Patent Description:
The invention has been developed with particular reference to microfluidic devices designed to be subjected to centrifugation, as well as to devices and methods for conducting examinations or analyses on fluid samples, preferably containing organic or biological particles or bacteria or micro-organisms, for example for rapid execution of antibiograms.

The invention may in any case also be applied to the detection of other types of particles that may be present in a fluid sample, not necessarily organic or biological fluids or particles, and not necessarily via centrifugation.

Various techniques are known for counting particles, for example cells, present in a sample of a fluid, for example a biological fluid. The systems most commonly used are of an optical type (with or without fluorescence), of an impedancemetry type, or of a static type by means of image recognition. These known systems in general require relatively large sample amounts and do not enable an efficient parallelisation of the measurement, such as a number of measurements carried out at the same time, i.e., they presuppose a considerable amount of the starting sample to be able to carry out many measurements in parallel and/or simultaneously.

Known systems based upon image-recognition techniques may be used for the analysis of small fluid samples, but do not enable parallelisation of a number of samples, with consequent lengthening of the measurement times unless investments are made, which, however, frequently prove anti-economic.

<CIT> discloses a device having the characteristic of the preamble of claim <NUM>.

<CIT> discloses disposable microfluidic cartridges that can receive a sample, such as a blood sample, that is suspected of containing bacterial cells, and separate the bacterial cells from the blood sample. Once the bacterial cells are separated from the blood, the system can introduce the recombinant detector bacteriophages into the system that can infect the bacterial cells.

In its general terms, the present invention has the aim of indicating devices and methods that make it possible to carry out, in a simple, rapid, and inexpensive way, quantification and/or identification of particles present at low concentrations and/or in small volumes in fluid samples, enabling in an equally simple and inexpensive way parallelisation between a number of samples, with advantages in terms of time and costs, as well as in terms of efficiency as regards sensitivity and reproducibility.

A further aim of the invention is to indicate methodologies that make it possible to carry out antibiograms (when micro-organisms are being measured), i.e., to obtain susceptibility profiles of at least one micro-organism, or microbe, or bacterium to antibiotics, in relatively short times, indicatively of some hours; an auxiliary aim of the invention is to indicate methodologies that enable simultaneous execution of a plurality of antibiograms.

The above aims are achieved, according to the present invention, by a microfluidic device for the concentration of particles, and by a corresponding method, which present the characteristics specified in the annexed claims. The invention likewise regards centrifugation and/or detection systems, which comprise the aforesaid microfluidic device, as well as methodologies of analysis based upon the use of such a device.

As will emerge clearly hereinafter, the invention makes it possible to carry out in a simple and rapid way effective detections of amounts of particles in samples of relatively modest volume of the fluid of interest.

Further aims, characteristics, and advantages of the invention will emerge clearly from the ensuing detailed description, with reference to the annexed drawings, which are provided purely by way of non-limiting example and in which:.

Hence, phrases such as "in an embodiment", "in one embodiment", "in various embodiments", and the like, that may be present in various points of this description do not necessarily refer to one and the same embodiment. Moreover, particular conformations, structures, or characteristics defined in the framework of the present description may be combined in any adequate way in one or more embodiments, even different from the ones represented. The reference numbers and spatial references (such as "top", "bottom", "upper", "lower", etc.) used herein are provided merely for convenience and hence do not define the sphere of protection or the scope of the embodiments. The same reference numbers are used in the figures to designate elements that are similar or technically equivalent to one another.

With initial reference to <FIG> and <FIG>, designated as a whole by <NUM> is a centrifugation and/or detection device, having a structure <NUM> that defines a treatment and/or detection chamber <NUM>.

In various embodiments, the device <NUM> includes a lid or door <NUM>, preferably hinged to the structure <NUM>, for closing the chamber <NUM>. The device <NUM> has a driving or movement system, designated as a whole by <NUM> in <FIG>, which includes a rotating member 5a within the chamber <NUM>, designed to set in rotation one or more microfluidic devices, preferably devices of a centrifugable type.

Possibly, the lid <NUM> may comprise a corresponding part 4a of a positioning and/or guide system of a centrifugable microfluidic device, or else of a support configured for supporting a plurality of centrifugable microfluidic devices. In the example, the part 4a includes a seat for a blocking and guiding element, designated by 5b, which can be coupled to the member 5a with the aforesaid centrifugable device or the aforesaid support set in between, in order to ensure mutual fixing in rotation between the parts referred to.

The actuation system <NUM> preferentially comprises an electric motor (partially visible in <FIG>, where it is designated by 5c), possibly provided with a motor reducer and/or an electronic control circuit. The centrifugation speed may indicatively be comprised between <NUM> and <NUM> rpm, preferably between <NUM> and <NUM> rpm, for times preferably comprised between <NUM> and <NUM>, very preferably between <NUM> and <NUM>.

In various embodiments, the device <NUM> comprises a system for control of the temperature and/or humidity within the treatment chamber <NUM>. In various embodiments, this system is configured for maintaining a temperature higher than <NUM>, preferably between <NUM> and <NUM>, and/or a humidity that is preferably higher than <NUM>%. In various preferred embodiments the device <NUM> comprises a suction system and/or a system for regulation of the pressure, pre-arranged for keeping the centrifugation area, or the chamber <NUM>, at a pressure lower than ambient pressure and/or for forcing a flow of air at output from the aforesaid area or chamber into a filtering system configured for preventing diffusion of potentially contaminated aerosols into the environment.

In various embodiments, the device <NUM> includes a control panel, such as the one represented only in <FIG> and <FIG>, designated by <NUM>, located on which are suitable control elements 6a for starting and/or stopping a process of centrifugation, and/or conditioning, and/or pressure regulation, and/or detection, and possibly for setting parameters of the aforesaid process (for example, centrifugation speed and/or time, and/or temperature, and/or humidity, and/or pressure in the chamber <NUM>), as well as possible display and/or warning elements. The aforesaid control elements may be of any suitable type (pushbuttons, knobs, sliders, a touch display, etc.).

With reference also to <FIG>, designated by <NUM> is a microfluidic device according to possible embodiments of the invention. In various embodiments, such as the one exemplified, the device <NUM> is configured for integrating or housing at least one arrangement designed to concentrate, via centrifugation, particles contained in a sample of a fluid substance. For this purpose, the device <NUM> includes or integrates at least one microfluidic arrangement, designated by M in <FIG>, preferably a plurality of microfluidic arrangements. In what follows, for simplicity, initial reference will be made to the case of a device <NUM> provided with a plurality of microfluidic arrangements M, but in other embodiments described hereinafter the microfluidic device <NUM> according to the invention may include just one microfluidic arrangement.

In various embodiments, and as exemplified in <FIG>, the microfluidic device comprises a substrate <NUM> and a covering element <NUM>, which define respective parts of a microfluidic arrangement M, or of each microfluidic arrangement M.

