GB2430393A

GB2430393A - Micro Device for Automatic Spermatozoa Selection and Cell Sorting

Info

Publication number: GB2430393A
Application number: GB0519473A
Authority: GB
Inventors: Xianghong Ma; Mark Prince; Peter Docker; Michael Ward
Original assignee: Aston University
Current assignee: Aston University
Priority date: 2005-09-23
Filing date: 2005-09-23
Publication date: 2007-03-28
Also published as: GB0519473D0

Abstract

A passive micro filtration device (10) and a method for producing same, for filtering particulate matter, such as cells, from a fluid sample comprising a body (12) having a sample reservoir (14) and a capillary (18) enabling fluid flow from the sample reservoir (14), wherein the capillary (18) is defined in the body and comprises an array (20) of filtration elements (22) between the sample reservoir and a filtrate reservoir (16). The array (20) contains filtration elements (22) shaped as cylindrical pillars or planar columns that define sub-capillaries within the capillary. In one embodiment the capillary (18) comprises multiple filtration arrays (20), each separated from the next by a sample reservoir (14) and the distance between filtration elements (22) decreases with increased distance from the sample reservoir (14). In another embodiment the device (10) comprises multiple filtration capillaries (18), aligned radially away from a common sample reservoir (14).

Description

A Passive Filtration Device The invention relates to a filtration device

particularly adapted for filtering particulate matter from a fluid sample. In particular, the device is adapted to assist in the selection, manipulation and detection of spermatogenic cells from testicular biopsies for the purposes of assisted reproduction therapy.

Current clinical procedures for selecting cells from testicular sperm extraction pellets include manually mincing the tissue under a microscope and subjecting the resultant tissues to multiple centrifugal operations through density media, separating the cells from other debris according to size, and then selecting potentially viable spermatozoa or late-stage spermatids manually for an IntraCytoplasmic sperm injection process.

According to one object the invention seeks to provide a device which provides a more passive selection process which would better enable selection of more viable cells from a sample.

According to one aspect of the invention there is provided a passive filtration device for filtering particulate matter, such as cells, from a fluid sample, comprising a body having a sample reservoir and a capillary enabling fluid flow from a sample reservoir, wherein the capillary is defined in the body and comprises an array of filtration elements between the sample reservoir and a filtrate reservoir.

Beneficially, the capillary acts to enable movement of the fluid sample between the sample reservoir and a filtrate reservoir due to an imbalance in forces arising for example through gravity, pressure differential, surface tension effects and/or capillary action whereby the array of filtration elements is able to prevent passage of larger particulate matter thereby enabling collection of a filtrate in a filtrate reservoir comprising only particulate matter below a certain size.

According to another aspect of the invention there is provided a method of fabricating a filtration device for filtering particulate matter comprising the steps of: forming a sample reservoir in a body, forming a capillary which in use enables flow of a sample away from the sample reservoir, and forming filtration elements in the capillary to enable filtration of sample flowing along the capillary in use.

Further aspects, beneficial features and inventive features of the present invention are set out in the appended claims.

Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which: Figures 1 a and b are a schematic view and partial a sectional side elevation view of the device according to the invention, Figures 2,3 and 4 are a schematic plan view and two microscopic views of part of a device according to a second embodiment, Figure 5 is a schematic plan view of part of a device according to a third embodiment of the invention, Figure 6 is a schematic plan view of part of a device according to a fourth embodiment of the invention and, Figures 7 and 8 are microscopic views of further parts of the embodiment in figure 6, Figure 9 is a schematic plan view of part of a fifth embodiment of the invention, Figures 10 a, b and c, 11 and 12 are a schematic plan and elevational views and two microscopic views of a sixth embodiment of the device according to the invention, Figure 13 is a schematic plan view of a seventh embodiment of a device according to the invention, Figure 14 is a schematic plan view of an eighth embodiment of a device according to the invention, Figure 15 is a plan view of an ninth embodiment of a device according to the invention, Figure 16 is a plan view of part of a device according to a tenth embodiment of the invention, Figure 17 is a schematic plan view with an enlarged portion of a mask used in manufacture a device according to an embodiment shown in figures 2,3 and 4, Figure 18 provides schematic representations of steps A to H in the manufacturing process to form a device according to the invention, Figure 19 is a microscopic view of a device formed according to the invention, Figure 20 is an enlarged microscopic view of some of the filtration elements formed in the device shown in figure 19, Figure 21 is a schematic plan view of elements used to determine the fluid dynamic results provided in figure 23, Figure 22 provides further details of variables used to determine the various characteristics shown in figure 23, and Figure 23 comprises three graphs showing different flow characteristics dependent on the arrangement of elements shown in figures 21 and 22, Referring to figures 1 A and B there is shown a first embodiment of a device 10 according to the invention comprising a body 12 for example etched from a silicon wafer. The device 10 comprises a sample reservoir 14 which in this case is substantially a cylindrical bore through the body 12. Further reservoirs 16 are defined at spaced intervals and again in this example comprise substantially cylindrical bores through the body 12.

A capillary or channel 18 is defined in body 12 extending away from sample reservoir 14 and passing each of the filtrate reservoirs 16. An array 20 of filtration elements 22 is provided in the capillary 18 between each of the reservoirs. The base of the body 12 abuts a substrate 24 such a glass plate.

In one form capillary 18 extends radially away from a central sample reservoir 14 located centrally within device 10 enabling a head of sample to be created within sample reservoir 14 shown in figure 1A as minisus M. The sample is able to flow through one or more capillaries 18 extending away from the sample reservoir 14. In figure 1 A eight capillaries 18 are shown each comprising three filtrate reservoirs 16.

The sample flows through the array 20 of filtration elements 22 along a capillary 18 towards a first or inner filtrate reservoir 16. Particles which are too large to pass through the first array 20 are held within the sample reservoir 14. A further array 20 of more closely packed filtration elements 22 is provided holding further particulate matter in the first filtrate reservoir 16. A third array 20 is also provided, again separating out particulate matter from the sample, collecting smaller particulate matter within the sample fluid at the final filtrate reservoir 16. The filtrate within the third filtrate reservoir 16 at the end of capillary 18 will contain fluid having particulate matter of the smallest dimensions. In this regard, the expression particulate matter is used to refer to all manner of objects suspended within a fluid such as cells and living organisms.

As can be seen in figure 1, the centre of the bores which define the sample and filtrate reservoirs are separated by a dimension R where ROl is the separation of the centres of sample reservoir 14 and inner filtrate reservoir 16, R12 is the separation of the centres of the inner filtrate reservoir 16 and the central filtrate reservoir 16, and R23 is the separation of the central filtrate reservoir 16 and the outer filtrate reservoir 16. In this embodiment, ROl is substantially equal to R12 which is substantially equal to R23, in other foms of separation can be decreased as one moves away from the sample reservoir.

Moreover, it can be seen that array 20 has a length L along capillary 18. In this example LOl is the length of the array 20 between sample reservoir 14 and inner filtrate reservoir 16, L12 is the length of array 20 between inner and central filtrate reservoirs 16, and L23 is the length of array 20 between central and outer filtrate reservoirs 16.

The lengths of the arrays along capillary 18 can be substantially equal or in one form the length decreases the further away the array is located from the sample reservoir.

Preferably the filtration elements 22 are located within the entire width, say 200 microns, of capillary 18 at each of the locations of an array 20 and also substantially equal to the height, say 50 microns, of capillary 18 between the body 12 and the substrate 24.

Moreover, preferably filtration elements 22 are provided substantially adjacent to the edges of each of the adjacent reservoirs such that the length of an array 20 along capillary 18 is the maximum possible length between adjacent reservoir.

