阅读说明：本技术 用于颗粒浓缩的微流体装置 (Microfluidic device for particle concentration ) 是由 M·皮济 G·麦罗利 V·加洛 M·扎宁 于 2019-06-05 设计创作，主要内容包括：一种用于浓缩流体样本中包含的颗粒的微流体装置包括具有表面的基板(11),在那里限定有至少一个微流体布置结构(M),所述微流体装置包括：-装载腔室(14),其用于将所述流体样本装载到所述至少一个微流体布置结构(M)中；-多个微通道(13),其具有连接到所述装载腔室(14)的相应的入口端部；以及-覆盖元件(12),其基本上不能渗透所述流体样本,并且至少部分地在所述多个微通道(13)上方延伸。所述装载腔室(14)和所述微通道(13)基本上按照由所述基板(11)确定的平面延伸,并且所述微通道(13)特别是在其与相应的入口端部大致相对的积聚区域(CA)处由至少可渗透空气的过滤装置(17)部分地界定,所述过滤装置(17)被构造成用于在每个微通道(13)内保留可能存在于所述流体样本中的任何可能的颗粒。(A microfluidic device for concentrating particles contained in a fluid sample comprises a substrate (11) having a surface, at least one microfluidic arrangement (M) being defined therein, the microfluidic device comprising: -a loading chamber (14) for loading the fluid sample into the at least one microfluidic arrangement (M); -a plurality of microchannels (13) having respective inlet ends connected to the loading chamber (14); and-a covering element (12) substantially impermeable to the fluid sample and extending at least partially over the plurality of microchannels (13). The loading chamber (14) and the microchannels (13) extend substantially according to a plane determined by the substrate (11), and the microchannels (13) are partially delimited, in particular at their accumulation areas (CA) substantially opposite to the respective inlet ends, by at least air-permeable filtering means (17), the filtering means (17) being configured for retaining within each microchannel (13) any possible particles that may be present in the fluid sample.)

1. A microfluidic device for concentrating particles contained in a Fluid Sample (FS), comprising a substrate (11) having a surface (11 b), at least one microfluidic arrangement (M) being defined at said surface (11 b), the microfluidic device comprising:

-a loading chamber (14) for loading the Fluid Sample (FS) into the at least one microfluidic arrangement (M);

-a plurality of microchannels (13) having respective inlet ends (13 a) connected to the loading chamber (14), in particular having a parallel fluidic connection with the chamber (14);

-a cover element (12) substantially impermeable to the Fluid Sample (FS) and extending at least partially over the plurality of microchannels (13),

wherein the loading chamber (14) and the microchannels (13) extend substantially according to a plane defined by the substrate (11),

wherein the microchannels (13) are at least partially delimited, at least at their accumulation area (CA) substantially opposite to the respective inlet end (13 a), by an at least air-permeable filtering means (17), the filtering means (17) being configured for retaining possible particles possibly present in the Fluid Sample (FS) within each microchannel (13),

namely: such that due to a force applied to at least one of the microfluidic device (10) and the Fluid Sample (FS) loaded into the loading chamber (14), such as a centrifugal force caused by the rotation of the substrate (11) about a center of rotation (5 a), or a positive pressure exerted on the Fluid Sample (FS) at the loading chamber (14), or a negative pressure exerted on the Fluid Sample (FS) at the accumulation area (CA) of the microchannels (13), particles possibly contained in a fluid volume of the Fluid Sample (FS) that permeates into each microchannel (13) tend to concentrate in the respective accumulation area (CA).

2. The microfluidic device according to claim 1, wherein the cover element (12) is sized or shaped to define at least one of:

-a port (15) for introducing the Fluid Sample (FS) into the loading chamber (14);

-a passage (16) for discharging air from the microchannel (13) through the filtering means (17);

-a passage (16) for the exit of a liquid from the microchannel (13), the filtering means (17) being permeable to the liquid.

3. The microfluidic device according to claim 1 or claim 2, wherein the filtering device (17) comprises a same filtering element at least partially delimiting a plurality of microchannels (13) at an accumulation area (CA) of said plurality of microchannels (13).

4. The microfluidic device of any one of claims 1-3, wherein:

-at least one of said base plate (11) and said covering element (12) is configured to define at least a portion of a seat (18) for said filtering device (17); and/or

-the covering element (12) is configured for maintaining the filtering means (17) in the corresponding operating position.

5. The microfluidic device according to any one of claims 1 to 4, wherein each microchannel (13) is closed at its longitudinal end opposite to the corresponding inlet end (13 a), the filtering means (17) being preferably provided at least one side wall of the microchannel (13) or above a terminal extension of the microchannel (13) defined in the substrate (11).

6. The microfluidic device according to any one of claims 1-5, wherein each microchannel (13):

-has a width comprised between 5 μm and 200 μm, preferably between 15 μm and 50 μm; and/or

-having a depth or height comprised between 2 μm and 100 μm, preferably between 5 μm and 40 μm; and/or

-has a length comprised between 5 mm and 50 mm; and/or

-has a substantially constant through-opening profile.

7. The microfluidic device of any one of claims 1-6, wherein:

-each microchannel (13) has at least one surface portion defined by at least one of a hydrophilic material and a hydrophobic material, preferably belonging to at least one of said substrate (11), said covering element (12) and said filtering means (17); and/or

-said device (10) is at least partially made of a transparent material belonging to at least one of said substrate (11), said covering element (12) and said filtering means (17).

8. The microfluidic device according to any one of claims 1-7, wherein the substrate (11):

-configured for mounting on a rotating member (5 a) of a centrifugation and/or detection device (1), in particular by means of an adapter support (30, 40); and/or

Is substantially disc-shaped.

9. The microfluidic device according to any one of claims 1-8, wherein a plurality of the microfluidic arrangement structures (12) are defined in the surface (11 b) of the substrate (11), preferably arranged in an at least approximately radial direction with respect to a center of rotation (11 a) of the substrate (11).

10. The microfluidic device according to any one of claims 1-9, wherein the microchannels (13) are arranged side by side, preferably at least partially identical to each other, and/or extend at least partially substantially parallel or equidistant to each other, and/or comprise: a first microchannel (13) arranged radially with respect to the center of rotation (11 a); and a second microchannel (13) arranged parallel or equidistant to the first microchannel (13).

11. The microfluidic device according to any one of claims 1 to 10, wherein the filtration device (17) comprises a membrane having a porosity or mesh size comprised between 0.02 μ ι η and 0.45 μ ι η, preferably made at least in part of a membrane of ceramic material, in particular of porous alumina.

12. A microfluidic device for concentrating particles contained in a Fluid Sample (FS), comprising a substrate (11) having a surface (11 b), at least one microfluidic arrangement (M) being defined at said surface (11 b), the microfluidic device comprising:

-a loading chamber (14) for loading the Fluid Sample (FS) into the at least one microfluidic arrangement (M);

-a plurality of microchannels (13) having respective inlet ends (13 a) connected to the loading chamber (14),

wherein the loading chamber (14) and the microchannel (13) extend at least partially in at least one of the substrate (11) and a covering element (12) of the substrate (11),

wherein the microchannels (13) are at least partially delimited, at least at or upstream of a respective accumulation area (CA) substantially opposite to the respective inlet end (13 a), by at least air-permeable filtering means (17), the filtering means (17) being configured for retaining, within each microchannel (13) and/or each accumulation area (CA), any possible particles that may be present in the Fluid Sample (FS).

13. A centrifugal device comprising a rotation member (5 a), the rotation member (5 a) being configured for rotating the microfluidic device (10) according to any one of claims 1-12.

14. A detection device, comprising: a rotation member (5 a) configured for subjecting a microfluidic device (10), in particular a microfluidic device (10) according to any one of claims 1-12, to an angular movement; and an optical sensor device (20) configured for performing optical detection on the microfluidic device (10) and/or detecting particles that have accumulated in an accumulation area (CA) of the microfluidic device (10),

wherein the optical sensor device (20) is configured for acquiring optical signals or images of one or more accumulation areas (CA) of the microfluidic device (10),

wherein preferably the detection device (1) is pre-arranged for processing information based on the light signal or image, the information being indicative of the amount of particles that have accumulated in one or more accumulation areas (CA).

15. A method for detecting particles possibly present in a Fluid Sample (FS), comprising the steps of:

-providing a microfluidic device (10) according to any one of claims 1-12;

-introducing a volume of the Fluid Sample (FS) into a loading chamber (14) of at least one microfluidic arrangement (M) of the microfluidic device (10);

-subjecting the microfluidic device (10) to centrifugation or the corresponding Fluid Sample (FS) to positive or negative pressure respectively at the loading chamber (14) or at an accumulation area (CA) of the microchannel (13); and

-detecting, in particular optically and/or electrically, particles that may accumulate in the accumulation area (CA) of each microchannel (13).