In various embodiments, the device <NUM> is configured for being set in rotation with respect to a centre of rotation, which is here assumed as being identified by the member 5a of the device <NUM> of <FIG> and <FIG>. For this purpose, in various preferential embodiments, the device <NUM> is disk-shaped and preferentially includes means 11a for coupling to the actuation system of a corresponding centrifugation device, for example, for coupling to the member 5a of the device <NUM> of <FIG> and <FIG>. In the case exemplified, the aforesaid coupling means 11a comprise a central passage or hole in the disk-shaped substrate <NUM>. As will be seen, on the other hand, the disk shape of the substrate <NUM> does not constitute an essential characteristic, this not discounting the fact that the substrate, in various embodiments, is to be set in rotation with respect to a centre of rotation.

In various embodiments, the substrate <NUM> has a relatively small thickness, for example comprised between <NUM> and <NUM>. The substrate may, for example, be made of glass or plastic (for instance, polycarbonate, or polyethylene, or cycloolefin copolymers or COCs) and have a diameter indicatively comprised between <NUM> and <NUM>, hence possibly being similar to a classic compact disk. The materials used are preferentially electrically insulating materials, very preferably materials that are at least in part transparent.

In various embodiments, also the covering element <NUM> has a relatively small thickness, for example comprised between <NUM> and <NUM>. The covering element <NUM> may, for example, be made of polycarbonate or COC or polyethylene or glass, and have a diameter similar to that of the substrate <NUM>, for example indicatively comprised between <NUM> and <NUM>.

The material or materials used for the covering element is/are preferentially substantially impermeable to air and to liquids. Also the covering element <NUM> may be disk-shaped, preferably provided with a central passage 12a that is to occupy a position concentric with respect to the passage 11a of the substrate <NUM> (see, for example, <FIG>). The covering element <NUM> may, for example, be made of a flexible sheet material, which is glued or bonded on the substrate <NUM>.

With reference to <FIG> and <FIG>, in various embodiments, the at least one microfluidic arrangement of a device according to the invention comprises a respective set of microchannels <NUM>, defined in a surface 11b of the substrate <NUM> (which is here defined also conventionally as upper surface) on which the covering element <NUM> is applied.

In various preferential embodiments, the device <NUM> has a plurality of microfluidic arrangements, which are not necessarily the same as one another. For this purpose, on the substrate <NUM> a number of sets of microchannels <NUM> may be provided, each set belonging to a respective microfluidic arrangement. The microchannels <NUM> of different sets have preferably substantially the same length, even though this does not constitute an essential characteristic.

In various embodiments, a number of sets of microchannels <NUM> of various lengths are provided. For instance, in <FIG> and <FIG>, designated by <NUM><NUM> is a set the microchannels of which have a maximum length, by <NUM><NUM> is a set the microchannels of which have a minimum length, and by <NUM><NUM> a set the microchannels of which have an intermediate length. Sets of microchannels having different lengths may, for example, be useful for optimising the occupation of the space available on the substrate <NUM>, in particular with a substrate having the circular shape and/or with microfluidic arrangements or sets of channels <NUM> in substantially radial positions in order to have available on the substrate <NUM> a large number of microfluidic arrangements and hence be able to carry out in a convenient way parallelisation of a number of samples.

In various embodiments, such as the one exemplified, the microchannels <NUM> of each set extend in respective substantially radial directions with respect to the centre of rotation of the device <NUM>, i.e., with respect to the central passage 11a of the substrate <NUM>. The microchannels <NUM> of each set are arranged side by side, preferably parallel to one another, and/or are preferentially substantially rectilinear. The microchannels <NUM> of each set extend preferentially according to a plane identified by the substrate <NUM>, and for this purpose they can be defined on the surface 11b via a suitable technique, for example via micro-etching, or moulding, or polymerisation of resins by means of UV. In any case not excluded from the scope of the invention is formation of the microchannels via deposition of material on the substrate <NUM>.

According to the preferential embodiment represented, the microchannels <NUM> of each set comprise at least one intermediate microchannel set in a radial position with respect to the centre of the passage 11a of the substrate <NUM>, whereas the other microchannels of the same set are parallel to said intermediate microchannel, in a configuration in any case close to a radial arrangement, preferably parallel along both sides of the radial microchannel.

According to a further embodiment not represented, the microchannels <NUM> of each set comprise all the microchannels set radially with respect to the centre of the central passage 11a of the substrate <NUM>; i.e., the microchannels <NUM> of each set are slightly angled with respect to one another, preferably mutually divergent at the end further away from the central passage <NUM>, i.e., convergent at the end closer to the central passage 11a.

Each microchannel <NUM> has an inlet end and is pre-arranged for receiving a fluid sample. For this purpose, each microfluidic arrangement M also comprises at least one loading chamber (which may also be in the form of a duct or channel), connected in fluid communication to which is the inlet end of each microchannel of a corresponding set <NUM>.

Such a loading chamber is clearly visible, for example, in the details represented in <FIG>, where it is designated by <NUM>. From <FIG> it may clearly be noted how the microchannels <NUM> have their inlet ends - some of which are designated by 13a - that are in fluid communication with the respective chamber <NUM>, and how these inlet ends 13a are connected to the chamber <NUM> itself, with a connection or an arrangement of the ends 13a in parallel or where they are set side by side. Hence, the microchannels <NUM> directly extend from the chamber <NUM>.

In various embodiments, in particular those regarding microfluidic devices provided for centrifugation, the chamber <NUM> and the inlet ends 13a of the microchannels <NUM> of a given set are to be set in a position closer to the centre of rotation of the substrate <NUM>, the opposite end of the microchannels being instead designed to occupy a position further away from the centre of rotation.

The microchannels <NUM> of each set are preferably at least in part the same as one another and/or extend at least in part substantially parallel to or equidistant from one another, for example parallel to or equidistant from one another in a substantially radial direction of the substrate <NUM>. In various embodiments, the microchannels of one and the same set are substantially the same as one another in terms of shapes and size. According to other embodiments (not represented) sets may, instead, be provided the microchannels of which substantially have one and the same pattern, but have lengths different from one another.

From <FIG> it may be noted how both the chamber <NUM> and the microchannels <NUM> are obtained from cavities or surface etchings made in the substrate <NUM>, the microchannels <NUM> being in particular in the form of micro-grooves. In general terms, each microchannel <NUM> may have a width of between <NUM> and <NUM>, preferably between <NUM> and <NUM>, and/or a depth or height of between <NUM> and <NUM>, preferably between <NUM> and <NUM>. The length of each microchannel <NUM> - understood as the distance between its two ends - may indicatively be between <NUM> and <NUM>. It is preferable for the microchannels <NUM> of one and the same set to have a constant section of passage, for homogeneity of analysis. Indicatively, the walls or portions in relief that separate the microchannels <NUM> from one another - some of these walls or portions being designated by 11d in <FIG> - may have a width of between <NUM> and <NUM>, preferably between <NUM> and <NUM>.

In various preferential embodiments, the chamber <NUM> has a depth equal or close to that of the microchannels <NUM>, for example a depth of between <NUM> and <NUM>, preferably between <NUM> and <NUM>.

