Multi-well microfiltration apparatus

ES 2 212 652 T3

DESCRIPTION

Multi-well microfiltration apparatus. Field of the invention

The present invention relates to multi-well plates and column arrangements in which samples are processed and analyzed.

Background of the invention

In recent years, microtiter wells have played an important role in many biological and biochemical applications, such as sample preparation, genome sequencing, and drug screening programs. Today a wide variety of multi-well devices are common, built according to standard formats. For example, a tray or plate having ninety-six cylindrical depressions or wells arranged in a regular 12 x 8 rectangular arrangement is a very common arrangement.

In some multi-well constructions, a membrane or filter sheet is held against the lower ends, or lips, of open-bottomed wells. Such plates are often manufactured as a multilayer structure comprising a single sheet of filter material arranged to cover the bottom openings of all the wells, the filter sheet being sealed with the outer lip of one or more of the openings of the filter. wells. The use of a single sheet of filter material can thus lead to cross contamination between adjacent wells, due to the tendency of the liquid to disperse, that is, to soak the entire sheet.

In an attempt to overcome this problem, it has been proposed to provide each well with its own individual filter element, ie a disc. According to one of these designs, a pre-cut filter disc is inserted into the open upper end of each well and pushed down until it rests against the bottom of the well. An O-ring is then pressed into each well until it comes to rest on the top of the filter disc. The O-ring friction fits against the inside wall of the column, thereby holding the filter in place. Although the problems of cross contamination of the individual filter sheets are avoided, this arrangement is obviously cumbersome to manufacture. Also, the part of the disk that is clamped between the O-ring and the bottom of the well introduces considerable “dead volume” that can have a negative effect on sample purification. For example, a significant portion of the sample matrix can be trapped in these areas along the peripheral edge of each of the individual filter discs. When purifying DNA from blood samples, small amounts of hemoglobin (heme) are trapped on the edges of the stained cellulose membrane and can potentially contaminate the final product in the later stages of the purification process. The contaminating "heme" residue is a potent inhibitor of PCR and sequential reaction in DNA products.

Another multi-well arrangement, in which each well has its own individual filter element, is formed by placing a single sheet of filter material between a top plate, which has a plurality of mini-columns formed within it, and a bottom plate having a plurality of corresponding "drop collectors". By joining the plates and gluing them together using ultrasound, the filter sheet is cut into individual filter discs located below the respective mini-columns. For example, US Patent No. 4,304,865 discloses an apparatus comprising combinations, a micro-culture plate and a harvester plate, the harvester plate having a plurality of wells arranged in recesses on a surface thereof, said wells being adapted in size, number and disposition to the wells of the micro-culture plate and being suitable to hold filter elements to absorb liquid from the corresponding wells of the culture plate. The apparatus further comprises means for cutting individual filter paper discs from a filter paper sheet in order to supply the required filter elements in the wells of the harvester plate. Although this construction is simpler to manufacture than the previous arrangement, it suffers from similar disadvantages. Specifically, a significant portion of the peripheral edge of each filter disc is pinched between the column plate and the drip collector plate, resulting in considerable dead volume that can adversely affect sample purification.

There is thus a need for a multi-well microfiltration arrangement that is relatively simple to manufacture and that solves the problems accompanying the above provisions, relating to cross-contamination due to absorption by a common filter sheet, or by individual discs. that trap sample constituents within considerable dead volumes.

Most multiwell filter plates, and especially those with a single filter disc, lack adequate space under the filter element to allow the fluid flow to be evenly distributed through the filter. . In many arrangements, a droplet collector at the bottom of each well provides a large flat surface on which most of the filter elements rest. In this way, preferential flow paths are created that favor the areas of the filter element that are not in contact, or are not close to the surface of the drip collector. Such a preferential flow rate can have a negative impact on the elution of solutes. For example, preferred flow paths can prevent leaching of trapped components from the sample in areas away from the filter element.