16. A method for performing antibiogram detection, comprising:

-providing a microfluidic device (10) according to any one of claims 1-12;

-providing a liquid medium containing a microorganism or a microbe or a bacterium of at least one bacterial strain;

-introducing a volume of the liquid culture medium into a plurality of first microchannels (13) of at least one first microfluidic arrangement (M) of the microfluidic device (10);

-subjecting the microfluidic device (10) to centrifugation or the corresponding Fluid Sample (FS) to positive or negative pressure respectively at the loading chamber (14) or at an accumulation area (CA) of the microchannel (13); and

-quantifying the number of microorganisms or microbacteria or bacteria that have accumulated in the accumulation area (CA) of each microchannel (13).

17. The method of claim 16, comprising:

i) pre-treating the first microchannel (13) with at least one first antibiotic, preferably with a different type and/or concentration of lyophilized antibiotic;

ii) obtaining a mass of microorganisms or microbacteria or bacteria;

iii) inoculating at least a portion of the mass into the liquid culture medium, thereby preferably forming a homogeneous dispersion;

iv) introducing a volume of the liquid culture medium into the first microchannel (13);

iv) waiting for a period comprised between 10 minutes and 6 hours, preferably between 1 hour and 2 hours;

v) subjecting the microfluidic device (10) to centrifugation or the corresponding Fluid Sample (FS) to positive or negative pressure respectively at the loading chamber (14) or at an accumulation area (CA) of the microchannel (13);

vi) quantifying the number of microorganisms or micro-bacteria or bacteria that have accumulated in an accumulation area (CA) of the first microchannel (13), in particular by performing a relative quantification between the first microchannel (13) and a second microchannel (13) of the microfluidic device (10) that is not pretreated with the at least one first antibiotic, in order to obtain a sensitivity profile of the microorganisms or micro-bacteria or bacteria to the at least one first antibiotic.

18. Method according to claim 17, wherein the pellet of step ii) is taken from a petri dish or is obtained from a sample taken from a host organism, in particular by centrifugation or by filtration and centrifugation of the sample.

Technical Field

The present invention relates generally to techniques for detecting or estimating the amount of particles present in a fluid sample, particularly particles of low concentration and small volume.

The present invention has been developed with particular reference to microfluidic devices designed to undergo centrifugation, and with reference to devices and methods that: for the examination or analysis of fluid samples, preferably containing organic or biological particles or bacteria or microorganisms, for example for the rapid performance of antibiogram detection.

In any case, the invention is also applicable to the detection of other types of particles that may be present in a fluid sample, which are not necessarily organic or biological fluids or particles, and which are not necessarily separated by centrifugation.

Background

Various techniques are known for counting particles, such as cells, present in a sample of a fluid, such as a biological fluid. The most commonly used systems are of the optical type (with or without fluorescence), impedance measurement type (impedance measurement type) or static type by means of image recognition. These known systems generally require a relatively large amount of samples and do not enable efficient parallelization of measurements, e.g. performing multiple measurements simultaneously, i.e. they assume that a considerable number of starting samples can perform many measurements in parallel and/or simultaneously.

Known systems based on image recognition techniques can be used to analyze small fluid samples, but do not enable parallelization of multiple samples, thereby extending measurement time unless investments are made, which often prove uneconomical.

Disclosure of Invention

In summary, the invention has the object of indicating an apparatus and a method, namely: the device and method make it possible to perform in a simple, fast and inexpensive manner the quantification and/or identification of particles present in low concentrations and/or small volumes in a fluid sample, making it possible to achieve parallelization between a plurality of samples in an equally simple and inexpensive manner, with advantages in terms of time and cost, as well as in terms of efficiency with respect to sensitivity and repeatability.

Another object of the invention is to indicate a method, namely: this method enables the performance of antibiogram detection (when measuring microorganisms), i.e. the acquisition of a sensitivity profile of at least one microorganism or microbe or bacterium to antibiotics in a relatively short time expressed in hours; an auxiliary object of the invention is to indicate a method that enables a plurality of antibiogram detections to be performed simultaneously.

According to the present invention, the above object is achieved by a microfluidic device for particle concentration and a corresponding support and method having the features specified in the appended claims. The invention also relates to centrifugation and/or detection devices that can be used in combination with the aforementioned microfluidic devices, as well as to analytical methods based on the use of such devices.

As will appear clearly hereinafter, the invention enables an effective detection of the amount of particles in a relatively moderate volume of sample of a fluid of interest to be performed in a simple and fast manner.

Drawings

Further objects, characteristics and advantages of the present invention will appear clearly from the following detailed description, with reference to the attached drawings, which are provided as non-limiting examples only, and in which:

figures 1 and 2 are schematic perspective views of a centrifugation and/or detection device and of a microfluidic device according to a possible embodiment of the invention;

figure 3 is a schematic perspective view of a microfluidic device according to a possible embodiment of the invention;

figure 4 is an exploded schematic view of a microfluidic device according to a possible embodiment of the present invention;

FIG. 5 is a detail of FIG. 4 on a larger scale;

figure 6 is a partial and schematic perspective view of a part of a microfluidic device according to a possible embodiment of the invention;

fig. 7 is an enlarged detail view of an end portion of a microfluidic arrangement according to a possible embodiment of the present invention;

fig. 8 is a schematic perspective view intended to illustrate a possible step of loading a fluid sample into a microfluidic arrangement according to a possible embodiment of the present invention;

fig. 9 is a schematic perspective view, partly in section, of a microfluidic arrangement according to a possible embodiment of the present invention;

figure 10 is a detail of figure 9 on a larger scale;

figure 11 is an exploded schematic view of a microfluidic device according to a possible embodiment of the present invention;

figures 12 and 13 are schematic perspective views, partly in section, of a microfluidic arrangement according to a possible embodiment of the invention;

figure 14 is a detail of figure 13 on a larger scale;

fig. 15 is an enlarged detail view of an end region of a microfluidic arrangement according to a possible embodiment of the present invention;

fig. 16 is a perspective view, partly in section, of the device of fig. 1-2, with the corresponding microfluidic device in an operating state;

figure 17 is a detail of figure 16 on a larger scale;

fig. 18 is a schematic perspective view of a centrifugation and/or detection device and some microfluidic devices according to a possible embodiment of the invention;

figure 19 is a greater-scale detail of figure 18;

fig. 20 is a schematic perspective view, partly in section, of the centrifugal device of fig. 18, with the corresponding microfluidic device in an operating state;

figure 21 is a schematic perspective view of a microfluidic device according to a possible embodiment of the present invention;

figures 22 and 23 are exploded schematic views of a microfluidic device according to a possible embodiment of the invention;

fig. 24 is a schematic perspective view intended to illustrate a possible step of loading a fluid sample into a microfluidic device of the type shown in fig. 21;

figures 25 and 26 are schematic perspective views, partly in section, of a microfluidic device according to a possible embodiment of the invention;

fig. 26a is a schematic perspective view, partly in section, of a microfluidic device according to a possible further embodiment of the invention; and

figures 27 and 28 are schematic perspective views intended to illustrate possible modes of use of the microfluidic device according to a possible embodiment of the invention.

Detailed Description

Reference to "an embodiment" or "one embodiment" within the framework of the specification is intended to indicate that a particular configuration, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, phrases such as "in an embodiment," "in one embodiment," "in various embodiments," and the like, that may be present in different places throughout this specification are not necessarily referring to one and the same embodiment. Furthermore, the particular features, structures, or characteristics defined within the framework of the present specification may be combined in any suitable manner in one or more embodiments, even if not identical to the embodiments presented. Reference numerals and spatial references (e.g., "top," "bottom," "upper," "lower," etc.) used herein are provided merely for convenience and, thus, do not define the scope of protection or the scope of the embodiments. In the drawings, the same reference numerals are used to designate elements that are similar or technically equivalent to each other.

With reference first to fig. 1 and 2, a centrifugation and/or detection device is indicated as a whole by 1, having a structure 2 defining a processing and/or detection chamber 3.

In various embodiments, the device 1 comprises a lid or door 4, preferably hinged to the structure 2, for closing the chamber 3. The device 1 has a driving or moving system, indicated as a whole by 5 in figure 2, which comprises, inside the chamber 3, a rotating member 5a designed to rotate one or more microfluidic devices, preferably of the centrifugable type.