As already mentioned, each microfluidic arrangement comprises a covering element <NUM>, which at least partially covers the microchannels <NUM> of the corresponding set of microchannels. The covering element <NUM> may be made at least in part of a transparent material, for example glass or a plastic material, in order to enable viewing of the underlying microchannels <NUM>, for example for the purposes of optical detection or of lighting. This does not constitute, however, an essential characteristic of the invention, for example when the substrate <NUM> is made of transparent material, at least in a part thereof defining a set of microchannels <NUM> or in a part thereof defining an end region of the microchannels <NUM> of a given set.

In various embodiments, such as the ones so far described, one and the same covering element is configured for covering at least partially a plurality of sets of microchannels <NUM>. With reference, for example, to the case of <FIG> and <FIG>, provided on the substrate <NUM> are thirty-six sets of microchannels <NUM>, each comprising a plurality of microchannels set side by side or parallel to one another, which have different lengths and are oriented in respective substantially radial directions, which are all at least partially covered by one and the same covering element <NUM>.

According to other embodiments, each microfluidic arrangement may include one or more individual covering elements, with the element or each element that covers a single set of microchannels <NUM> at least partially.

The covering element <NUM> (or each covering element) is configured or sized for leaving at least one portion of each microfluidic arrangement M, and in particular at least one part of the chamber <NUM>, exposed. For this purpose, in various embodiments, the covering element <NUM> has at least one loading opening or passage that, in the assembled condition of the device <NUM>, is substantially at a corresponding chamber <NUM>. This characteristic may be fully appreciated, for example, in <FIG>, where some of the loading passages are designated by <NUM>. In the example, each loading passage <NUM> has a circular profile, but this shape is evidently not imperative. Likewise, the generally curved profile of the chamber <NUM> does not constitute an essential characteristic.

In various embodiments, the material of which the covering element <NUM> is made is hydrophilic to facilitate entry of the fluid by capillary into each microchannel <NUM> of one set, from the chamber <NUM> to the inlet ends 13a of the microchannels themselves. The material of which the microchannels <NUM> are made, or the material of the substrate <NUM>, may in this case also be hydrophobic.

It is also possible for at least one surface of the microchannel <NUM> that extends throughout the whole length thereof to be made of hydrophilic material: for example, in a microchannel <NUM> with rectangular or trapezial cross section, at least one of the four walls that define the cross section of the microchannel will preferably be made of hydrophilic material, for example the wall defined by the covering element <NUM>.

As already mentioned, both the substrate <NUM> and the covering element <NUM> may be transparent. For instance, the substrate <NUM> may be made at least in part of a transparent material to enable viewing of the microchannels <NUM>, and the covering element <NUM> may be transparent to enable back-lighting of the microchannels themselves.

In various embodiments, each microchannel <NUM> has, throughout its whole extent, at least a continuous portion of inner surface having hydrophilic characteristics. The continuity of a hydrophilic portion along the inner wall of the microchannel <NUM> may be useful during filling, which envisages, for example, deposition of a drop of the sample liquid in the chamber <NUM> (as represented schematically in <FIG>). Contact with the hydrophilic portion causes filling of the microchannels <NUM> by capillarity. For this purpose, in various embodiments, the bottom wall and the side walls of the microchannels <NUM>, and the corresponding chamber <NUM>, are made of a single hydrophobic material, whereas a prevalent part of the upper walls of the microchannels (for example, their part formed by the covering element <NUM>) is made of hydrophilic material. On the other side, as will be seen, each microfluidic arrangement is preferentially configured, at its end region opposite to the inlet end 13a of the microchannels <NUM>, for countering exit of the liquid in the absence of stresses. Consequently, once each microchannel <NUM> is entirely filled, it is no longer subject to the flow of liquid inside it unless it is subjected to external forces, as explained hereinafter.

As mentioned previously, the substrate <NUM> of a device <NUM> does not necessarily have to be disk-shaped. Such a case may be appreciated from <FIG>, which shows a microfluidic arrangement M having a substrate <NUM> with a shape sectioned substantially in the form of a parallelepiped, preferably planar, and a covering element <NUM> in the form of a foil that is also parallelepipedal.

As will be seen, substrates of this sort, i.e., not ones having a disk shape, may advantageously be pre-arranged for being treated - for example, via suitable supports or adapter elements - in a centrifugation device of a commercially available type, or else on a generic disk-shaped support that is to be coupled to the rotating member 5a of the device <NUM> of <FIG>. It should in any case be noted that <FIG> (as likewise the subsequent <FIG>, <FIG>) may in any case be also understood as representing the portion of a larger microfluidic device, for example the rectangular portion designated by M of the device <NUM> of <FIG>.

The microfluidic arrangement of a device according to the invention comprises, in an end region thereof generally opposite to the inlet ends of the microchannels, a passageway for enabling at least outlet of air from the microchannels themselves. According to the invention, provided between this passageway and the microchannels is a filter element permeable at least to air, which is configured for withholding within the microchannels themselves the particles of interest present in the fluid sample.

The meshes or the porosity of the filter element may hence be chosen, during production of the microfluidic device, according to the size of the particles that are to be analysed. In various embodiments, the filter element is also permeable to the liquid part of the sample fluid, for example to enable exit of the liquid part from the microchannels during centrifugation.

In various embodiments the covering element may also be configured to define at least part of a housing seat for the filter element; alternatively, a housing seat for the filter element could be obtained in the substrate <NUM>.

In various embodiments the position of the filter element is generally an apical one, in particular in a position which is substantially at the end opposite to the end through which the fluid enters the microchannels. The microchannels can end at a portion of the filter element, or else extend as far as a successive area, further closed by the covering element impermeable both to liquid and gases.

In the first case, each microchannel may be completely filled by capillarity, whereas in the second case the area of the channel which extend beyond the filter element will initially remain full of air (unless filling is performed under conditions of vacuum or negative pressure, or the microchannel contains initially at least in part a neutral fluid). During centrifugation, centrifugal force compresses the air (or other fluid), causing thereby partial or total outflow thereof through the filter element. At the end of the centrifugation the particles will be concentrated at the end of the channel, in an area not covered by the filter element. This configuration is advantageous when the filter element is not transparent or is subject to introduce optical distortions that may worsen displaying of the pellet, i.e., of the particle mass or concentrated. In this way, the displaying can occur through transparent and flat surfaces.

The covering element, which, as has been said, is configured for covering at least partially the microchannels of at least one microfluidic arrangement, may be sized or configured in order to define the aforesaid passageway. With reference, for example, to <FIG>, <FIG>, <FIG>, <FIG>, and <FIG>, in various embodiments, the covering element <NUM> defines a passageway <NUM> of a corresponding microfluidic arrangement M, where the passageway <NUM> is, for example, provided by a through opening of the element <NUM>. In the case exemplified in <FIG>, <FIG>, and <FIG>, given that on the substrate <NUM> thirty-six sets of microchannels <NUM> are provided, the covering element <NUM> defines a corresponding number of passageways <NUM>. In other embodiments, a single passageway <NUM> may be provided in a position corresponding to a plurality of sets of microchannels <NUM>.

Once again with reference to the example of the above figures, given that the microchannels <NUM> of the various sets are substantially rectilinear, the passageway <NUM> and the loading passage <NUM> of one and the same microfluidic arrangement are substantially aligned with one another in the direction of extension of the corresponding microchannels <NUM>.