On the other hand, the lack of adequate support under each filter element can also be problematic. The filter media used in multi-well trays are generally very thin and have relatively low mechanical characteristics. In certain situations with high stresses, that is, when filtering through vacuum or high pressure, it is possible that these membranes may not maintain their integrity. Filter discs that are supported only by their peripheral edges can sag, especially their central part, and can even come loose from the structure that supports their edges. For example, a filter disc can collapse inside the cavity of a drift collector. This would affect the porosity of the filter that would trap certain constituents of the sample.

ES 2 212 652 T3 inside the filter that would otherwise undergo elution. Also, if a bypass is formed along the edges of the filter, because the filter disk has been pulled out of the peripheral support structure, undesirable loss of sample can occur.

There is therefore a need for a multi-well microfiltration arrangement that adequately supports the filter media in each of the wells without creating a preferred flow path.

Some of the known multi-well microfiltration devices have a collection plate, which is positioned below the sample well plate having a plurality of closed-bottomed wells corresponding to the sample wells. In general, collection of the filtrate occurs by drawing a vacuum to drive the mobile phase through each well. In most of these arrangements, attempts to separately collect the filtrate from each sample well have yielded unreliable results due to cross contamination between the wells of the collection plate. One of the main causes of such cross contamination is related to the generation of aerosols when the filtrate leaves the droplet collectors. Aerosols can easily disperse and travel to nearby collection wells. Additionally, aerosols can expose technicians to potentially pathogenic microorganisms that may be present in samples.

Cross contamination due to the generation of aerosols is increased due to the typical flow pattern induced by the vacuum devices of such systems. Generally, the sample well plate is placed on top of the collection plate, which in turn is seated in a vacuum chamber. As the chamber is evacuated, the solution inside each well is drawn down through the filter element into the corresponding collection well. Normally, the vacuum draws in following the flow paths that extend from the interior of each mini-column, through the respective droplet collector and horizontally along the top of the collection plate, until it reaches one of the sides. from the collection plate where the flow paths rotate downward toward an outlet port. Except in the case of the droplet collectors located directly next to the chamber that has the outlet orifice, the substances (i.e. entrained aerosols, gases, etc.) are carried along each flow path, from each drop collector and must pass through neighboring collection wells as they travel over the top of the collection plate. Unfortunately, aerosols originating from filtrate exiting a droplet collector can be flowed along the collection plate and into nearby wells.

The potential risk of cross contamination is especially high when the top plate of the sample well and the droplet collector plate are removed from the collection plate. Drops hanging from the filtrate and remaining in the droplet collectors may inadvertently fall into neighboring wells when the droplet collectors are moved over the collection plate. With standard multi-well plates, due to the large number of wells, it is difficult, if not impossible, to achieve a manual "drop touch" of all hanging droplets so that they fall into the collection wells. Applying a powerful vacuum below the droplet collectors in an attempt to pull the hanging droplets out of the droplet collectors can atomize the hanging droplets, resulting in the aforementioned problem of contamination by the droplet. formation of an aerosol.

There is thus a need for a multi-well microfiltration device that facilitates separate collection of the filtrate from each well, while avoiding cross-contamination due to the formation of aerosols and / or hanging droplets.

Summary of the invention

The present invention provides an apparatus for microfiltration for processing a plurality of fluid samples according to claim 1, and a method for simultaneously forming a plurality of microfiltration wells according to claim 11. The forms of preferred embodiment.

According to one embodiment, the microfiltration apparatus of the invention includes a first plate provided with a plurality of columns and a second plate having a plurality of evacuation conduits. Each of the columns has a first inner hole that delimits an opening inside the column and a final part to house a filter medium inside the column. The end part of the column delimits a second inner hole having a diameter greater than that of the first inner hole, and a transition zone that joins the second inner hole with the first inner hole. Inside the end zone of each column and adjacent to the transition zone, a filter medium is placed to filter the sample. Each evacuation duct has at the end an upper area that protrudes and is aligned and housed inside the end area of ​​the corresponding column, so that a fluid-tight separating surface is formed between them. The upper end zone of the evacuation duct has a part with a terminal edge, to support a circular zone of the filter medium, so that each of the filter media is kept between the transition zone of the column and the zone with an edge. terminal of the corresponding evacuation duct.

