₁ . Thus, it is understandable that Particles of different sizes are differentiable based on their average speed, if the flow velocity of the fluid is so high that the velocity distribution of all particle populations are sufficiently focused, see. 3, and the differences in the hydrodynamic properties are significant enough for the velocity distributions to have maxima which are far enough apart from each other, cf. FIG. 4.

The inventors have also found that in addition to the size of the particles and their elasticity, shape and density as hydrodynamic properties have an influence on the arrangement in the velocity profile. These properties can cancel each other out so that particles with different hydrodynamic properties still quantitatively result in very similar or even average velocities and similar or equal velocity distribution profiles. Under these circumstances, no definite determination but only a classification of the particles is possible. For example, the hydrodynamic focusing of bacteria with a certain diameter and an elastic deformability takes place even at a lower flow velocity than the rigid spherical particles of the same size. In other words, a bacterium can assume the same average velocity as a much larger but rigid sphere.

How the invention utilizes the found effects will be explained below.

FIG. 6 shows a schematically greatly simplified device for differentiating, classifying or determining undissolved particles in a flowing fluid. Specifically, a measuring channel 40 is shown cut therein in the region of a measuring section 42, in which particles of two populations are transported in the direction 44 of the fluid flow. Again The particle populations are illustrated by small circular symbols 46 and large triangular symbols 48 and move after focusing at different distances to the central axis 50 of the channel 44 and at different speeds v _l and v ₂ . The measuring channel 44 has at least in the region of the measuring section 42 has a substantially constant cross section with a width w, not shown, and a height h, resulting in a

Aspect ratio e = ^, which is preferably less than or equal to 0.1.

The measuring section 42 comprises a time measuring device, which has a start sensor 52 at the beginning and a stop sensor 54 at the end of the measuring section in the flow direction 44. The sensors 52 and 54 are each arranged to detect the passage of a particle. For example, the start and stop sensors can be formed by light barriers. The velocity measurement of particle streams by means of such sensors is referred to in the literature as "particle imaging velocimetry" and is known per se.

With the measuring device shown in Figure 6, a raw signal could be detected, which has the simplified course shown in Figure 7. Here, two signals 56 and 58 are shown with different signal strength, each representing the start and stop signals and can be assigned due to their different signal level either the start sensor 52 or the stop sensor 54. The signals are recorded (sampled) in the chronological order of their occurrence, and a residence time of individual particles 46, 48 within the measurement path 42 in the measurement channel 40 is derived from the signal-time profile. Difficulties in the evaluation can manifestly occur when individual signals coincide in time and when an assignment of a start signal to a stop signal due to multiple simultaneous measuring section of the flowing through particles of different populations is not clearly possible. This measurement method therefore sets assume that the length of the measurement path 46 is not too large relative to the density of the particles 50, 52 in the fluid.

FIGS. 8 and 9 show a second embodiment of the device according to the invention, in which the measuring channel 60 with a flat rectangular profile is incorporated into the substrate of a microfluidic chip 62. The microfluidic chip is shown in Figure 8 in the side view in section and in Figure 9 in plan view. The side view shows the measuring channel 60 in the height direction, in which an extent or height h has. In the width direction, it has an extension or width w. For the aspect ratio, preferably e = - <0.1.

w

At the beginning, the measuring channel has a feed 64 and an outlet 66 for the fluid to be examined. The feed 64 and / or the discharge 66 can be connected to a pumping device, not shown, for conveying the fluid with the particles (this time from right to left) through the measuring channel 60, with which a pressure difference can be generated, which is assigned by means of a pumping device Control means can be adjusted so that the fluid flows through the measuring channel with a flow velocity v at which the channel Reynolds number

_ _ P ^■ v ^■ d _h yd

Values between 0.1 and 500, preferably between 0.1 and 200 and particularly preferably between 0.1 and 50.