The lid 4 may possibly comprise a corresponding portion 4a of a centrifugally centrifuge microfluidic device or of a positioning and/or guiding system configured as a support for supporting a plurality of centrifugally centrifuge microfluidic devices. In this example, the portion 4a comprises a seat for blocking and guiding an element, denoted by 5b, which can be coupled to the member 5a with the aforesaid centrifiable means or with the aforesaid support interposed, to ensure mutual fixing in rotation between the mentioned portions.

The actuation system 5 preferably comprises an electric motor (partially visible in figures 16-17, in which it is indicated by 5 c), possibly provided with a motor reducer and/or with an electronic control circuit. The centrifugation speed may be indicatively comprised between 200 and 1200 rpm, preferably between 400 and 1000 rpm, for a time comprised preferably between 3 and 30 s, very preferably between 5 and 15 s.

In various embodiments, the apparatus 1 comprises a system for controlling the temperature and/or humidity within the process chamber 3. In various embodiments, the system is configured for maintaining a temperature above 25 ℃, preferably between 36 ℃ and 38 ℃ and/or a humidity preferably above 95%. In various preferred embodiments, the device 1 comprises a suction system and/or a system for regulating the pressure, prearranged for maintaining the centrifugation zone or chamber 3 at a pressure lower than the ambient pressure, and/or for forcing the airflow output from the aforesaid zone or chamber into a filtering system configured for preventing the diffusion of potentially contaminated aerosols into the environment.

In various embodiments, the device 1 comprises a control panel, such as that presented only in fig. 1 and 2, indicated by 6, on which are suitable control elements 6a and possibly display and/or warning elements, the control elements 6a being used to start and/or stop the process of centrifugation and/or regulation and/or pressure regulation and/or detection, and possibly to set the parameters of the aforementioned process (for example, the speed and/or time of centrifugation, and/or the temperature and/or humidity and/or pressure in the chamber 3). The aforementioned control elements may be of any suitable type (push buttons, knobs, sliders, touch displays, etc.).

Referring also to fig. 3, designated by 10 is a microfluidic device according to a possible embodiment of the present invention. In various embodiments, such as the illustrated embodiment, the device 10 is configured for integrating or housing at least one arrangement designed to concentrate particles contained in a sample of a fluid substance by centrifugation. For this purpose, the device 10 comprises or integrates at least one microfluidic arrangement, denoted M in fig. 3, preferably a plurality of microfluidic arrangements. In the following, for the sake of simplicity, reference will first be made to the case of a device 10 provided with a plurality of microfluidic arrangements M, but in other embodiments described below, the microfluidic device 10 according to the invention may comprise only one microfluidic arrangement.

In various embodiments, and as illustrated in fig. 4, the microfluidic device comprises a substrate 11 and a cover element 12, which define respective portions of the or each microfluidic arrangement M.

In various embodiments, the device 10 is configured for being disposed for rotation relative to a center of rotation, which is assumed herein to be identified by the member 5a of the device 1 of fig. 1 and 2. For this purpose, in various preferred embodiments, the device 10 is disc-shaped and preferably comprises means 11a for coupling to an actuation system of a corresponding centrifugal device, for example, for coupling to the member 5a of the device 1 of fig. 1 and 2. In the illustrated case, the aforementioned coupling means 11a comprise a central through opening or hole in the disk-shaped substrate 11. On the other hand, as will be seen, the disc-like shape of the substrate 11 does not constitute a necessary characteristic, which does not hinder the fact that: in various embodiments, the substrate will be configured to rotate relative to a center of rotation.

In various embodiments, the substrate 11 has a relatively small thickness, for example comprised between 0.5 mm and 4 mm. The substrate may be made of glass or plastic (e.g. polycarbonate, or polyethylene, or cyclic olefin copolymer or COC), for example, and have a diameter comprised indicatively between 10 cm and 30 cm, and may therefore resemble a classical optical disc. The material used is preferably an electrically insulating material, very preferably an at least partially transparent material.

In various embodiments, the covering element 12 also has a relatively small thickness, for example comprised between 0.1 mm and 0.5 mm. The covering element 12 may be made, for example, of polycarbonate or COC or polyethylene or glass and has a diameter similar to that of the substrate 11, for example, comprised between 10 and 30 cm, as indicated.

The material or materials used for the cover element are preferably substantially impermeable to air and liquid. Moreover, the covering element 12 may be disc-shaped, preferably provided with a central through opening 12a, which central through opening 12a will occupy a concentric position with respect to the through opening 11a of the substrate 11 (see, for example, fig. 3). The covering element 12 may, for example, be made of a flexible sheet material which is glued or bonded to the substrate 11.

With reference to fig. 4 and 5, in various embodiments, the at least one microfluidic arrangement of the device according to the invention comprises a respective set of microchannels 13, which microchannels 13 are defined on the surface 11b of the substrate 11 on which the covering element 12 is applied (which is also generally defined herein as the upper surface).

In various preferred embodiments, the device 10 has a plurality of microfluidic arrangements, which are not necessarily identical to each other. For this purpose, a plurality of groups of microchannels 13 may be provided on the substrate 11, each group belonging to a respective microfluidic arrangement. The different sets of microchannels 13 preferably have substantially the same length, but this does not constitute a necessary characteristic.

In various embodiments, multiple sets of microchannels 13 of various lengths are provided. For example, in FIGS. 4 and 5, 13₁Indicated is a group whose microchannels have the greatest length, indicated by 13₂Indicated are the groups whose microchannels have the smallest length and are designated by 13₃Shown are groups whose microchannels have intermediate lengths. Sets of microchannels having different lengths may be useful, for example, for optimizing the occupation of the available space on the substrate 11, in particular for substrates such as: the substrate has a circular shape and/or has microfluidic arrangements or groups of channels 13 in substantially radial positions in order to have a large number of microfluidic arrangements available on the substrate 11 and, thus, parallelization of multiple samples can be performed in a convenient manner.

In various embodiments, such as the one illustrated, the microchannels 13 of each group extend in respective substantially radial directions with respect to the centre of rotation of the device 10, i.e. with respect to the central through opening 11a of the substrate 11. The microchannels 13 of each group are preferably arranged side by side, preferably parallel to each other, and/or preferably substantially rectilinear. The microchannels 13 of each group preferably extend according to a plane defined by the substrate 11 and, for this purpose, they may be defined on the surface 11b by a suitable technique, for example by microetching or moulding or by resin polymerization by means of UV. In any case, the formation of microchannels by deposition of material on the substrate 11 is not excluded from the scope of the present invention.

According to the preferred embodiment presented, the microchannels 13 of each group comprise at least one intermediate microchannel arranged in a radial position with respect to the centre of the through opening 11a of the substrate 11, while in a configuration in which in any case the radial arrangement is approached, the other microchannels of the same group are parallel to said intermediate microchannel, preferably on both sides of the radial microchannel.

According to another embodiment not represented, the microchannels 13 of each group comprise all the microchannels arranged radially with respect to the centre of the central through opening 11a of the substrate 11; i.e. the microchannels 13 of each group are slightly angled with respect to each other, preferably diverging from each other at the end further away from the central through opening 11, i.e. converging at the end closer to the central through opening 11 a.

Each microchannel 13 has an inlet end and is prearranged for receiving a fluid sample. For this purpose, preferably but not necessarily, each microfluidic arrangement M also comprises at least one loading chamber (which may also be in the form of a duct or channel) to which is connected in fluid communication the inlet end of each microchannel of the corresponding group 13.

Such a loading chamber is clearly visible, for example, in the detail views presented in fig. 6 and 7, in which it is indicated by 14. It is clear from fig. 7 how the microchannels 13 have their inlet ends in fluid communication with the respective chambers 14, some of these inlet ends being indicated by 13a, and how these inlet ends 13a are connected to the chambers 14 themselves, preferably with a connection or arrangement of the ends 13a parallel or they are arranged side by side. Thus, the microchannels 13 extend directly from the chamber 14.

In various embodiments, particularly those relating to microfluidic devices configured for centrifugation, the chambers 14 and inlet ends 13a of a given set of microchannels 13 will be disposed closer to the center of rotation of substrate 11, whereas the opposite ends of these microchannels are designed to occupy positions further away from the center of rotation.

The microchannels 13 of each group preferably are at least partly identical to each other and/or extend at least partly substantially parallel or equidistant to each other, for example in a substantially radial direction of the substrate 11. In various embodiments, the microchannels of the same group are substantially identical to each other in shape and size. Alternatively, according to other embodiments (not presented), the following groups may be provided, namely: the microchannels thereof have substantially the same pattern but different lengths from each other.