In one embodiment, the transition zone of each column has a conical annular portion. The circumference of the conical annular part decreases in a practically constant way following the direction from the second inner hole to the first inner hole. In a related embodiment, a line running along the conical portion, longitudinally relative to the column, forms an acute angle with a plane perpendicular to the longitudinal axis of the column and intersects the column through a junction of the transition zone with the second inner hole. In one embodiment, the acute angle is between 30 and 70 degrees. Preferably, the acute angle is between 30 and 60 degrees. In a certain embodiment, the acute angle is about 45 degrees.

ES 2 212 652 T3

According to one embodiment, the area of ​​the terminal edge of each of the evacuation ducts is in contact with no more than 15%, and preferably less than approximately 10%, and more preferably with less than 5% of the area of ​​the bottom surface of the corresponding filter medium.

One embodiment provides a plurality of fin-shaped support buttresses in each of the exhaust ducts. In this embodiment, each of the supporting buttresses has an elongated and narrow upper surface that is practically coplanar with a plane defined by the area of ​​the terminal edge of the corresponding evacuation duct. In a related embodiment, the horizontal area of ​​the upper section of each supporting buttress decreases in a direction extending toward its highest surface so that the intersection of the highest surface with the plane of the terminal edge zone is practically tangent by nature, forming a line.

According to another embodiment, the microfiltration apparatus is provided with a gas-permeable matrix composed, at least in part, of a porous, hydrophilic, polymeric material. The die is attached to the second plate on the face opposite the first plate. Also, in this embodiment, the matrix surrounds a plurality of exhaust ducts.

A further embodiment provides means for moving the first and second plates in either one of two directions, from an "origin" reference position, along a substantially horizontally extending axis and then returning the plates to the reference "origin" position. The displacement means may include a stepping motor arranged in mechanical connection with the plates so that angular rotation of the stepping motor produces linear displacement of the plates.

According to another embodiment, means are provided for producing the vacuum in order to extract adhering drops of fluid hanging from the evacuation conduits in a direction away from the collection wells and up to the evacuation conduits.

In another of its aspects, the present invention provides a method for forming a plurality of microfiltration wells according to claim 11. In one embodiment, a sheet of filter media is positioned between a first plate containing a plurality of columns and a second plate having a plurality of evacuation ducts. Each of the columns has a first internal hole that delimits an opening inside the column and an end zone that delimits a second internal hole that has a diameter greater than that of the first internal hole, and a transition zone that joins the second inner hole with the first inner hole. Each of the evacuation conduits has an upper extreme area that protrudes and is facing the first plate and aligned with the corresponding area at the end of the column. The plates are pressed together in a way that is effective in punching out portions of the filter media from the sheet, to achieve a plug of the filter media located inside the end zone of each column abutting the transition zone of the column and with the zone with a terminal edge of the corresponding evacuation duct of the upper end zone.

The process of the invention also provides compression fit sealing of each of the filter elements. In one embodiment, the compression of each filter element between the transition zone of the column and the zone of the terminal edge of the corresponding exhaust duct, serves to fix and seal the filter element to an inner side wall of the column.

In another embodiment, the method further includes the step of attaching the first plate to the second plate. The clamping step can be carried out by forming a joint such as an ultrasonic weld between the inner side wall of each second inner hole and the outer circular surface of the corresponding upper end zone.

These and other features and advantages of the present invention will become clear from the following description.

Brief description of the figures

The structure and mode of operation of the invention, together with the additional objectives and advantages thereof, may be better understood by referring to the following description taken in conjunction with the accompanying drawings, in which the same reference numerals identify similar elements. , and in which:

Figure 1 is a perspective view of a multiwell microfiltration apparatus manufactured in accordance with an embodiment of the present invention.