In FIG. 9, it can be seen that the measuring channel 60 is further provided with two further connections 68, 70, through which a sheath liquid viewed in the width direction can be supplied from both sides. The one by the feeder 64 introduced into the measuring channel 60 flow of the fluid with the particles is hydrodynamically focussed in the flow direction v behind an intersection 71 with the connections 68, 70 for the sheath liquid so that it flows through the measuring channel 60 in the width direction w substantially in the center, while only the Having been provided by the openings 68, 70 sheath liquid contact with the channel wall in the width direction w. In this way, on the one hand, the flow of the fluid with the particles is adjusted to a desired width so that all particles pass through the excitation zone described below. On the other hand, this ensures that a substantially constant flow profile is formed in the width direction of the measuring channel and the flow profile varies substantially only in its vertical direction according to the parabolic shape shown in FIG.

Furthermore, the microfluidic chip 62 has an entrance window 72 for a laser beam 74, which is arranged to the direction of the laser beam such that it completely or predominantly into the substrate medium of the microfluidic chip 62, which is preferably made of plastic and particularly preferably PMMA. broken in and passed under total internal reflection between the two interfaces of the microfluidic chip 62 to its environment. The laser beam passes through the measuring channel 60 and excites the particles in the fluid or irradiates them. Accordingly, section 76 within the meaning of the claims forms an excitation zone in the measurement channel, to which the radiation source (not shown) for electromagnetic radiation 74 is aligned in the sense of this invention.

In the flow direction v behind the channel intersection 71 starts a run-up path 78 of length L. In this section, the particles flowing in the fluid have an opportunity to locate within the airfoil as shown in Figure 5 and to assume a corresponding migration rate. In the flow direction v behind the run-up path 78 is located the measuring section 80, which is characterized in that a detector 82 is aligned thereon. In the region of the measuring section 80, a mask 84 is also located between the measuring channel 60 and the detector 82. This so-called shadow mask has spatially consecutive transparent and non-transparent sections in the flow direction v. These sections block emission from the particles as a result of the excitation and / or scattered light emanating from the particles on the way to the detector 82 or allow it to pass through. This is exemplified at wavy lines 86 and 88, respectively, for two of the sections.

When passing an emitting or scattering particle, this measuring arrangement generates the time-modulated detector signal shown in FIG. It should be noted that the diagram of Figure 10 shows a schematically idealized representation and no real measurement. The duty cycle of the signal represents the sequence of the transparent and non-transparent portions of the shadow mask 84. The signal is recorded in a manner known per se with a sufficient sampling rate. The modulated signal of a particle has a time length (residence time) which results from its velocity and the length of the measuring channel. Time duration and speed are equivalent. From the residence time and the sampling rate, ie the number of measurement points per time unit, the total number of measurement points that make up the signal results. Also, this total number of measurement points occupied by the modulated signal thus depends on the velocity of the particle. The scanning sequence or the successive sections can in the simplest case be in the form of a periodic sequence of transparent and non-transparent sections. However, they are preferably arranged as so-called "pseudo random binary sequence", that is to say in an order with irregular distances and lengths, in particular during measurement several particles that overlap the measuring path 80 overlap, to allow a clear identification.

This evaluation is done using a correlation analysis. The pattern of the shadow mask is known. From this, the evaluation device computationally forms a reference sequence. It is scaled in absolute length and compared to the sampled detector signal, shifting it over the entire sampling. The process is iteratively repeated at other scales until a match is found. This process can also be referred to as a query of velocity channels or filtering and takes place at discrete values, whereby here too attention must be paid to a sufficient resolution.

The result of this is shown in FIG. 11. The correlation signal always grows approximately linearly from the two sides towards the middle, only exactly in the middle does a significant correlation peak form, which signals the agreement. For the exact determination of the position of the correlation peak, the correlation diagram according to FIG. 11 can be derived mathematically, from which the diagram according to FIG. 12 results. Therein, the zero crossing between the maximum and the minimum of the derivative signal marks the exact value of the correlation signal.