It can be noted from fig. 7 how, in various preferred embodiments, the chamber 14 and the microchannels 13 are obtained by cavity or surface etching carried out in the substrate 11, the microchannels 13 being in particular in the form of microgrooves. In general, each microchannel 13 may have a width of between 5 μm and 200 μm, preferably between 15 μm and 50 μm, and/or a depth or height of between 2 μm and 100 μm, preferably between 5 μm and 40 μm. The length of each microchannel 13, understood as the distance between its two ends, may be indicatively between 5 mm and 50 mm. For uniformity of analysis, it is preferable to have the microchannels 13 of the same group have a constant through-opening cross section. Illustratively, the walls or raised portions separating the microchannels 13 from each other, some of which are indicated at 11d in fig. 7, may have a width of between 5 μm and 200 μm, preferably between 15 μm and 100 μm.

In various preferred embodiments, the chamber 14 has a depth equal to or close to the depth of the microchannel 13, for example, a depth between 2 μm and 100 μm, preferably between 5 μm and 40 μm.

As already mentioned, each microfluidic arrangement comprises a cover element 12, which at least partially covers a microchannel 13 of the corresponding set of microchannels. The cover element 12 may be at least partially made of a transparent material, such as glass or a plastic material, in order to enable the observation of the underlying microchannels 13, for example for optical detection or illumination purposes. However, this does not constitute an essential feature of the invention, for example when the substrate 11 is made of a transparent material, defining at least in a portion thereof a group of microchannels 13 or defining in a portion thereof the end regions of a given group of microchannels 13.

In various embodiments, such as the embodiments described so far, the same covering element is configured for at least partially covering a plurality of groups of microchannels 13. For example, with reference to the case of fig. 3 and 4, on the substrate 11 thirty-six groups of microchannels 13 are provided, each group comprising a plurality of microchannels arranged side by side or parallel to each other, having different lengths and oriented in respective substantially radial directions, all of which are at least partially covered by the same covering element 12.

According to other embodiments, each microfluidic arrangement may comprise one or more individual covering elements, while the or each element at least partially covers a single group of microchannels 13.

The cover element 12 (or each cover element) is configured or dimensioned for exposing at least a portion of each microfluidic arrangement M, and in particular at least a portion of the chamber 14. For this purpose, in various embodiments, the covering element 12 has at least one loading opening or through opening, which is substantially at the corresponding chamber 14 in the assembled state of the device 10. This feature is best understood, for example, in fig. 8-10, where some of the loading ports are indicated at 15. In this example, each loading through-opening 15 has a circular profile, but this shape is obviously not essential. Likewise, the generally curved profile of the chamber 14 does not constitute an essential feature.

In various embodiments, the material of which the covering element 12 is made is hydrophilic to facilitate the entry of the fluid into each microchannel 13 of a group by capillary from the chamber 14 to the inlet end 13a of the microchannel itself. In this case, the material of which the microchannels 13 are made or the material of the substrate 11 may also be hydrophobic.

It is also possible to make at least one surface of the microchannel 13 extending over its entire length of hydrophilic material: for example, in a microchannel 13 having a rectangular or trapezoidal cross section, at least one of the four walls defining the cross section of the microchannel will preferably be made of a hydrophilic material, such as the wall defined by the covering element 12.

As already mentioned, both the substrate 11 and the cover element 12 may be transparent. For example, the substrate 11 may be at least partially made of a transparent material to enable viewing of the microchannels 13, and the cover element 12 may be transparent to enable backlighting of the microchannels themselves.

In various embodiments, each microchannel 13 has at least one continuous inner surface portion with hydrophilic properties throughout its extent. The continuity of the hydrophilic part along the inner wall of the microchannel 13 may be useful during filling, which envisages, for example, the deposition of a drop of sample liquid in the chamber 14 (as schematically represented in fig. 8). Contact with the hydrophilic part causes filling of the microchannel 13 by capillary action. To this end, in various embodiments, the bottom and side walls of the microchannels 13 and the corresponding chambers 14 are made of a single hydrophobic material, while the main part of the upper walls of the microchannels (for example, their part formed by the covering element 12) is made of a hydrophilic material. On the other hand, as will be seen, each microfluidic arrangement is preferably configured at its end region opposite to the inlet end 13a of the microchannel 13 for resisting the discharge of liquid in the absence of stress. Thus, once each microchannel 13 is completely filled, it is no longer subject to the flow of liquid within it unless it is subjected to an external force, as explained below.

As previously mentioned, the base plate 11 of the device 10 does not necessarily have to be disc-shaped. Such a situation can be understood from fig. 8, which fig. 8 shows a microfluidic arrangement M having: a base plate 11, the base plate 11 having a shape cut substantially in the form of a parallelepiped, preferably in the form of a plane; and a covering element 12 in the form of a foil, which is also parallelepiped.

As will be seen, such a substrate, i.e. not a substrate having a disc-like shape, may advantageously be prearranged for processing, for example in a centrifugal device of the commercially available type, by means of a suitable support or adapter element, or on a universal disc-like support to be coupled to the rotating member 5a of the device 1 of fig. 1. In any case it should be noted that fig. 8 (as in the subsequent fig. 9, 12 and 13) can also be understood to represent in any case a portion of a larger microfluidic device, for example the rectangular portion denoted by M of the device 10 of fig. 3.

The microfluidic arrangement of the device according to the invention comprises, in its end region substantially opposite to the inlet end of the microchannel, a passage for enabling at least the outlet of air from the microchannel itself. According to the invention, between the passage and the microchannel there is provided a filter element permeable at least to air, configured for retaining the particles of interest present in the fluid sample within the microchannel itself.

Thus, during production of the microfluidic device, the mesh or porosity of the filter element may be selected according to the size of the particles to be analyzed. In various embodiments, the filter element may also be permeable to the liquid portion of the sample fluid, for example, to enable the liquid portion to be drained from the microchannel during centrifugation.

In various embodiments, the cover element may be further configured to define at least a portion of a receptacle for the filter element; alternatively, a housing seat for the filter element can be obtained in the base plate 11.

In various embodiments, the location of the filter element is generally the location of the tip, particularly at an end substantially opposite the end through which fluid enters the microchannel. The microchannels may terminate at a portion of the filter element or extend to a contiguous region that is further enclosed by a cover element that is both liquid and gas impermeable.

In the first case, each microchannel may be completely filled by capillary action, while in the second case, the region of the channel extending beyond the filter element will initially remain filled with air (unless filling is performed under vacuum or negative pressure conditions, or the microchannels initially contain at least partially a neutral fluid). During centrifugation, centrifugal forces compress the air (or other fluid) causing it to partially or fully flow out through the filter element. At the end of centrifugation, the particles will collect in the area at the end of the channel not covered by the filter element. This configuration is advantageous when the filter element is not transparent or is prone to introduce optical distortions that may deteriorate the appearance of aggregates, i.e. particle quality or concentrated aggregates. In this way, display can occur through a transparent and flat surface.

As already said, a cover element configured for at least partially covering a microchannel of at least one microfluidic arrangement may be dimensioned or configured to define the aforementioned passage. For example, with reference to fig. 3, 4, 8, 9 and 11, in various embodiments, the cover element 12 defines a passage 16 of the corresponding microfluidic arrangement M, wherein the passage 16 is provided, for example, by a through opening of the element 12. In the case illustrated in fig. 3, 4 and 11, it is assumed that thirty-six groups of microchannels 13 are provided on the substrate 11, the covering element 12 defining a corresponding number of passages 16. In other embodiments, a single passage 16 may be provided at a location corresponding to multiple sets of microchannels 13.

Referring again to the example of the above figures, assuming that the microchannels 13 of each group are substantially rectilinear, the passages 16 and the loading through openings 15 of the same microfluidic arrangement are substantially aligned with each other in the extension direction of the corresponding microchannels 13.

In fig. 3, 4, 8, 9 and 11, designated 17 are some of the aforesaid filter elements, permeable at least to air, which will be located between the groups of microchannels 13 and the corresponding passages 16. As can be seen in particular in fig. 11 and 12, in various embodiments, the covering element 12 may advantageously define a seat 18, this seat 18 being configured for at least partially housing the corresponding filtering element 17. As illustrated (see, for example, fig. 11), such seats 18 may be defined at the corresponding passages 16, in particular in the side of the covering element 12 to be facing the substrate 11.

Thus, in various embodiments, the microfluidic arrangement is configured in such a way that: so that the corresponding filter element is held in the operating position by the same covering element that at least partially covers the corresponding microchannel.