Figure 2 is an exploded view of the multiwell microfiltration apparatus of Figure 1.

Figure 3 is a partial sectional view of the multiwell microfiltration apparatus of Figures 1 and 2.

Figure 4 is an enlarged illustration of a detail of a microfiltration well from the sectional view of Figure 3.

Figure 5 is a side view, partially in section, showing a microfiltration well made in accordance with an embodiment of the present invention.

Figure 6 is an exploded view of a microfiltration well having a support membrane structure in the form of three fin-shaped support buttresses, manufactured in accordance with an embodiment of the present invention.

Figure 7 is an end elevational view of a carriage assembly that performs relative movement between the drop collectors of a drop collector plate and the collection wells of a collection plate, according to an embodiment of the present invention.

Figure 8 is a partially exploded perspective view, illustrating the assembly of a carriage for performing relative movement between the drop collectors of a drop collector plate and the collection wells of a collection plate, according to a shape of embodiment of the present invention.

Figures 9 (A) to 9 (C) are sectional side views showing a peeling operation, in which a plurality of drop collectors are moved laterally to the right and to the left, so that the outlet zones from colec4

ES 2 212 652 T3 tor of droplets simultaneously contact the inner side walls of a plurality of corresponding collection wells.

Figure 10 (A) is a partially schematic top plan view illustrating the spring loaded drop release mechanism in its normal or neutral position.

Figure 10 (B) is a partially schematic top plan view illustrating the spring loaded drop release mechanism of Figure 10 (A) in a first offset position.

Figure 10 (C) is a partially schematic top plan view illustrating the spring loaded drop release mechanism of Figures 10 (A) through 10 (B) in a second offset position.

Figure 11 is a perspective view showing a high-throughput automated sample preparation workstation, including for example a microfiltration apparatus, cross-contamination control devices, and well cap assemblies. collecting and heat sealing, and associated components and reagents according to the teachings of the present invention.

Detailed description of the invention

The following comments on the preferred embodiments of the present invention are merely exemplary in kind. Accordingly, the comments are not intended to limit the scope of the invention, the application of the invention, or the uses of the invention in any way.

Figures 1 to 3 present in perspective, exploded and partial exploded section views, respectively, an embodiment of the multi-well microfiltration apparatus manufactured according to the present invention. In the assembly phase of its manufacture, a filter sheet or membrane, indicated in Figure 2 with reference number 8, is placed between a tray of columns or plate 10 that presents an arrangement of mini-columns with an open bottom, such as at 12, a drop collector tray or plate 14, presenting an arrangement of drop collectors such as at 16, corresponding to the mini-columns. By exactly matching and fitting the mini-columns 12 with the droplet collectors 16, an arrangement of microfiltration wells is formed, referred to as a whole in Figure 3 with reference numeral 18, each having a filter element or medium. independent (that is, a plug, disk or the like) such as 8a and 8b located inside. The interior walls of each mated mini-column / drop collector pair limit a path for flow extending downward through well 18.

As shown in Figures 2 and 3, each microfiltration well has an inner zone or an opening with a substantially circular horizontal section. It will be appreciated, however, that any desired geometric section (ie, oval, square, rectangular, triangular, etc.) could be used for the microfiltration wells. Similarly, the wells could be any desired shape when viewed along their longitudinal axis, that is, straight, conical, or otherwise. In one embodiment, the walls of each well have a slightly increasing taper (that is, the diameter of the well increases) along the direction from the upper loading end of the well to the filter medium.

The plates of the microfiltration apparatus can be made of any material that is substantially rigid, insoluble in water, impervious to fluids, and which is in practice chemically inert towards the samples to be tested. The term "substantially rigid" as used herein is intended to mean that the material resists deformation or warping under low mechanical or thermal loads, although the material may be somewhat elastic. Suitable materials include acrylates, polycarbonates, polypropylenes, and polysulfones. Also, it should be noted that the terms "tray" or "plate" are used here synonymously and are interchangeable.