Referring to figures 2, 3 and 4, there are shown various views of the second embodiment of a device 10 according to the invention. In this embodiment, a central sample reservoir 14 is provided in a body 12 and eight (not all shown) regularly extending capillaries 18. Each of the capillaries tapers inwardly away from the sample reservoir 14, and hence as can be seen in the micrographs given in figures 3 and 4, the radius of the filtrate reservoirs also decreases the more radially outward the filtrate reservoir is.

In this embodiment the filtration elements are arranged in circumferential columns, each column being equally distant from the centre of the device. However, the columns are not aligned radially with adjacent columns and hence the filtration elements are staggered. Moreover in this embodiment the arrays 20 of filtration elements 22 occupy substantially only 50% of the available length between reservoirs. However, further structural pillars or balance pillars 26 are provided which are useful both in the fabrication and use of the device 10.

Referring to figure 5 there is shown a different embodiment of a device 10 according to the invention, again comprising eight radially extending capillaries 18 and a central sample reservoir 14. In this embodiment the filtration elements are again circumferentially arranged in columns C but in contrast to the previous embodiment, the filtration elements are radially aligned with filtration elements in adjacent columns and not staggered. Accordingly, it is possible to see in figure 5 clear radial lines of pathways extending outwardly from sample reservoir 14 through to each of the filtrate reservoir 16. Again, the capillaries taper inwardly or narrow the further away from the sample reservoir 14 they are.

Referring to figure 6, there is shown a further embodiment of device 10 according to the invention. Again, a number, such as eight, capillaries 18 are provided extending away from a central sample reservoir 14. In this example the capillaries do not taper but have substantially uniform width W along their length except in the very end adjacent outer filtrate reservoir 16. Again circumferential columns of filtration elements are provided.

However, adjacent columns are staggered and moreover the elements 22 can clearly be seen to be more tightly packed, or less separated, in the radially outer array 20, as in all previous embodiments but more visibly so. Moreover, the separation (R23) of the central and outer filtrate reservoir 16 is less than the separation of the other reservoirs whereby the length (L23) of array 20 between the central and outer filtrate reservoir 16 is reduced compared to the inner array. Micrographs of a device according to this embodiment are shown in each figures 7 and 8.

In the embodiments used so far, typically each filtration element has a cylindrical shape wherein the diameter of each element is approximately 10 to 20 microns and a height of about 50 microns. Typically the filtration elements 22 are spaced from one another by a separation of approximately 25 microns in the inner array 20, by 15 microns in the central array 20 and by only 8 microns in the outer array 20 (being radially inward of the outennost filtrate reservoir 16). However, other separations are possible and for example the separation in the inner array 20 might be 15 microns, the central array 10 microns and the radially outer array 4 microns.

In another form, as shown in figure 9, the filtration elements do not have circular cross sections but square or rectangular cross sections. Moreover, the capillary 18 can be seen to be defined in a more rectangular manner and does not taper along its length.

In another form of a device according to the invention as shown in figures 1 Oa, b and c, the filtration elements 22 are in a form of planar structures or sheets. Accordingly, an array of planar filtration elements 22 provides a series of sub-capillaries between adjacent reservoirs. A micrograph of this embodiment is shown in figures 11 and 12.

Referring in more detail to figures bA, B and C, it can be seen that device 10 according to this embodiment comprises a central sample reservoir 14 and eight capillaries 18 extending radially away from the sample reservoir 14. Each of the capillaries 18 comprises an inner, central and outer array (or stage) of filtration elements 22. The width of each capillary is approximately 300 microns and the length of each array and each filtrate reservoir 16 along each capillary is approximately 300 microns. Accordingly, the length of each capillary away from the central reservoir 14 is approximately 1.8 mm. The central sample reservoir 14 has a diameter of approximately 0.8 mm (800 microns) and can hold a reservoir of approximately 0.13 microlitres.

The height of the filtration elements 22 is approximately 50 microns and accordingly, each filter stage has a capacity to hold approximately 0. 0054 microlitres of sample.