The actual length of the measuring section is known. Therefore, by means of a calibration measurement with a known position of the correlation peak, i. the velocity of the particle is determined absolutely at the determined length of the reference sequence. Otherwise, the result is used for a relative comparison with the speed of other particles.

In reality, multiple particles become the measurement path 80 simultaneously or at least sequentially and partially in an overlapping time interval happen, which is why the time-modulated measurement signal at first glance does not allow a clear statement about the number of particles and their residence time within the measuring section 80. Such a measurement of eight partially time overlapping particles is shown in the sample of FIG. At the same time, for illustrative purposes, for each identified particle, the shadow mask 84 is shown, from which it can be seen that the events of passing particles partially overlap in the right half of the diagram. Nevertheless, the particles can be determined exactly in the manner described above, as can be seen from the derivations of the correlation peaks in FIG. 14, because the use of an unperiodic shadow mask ensures significant discrete correlation results despite partial overlapping of the residence times of the particles in the region of the measurement path. If the particles do not have an identical migration speed, which is basically the case in reality, the correlation analysis yields different scaling values for the reference sequence. This is preceded by a high computational effort, since the entire sampling must be correlated with a large number of scaled reference sequences, depending on the required resolution and width of the velocity interval to be scanned. To reduce this effort, the device according to the invention and the method according to the invention make it possible to select the velocity distribution so narrow that the scaling interval can be considerably reduced without loss of measurement data.

Accordingly, both velocity distributions of identical particles and velocities or their equivalents of particles of different populations can be determined. Both together is done by way of example in the diagram of FIG. The diagram shows the velocity distributions of three particle populations which were simultaneously conveyed through a measurement channel under the experimental conditions according to the invention at a channel Reynolds number R _K of 14. The solid line shows the velocity distribution of the smallest particles with an average particle diameter α = 0.84μ / «. The velocity of these particles is distributed from about 340 mm / sec to about 620 mm / sec over a large interval and has approximately the curve shape of the second distribution curve 21 in the three-dimensional diagram according to FIG. The curve is normalized to the maximum value of the right edge peak.

Furthermore, particles having an average particle diameter a _p of 2.1 μιι are contained in the fluid. At the same channel Reynolds number, these have a very sharp distribution in the range of about 410-415 mm / sec, as can be seen from the dashed curve. Finally, even larger particles with a mean particle diameter a _p of 6.42 μιι are contained in the fluid whose dotted-line distribution is narrower again and comprises a range of about 425 to about 428 mm / sec. The two latter curves are also normalized to the maximum value of the respective peaks. These both have the shape of the distribution according to curve 25 in the diagram in FIG. The diagram confirms the effects discussed above, namely that particles of different size concentrate in the fluid at different equilibrium velocities, having a very narrow velocity distribution at a sufficient flow velocity and thus being easily differentiable or identifiable. However, FIG. 15 shows yet another effect that has not been described in detail above. The fact that the smallest particles apparently have not yet reached their equilibrium position and therefore show a broad distribution in which a maximum value only slowly begins to form at the left edge, shows that the flow velocity and 5, the parabolic distribution profile of the fluid velocity is not yet steep enough that the forces acting on the small particles are sufficient to move them completely into an equilibrium position. This shows that specifying an upper limit for the channel Reynolds number has no generality for achieving a narrow, significant velocity distribution. Therefore, the invention according to an advantageous embodiment, that in a system with particles having a maximum particle diameter a _p and a measuring channel, at least in the region of

Measuring section, but preferably over its entire length, a substantially constant height h, the flow rate is adjusted so that the particle Reynolds number R _P = R _K ^{■ is} greater than 2.5-10 ^"6th

The correctness of this assumption is confirmed by the diagram in FIG. 16, which shows the velocity distributions of the same three particles in the same fluid and in the same device but at a mean flow velocity v of the fluid corresponding to a channel Reynolds number of 0.66. Under these conditions, neither particles with a mean particle diameter α = 0.84μ / "nor those with a mean particle diameter of 2.1 μιτι and not even the largest particles with a mean particle diameter of 6.42 μιτι arrange in the fluid.