Optionally, the surfaces of the drip collectors that are in contact with the fluids can be composed of a single material and / or be provided with a coating that renders these surfaces hydrophobic, reducing the possibilities of cross contamination. For example, low surface energy materials can be used to shape and / or coat the droplet collectors. Of course, these materials must be compatible with the samples to be tested.

The plates can be shaped by any common means, injection molding being a particularly suitable technique. One of the embodiments of the invention contemplates the use of injection molded rectangular plastic plates, the length and width of which are adapted to those commonly used of 5.03 "x 3.37" (127.8 mm x 85.5 mm). In the embodiment of Figures 1-3, the wells are integrally formed with such a plate, arranged in a regular 12x8 rectangular arrangement with a 0.9 cm center to center spacing. Alternatively, the wells may be constituted as individual units (not shown) interconnected by a plastic web to provide an arrangement. In another embodiment the wells are arranged in the form of bands (not shown). For example, a plurality of wells could be arranged in a row with adjacent wells connected to one another by suitable means, ie, perishable plastic webs. A plurality of bands could then be arranged side by side, within a frame designed to support said bands. For example, twelve 8-well bands could be placed side by side in a rectangular frame to form a 96-well arrangement. In another embodiment, each well is formed as a separate unit removably located within the corresponding opening formed in a support plate (not shown). For example, a tray could be provided with an arrangement of 12 x 8 circular openings in which cylindrical wells would be housed and held, in a manner similar to how test tubes are kept in a standard holder.

Although the illustrated embodiments have arrangements configured according to the popular 96-well format, the invention also contemplates any reasonable number of wells (ie, 12, 24, 48, 384, etc.) arranged in any suitable configuration.

ES 2 212 652 T3

Referring once again to Figures 1 to 3, a vacuum chamber 20 is located on the column plate 10. The upper vacuum chamber 20 is adapted to move between (i) a mounting position in which four inclined circular walls, designated 20a, form a practically watertight joint with the upper peripheral surface of the column plate 10 by means of an elastic joint. sandwiched 21, and (ii) a rearward position in which chamber 20 is separated from column plate 10. The hollow interior of chamber 20 may be pneumatically connected to an exterior source of vacuum via a hose connection 23 that passes through the top of chamber 20. A reduced pressure may be established above the sample wells by placing chamber 20 in the mounting position on column plate 10 and then evacuating chamber 20.

In some cases, it may be advisable to set a pressure boost over the sample wells (for example, to facilitate the flow of samples through the filter media via the lower evacuation lines). In such cases, chamber 20 can be pressurized by a suitable pressure source (eg, a pump).

A collection or receiving plate 24 is located below the drop collector plate 14. The collection plate 24 includes a flat top surface indicated at 25, and an array of closed bottom wells, such as 26 hanging from it. The arrangement of the collection wells corresponds to the arrangement of the drop collectors, which allows separate collection of the filtrate from each of the sample wells. The collection plate is adapted to fit within an open reservoir of a lower vacuum chamber, indicated 29, with the collection wells extending down into the well.

Openings or vents such as 28 pass through the flat top surface 25 of the collection plate 24. For obvious reasons, at least one of the openings should be located adjacent to each collection well. The openings 28 allow fluid communication between the areas above and below the plate 24. By this construction, a vacuum drawn from below the collection plate will propagate to the areas above the plate and into the wells.

Although not shown in the figures, the present invention also provides a plate like the collection plate 24, except that it has open bottom wells as opposed to the closed bottom wells of plate 24. Other than that, the collection plate The open bottom well is configured as the collection plate 24. That is, the open bottom well plate provides a structure for filtration and washing, while avoiding cross contamination. However, instead of collecting the filtrates in the various wells separately, the filtrate passes through the wells and exits the open bottom. It is contemplated that the open bottom well plate will be used in a manner similar to that described herein for plate 24, except that this situation does not require separate collection of the filtrate. For example, the open-bottom well plate is especially useful when performing intermediate washes. As used herein, "collection plate" and "receiving plate" are used synonymously and are interchangeable, each term referring to a plate intended to be placed underneath a droplet collecting device having both open bottom wells, as closed bottom wells, as appropriate for the task being performed. When separate collection of filtrates is to be carried out, it is understood that the wells are of the closed bottom type. Optionally, a collection plate can be formed having open bottom wells without the feature of vents (such as at 28) where the vacuum can pass directly down through the bottom of each well.