The or each filter element 17 is preferably shaped like a membrane having a porosity or mesh size of between 0.02 μm and 0.45 μm, preferably about 0.2 μm. One class of materials that is advantageous in this sense is ceramic materials, such as alumina, which can be obtained with controlled porosity. In particular, alumina has a very low tendency to bind in a non-specific manner to dyes or fluorescent dyes that are commonly used to label cells. It is clear that other porous materials suitable for this purpose can be used, for example plastic materials, which, although generally showing advantages in terms of cost, must be evaluated case by case with regard to the tendency to bind to the aforementioned marker dyes or fluorescent dyes and also on the basis of the fluorescence of the polymeric material itself. Generally, in the case where the microorganisms or cells to be analyzed are previously labeled with a fluorescent dye, the filter element 17 will preferably be made of a material such as: the material does not bind in a non-specific manner to the fluorescent dye used and does not exhibit auto-fluorescence which would result in a reduction of the signal to noise ratio.

In various embodiments, the thickness of the filter element used is comprised between 20 μm and 1000 μm, preferably between 100 μm and 600 μm. The filter element is preferably optically transparent. Porous alumina tends to scatter light and therefore appears opaque and is not at all suitable as a substrate of optical quality, but in certain cases the effect of scattering is greatly reduced when its nanopores are filled with water (refractive index of about 1.33) or other fluids having a refractive index more similar to that of alumina (refractive index of about 1.63 measured at 550 nm), and the quality of the image obtainable by wetting the alumina membrane is sufficient for detecting particles or cells in a clear field or fluorescence.

In the illustrated case, the element 17 has a quadrangular shape, but this shape should not be considered essential: the shape of the filter element 17 may in fact vary according to the needs or to the type of microfluidic device obtained.

Fig. 8 schematically shows a possible mode of introducing a fluid sample into a microfluidic arrangement M of a device according to a possible embodiment of the invention. In the illustrated case, via a suitable tool T (for example, a pipette designed to dispense a controlled quantity of fluid, which is indicatively of the order of microliters or tens of microliters), a sample FS of the fluid to be subjected to inspection is deposited in the chamber 14 through a corresponding loading port 15, which loading port 15 is preferably at least partially defined in the covering element 12.

As in the illustrated case, the sample FS may be a simple drop of fluid, or may even have a larger volume.

The chamber 14 and the port 15 facilitate the introduction of the fluid sample into the microfluidic arrangement M. Furthermore, assuming that the arrangement M comprises a plurality of microchannels 13, the chamber 14 essentially acts as a collector for introducing the fluid into the plurality of microchannels in parallel. In other words, providing the chambers 14 connected in parallel to the respective ends 13a of the plurality of microchannels 13 has the advantage of: the need to introduce respective portions of the sample into a single microchannel is avoided.

It should be noted that, as mentioned previously, the chamber 14 may be provided by a duct or channel via which the fluid sample is delivered to the inlet end 13a of the microchannel.

The possibility of connecting a plurality of microchannels to the same inlet, whether it be a chamber, a port or a duct, makes it possible to increase the statistical basis of the detection, i.e. to obtain a number of repetitions of the same nominal conditions.

The number of microchannels to be used under the same nominal conditions will depend on the type of use of the device and on the volume of each microchannel: for example, if the microchannel 13 is 2 cm long, with a width of 50 μm and a depth of 5 μm, the total volume will be 5 ∙ 10⁶ μm³. At a concentration of 10⁵bacteria/mL, there will be 10 per cubic micron^-7And (4) bacteria. This means that there will be an average of 0.5 bacteria per microchannel. This also means that in a microchannel containing at least one bacterium, in the case of proliferation, the signal may double after a very short time (about 20-40 minutes), while remaining constant in a microchannel in which there is no proliferation.

This type of use may be referred to as "digital antibiogram". Since microchannels are very small and may be defined in similar patterns in close proximity to each other, there may be multiple channels, for example between 250 and 500 microchannels, over a very limited area (e.g., the area of a single microscope slide).

At concentrations like the one just mentioned, it will be convenient to allocate a plurality of microchannels comprised between 100 and 200 for each n-replicate (n-replicate), i.e. the group of n microchannels used under the same nominal conditions, at the same nominal concentration in order to have a sufficient statistical basis. Thus, on a single centrifugable device, for example a disc-shaped centrifugable device, it will be possible to test a plurality (tens) of different conditions, each of which is repeated n times (n-configured), where n is comprised between 100 and 200. Conversely, for higher concentrations, it will be possible to group nominally identical conditions in a smaller number of microchannels. For example, at a concentration of about one million bacteria per milliliter, an n-tuple of 10-20 microchannels would be usable for each nominally identical condition.

In various embodiments, each microchannel 13 is closed at its longitudinal end opposite to the inlet end 13a, for example, as presented in fig. 15, fig. 15 illustrates an end region CA of a group of microchannels 13, with the corresponding filtering element 17 cut away. Such an embodiment may be used when disposed on top of at least the end extensions of the microchannels 13 is a filter element 17, as can be seen for example in fig. 13-15: thus, the fluid can penetrate from the inlet end of the microchannel 13 (fig. 10, 13 a) due to the fact that: the air contained in the microchannels 13 can be gradually discharged through the filter element 17. It should be noted that the fluid that initially at least partially fills the microchannel 13 may not be air (e.g., a neutral liquid or gas, i.e., it does not alter subsequent operations, this initial fluid being subsequently replaced by a liquid containing a possible particle analysis object).

It can be well noted from figures 13-15 how the same filtering element 17 can be superimposed to the plurality of microchannels 13, preferably but not necessarily in a position corresponding to the end regions CA thereof, which are parallel to each other and closed at the end thereof opposite to the inlet end 13 a.

As already said, in various embodiments each microchannel 13 is preferably filled by capillary action or by exploiting the hydrophilicity of at least one of the walls or surfaces delimiting the microchannel itself. On the other hand, as will be seen, in other embodiments (not represented), the fluid sample may be urged into the microchannel under pressure, for example using a positive or overpressure at the inlet, or a negative pressure (always with respect to the ambient pressure) at the outlet. As already said, during the filling of the microchannels 13, the air initially contained therein may be expelled through the respective filtering element 17 and the respective passage 16, which passage 16 is defined here in the covering element 12.

For example, with reference to the device 10 of fig. 3, after the introduction of a corresponding fluid sample into the chamber 14 of at least one arrangement M (for example, as shown in fig. 8), the microfluidic device is subjected to centrifugation, for example by means of a device 1 of the same type as presented in fig. 1 and 2. Fig. 16 illustrates the installation of a device 10 of the disc type, for example of the device of fig. 3, in a centrifugation and/or detection device 1, in which the latter door 4 is in the closed condition.

After rotation of the device 10, and due to centrifugal forces, the particles present in the liquid volume occupying the microchannels 13 will tend to accumulate at their end regions CA, remaining mainly within the channels themselves; in particular, in the vicinity of the filter element 17, the particles will tend to accumulate at or near the closed end of the corresponding microchannel and/or on its bottom wall and/or on its side walls in the end region CA.

In case the filter element 17 is also permeable to liquids, the same liquid in the sample fluid will be able to exit from the microchannel 13 due to centrifugal forces, passing through the element 17 and the corresponding passage 16, but in any case retaining the particles of interest in the end region CA of the microchannel.

According to various embodiments, in particular in the case of a filter element 17 permeable to liquids, at least a portion of the particles present in the liquid volume contained in each microchannel 13 will tend to accumulate on at least a portion of the filter element 17 located in the end region CA of the respective microchannel 13, or on the portion of the wall of the microchannel delimited by said portion of the filter element 17.

Of course, the size of the microchannels 13 must be sufficient to allow the particles of interest to enter therein. In general, relatively shallow microchannels are preferred, i.e., microchannels having a size of about the particle of interest or only slightly greater height or depth. The reason for this is that, given the same number and size of particles, in the end regions CA of the shallow microchannels 13, the amount of particles accumulated beside each other will form an image in a plane having a larger area than the deeper microchannels, wherein these particles may be located on top of each other and, thus, to some extent skew the detection of the amount of particles and/or their type. Thus, the use of shallow microchannels, preferably with an approximately rectangular cross-section, facilitates and improves the quality of the number and/or type of readings taken using the optical system.

For example, if the device 10 has to be used to separate different types of whole blood cells, it is preferred to have the micro-channels 13 with a height (depth) between 10 μm and 40 μm, preferably between 10 μm and 20 μm. Alternatively, if the analysis object is a bacterium, the microchannel may have a height (depth) of between 3 μm and 10 μm, preferably between 4 μm and 8 μm. Also, in case yeast is to be measured, the height (depth) of the micro-channels will preferably be between 5 μm and 20 μm, most preferably between 8 μm and 12 μm.