FIG. 17 shows a two-dimensional diagram in which the signal intensity in the vertical direction is plotted against the velocity distribution in the horizontal direction. Again, this measurement contains two particle populations that are clearly separated from one another in both projection on the velocity axis and projection onto the intensity axis. If the measurement is carried out in such a way that, in addition to the velocity distribution, the intensity of the emitted or scattered radiation is registered as a further property of the particles and measured coincidentally, then a measurement determines a further particle property and therefore the particles are classified or determined more precisely. The projection on the velocity axis is shown in FIG. 4 described above. The projection onto the intensity axis is shown in FIG.

FIG. 19 shows a velocity distribution diagram of two populations under the same flow conditions. The populations contain on the one hand spherical particles with a diameter of 6.42 μιτι and on the other hand in pairs contiguous spheres of this type, with indicated in the diagram form. If one imagines the latter particles as ellipsoid, they have a length of 12.84 μm in the orientation of their main axis and therefore behave similarly to larger particles, but not equal. In particular, they arrange closer to the center of the channel than the simple, smaller spheres and are therefore faster than these. This specific behavior shows that besides the size, the shape of the particles also represents a hydrodynamic property and contributes to differentiation, classification or identification.

List of reference cylindrical channel

center line

flow direction

contour

Height line velocity distribution curve

Speed distribution curve

Velocity distribution curve measuring channel

center line

flow direction

Flow profile of the fluid

first particle population

second particle population measuring channel

measuring distance

flow direction

first particle population

second particle population centerline

start sensor 4 stop sensor

6 start signal

8 Stop signal 0 Measuring channel

2 microfluidic chip

4 access, inlet

6 outlet, outlet

8 connection 0 connection

1 channel crossing

2 entrance windows

4 laser beam

6 excitation zone

8 start-up distance 0 measuring distance

2 detector

4 mask

6 light emission or scattering

8 Light emission or scattering b Width of the measuring channel

h Height of the measuring channel

L Length of the start-up route

r radius or half height of the channel

v flow velocity or direction v mean flow velocity of the fluid v _max maximum flow velocity of the fluid v _l Particle velocity of the first particle population v ₂ Particle velocity of the second particle population

Claims

Patent claims

1 . Method for differentiating, classifying or determining undissolved particles located in a flowing fluid, which fluid has the density p and the viscosity μ, in which the fluid with the particles is conveyed through a measuring channel (30, 40, 60), wherein the fluid with the particles flows through a measuring section (42, 80), characterized in that the fluid is conveyed through the entire measuring channel (30, 40, 60) at an average flow velocity v at which the channel Reynolds number

_ p - v ^■ d _hyd

values between 0.1 and 500, where d _hyd denotes the hydraulic diameter of the measuring channel (30, 40, 60), that the migration speed of the particles over the measuring section (42, 80) is determined and that the flow behavior of the particles is determined based on the determined Migration speed is analyzed and used to differentiate, classify or determine the particles.

2. Method according to claim 1,

characterized in that the particles have a maximum particle diameter a _p and that the fluid with the particles in the measuring channel (30, 40, 60) in the direction of flow in front of the measuring section (42, 80) has a run-up section (78) with a length L of at least 1 hour ⁴

L = 37Γ—g

°K,a ^a p flows through, where R _{K a} denotes the channel Reynolds number in the approach section (78) and the measuring channel (30, 40, 60) has a height h at least in the area of the approach section (78).

3. Method according to claim 1 or 2,

characterized in that the measuring channel (30, 40, 60) has a substantially constant cross section with a width w and a height h at least in the area of the measuring section and that the measuring channel (30, 40, 60) has an aspect ratio e = ^ < 0 .1 has.

4. Method according to one of the preceding claims,

characterized in that the particles have a maximum particle diameter a _p and that the measuring channel (30, 40, 60) has a substantially constant height h at least in the area of the measuring section (42, 80), the ratio γ = - < 0, 75 is.