A cross flow restrictor (also referred to as aerosol shield) designated 30, which is generally permeable to gases but practically impermeable to aerosols, is sandwiched between the upper surface of the collection plate 24 and the lower surface of drip collector plate 14. In the illustrated embodiment, the cross flow restrictor 30 has a plurality of passages, such as 32, arranged in a configuration that complements the configuration of the collection wells and the drop collectors. Passages 32 allow filtrate to pass from each drop collector 16 to the corresponding collection well 26. In the illustrated arrangement, each drop collector 16 extends through the corresponding passage. Except for such passages, the cross flow restrictor 30 substantially fills the space between the facing faces of the drop collector plates and collection wells (14, 24).

Preferably, means are provided to support the assembled device of mini-columns and drop collector plates and to assist in the formation of a watertight seal between this arrangement and the lower vacuum chamber 29. In the illustrated embodiment, a carriage With a rectangular frame, indicated with the number 38, it is configured to support the set of mini-columns and the plate of the drip collector. Staples 34, 36 are rotatably mounted on generally vertically extending shafts at opposite ends of frame 38. The staples 34, 36 can be actuated to engage and hold the assembly of the columns and the drop collector on the frame 38, the lower peripheral edge 40 of the column plate and the drop collector being pressed against a joint 42 arranged on the upper surface of frame 38 around the central opening of the frame.

A spring loaded centering pin, such as 37 and 39, can pass through each clip 34, 36. In the embodiment of Figure 3, the centering pin 37 has a stem that is spring biased. 41 to be housed inside a complementary hole or a depression 43 formed in one of the side walls of the column plate 10. In another embodiment (not shown) three spring-loaded centering pins are used, two of the pins being located at positions on the longer side of the device and one pin located at a position on one of the short sides, the which can be actuated to push the tray against a corner. In this way, the components can be easily centered (on the axis).

ES 2 212 652 T3

Around the central opening of the frame, next to the lower surface of the frame 38, is disposed a stepped joint designated in conjunction with number 44. The gasket 44 has (i) an upper portion with an inwardly projecting fin, indicated at 44a, which has its bottom surface suitable for engaging an upwardly projecting edge 48, disposed around the periphery of the plate. pickup 24, and a (ii) fin at the bottom, indicated 44b, extending diagonally downward and outward to engage an upper surface 50 surrounding the open reservoir of the lower vacuum chamber 29. A central area of ​​the gasket 44 in the form of a flat plateau, indicated 44c, is attached to the frame 38 by any suitable means. For example, the central flat land area 44c can be secured using an adhesive and / or fasteners. In one embodiment, gasket 44 is sandwiched between frame 38 and a rectangular clamp frame (not shown). In this embodiment, the rectangular clamping frame is arranged adjacent to the region 44c of the flat land of the seal 44 on one of the sides of the seal 44, opposite the frame 38. The clamping frame is perfectly fixed to the frame 38 using threaded clamping elements that pass through aligned passages (not shown) formed in the clamping frame and the gasket, and which are housed in internally threaded holes that penetrate partially into frame 38 from the bottom surface of the frame. At the same time, the upper gasket 42 and the lower gasket 44 help to form an airtight seal between (i) the upper set of microfiltration wells and the frame carriage, and (ii) between the carriage frame and the set of the lower vacuum chamber, respectively.