In <FIG>, <FIG>, <FIG>, <FIG>, and <FIG>, designated <NUM> are some of the aforementioned filter elements permeable at least to air, which are to be positioned between the microchannels <NUM> of the various sets and the corresponding passageways <NUM>. As may be seen in particular in <FIG> and <FIG>, in various embodiments, the covering element <NUM> may advantageously define a seat <NUM> configured for at least partial housing of a corresponding filter element <NUM>. As exemplified (see, for instance, <FIG>), such a seat <NUM> may be defined at a corresponding passageway <NUM>, in particular in the side of the covering element <NUM> that is to face to the substrate <NUM>.

Hence, in various embodiments, a microfluidic arrangement is configured in such a way that a corresponding filter element is kept in the operative position by the same covering element that covers at least partially the corresponding microchannels.

The filter element <NUM>, or each filter element, is preferentially shaped like a membrane, with a porosity or mesh size of between <NUM> and <NUM>, preferably approximately <NUM>. A class of materials favoured in this sense are ceramic materials, for example alumina, which can be obtained with controlled porosity. In particular, alumina has an extremely low tendency to bind in a non-specific way with the dyes or fluorochromes typically used for marking cells. It is obviously possible to use other porous materials suitable for the purpose, such as plastic materials, which albeit generally presenting advantages in terms of costs, must be evaluated on a case-by-case basis in relation to the tendency to bind to the aforesaid marking dyes or fluorochromes and on the basis of the fluorescence itself of the polymeric material. In general, in the case where the micro-organisms or cells being analysed are previously marked with fluorochrome, the filter element <NUM> will be preferentially made of a material that does not bind in a non-specific way to the fluorochrome used and does not present autofluorescence that would cause a lowering of the signal-to-noise ratio.

In various embodiments, the thickness of the filter elements used is comprised between <NUM> and <NUM>, preferably between <NUM> and <NUM>. The filter element is preferably optically transparent. Porous alumina tends to scatter the light and hence appears opaque and far from suitable as substrate of optical quality, but in the specific case, when its nanopores are full of water (refractive index of approximately <NUM>) or other fluid with a refractive index more similar to that of alumina (refractive index of approximately <NUM> measured at <NUM>), the effect of scattering is considerably reduced, and the quality of the image that can be obtained through the wet alumina membrane is sufficient for detecting particles or cells in clear field or in fluorescence.

In the case exemplified, the elements <NUM> have a quadrangular shape, but this shape is not to be deemed essential: the shape of the filter element <NUM> may, in fact, be different according to the needs or of the type of the microfluidic device obtained.

<FIG> shows schematically a possible mode of introduction of a fluid sample into the microfluidic arrangement M of a device according to possible embodiments of the invention. In the case exemplified, via a suitable tool T (such as a pipette designed to dispense a controlled amount of fluid, indicatively of the order of microlitres or tens of microlitres) a sample FS of the fluid that is to undergo examination is deposited in the chamber <NUM>, through the corresponding loading passage <NUM>, preferably defined at least in part in the covering element <NUM>.

The sample FS may be a simple drop of the fluid, as in the case exemplified, or may even have a larger volume.

The chamber <NUM> and the passage <NUM> facilitate introduction of the fluid sample into the microfluidic arrangement M. Moreover, given that the arrangement M includes a plurality of microchannels <NUM>, the chamber <NUM> basically functions as collector for introduction in parallel of the fluid into a number of microchannels. In other words, provision of a chamber <NUM>, connected in parallel to which are the homologous ends 13a of a number of microchannels <NUM>, presents the advantage of avoiding the need to introduce individually respective fractions of the sample into the single microchannels.

It should be noted that, as mentioned previously, the chamber <NUM> could be provided by a duct or a channel, via which the fluid sample is delivered to the inlet ends 13a of the microchannels.

The possibility of connecting a number of microchannels to one and the same inlet - whether it is a chamber, a passage, or a duct - makes it possible to increase the statistical basis of detection, i.e., to have available a number of repetitions of the same nominal conditions.

The number of microchannels to be used in the same nominal conditions will depend upon the type of use of the device and upon the volume of each microchannel: if, for example, a microchannel <NUM> were <NUM> long, with a width of <NUM> and a depth of <NUM>, the total volume would be <NUM>·<NUM><NUM> µm<NUM>. With a concentration of <NUM><NUM> bacterialmL, there would be <NUM>-<NUM> bacteria per cubic micrometre. This means that in each microchannel there would be on average <NUM> bacteria. This also means that, in the microchannels that contain at least one bacterium, the signal could double after a very short time (approximately <NUM>-<NUM>) in the cases of proliferation, and remain constant in those in which there is no proliferation.

This type of use may be referred to as "digital antibiogram". Since the microchannels are very small and may be defined in positions very close to each other, with a similar pattern, it is possible to have, on a very limited area (such as that of a single microscope slide), a multitude of channels, for example between <NUM> and <NUM> microchannels.

At concentrations like the ones just referred to, it would be expedient to dedicate to each n-tuplicate (i.e., set of n microchannels used in the same nominal conditions) at the same nominal concentration a number of microchannels comprised between <NUM> and <NUM> in order to have a sufficient statistical basis. On a single centrifugable device, for example a disk-shaped one, it would hence be possible to test a multitude (various tens) of different conditions, each of which is n-tuplicated, where n is comprised between <NUM> and <NUM>. For higher concentrations, it will, instead, be possible to group in a smaller number of microchannels the conditions that are nominally the same. For instance, in the case of concentrations of the order of one million bacteria per millilitre it will be possible to use n-tuples of <NUM>-<NUM> microchannels for each nominally identical condition.

Each microchannel <NUM> is closed at its longitudinal end opposite to the inlet end 13a, for example as represented in <FIG>, which illustrates an end region CA of a set of microchannels <NUM>, with a corresponding filter element <NUM> sectioned. Embodiments of this sort may be used when set on top of at least the terminal stretch of the microchannels <NUM> is a filter element <NUM>, as may be seen, for example, in <FIG>: the fluid can thus penetrate from the inlet end (13a, <FIG>) of the microchannel <NUM>, thanks to the fact that the air contained in the latter can progressively vent through the filter element <NUM>. It should be noticed that the fluid which initially fills at least in part the microchannels <NUM> may be other than air (for instance a neutral liquid or gas, i.e., which does not alter the subsequent operations, this initial fluid being then replaced by the liquid containing the possible particles object of the analysis).

From <FIG> it may be well noticed how one and the same filter element <NUM> may be superimposed to the plurality of microchannels <NUM>, preferably but not necessarily in a position corresponding to the end regions CA thereof, which are parallel to one another and which are closed at their end opposite to the inlet ends 13a.

As has been said, in various embodiments, each microchannel <NUM> is preferably filled by capillarity or by exploiting the hydrophilicity of at least one of the walls or surfaces that delimit the microchannel itself. On the other hand, as will be seen, in other embodiments (not represented), the fluid sample could be forced under pressure into the microchannels, for example using a positive pressure or over-pressure at inlet or a negative pressure at outlet (always with respect to ambient pressure). As has been said, in the course of filling of a microchannel <NUM>, the air originally contained therein can vent through the corresponding filter element <NUM> and the corresponding passageway <NUM>, here defined in the covering element <NUM>.