In any case, due to the arrangement mentioned, the particles that may be contained in the fluid volume that permeates into the microchannel 13 tend to concentrate at the corresponding end regions CA, both due to the centrifugal force to which the particles are directly subjected and caused by the rotation of the device 10 about the centre of rotation 5a, and due to the flow of the fluid and/or the evacuation of the microchannel with which the suspended particles are entrained.

The detection or reading can be performed by optically quantifying the size of the particle mass formed in each end accumulation region CA due to the centrifugation. Such amount and/or type of detection may also be performed by measuring the intensity of fluorescence in the case where the particles have been previously labeled with a fluorescent dye.

In various embodiments, the apparatus 1 itself may incorporate an optical detection arrangement. The optical arrangement may comprise a single sensor or an array of sensors (e.g. as in an optical scanner), or a rectangular array of sensors, such as e.g. CCD or CMOS sensors, by which an image of the end region CA of the microchannel may be captured and analyzed in various ways, e.g. with an automated processing program for counting particles. In general, then, the same device 1 can integrate a centrifugation function and a detection or reading function, in particular by using the rotation of the support 10 for the aforementioned centrifugation, as well as for the aforementioned reading using an optical detection arrangement.

For example, fig. 16 and 17 illustrate a centrifugation device 1 having a detection arrangement comprising at least one optical sensor 20, the optical sensor 20 preferably itself being constituted by an array of optical sensors. In this example, the sensor 20 is fixedly mounted, in particular at the bottom wall 3a of the process chamber 3. The sensor 20 is at a distance from the centre of rotation 5a of the device 10 such that it can pass through the end regions CA (fig. 6 and 15) of all microfluidic arrangements present on the disc-shaped device 10 before the sensor itself. In the illustrated case, the sensor 20 faces the side of the substrate 11 opposite to the covering element 12, and the substrate 11 is made of transparent material at least at the aforesaid end regions CA of the various microfluidic arrangements M: in this way, the sensor 20 is able to perform the necessary optical detection in any case. The optical sensor may be provided with suitable optics designed to focus and magnify the area of interest.

Possibly, at a portion substantially opposite to the optical sensor 20, a light source may be provided for optical detection, or another optical detection sensor may be provided. In the illustrated case, the light source 21 is associated with the inside of the door 3 of the device 1, in a position such that: so that the source 21 illuminates at least the end area CA each time it is exposed to the sensor 20 in the condition of door closed as presented in fig. 16-17. Also for this purpose, the filter element 17 can be made of a transparent material or of a material which is transparent when it comes into contact with a liquid. As already mentioned, a preferred material for providing the filter element 17 is porous alumina.

The control system of the device 1 may be prearranged for controlling the angular position of the microfluidic device 10 as a function of the optical detection to be performed each time. The control system may also be prearranged to perform the optical detection after the end of the centrifugation step by driving and stopping the support 10 each time in a different angular reading position, or to make the optical detection be performed as the support 10 is moved, preferably at a low speed, for example a speed lower than the centrifugation speed during the detection or reading, or by synchronizing the rotation with the reading.

In other embodiments, for example for microfluidic devices provided with microfluidic arrangements that are oriented in a different way than in the case previously illustrated with reference to fig. 1-3, the optical sensor 20 may be movably mounted on a corresponding guide, for example by means of its own actuator, such that it may be displaced in a radial direction, for example with respect to the device 10, for performing the necessary optical detection on a plurality of microfluidic arrangements. For such a case, the control system of the device 1 will be prearranged for controlling the position of the sensor 21 according to the optical detection to be performed each time.

In various embodiments of the present invention, the optical sensor device 20 of the centrifugation and/or detection device of the mentioned type is configured for acquiring cumulative light signals or cumulative images of a plurality of accumulation regions of the microfluidic device, i.e. signals or images relating to all accumulation regions CA of the microchannels 13 of the corresponding microfluidic arrangement M. Then, for example by means of suitable software, the centrifugation and/or detection means are prearranged for processing, on the basis of the aforementioned optical signals or images, information representative of the amount of particles that have accumulated in each of the individual accumulation regions CA of the respective microchannels of the same microfluidic arrangement, in particular with the following processing, namely: this process enables the number of particles per individual microchannel 13 to be estimated.

In other embodiments, for example, when the optical sensor 20 comprises an array of sensors, such as in an optical scanner, the sensor itself may be configured for acquiring individual optical signals or individual images of the accumulation region CA of each individual microchannel 13 of the corresponding microfluidic arrangement M. Also in this case, the centrifugation and/or detection means are arranged in advance for processing, based on the aforementioned optical signals or images, information indicative of the amount of particles that have accumulated in each of the respective accumulation regions CA of the respective microchannels of the microfluidic arrangement.

Of course, the device 1 can also be arranged so as to be able to employ both of the mentioned techniques of optical detection (i.e. collective and individual).

Fig. 18-20 illustrate possible variant embodiments of the centrifugation and/or detection device 1 and of the microfluidic device 10.

In the illustrated case, the devices 10 have a substantially quadrangular outline and preferably each comprise a single microfluidic arrangement. However, a device of this shape may also comprise a plurality of microfluidic arrangements substantially parallel to each other.

As can be seen in particular in fig. 18, in various embodiments, the device 1 may be equipped with a centrifugal support 30, the centrifugal support 30 defining one or more seats 31, the seats 31 being preferably oriented in a substantially radial direction with respect to the centre of rotation 5a, the one or more seats 31 each being intended to receive at least one microfluidic device 10. In various embodiments, the support 30 has, at each seat 31, a through opening 32 (for example, an opening or window or an optically transparent area), which through opening 32 is located in a position corresponding to the position assumed by the end detection area CA of the microchannel when the corresponding device 10 is mounted on the support itself, as illustrated in fig. 19. The aforementioned position of the through opening 32 on the support also corresponds in the radial direction to the position of the sensor 20 of the device 1, so that this sensor can perform the necessary optical detection. The through openings 32 make it possible to make the centrifugal support 30 of a non-transparent material, but there may be centrifugal supports 30 that are at least partly made of a transparent material.

In the non-limiting example presented, four seats 31 are provided, one for each device 10, each seat 31 being provided with a corresponding through opening to enable detection by the optical sensor 20.

In various embodiments, in order to ensure the positioning of the microfluidic device 10 on the centrifugal support 30, the latter may be provided with an upper element, indicated by 40 in fig. 18, which closes the seat 31 from above, thus ensuring that the microfluidic device 10 remains in position. Furthermore, the upper element 40 may be provided with through openings 41, for example openings or windows or optically transparent areas, at positions substantially corresponding to end regions of the microfluidic arrangement of the device 10, in order to enable illumination thereof by the light source 21.

Fig. 20 schematically illustrates the mounted state of the support 30, the support 30 having corresponding upper elements 40 and having the microfluidic device 10 disposed therebetween, only one of which is visible at a cut-away portion of the upper element 40. The operation of the device 1 of fig. 18-20 is similar to the operation of the device 1 described with reference to fig. 1-2 and 16-17 in terms of its centrifugation and/or detection functions.

Fig. 21 illustrates a microfluidic device 10 having a quadrangular outline, which is for example suitable for use on a centrifugation and/or detection device 1 of the type shown in fig. 18-20, i.e. designed for mounting on a corresponding centrifugation support 30. In this case, the device comprises a single microfluidic arrangement M, which in turn comprises a set of microchannels 13 defined on a substrate 11, as can be appreciated from fig. 22 or 23, as well as a chamber 14, a cover element 12 and a filter element 17.

In the case illustrated in fig. 22, the filtering element 17, having a substantially rectangular profile, is sized to completely or practically completely cover the microchannel 13, leaving at least a portion of the chamber 14 exposed. The material constituting the filter element 17 may advantageously be a hydrophilic material or a hydrophobic material, as required, based on what has been explained previously. The filter element 17 may be fixed in place on the base plate 11, for example by gluing or bonding. The cover element 12, which here also has a substantially rectangular contour, is then fixed to the filter element 17 and possibly partially to the base plate 11.

In the case illustrated in fig. 23, the filtering element 17, having a substantially rectangular profile, is sized to cover only the end region of the microchannel 13 opposite the chamber 14. Also in this case, the filter element 17 can be fixed in place on the base plate 11, for example by gluing or bonding. Then, on at least a part of the filter element 17 and possibly partly on the substrate 11, a covering element 12 is fixed, which covering element 12 here also has a substantially rectangular profile, and in this case directly delimits the microchannel 13 at least in the upper part of the microchannel 13, at least for the main extension of this microchannel 13 extending between the filter element 17 and the chamber 14.