¹ hour

5. Method according to claim 4,

characterized in that the height h of the measuring channel (30, 40, 60), at least in the area of the measuring section (42, 80), does not exceed 200 times, preferably 50 times, the maximum particle diameter a _p .

6. Method according to one of the preceding claims,

characterized in that the speed distribution and/or a speed average from the determined migration speed speed is obtained and used for differentiation, classification or determination of the particles.

Method according to one of the preceding claims,

characterized in that the average flow velocity v is changed in a predetermined manner during the flow without the channel Reynolds number leaving the value range between 0.1 and 500.

Method according to claim 7,

characterized in that a change in the measured migration speed and/or a change in the distribution of the migration speed as a result of the change in the flow speed v is analyzed and used to differentiate, classify or determine the particles.

Method according to one of the preceding claims,

characterized in that determining the migration speed includes measuring the time that the particles need to pass the measuring section (42, 80).

Method according to one of the preceding claims,

characterized in that the fluid contains at least two populations of particles (46, 48) with at least one different hydrodynamic property and that the particle velocity is used to differentiate the at least two particle populations (46, 48).

Method according to one of the preceding claims, characterized in that the flow velocity v is chosen so that the particles assume a velocity distribution whose half-width is less than 4% of the average velocity of the particles

12. Device for differentiating, classifying or determining undissolved particles located in a flowing fluid, which fluid has the density p and the viscosity μ, with a measuring channel (30, 40, 60) through which the fluid with the particles can be conveyed is and which comprises a measuring section (42, 80), and with a pump device for conveying the fluid with the particles, characterized by a control device assigned to the pump device, which is set up to provide a flow velocity v for the fluid through the entire measuring channel (30, 40 , 60) at which the channel Reynolds number

_ p - v ^■ d _hyd

Assumes values between 0.1 and 500, where d _hyd denotes the hydraulic diameter of the measuring channel (30, 40, 60), a measuring device arranged along the measuring section (42, 80), which is set up to measure the migration speed of the particles over the measuring section ( 42, 80), and an analysis device that is set up to analyze the flow behavior of the particles based on the determined migration speed and to differentiate, classify or identify the particles.

13. Device according to claim 12,

characterized in that the measuring channel (30, 40, 60) has a start-up section (78) with a length L of at least

1 hour ⁴

L = 3π— _g

°K,a ^a p, where R _{K a} denotes the channel Reynolds number in the approach section (78) and wherein the measuring channel (30, 40, 60) has a substantially constant height h at least in the area of the approach section (78).

14. Device according to claim 12 or 13,

characterized in that the measuring channel (30, 40, 60) has a substantially constant cross section with a width w and a height h at least in the area of the measuring section (42, 80) and that

Measuring channel (30, 40, 60) has an aspect ratio e = ^ <0.1.

15. Device according to one of claims 12 to 14,

characterized in that the measuring device comprises a time measuring device with a start sensor at the beginning and a stop sensor at the end of the measuring section (42, 80), each of which is set up to detect the passage of a particle.

16. Device according to one of claims 12 to 14, characterized in that the measuring device has a radiation source for electromagnetic radiation, which is aligned with an excitation zone (76) in the measuring channel (30, 40, 60), a detector (82) aligned with the measuring section (42, 80), which is set up, to detect emission emanating from the particles as a result of the electromagnetic radiation and/or scattered light emanating from the particles, a mask ( 84) for spatial modulation of the emission and/or scattered light and an evaluation device for determining the migration speed from the modulated signal.

17. Device according to claim 16,

characterized in that the evaluation device has means for correlation analysis.

18. Device according to claim 16 or 17,

characterized in that the detector is set up to quantitatively detect the intensity of the emission emanating from the particles and/or the scattered light emanating from the particles and that the analysis device is set up to output the intensity for differentiation, classification or determination of the particles.