With reference, for example, to the device <NUM> of <FIG>, after introduction of the corresponding fluid sample into the chamber <NUM> of at least one arrangement M (as shown, for example, in <FIG>), the microfluidic device is subjected to centrifugation, for example by means of a device <NUM> of the same type as the one represented in <FIG> and <FIG>. <FIG> illustrates the condition of installation of a device <NUM> of a disk-shaped type, such as the device of <FIG>, in a centrifugation and/or detection device <NUM>, with the door <NUM> of the latter in a closed condition.

Following upon rotation of the device <NUM>, and as a result of the centrifugal force, the particles present in the volume of liquid that occupies a microchannel <NUM> will tend to accumulate at its end region CA, remaining prevalently within the channel itself; in particular, the particles will tend to accumulate at, or in the proximity of, the closed end of the corresponding microchannel and/or on its bottom wall and/or on its side walls in the end area CA, in the proximity of the filter element <NUM>.

In the case where the filter element <NUM> is also permeable to liquid, the same liquid of the sample fluid will be able to exit from the microchannel <NUM>, as a result of the centrifugal force, passing through the element <NUM> and the corresponding passageway <NUM>, but in any case withholding 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 <NUM> permeable to liquid, at least part of the particles present in the volume of liquid contained in each microchannel <NUM> will tend to accumulate on at least part of the filter element <NUM> located in the end region CA of the respective microchannel <NUM>, or on the portion of wall of the microchannel delimited by said portion of filter element <NUM>.

Of course, the dimensions of the microchannels <NUM> must be sufficient to allow entry of the particles of interest therein. In general terms, relatively shallow microchannels are preferable, i.e., ones having a height or depth of the order of the size of the particles of interest or just slightly greater. The reason for this is that - given the same number and size of the particles - in the end region CA of a shallow microchannel <NUM> the amount of particles accumulated alongside one another will form an image in the plane having a larger area than a deeper microchannel, where the particles could lie on top of one another and thus falsify to a certain extent detection of the amount of particles and/or type thereof. The use of shallow microchannels, preferably with an approximately rectangular section, hence facilitates and improves the quality of reading of the amount and/or type using optical systems.

For instance, if a device <NUM> has to be used for separation of different types of whole blood cells, it is preferable for there to have a height (depth) of the microchannels <NUM> of between <NUM> and <NUM>, preferably between <NUM> and <NUM>. If the object of analysis are, instead, bacteria, the microchannels may have a height (depth) of between <NUM> and <NUM>, preferably between <NUM> and <NUM>. Again, in the case where yeasts are to be measured, the height (depth) of the microchannels will preferably be between <NUM> and <NUM>, most preferably between <NUM> and <NUM>.

In any case, thanks to the arrangement referred to, the particles possibly contained in a volume of the fluid that penetrates into a microchannel <NUM> tend to concentrate at the corresponding end region CA, both as a result of the centrifugal force undergone directly by the particles and caused by a rotation of the device <NUM> about the centre of rotation 5a and possibly as a result of the flow of the fluid and/or of emptying of the microchannel that entrains along with it the particles in suspension.

Detection or reading may be carried out by quantifying in an optical way the size of the mass of particles that, as a result of centrifugation, is formed in each end accumulation region CA. It is also possible to carry out such a detection of the amount and/or type by measuring the intensity of fluorescence, in the case where the particles have previously been marked with fluorochromes.

In various embodiments, the device <NUM> itself can integrate an optical detection arrangement. The optical arrangement may include a single sensor or else an array of sensors (for example, as in an optical scanner), or else a rectangular array of sensors, as for example a CCD or a CMOS sensor, with which it is possible to capture the image of the end area CA of the microchannel and analyse it in various ways, for example with automatic processing programs for counting particles. In general, then, one and the same device <NUM> can integrate functions of centrifugation and functions of detection or reading, in particular by exploiting rotation of the support <NUM> both for the aforesaid centrifugation that for the aforesaid reading using the optical detection arrangement.

For instance, <FIG> exemplify a centrifugation device <NUM> having a detection arrangement that includes at least one optical sensor <NUM>, which preferably is itself constituted by an array of optical sensors. In the example, the sensor <NUM> is mounted stationary, in particular at a bottom wall 3a of the treatment chamber <NUM>. The sensor <NUM> is at a distance from the centre of rotation 5a of the device <NUM> such that in front of the sensor itself there can pass the end regions CA (<FIG> and <FIG>) of all the microfluidic arrangements present on the disk-shaped device <NUM>. In the case exemplified, the sensor <NUM> faces the side of the substrate <NUM> opposite to the covering element <NUM>, and the substrate <NUM> is made of transparent material, at least at the aforesaid end regions CA of the various microfluidic arrangements M: in this way, the sensor <NUM> is in any case able to carry out the necessary optical detections. The optical sensor may be provided with the appropriate optics designed to focus and magnify the area of interest.

Possibly, at a part generally opposite to the optical sensor <NUM> a light source may be provided, in order to facilitate optical detection, or else another optical detection sensor. In the case exemplified, a light source <NUM> is associated to the inner side of the door <NUM> of the device <NUM>, in a position such that - in the condition where the door is closed as represented in <FIG> - the source <NUM> illuminates at least the end region CA each time exposed to the sensor <NUM>. Also for this purpose the filter element <NUM> may be made of a transparent material, or a material that it is transparent when it comes into contact with a liquid. A preferred material for providing the filter element <NUM> is, as has been said, porous alumina.

The control system of the device <NUM> may be pre-arranged for controlling the angular position of the microfluidic device <NUM> according to the optical detections to be carrying out each time. This control system may also be pre-arranged so as to carry out optical detections after the end of the centrifugation step, by driving and stopping each time the support <NUM> in the various angular reading positions, or else so that the optical detections are performed with the support <NUM> moving, preferably at low speed, such as a speed during detection or reading lower than the centrifugation speed, or by synchronising rotation with reading.

In other embodiments, for example with microfluidic devices provided with microfluidic arrangements oriented in a way different from the cases previously exemplified with reference to <FIG>, the optical sensor <NUM> can be mounted movable, for example via an actuator of its own, on a corresponding guide so that it can be displaced, for example in the radial direction relative to the device <NUM>, for carrying out the necessary optical detections on a number of microfluidic arrangements. For such cases, the control system of the device <NUM> will be pre-arranged for controlling the position of the sensor <NUM> according to the optical detections to be carried out each time.

In various embodiments of the invention, the optical sensor means <NUM> of a centrifugation and/or detection device of the type referred to are configured for acquiring a cumulative optical signal or a cumulative image of a plurality of accumulation regions of the micro-fluidic device, i.e., a signal or image regarding all the accumulation regions CA of the microchannels <NUM> of a corresponding microfluidic arrangement M. The centrifugation and/or detection device is then pre-arranged, for example via suitable software, for processing, on the basis of the aforesaid optical signal or image, information representing an amount of particles that have accumulated in each of the individual accumulation regions CA of the various microchannels of one and the same microfluidic arrangement, in particular with a processing that enables estimation of the number of particles for each individual microchannel <NUM>.