Each of the filtrate reservoir 16 has a capacity to hold approximately 0. 03 microlitres.

Accordingly, the total saturation capacity of device 10 shown in figure 1 OC is approximately 1 microlitre. It can be found that an overfill of the sample reservoir 14 with.3 microlitres conveniently provides an additional head for encouraging flow of sample along capillaries 18.

As can be seen from the perspective view of figure lOB, the separation of the filtration elements 22 progressively narrows from one stage to the next. Accordingly, the inner array 20 (stage 1) comprises a coarse filter with separation of filtration elements 22 of approximately 25 microns. The central array 20 comprises filtration elements having a separation of approximately 12 microns and the radially outer array 20 of filtration elements 22 has a separation of approximately 3 microns.

Preferably each of the planar filtration elements 22 are structurally parallel with an adjacent element 22. As before, the separation of filtration elements can be varied the further out the array is from the central sample reservoir 14.

Referring to figure 13 there is shown a further embodiment of a device 10 according to the invention. In this embodiment a central sample reservoir 14 is provided having five sets of capillaries 18 extending to inner central and outer filtrate reservoir 16. In this embodiment each set of capillaries 18 for each of the five outer filtrate reservoir 16 are substantially similarly shaped to that of the inner part of the device so it has five fold rotational symmetry. A first capillary 18 passes through each of the inner, central and outer filtrate reservoirs 16 whereas a second capillary 18 extends simply from the sample reservoir 14 to the central filtrate reservoir 16 and a third capillary 18 extends simply from the sample reservoir 14 and the outer reservoir 16. The darker regions adjacent to the outer filtrate reservoir 16 comprise arrays of filtration elements 22 which are the most fine and densely packed. For example each filtration element can comprise a column having approximately 6 microns diameter and being spaced only 3 microns from an adjacent filtration element.

Further in this embodiment a drain 28 is provided to facilitate further escape to either an absorbent medium placed at the exit of drain 28 or to the atmosphere. The annular outer path 30 and radial path 32 leading to drain 28 comprise substantially uniform support structures 26.

Referring to figure 14 there is shown a further embodiment of a device 10 according to the invention. Again, eight radially extending capillaries 18 are provided. In this embodiment the capillaries expand or taper outwardly away from the central sample reservoir 14. Each of the arrays 20 of filtration elements 22 can provide progressive filtration as before and for example might provide filtration of 25, 15 and 8 micron particulate size respectively.

Referring to figure 15, there is shown a further embodiment of a device 10 according to the invention. In this embodiment 3 sample reservoirs 14 are provided in a single body 12 having linear capillaries 18 extending, two from each sample reservoir 14 thereby providing 6 outer filtrate reservoirs 16. In this embodiment five arrays 20 of filtration elements 22 are provided along each capillary 18 these can for example be used to select particulate size progressively from 25, 15, 12, 8 and 3 microns for example.

Referring to figure 16, there is shown a further embodiment of a device 10 according to the invention wherein an annulus of filtration elements 22 provide an inner array 20 to each of the capillaries 18 extending radially away from the central sample reservoir 14.

The inner array 20 comprises planar filtration elements which extend radially away from the centre of the sample reservoir 14 and hence not parallel to one another.

However, similar planar filtration elements 22 in each of the arrays 20 along the capillaries are provided substantially similarly to the elements shown in figures 10 to 12 for example, and hence whilst substantially planar they are parallel to adjacent elements and the wide walls defining the capillary 18.

In figure 17 there is shown a mask for use in fabricating a device according to the embodiment shown in figures 2, 3 and 4. The mask is placed above a body 12 of materials such as silicon for fabrication of a device 10 according to the invention. The steps according to a preferred manufacturing process are set out in figure 18.

In step A, a single crystal silicon wafer is cleaned with Isopropanol and acetone ready for patterning.

At step B, AZ5214 photo resist is spun onto both sides of the wafer and both sides are exposed using a double side mask alignment process, and developed using AZ514 developer. The darkened area indicates areas to be etched.