In the case of fig. 22 and in the case of fig. 23, the element 12 is dimensioned in such a way as to expose in any case at least one portion of the chamber 14 and at least one end portion of the filter element 17 in such a way that: so that, according to what has been explained previously, in any case a passage 16 will be defined for the outflow of air and possibly liquid of the fluid sample.

Then, also in an embodiment of the type shown in fig. 22 and 23, the covering element 12 extends at least partially over the microchannels 13, but with the filter element 17 disposed at least partially between the microchannels 13 and the covering element 12. Since the element 12 is substantially impermeable to fluids, it makes it possible in this way to confine the fluid itself within the microchannel 13, at least between its inlet end 13a (i.e. the chamber 14) and its accumulation portion CA where the filter element 17 is not covered by the covering element 12. It should be noted, however, that with reference to an embodiment of the type shown in fig. 23, the covering element 12 does not necessarily have to at least partially cover the filter element 17, and both elements may be fixed to the base plate in an adjacent position.

Fig. 24-25 illustrate possible modes of introducing the fluid sample FS into the microfluidic device according to fig. 21-22 using a suitable tool T, as already described with reference to fig. 8. The inlet end 13a of the microchannel 13 can be understood in particular from fig. 24, which inlet end 13a can be covered at the top by a filter element 17, as in the illustrated case. In contrast, from the next figure 26, it is possible to see the opposite end portions of the microchannel 13, while the longitudinal ends thereof are closed, so as to define an end region CA where the particles accumulate after centrifugation.

As can be appreciated, in this case, the concentration of the particles of interest at the end region CA of the microchannel 13 is obtained by rotating the device 10 with respect to the centre of rotation, for example using a device 1 of the type shown in fig. 18-20.

As already mentioned, in various embodiments, the microchannels 13 may extend beyond the filter element 17 in the area enclosed in any case by the cover element 12. One such case is illustrated in fig. 26a, in which the end area CA of the microchannel 13 is highlighted, which extends beyond the filtering element 17, but is in any case covered by the covering element 12 provided with the passages 16. In this case, therefore, the filter element 17 and the passage 16 are in the middle region of the microchannel, i.e. upstream of the corresponding closed end region CA. The area CA is initially filled with air (or other gas or neutral liquid) which is compressed during centrifugation and can partially or completely exit from the filter element 17 and the passage 16. At the end of centrifugation, the particles are concentrated at the bottom end of the microchannel 13, i.e. in the area CA not covered by the filter element 17. Thus, in this type of embodiment, the filter element may be opaque, while at least one of the substrate 11 and the cover element 12 will be transparent to allow the required detection.

As mentioned, in other embodiments, for example, an overpressure at the inlet or a negative pressure at the outlet is used with respect to the ambient pressure, and thus, even without centrifugation, the fluid sample may be forced under pressure through the microchannels of the microfluidic device according to the invention. Such an example is illustrated in fig. 27 and 28 relative to the device 10 as shown in fig. 21.

In the case of fig. 27, for this purpose a pressure generator system is provided, only partially visible and indicated by 60, and prearranged for generating and directing a pressurized flow of liquid to be treated or of air or other gas a to the chamber 14, where a sample of fluid has been prearranged. In this way, the fluid sample in the chamber 14 is first actuated to penetrate into the microchannels 13 and then through them up to their closed ends and then possibly to exit from the passages 16 through the corresponding portion of the filter element 17, in which case this filter element 17 will also be permeable to liquids. In this way, according to what has been described previously, the pressurized gas or fluid will cause the liquid part of the sample to be expelled from the microchannel 13, at the end region of the microchannel 13 will instead accumulate possible particles to be analyzed.

Alternatively, fig. 28 illustrates the case of a system for generating a negative pressure or vacuum, only partially visible and indicated by 70, such as a suction syringe or pump, predisposed for generating a vacuum or suction pressure V at the passage 16 defined by the terminal extension of the filter element 17, in which case the filter element 17 will also be permeable to the liquid.

In this way, the fluid sample previously set in the chamber 14 is attracted by the negative pressure or vacuum generated and first penetrates into the microchannels 13 and then passes through them until their closed ends and finally, also in this case, exits from the passage 16 through the corresponding portion of the filter element 17. In this way, possible particles to be analyzed will instead accumulate at the end regions of the microchannels, while the liquid fraction will be discharged from the device 10.

It will be understood that in the case of a microfluidic device 10 of the type shown in fig. 3, a pressure generator system 60 and/or a suction system 70 may also be used.

In various embodiments, the microchannels of the microfluidic arrangement M are only used for detecting particles of interest contained in the fluid sample, while in other embodiments, in particular in case the particles to be detected are microorganisms capable of propagating, these microchannels may also be used as culture wells (culture wells). Alternatively, some microchannels may be "loaded" with biological material (e.g., bacteria) that has been induced to proliferate outside the device or that has been inhibited by antibiotics.

In various embodiments, at least one microchannel or each microchannel may be associated with at least two electrodes, in particular at least at the respective end regions CA. These electrodes may be electrodes for detection or electrodes for manipulation of particles.

For example, in various embodiments, at least one pair of electrodes at the end region CA may be used to perform a reading of the amount of particles via the detection of electrical impedance. Differential reading (differential reading) can also be performed by positioning further pairs of electrodes in the portion of the microchannel comprised between the corresponding ends, so as to make it possible to distinguish between the contribution to the electrical impedance represented by the particles and the contribution to the fluidic representation of the sample. In case the fluid sample is a culture medium or a physiological solution, the electrical conductivity is relatively high due to the ions dissolved in the fluid.

An electrode pair positioned in such a way that: one electrode of the pair is in a position corresponding to the portion of the microchannel closer to the corresponding inlet end 13a, while the other electrode of the pair is in the vicinity of the end region, also making it possible to verify whether the microchannel is properly filled with the fluid containing the particles to be counted (this verification is relatively easy considering that the fluid has a conductivity that is generally much higher than that of air as an insulator). Preferably, when envisaged, these electrodes are also at least partially made of an electrically conductive transparent material.

Given that the device 10 according to the invention can be used to accumulate cells in a precise position (i.e. at the end accumulation area CA), the electrodes of the mentioned type can also be used to perform manipulations of the cells themselves, such as electroporation, or to hold them in place by means of dielectrophoresis.

As already mentioned, the microfluidic device 10 according to the invention may be used for the purpose of simple counting and/or type detection of particles contained in a fluid sample, or also for more complex analytical functions, for example for performing antibiogram detection (in which case the microchannels may also be pretreated, for example by introducing antibiotics therein).

The microfluidic device and the centrifugation and/or detection device according to the invention can be advantageously used for the purpose of assessing the proliferative capacity of bacteria and microorganisms and, from the generic point of view, for the purpose of determining its sensitivity profile (antibiogram) to antibiotics in a short time and with a small amount of sample fluid.

The methods known for this purpose are based on an assessment of the ability of the microorganism or bacteria to form colonies in a medium suitable for their growth, or on an assessment of the turbidity of the culture broth after the proliferation of the microorganism. The ability of antibiotics to inhibit the proliferation of microorganisms or bacteria is classically assessed by counting the turbidity levels of the corresponding colonies or of the corresponding culture broth, characteristics that vary according to the sensitivity of the microorganisms or bacteria to the antibiotics.

This sensitivity is related to the ability of the antibiotic to inhibit the efficient proliferation of the bacterial strain, and it is clear that the time associated with this type of analysis depends on the rate of proliferation of the microbe or bacteria. The methods adopted according to the prior art are essentially based on the following facts: a "two-dimensional" layer of bacteria or micro-organisms (colonies) can grow until it becomes visible to the naked eye, or on the basis of the fact that: the proliferation of bacteria or microorganisms in a liquid enables the turbidity of the liquid itself to be altered in a statistically significant manner, which turbidity can be measured by means of photometry in the turbidity range (readings are usually made at wavelengths between 500 nm and 600 nm).

Instead, the techniques presented herein that utilize the previously described microfluidic devices are based on certain parameters that do not take into account the two-dimensional growth of bacteria or layers of microorganisms or growth in a liquid, which is read as an increase in turbidity.

More specifically, the method presented herein contemplates:

i) obtaining short-term growth of biological material (e.g., urine collected directly by a patient) with or without the addition of growth factors (e.g., a bacterial culture, such as BH);

ii) introducing the culture obtained in the preceding step into a microchannel 13, which microchannel 13 is seeded with the same concentration of biological material and/or culture medium (for which purpose it may be particularly advantageous to provide micropores in the microchannel 13);

iii) measuring the proliferation of bacteria in the microchannel 13;

iv) identifying one or more "negative" microchannels 13, i.e. microchannels in which only medium will be added (e.g. 50% in the case of PBS buffer or physiological solution);

v) identifying one or more "positive" microchannels 13, i.e. microchannels capable of verifying the proliferative capacity of the bacteria or microbacteria strains present in the system of microchannels 13;

vi) identifying a series of micro-channels 13 containing antibiotics in the following manner, namely: verifying the resistance or sensitivity to antibiotics of the bacterial or microbacterial strains present in the seeded biological material.