In other embodiments, for example when the optical sensor <NUM> includes an array of sensors, such as in an optical scanner, the sensor itself may be configured for acquiring an individual optical signal or an individual image of the accumulation region CA of each individual microchannel <NUM> of a corresponding microfluidic arrangement M. Also in this case, the centrifugation and/or detection device is pre-arranged for processing, on the basis of the aforesaid optical signal or image, information representing an amount of particles that have accumulated in each of the individual accumulation regions CA of the various microchannels of the microfluidic arrangement.

Of course, a device <NUM> may also be provided in order to be able to employ both of the techniques of optical detection (i.e., collective and individual) referred to.

<FIG> illustrate possible variant embodiments of a centrifugation and/or detection device <NUM> and of microfluidic devices <NUM>.

In the case exemplified, the devices <NUM> have a generally quadrangular profile and preferentially each include a single microfluidic arrangement. Devices of this shape may, however, include also a number of microfluidic arrangements generally parallel to one another.

As may be seen in particular in <FIG>, in various embodiments, the device <NUM> may be equipped with a centrifugation support <NUM>, which defines one or more seats <NUM> - preferably oriented in a substantially radial direction with respect to the centre of rotation 5a - that are each to receive at least one microfluidic device <NUM>. In various embodiments, the support <NUM> has, at each seat <NUM>, a passage <NUM> (such as an opening or a window or an optically transparent area) that is located in a position corresponding to the one assumed by the end detection region CA of the microchannels, when the corresponding device <NUM> is mounted on the support itself, as exemplified in <FIG>. The aforesaid position of the passage <NUM> on the support likewise corresponds - in a radial direction - to the position of the sensor <NUM> of the device <NUM>, so that the sensor is capable to carry out the necessary optical detections. The passages <NUM> make it possible to make the centrifugation support <NUM> of a non-transparent material, but there may possibly be present in a centrifugation support <NUM> made at least in part of transparent material.

In the non-limiting example represented, four seats <NUM> are provided, one for each device <NUM>, each seat <NUM> being provided with a corresponding passage to enable detection by the optical sensor <NUM>.

In various embodiments, in order to ensure positioning of the microfluidic devices <NUM> on the centrifugation support <NUM>, the latter may be provided with an upper element, designated by <NUM> in <FIG>, which closes the seats <NUM> from above ensuring maintenance of the position by the microfluidic devices <NUM>. Also the upper element <NUM> may be provided with passages <NUM>, such as openings or windows or optically transparent areas, in positions substantially corresponding to the end regions of the microfluidic arrangements of the devices <NUM>, in order to enable lighting thereof by the light source <NUM>.

<FIG> illustrates schematically the mounted condition of the support <NUM> with the corresponding upper element <NUM>, and with the microfluidic devices <NUM> set in between, only one of which is visible at the sectioned part of the upper element <NUM>. Operation of the device <NUM> of <FIG> is similar, in relation to its functions of centrifugation and/or detection, to that of the devices <NUM> described with reference to <FIG> and <FIG>.

<FIG> illustrates a microfluidic device <NUM> with quadrangular profile, for example suitable for use on a centrifugation and/or detection device <NUM> of the type illustrated in <FIG>, i.e., designed for installation on the corresponding centrifugation support <NUM>. In this case, the device includes a single microfluidic arrangement M which, as may be appreciated from <FIG> or from <FIG>, in turn includes a set of microchannels <NUM> defined on a substrate <NUM>, as well as a chamber <NUM>, a covering element <NUM>, and a filter element <NUM>.

In the case exemplified in <FIG>, the filter element <NUM>, having a substantially rectangular profile, is sized so as to coat the microchannels <NUM> completely or practically completely, leaving at least part of the chamber <NUM> exposed. The material that constitutes the filter element <NUM> may advantageously be a hydrophilic material or a hydrophobic material, according to the needs, on the basis of what has been explained previously. The filter element <NUM> may be fixed in position on the substrate <NUM>, for example, via gluing or bonding. On the filter element <NUM>, and possibly in part on the substrate <NUM>, the covering element <NUM>, which here also has a substantially rectangular profile, is then fixed.

In the case exemplified in <FIG>, the filter element <NUM>, having a substantially rectangular profile, is sized so as to coat only an end area of the microchannels <NUM> that is opposite to the chamber <NUM>. Also in this case, the filter element <NUM> can be fixed in position on the substrate <NUM>, for example via gluing or bonding. On at least part of the filter element <NUM>, and possibly in part on the substrate <NUM>, there is then fixed the covering element <NUM>, which here also has a substantially rectangular profile and in this case directly delimits the microchannels <NUM> in their upper part, at least for a substantial stretch thereof that extends between the filter element <NUM> and the chamber <NUM>.

Both in the case of <FIG> and in the case of <FIG>, the element <NUM> is sized so as to leave in any case exposed at least part of the chamber <NUM>, as well as at least one end part of the filter element <NUM> in such a way that a passageway <NUM> will in any case be defined for outflow of the air and possibly of the liquid of the fluid sample, according to what has already been explained previously.

Also in embodiments of the type illustrated in <FIG> and <FIG>, then, the covering element <NUM> extends at least partially over the microchannels <NUM>, but with the filter element <NUM> that is at least in part set between the microchannels <NUM> and the covering element <NUM>. Since the element <NUM> is substantially impermeable to the fluid, it makes it possible in this way to confine the fluid itself inside the microchannels <NUM>, at least between their inlet end 13a (i.e., the chamber <NUM>) and their accumulation portion CA, at which the filter element <NUM> is not overlaid by the covering element <NUM>. It should, however, be noted, with reference to embodiments of the type illustrated in <FIG>, that the covering element <NUM> does not necessarily have to be overlaid at least partially on the filter element <NUM>, it being possible for these two elements to be fixed on the substrate in adjacent positions.

<FIG> exemplify a possible mode of introduction of a fluid sample FS into a microfluidic device according to <FIG>, using a suitable tool T, as has already been described with reference to <FIG>. From <FIG> there may in particular be appreciated the inlet ends 13a of the microchannels <NUM>, which can be covered at the top by the filter element <NUM>, as in the case exemplified. From the next <FIG> there may instead be seen the opposite end part of the microchannels <NUM>, with their longitudinal end closed, in order to define the end region CA of accumulation of the particles following upon centrifugation.

As may be appreciated, also in this case, the concentration of the particles of interest at the end regions CA of the microchannels <NUM> is obtained by setting in rotation the devices <NUM> with respect to a centre of rotation, for example using a device <NUM> of the type illustrated in <FIG>.