At step C, the side to be populated by the fine filter structure features is etched a suitable DRIE etcher and etched to a depth of 60 microns. This creates the "feature" side of the device.

At step D, the "device side" of the wafer is then cleaned and stripped of photoresist in oxygen plasma within the etching machine.

At step B, a "handle" wafer is cleaned and coated with AZ5214 photo resist. Before curing with heat the device wafer is placed onto the handle wafer with the etched structures in contact with the photoresist. The cured resist then serves to bond the wafers together and protect the delicate etched surface.

At step F, the deep wells in the device wafer are then etched through to the handle wafer.

At step G, the remaining photoresist is cleaned off using fuming nitric acid, which also serves to release the device wafer from the handle wafer. The device wafer is then washed using distilled water.

At step H, the freed device wafer is again inverted and the feature side is anodically bonded to glass, sealing the capillary trenches. Finally the devices are diced into the discrete chips using a resin blade.

For example a device formed in this manner can be seen in figure 19 which is a SEM (scanning electron microscope) micrograph showing the fine level of accuracy with which the arrays and filtration elements can be formed. Indeed, the individual filtration elements 22 are shown in further detail in an enlarged micrograph shown in figure 20.

Referring to figures 21 to 23, there is shown some data regarding the effect of the filtration spacing or pitch, and the number of stages on the fluid flow velocity along a capillary. Figure 23A, a fluid velocity at the data collection point, (see figures 21 and 22) is shown in meters per second depending on the number of stages (or columns) of filtration elements along a capillary.

The data shown in figure 22 was acquired using a capillary 18 having a width of 100 microns and a length of 300 microns. As can be seen in figure 21 the flow of sample was from left to right the sample passed through a filter model element F and data collection point for assessing flow characteristics was made at data point D 150 microns along the capillary 18. The characteristics of the filter element array are shown more clearly in figure 22 wherein the separation of columns of filter elements is referred to as the filter stage pitch or SP and the circumferential separation of the filtration elements 22 is referred to as the filter pitch FP. As can be seen from figure 23 there is a marked decrease in the fluid velocity and therefore the time required to effect separation into the various filtrate reservoir 16 according to the number of stages (or arrays) of filtration elements which are used. As can be seen in figures 23B and C, the wider the filter pitch FP the greater the sample flow, as would be predicted, but there is a marked decreased in this effectiveness above a pitch of approximately 20 microns using the silicon based body of the preferred embodiment. However, there is a fairly monatomic decrease in fluid velocity with increasing filter stage pitch (SP).

Claims

Claims 1 A passive filtration device for filtering particulate matter,

such as cells, from a fluid sample comprising a body having a sample reservoir and a capillary enabling fluid flow from the sample reservoir, wherein the capillary is defined in the body and comprises an array of filtration elements between the sample reservoir and a filtrate reservoir.

2 A device according to claim 1 wherein the filtration elements are integrally formed in the device body.

3 A device according to claim 1 or 2 wherein the filtration elements comprise substantially monolithic structures.

4 A device according to claim 1,2 or 3 wherein the filtration elements are substantially cylindrical structures.

A device according to any of claims 1 to 3 wherein the filtration elements are substantially planar structures.

6 A device according to claim 5 wherein the array of filtration elements defines two or more sub-capillaries within the capillary.

7 A device according to any preceding claim wherein the array of filtration elements comprises elements aligned radially away from the sample reservoir.

8 A device according to any preceding claim wherein the array of filtration elements comprises elements aligned circumferentially away from the sample reservoir.

9 A device according to any of claims 1 to 6 wherein the array of filtration elements comprises elements aligned on a rectangular array A device according to any of claims 1 to 6 wherein the array of filtration elements comprises elements which are unaligned, and preferably substantially randomly arranged.

11 A device according to any preceding claim wherein the array of filtration elements comprises elements of substantially equal width.