The measurement of the sensitivity to antibiotics can be carried out using different strategies, starting from the sedimentation of the bacteria or microorganisms after their proliferation in the accumulation area CA of the microchannel 13, which can be obtained by centrifugation of the device 10 or by overpressure and/or underpressure as explained in relation to fig. 26-27. The method advantageously makes it possible to make the necessary comparisons between:

-the amount of bacteria or microbacteria present in the starting material;

-the amount of bacteria or microbacteria present at the end of the incubation; and

the amount of bacteria or microbacteria present in the microchannel 13 treated with antibiotics; the use of suitable fluorescent dyes may enable selective identification of live and dead bacteria.

For such an analysis, a support 10 provided with a plurality of microfluidic arrangements, for example the support of fig. 3, may be particularly advantageous. These microfluidic techniques have a higher sensitivity compared to other techniques (e.g. turbidity), which takes into account that external stresses (centrifugation, overpressure, negative pressure) cause the microorganisms to "concentrate" in a small space, thus making them visible both in transmission and reflection in a clear field with visible light, or in fluorescence on labeled cells. With the proposed concentration technique and suitable image analysis, the +/-20% change in cell number is measured in a reliable and accurate manner by means of a linear array of sensors or by means of a rectangular array of sensors (e.g. a CCD or CMOS camera or any other technique for image acquisition). Even after a short growth time (for example comprised between 20 and 40 minutes), it is possible to determine the variation of this degree, which can be detected using the proposed method, and not using classical turbidity techniques. As already mentioned, the quantification or estimation can be performed by optical detection at least in the accumulation area CA of each microchannel 13 of interest.

Additionally or alternatively, the counting of the bacterial bodies can be performed using electrodes provided in the accumulation area CA, in order to detect the impedance variations of the electric field containing the "proliferating" bacterial or micro-bacterial population: this change can be used as a signal for the sensitivity (or resistance) of the bacterial strain under examination. Also in this case, the detection time may be extremely short.

The above method can be advantageously used in very different situations from a clinical point of view.

For example, the "absolute" number of bacteria or microbes in a sample of relatively common biological material (e.g., urine for urine culture) can be measured. For example, if a count above 100000 bacteria/mL indicates a urinary tract infection, only a "digital" record of the bacterial charge (charge) indicates a pathological condition with high accuracy.

Furthermore, without the identification of micro-bacteria or bacteria (which can in any case be performed with standard techniques, if required), the profile of sensitivity/resistance to the antibiotic group can be easily assessed, thus providing the patient with the opportunity to receive "non-empirical" treatment, but this is based on studies on the actual antibiotic sensitivity. In this case, it is important to remember that most positive urine cultures are characterized by a single isolated microbe, whereas in hospitalized patients, or for pre-analysis reasons, in patients complicated by the reasons associated with sampling techniques, multiple bacterial infections are more frequent.

In more complex situations (e.g. in hospitalised patients), the identification of bacteria leads not only to an improvement in the treatment strategy of patients, but also to an improvement in the treatment strategy of nosocomial infections that may be associated therewith. On the other hand, as already said, for less "expensive" materials such as urine, the identification of pathogens may follow different routes, while profiling of the sensitivity to antibiotics, which is not performed in a very short time, may lead to delays in establishing life-saving antibiotic therapy. For this reason, the device 10 (in particular with micro-wells, as already mentioned) can be loaded with a single colony (for example, isolated from a blood culture) which has not been identified yet, but for this reason an immediate treatment method becomes necessary. In this latter case, bacteria isolated from composite materials (complex materials) can be seeded, and an antibacterial spectrum can be obtained within tens of minutes.

The features of the invention and its advantages are also apparent from the foregoing description.

The proposed device and method enable operation with a relatively small starting sample volume, e.g. containing between 0.05 mL and 1 mL. For example, in paediatrics, in studies on small animals, and in any situation where it is useful to reduce the amount of (biological and reagent) material, it is advantageous to be able to use relatively small volumes, also for economic reasons. When a plurality of particles were counted with almost no reproducibility problem, the measurement of the particulate component (coprinused component) was terminated: typically, 16000 particles are counted to obtain an accurate estimate of the subpopulations, which comprise 1% to 5% of the population. Thus, if for example it is assumed that starting from a concentration of one hundred thousand particles per mL, an amount of starting sample comprised between 0.2 mL and 0.4 mL will be sufficient according to the invention, whereas for higher concentrations, for example one million particles per mL, the amount of starting sample may be reduced, for example to between 0.02 mL and 0.06 mL.

The device according to the invention is particularly advantageous for performing antibiogram detection.

Generally, for this purpose, a bacterial culture can be inoculated into the microchannels 13 of at least one arrangement M of the device 10. Then, as mentioned, the device 10 is subjected to centrifugation, overpressure or negative pressure and, subsequently, the bacterial count that has accumulated in the region CA of the microchannel 13 is quantified or estimated. In such applications, the microfluidic device 10 may be used exclusively for quantifying microorganisms, such as bacteria, provided that the proliferation under the different conditions to be compared can be obtained beforehand using common laboratory equipment and devices.

In other applications, the antibiogram detection may be performed starting from a two-dimensional culture of bacteria on a solid support. In this case, the method may envisage the following steps:

i) taking out bacterial colonies from the solid culture dish;

ii) inoculating the colonies or a part thereof into a liquid medium, such as a culture broth, to preferably form a homogeneous dispersion; and

iii) loading a liquid culture medium containing bacteria into the microchannels 13 of at least one arrangement M of the device 10, wherein at least some of the aforementioned microchannels have previously been provided with antibiotics, preferably different types and/or different concentrations of lyophilized antibiotics, and other microchannels have not been provided with antibiotics;

iv) incubation for a period ranging from 10 minutes to 6 hours, preferably between 1 and 2 hours;

v) treating the device 10 by centrifugation or by overpressure or underpressure;

vi) quantifying the bacteria that have accumulated in the area CA of the microchannel 13, in particular by performing a relative quantification between the microchannel 13 pretreated with antibiotics and the microchannel 13 not pretreated, in order to obtain a profile of the sensitivity of the bacteria in question to the antibiotic or antibiotics used.

In other applications, however, the device according to the invention may advantageously be used to perform antibiogram detection starting from an original sample, i.e. a sample taken directly from a subject or host organism (human or animal). In this case, the method may envisage the following steps:

i) obtaining a concentrate or pellet (or pellet) of bacteria from a raw sample such as urine; for this purpose, the original sample may be subjected to centrifugation, for example using common laboratory equipment and devices, in order to separate the aforementioned bacterial mass from the surfactant; centrifugation is preferably performed in two steps: the first step is to remove cells at a low speed; and a second step of concentrating the bacteria at high speed; alternatively, the first centrifugation step may be replaced by filtration to remove cells;

ii) inoculating the obtained bacterial mass or a part thereof into a liquid culture medium, such as a culture broth, to preferably form a homogeneous dispersion;

iii) loading a liquid culture medium containing bacteria into the microchannels 13 of at least one arrangement M of supports 10, wherein at least some of the aforementioned microchannels have previously been provided with antibiotics, preferably with different types and/or concentrations of lyophilized antibiotics, and other microchannels are not provided with antibiotics;

iv) incubation for a period ranging from 10 minutes to 6 hours, preferably between 1 and 2 hours;

v) treating the device 10 by centrifugation or by overpressure or underpressure; and

vi) quantifying the bacteria that have accumulated in the end regions CA of the microchannels 13, in particular by performing a relative quantification between the microchannels 13 pretreated with antibiotics and the microchannels 13 not pretreated, in order to obtain a profile of the sensitivity of the bacteria in question to the antibiotic or antibiotics used.

It is clear that a person skilled in the art may carry out numerous variations to the support and to the substrate, to the device and to the method described herein as examples, without thereby departing from the scope of the present invention. It will also be apparent to those skilled in the art that various features described in relation to one embodiment may be used in other embodiments described herein, even if different from the previous examples.

The application of the invention is not limited to the medical or veterinary field, but the described supports and devices can be used for concentrating and/or quantifying particles present in any type of fluid, for example particles also present in the industrial or agricultural field.