As already mentioned, in various embodiments, microchannels <NUM> may extend beyond the filter element <NUM>, in an area which is in any case closed by the covering element <NUM>. One such case is exemplified in <FIG>, wherein the end regions CA of the microchannels <NUM> are highlighted, which extend beyond the filter element <NUM> but are anyway covered by the covering element <NUM>, provided with the passageway <NUM>. Hence, in this case, the filter element <NUM> and the passageway <NUM> are in an intermediate area of the microchannels, i.e., upstream of the corresponding closed end regions CA. The regions CA are initially full of air (or other gas or neutral liquid) which, during centrifugation, is compressed, and is able to exit partially or completely from through the filter element <NUM> and the way <NUM>. At the end of the centrifugation the particles are concentrated at the bottom ends of the microchannels <NUM>, i.e., in the regions CA not covered by the filter element <NUM>. Hence, in embodiments of this type, the filter element may be not transparent, while at least one of the substrate <NUM> and the covering element <NUM> will be transparent, to allow for the required detections.

As mentioned, in other embodiments, a fluid sample could be forced under pressure through the microchannels of a microfluidic device according to the invention, for example using an over-pressure at inlet or a negative pressure at outlet, with respect to ambient pressure, and hence even in the absence of centrifugation. Examples of this sort are illustrated in <FIG>, in relation to devices <NUM> as that of <FIG>.

In the case of <FIG> a pressure-generator system is provided for this purpose, which is only partially visible and is designated by <NUM> and is pre-arranged for generating a pressurized flow of the liquid to be treated or else a pressurized flow of air or other gas A and directing it at the chamber <NUM>, where a sample of the fluid has previously been set. In this way, the fluid sample in the chamber <NUM> is forced first to penetrate into the microchannels <NUM> and then pass through them as far as their closed end, and then possibly exit from the passageway <NUM> through the corresponding portion of the filter element <NUM>, which in this case will be permeable also to the liquid. In this way, the pressurized aeriform or fluid will bring about exit of the liquid fraction of the sample from the microchannels <NUM>, at the end regions of which there will instead be accumulated the possible particles that are to be analysed, according to what has been described previously.

<FIG> exemplifies, instead, the case of a system for generating a negative pressure or vacuum, visible only partially and designated by <NUM>, such as an aspirating syringe or a pump, which is pre-arranged for generating the vacuum or suction pressure V at the passageway <NUM> defined by the terminal stretch of the filter element <NUM>, which also in this case will be permeable to the liquid.

In this way, the fluid sample previously set in the chamber <NUM> is drawn in thanks to the negative pressure or vacuum generated and first penetrates into the microchannels <NUM> and then passes through them as far as their closed end, and finally exits also in this case from the passageway <NUM> through the corresponding portion of the filter element <NUM>. In this way, at the end regions of the microchannels there will instead be accumulated the possible particles that are to be analysed, whereas the liquid part will be evacuated from the device <NUM>.

It will be appreciated that pressure-generator systems <NUM> and/or suction systems <NUM> may be used also in the case of microfluidic devices <NUM> of the type illustrated in <FIG>.

In various embodiments, the microchannels of the microfluidic arrangements M are used only for detection of particles of interest contained in the fluid sample, whereas in other embodiments the microchannels can be exploited also as culture wells, in particular in the case where the particles that are to be detected are micro-organisms capable of reproduction. Alternatively, some microchannels may be "loaded" with biological materials (for example, bacteria) that the device have been induced to proliferate outside the device or have been inhibited by antibiotics.

In various embodiments, to at least one microchannel, or to each microchannel, there can be associated at least two electrodes, in particular at least at a respective end region CA. These electrodes may be electrodes for detection or else electrodes for manipulation of the particles.

For instance, in various embodiments, at least one pair of electrodes at an end region CA may be used to carry out reading of amounts of particles via detection of an electrical impedance. It is also possible to carry out differential readings by positioning further pairs of electrodes in portions of the microchannel comprised between the corresponding ends in order to make it possible to distinguish the contribution to the electrical impedance represented by the particles from the contribution represented by the fluid of the sample. In the cases where the fluid sample is a culture medium or a physiological solution, the electrical conductivity is relatively high on account of the ions dissolved in the fluid.

Pairs of electrodes positioned in such a way that an electrode of the pair is in a position corresponding to the part of the microchannel closer to the corresponding inlet end 13a and the other electrode of the pair is in the proximity of the end region, also enable verification of whether the microchannel is filled properly with the fluid containing the particles to be counted (this verification is relatively easy, considering that the fluid has in general a conductivity much higher than that of air, which is an insulator). Preferably, also the electrodes, when envisaged, are made at least in part of an electrically conductive transparent material.

Given that the device <NUM> according to the invention can be used for accumulating cells in a precise position (i.e., at the end accumulation regions CA), electrodes of the type referred to can be used also for carrying out manipulations on the cells themselves, for example electroporation, or else to keep them in position by means of dielectrophoresis.

As already mentioned, a microfluidic device <NUM> according to the invention may be used for the purposes of simple counting and/or detection of the type of the particles contained in the fluid sample, or also for more complex functions of analysis, for instance for carrying out antibiograms (in which case the microchannels could also be pre-treated, for example by introducing antibiotics therein).

The microfluidic devices and the centrifugation and/or detection devices according to the invention may advantageously be used for the purposes of evaluation of the capacity for proliferation of bacteria and microbes and, subordinately, for the purposes of determining a profile of susceptibility thereof to antibiotics (antibiogram) in short times and with small volumes of the sample fluid.

The methodologies known for this purpose are based upon evaluation of the capacity of a microbe or of a bacterium to form colonies in a medium suited to its growth, or upon evaluation of the turbidity of a culture broth following upon proliferation of the microbe. The capacity of an antibiotic to inhibit proliferation of a microbe or of a bacterium is evaluated classically by counting the corresponding colonies or on the level of turbidity of the corresponding culture broth, which are characteristics that vary as a function of the susceptibility of the microbe or bacterium to antibiotics.

This susceptibility is linked to the capacity of the antibiotic to inhibit efficient proliferation of a bacterial strain, and it is evident that the times linked to this type of analysis depend upon the speed at which the microbe or bacterium proliferates. The approach followed according to the prior art is essentially based upon the fact that a "two-dimensional" layer of bacteria or microbes (a colony) can grow until it becomes visible to the naked eye, or upon the fact that proliferation of the bacteria or microbes in a liquid can be such as to modify, in a statistically significant way, the turbidity of the liquid itself, this turbidity being measurable by means of photometry in the turbidity range (the reading is typically made at a wavelength of between <NUM> and <NUM>).

The techniques proposed herein, which exploit the microfluidic devices described previously, are based, instead, upon some parameters that do not consider either the two-dimensional growth of the layer of bacteria or microbes or the growth in the liquid, which is read as increase in turbidity.

More in particular, the methodologies proposed herein envisage:.

The measurement of susceptibility to antibiotics may be carried out using different strategies, starting from sedimentation of the bacteria or microbes after proliferation in the accumulation regions CA of the microchannels <NUM>, which can be obtained via centrifugation of a device <NUM>, or else via over-pressure and/or negative pressure as explained in relation to <FIG>. This approach advantageously makes it possible to carry out the necessary comparisons between:.