12 A device according to any preceding claim wherein the array of filtration elements comprises elements substantially equally spaced from one another.

13.A device according to any preceding claim s wherein the array of filtration element capillaries element of substantially equal height and preferably wherein the height is less that 100 microns and more preferably about 50 microns.

14 A device according to any preceding claim wherein the body comprising the sample reservoir and the body comprising the capillary are integrally formed with one another.

A device according to any preceding claim wherein the capillary has a substantially rectangular cross-section substantially normal to the direction of flow of sample along the capillary in use, and or the width is preferably in the order of or less than 200 microns.

16 A device according to any preceding claim wherein the width of the capillary substantially normal to the direction of flow of sample along the capillary in use is substantially constant 17 A device according to any of claims 1 to 15 wherein the width of the capillary substantially normal to the direction of flow of sample along the capillary in use decreases, preferably monotomcally, away from the sample reservoir.

18 A device according to any of claims 1 to 15 wherein the width of the capillary substantially normal to the direction of flow of sample along the capillary in use increases, preferably monotonically, away from the sample reservoir.

19 A device according to any preceding claim comprising two or more arrays of filtration elements along the capillary.

A device according to claim 19 comprising a filtrate reservoir after each array of filtration elements.

21 A device according to claim 20 wherein a filtrate reservoir comprises a drainage path preferably to an external absorbent medium.

22 A device according to claim 19, 20 or 21 wherein the average separation of filtration elements in an array decreases the further along the capillary away from the sample reservoir it is, and or the average separation of filtration elements is substantially constant for each array but decreases from one array to the next along the capillary.

23 A device according to claim 22 wherein filtration elements in an array abut substantially the edge of an adjacent reservoir.

24 A device according to any of claims 19 to 24 comprising at least three arrays of filtration elements.

25 A device according to claim 19 to 24 comprising at least five arrays of filtration elements.

26 A device according to any of claims 19 to 25 wherein an array is designed to prevent flow of particulate matter having a size equal to or greater than 25, 15, 12, 10, 8, or 4 microns along the capillary.

27.A device according to any of claims 19 to 25 wherein the length of an outer array of filtration elements along the direction of flow of the sample in the capillary, is less than an inner array which is closer to the sample reservoir.

28 A device according to any preceding claim comprising two or more capillaries enabling fluid flow away from the sample reservoir.

29 A device according to claim 28 comprising an array of capillaries extending substantially radially away from the sample reservoir.

A device according to claim 28 or 29 wherein the capillaries are arranged substantially symmetrically around the sample reservoir.

31 A device according to claim, 28, 29 or 30 comprising 2, 5, 6 preferably 8 capillaries extending away from the sample reservoir.

32 A device according to any preceding claim wherein the filtration elements are formed by removing part of the body for example by etching.

33 A device according to any preceding claim wherein the device is made substantially of silicon.

34 A device according to any preceding claim wherein the surface of the body which defines a capillary has a hydrophilic coating, and preferably over substantially all of the surface of the filtration elements within the capillary, and more preferably the coating in an oxide layer such as an oxide of silicon.

A method of fabricating a filtration device for filtering particulate matter comprising the steps of: forming a sample reservoir in a body, forming a capillary which in use enables flow of a sample away from the sample reservoir, and forming filtration elements in the capillary to enable filtration of sample flowing along the capillary in use.

36 A method according to claim 35 wherein the capillaries and formed by removal of part of the body thereby also to form the filtration elements which are integral with the body.

37 A method according to claim 35 or claim 36 comprising the step of etching the capillary from the body.

38 A method according to claims 35 to 37 comprising the steps of forming fine features, such as filtration elements, in one side of the body and forming larger features such as a sample reservoir andlor filtrate reservoir, from the other side of the body.

39 A method according to any of claims 35 to 38 comprising the step of forming a hydrophilic surface along the capillary, preferably comprising the step of using an oxygen plasma to form a hydrophilic oxide coating.