For analyses of this sort, particularly advantageous may be supports <NUM> provided with a number of microfluidic arrangements, such as the support of <FIG>. These microfluidic techniques have a higher sensitivity as compared to other techniques (for example, turbidity), given that the external stress (centrifugation, over-pressure, negative pressure) enables "concentration" of the micro-organisms in a small space, hence rendering them visible either in clear field with visible light, both in transmission and in reflection, or in fluorescence on marked cells. With the concentration technique proposed and an appropriate analysis of the image, either by means of linear arrays of sensors of by means of rectangular arrays of sensors (for example, CCD or CMOS cameras or any other technique used for image acquisition) a modification of +/-<NUM>% of the number of the cells is measured in a reliable and accurate way. Variations of this degree, which can be detected using the methodology proposed and instead cannot be detected using classic turbidity techniques, can be determined even after short growth times, for example comprised between <NUM> and <NUM>. The quantification or estimation may be made, as has been said, via optical detections at least in the accumulation regions CA of the various microchannels <NUM> of interest.

In addition or as an alternative, counting of the bacterial bodies can be carried out using electrodes set in the accumulation regions CA in order to detect the modification of the impedance of an electrical field that contains a "proliferating" population of bacteria or microbes: this modification may be used as signal of the susceptibility (or resistance) of the bacterial strain under examination. Also in this case, the detection times may be extremely short.

The methodologies described above can be profitably used in situations that are extremely different from a clinical standpoint.

For instance, it is possible to measure the "absolute" number of bacteria or microbes in a sample of relatively common biological material (for example, urines for urinoculture). If, for example, a count higher than <NUM> bacterialmL is indicative of infection of the urinary ways, the mere "numerical" documentation of the bacterial charge indicates the pathological situation with great accuracy.

Also in the absence of identification of the microbe or bacterium (which can in any case be carried out with standard techniques, if necessary), the profile of susceptibility/resistance to an antibiotic panel may be easily evaluated, offering the patient the opportunity of undergoing a "non-empirical" treatment, but one based upon the study of the real antibiotic susceptibility. In this case, it is important to recall that the majority of positive urinocultures are characterised by a single isolated microbe, whereas polymicrobism is more frequent in hospitalised patients or, owing to pre-analytical causes, in patients that are complex for reasons linked to the sampling technique.

In a more complex situation (for example, in hospitalised patients), identification of the bacterium leads to an improvement in the strategies of treatment not only of the patient, but also of nosocomial infections that may be associated thereto. On the other hand, as has already been said, for less "noble" materials, like urines, identification of the pathogen can follow different pathways, whereas the profile of susceptibility to antibiotics that is not carried out in extremely short times could lead to a delay in setting up a life-saving antibiotic therapy. For this reason, the device <NUM> (in particular with micro-wells, as already mentioned) could be loaded with a single colony (for example, isolated from a haemoculture), which has not yet been identified but for which an immediate therapeutic approach becomes necessary. In this latter case, bacteria isolated from complex materials may be seeded, and the antibiogram could be available within some tens of minutes.

From the foregoing description, the characteristics of the present invention are clear, as likewise are its advantages.

The devices and the methodologies proposed enable operation with relatively small starting sample volumes, for example comprised between <NUM> and <NUM>. For instance, in paediatrics, in research conducted on small animals and in any case where it is useful to reduce the amount of (biological and reagent) material, also for economic reasons, it is advantageous to be able to use relatively small volumes. The measurement of a corpusculated component terminates when a number of particles are counted such as to render the problem of reproducibility virtually absent: in general, <NUM> particles are counted to obtain an accurate estimate of sub-populations that are represented by <NUM> to <NUM>% of the total. Hence, if it is assumed, for example, to start from a concentration of one hundred thousand particles per millilitre, according to the invention an amount of starting sample comprised between <NUM> and <NUM> will be sufficient, whereas, for higher concentrations, for example one million particles per millilitre, the amount of starting sample may drop, for example, to between <NUM> and <NUM>.

The devices according to the invention are particularly advantageous for carrying out antibiograms.

In general terms, for this purpose, a bacteria culture can be inoculated into the microchannels <NUM> of at least one arrangement M of a device <NUM>. The device <NUM> is then subjected to centrifugation, or to over-pressure, or to negative pressure, as mentioned, and the number of bacteria that have accumulated in the regions CA of the microchannels <NUM> is then quantified or estimated. In applications of this sort, the microfluidic device <NUM> may be used exclusively for quantification of micro-organisms, for example bacteria, in so far as the proliferation in different conditions to be compared may be obtained previously, using ordinary laboratory equipment and devices.

In other applications, an antibiogram can be carried out starting from a two-dimensional culture of the bacteria on a solid support. In this case, the methodology may envisage the following steps:.

Yet in other applications, the devices according to the invention can be advantageously used for carrying out an antibiogram starting from a primary sample, i.e., a sample taken directly from a subject or host organism (human or animal). In this case, the methodology may envisage the following steps:.

It is clear that numerous variations may be made by the person skilled in the art to the supports and substrates, the devices, and the methods described herein by way of example, without thereby departing from the scope of the invention. It will likewise be evident to the person skilled in the art that individual characteristics described in relation to one embodiment may be used in other embodiments described herein, even different from the previous examples.

Claim 1:
A microfluidic device for concentrating particles contained in a fluid sample (FS), comprising a substrate (<NUM>) having a surface (11b) at which at least one microfluidic arrangement (M) is defined, which comprises:
- a loading chamber (<NUM>), for loading the fluid sample (FS) into the at least one microfluidic arrangement (M);
- a plurality of microchannels (<NUM>), which have respective inlet ends (13a) extending from the loading chamber (<NUM>), the microchannels (<NUM>) of the plurality being fluidically connected in parallel to said chamber (<NUM>) and set side by side;
- a covering element (<NUM>), which is substantially impermeable to the fluid sample (FS) and extends at least partially over the plurality of microchannels (<NUM>),
wherein the loading chamber (<NUM>) and the plurality of microchannels (<NUM>) extend substantially according to a plane identified by the substrate (<NUM>),
wherein:
- the loading chamber (<NUM>) and the plurality of microchannels (<NUM>) are in the form of cavities or surface etchings of the substrate (<NUM>),
- each microchannel (<NUM>) of the plurality is closed at its longitudinal end opposite to the inlet end (13a), and
- the microchannels (<NUM>) of the plurality are partially delimited, at least at an accumulation region (CA) thereof generally opposite to the respective inlet ends (13a), by a filter element (<NUM>) which is permeable at least to air, is set on top of at least a stretch of the microchannels (<NUM>) of the plurality, and is configured for withholding within each microchannel (<NUM>) possible particles that may be present in the fluid sample (FS),
in such a way that particles possibly contained in a volume of fluid of the fluid sample (FS) that penetrates into each microchannel (<NUM>) tend to concentrate in the respective accumulation region (CA) as a result of a force applied to at least one of the microfluidic device (<NUM>) and the fluid sample (FS) loaded into the loading chamber (<NUM>), such as a centrifugal force caused by a rotation of the substrate (<NUM>) about a centre of rotation (5a), or a positive pressure applied on the fluid sample (FS) at the loading chamber (<NUM>), or a negative pressure applied on the fluid sample (FS) at the accumulation regions (CA) of the microchannels (<NUM>).