The gaskets (21, 42 and 44) ​​can be formed of any deformable, elastic and practically inert material capable of forming a gasket. Examples of such materials are silicone, rubber, polyurethane elastomers, and polyvinyl chloride. The thickness of each of the joints is not critical, as long as it is sufficient to form the joint. Normal joint thicknesses range from about 1mm to about 5mm.

Once the appropriate seals have been formed, vacuuming the lower chamber 29 achieves a nearly uniform drop in pressure across all sample wells 18, allowing a plurality of individual samples (i.e., up to ninety and six in the illustrated embodiment) are processed simultaneously on the chosen membrane.

Those skilled in the art will agree that the selection of the filter media will depend on the intended use of the well. For example, the filter medium can serve as a size exclusion filter, or it can serve as a solid phase that interacts with a species in the liquid phase to immobilize such species in contact with it, such as an immunological interaction reaction or any other. type of affinity interaction. Examples of suitable filters include but are not limited to nitrocellulose, regenerated cellulose, nylon, polysulfones, fiberglass, blown microfibers, and paper. Suitable filters can be found from a variety of vendors such as Schleicher & Schuell, Inc. (Keene, NH) and Millipore.

Corp. (Bedford, Mass.).

Other examples of suitable filters include ultra-pure quartz (SiO2) microfiber filters, for example those manufactured by Whatman Inc. (Tewksbury, MA) and sold under the trademarks QM-A and QM-B. The QM-A filters are about 0.45mm thick and retain particles down to about 0.6μm. The QM-B filters are of the same composition as the QM-A filters, but they are twice as thick and therefore provide a longer and more tortuous flow path. In one embodiment, a quartz or fiberglass element is heated (for example to about 400 ° C) before placing it in a microfiltration well in order to reduce the generation of particles, thus reducing the possibility of obstruction of the drip collectors.

In another embodiment, the filter media is a porous element that acts as a binder that serves to contain a column of packed material (ie, reverse phase or size exclusion packets).

Certain aspects of the invention that refer to the problems mentioned above, corresponding to (i) cross contamination due to absorption by a common filter sheet and (ii) individual filter elements that trap sample constituents inside considerable dead volumes will be described in more detail below.

Figure 4 shows a micro-filtration well enlarged in detail according to the sectional view of Figure 3. The mini-column 12 and the droplet collector 16 are axially aligned and nested with a part of the droplet collector 16 that protrudes upwards. , perfectly fitted inside the lower area of ​​the mini-column opening to form a well 18 that is practically fluid-tight.

A means is provided to hold the droplet collector and the mini-column together. In one embodiment, ultrasonic or glue welds (not shown) are performed along an annular contact area indicated in Figure 4 with reference number 48, which hold together the mini-column 12 and the drip collector 16. It will be appreciated that such a solder or glue helps to achieve a fluid-tight interface between these elements. In another embodiment, the mini-column 12 and the drop collector 16 are held together by a device with a tab within a groove (not shown) formed along the surfaces of the plates 10 and 14 that face each other. . For example, the column plate could be formed with stripes or striations along its bottom surface surrounding each well. The upper surface of the drop collector plate could be provided with upwardly projecting ribs arranged in an arrangement complementary to the configuration of the splines of the column plate and configured to fit snugly into said splines. As a variant, the coupling of the droplet collectors with the mini-columns may be tight enough to hold the plates together solely by frictional coupling.

Means are provided to hold each individual filter element within its respective

ES 2 212 652 T3 microfiltration well assembly. In this regard, each filter element is arranged inside the opening of the mini-column, so that a part of its peripheral edge is kept between (i) a zone of a decreased diameter inside the lower part of the mini-column and (ii) between an upper part of the droplet collector. The central area of ​​the filter element extends completely through the opening of the mini-column.

In the embodiment of Figure 4, the mini-column 12 is formed with a hole 12a and a widened hole 12b, the latter extending upwardly from the lower end or lip 12c of the mini-column. Between the hole 12a and the widened hole 12b there is a transition zone. The transition zone provides a zone with a reduced diameter, or a protrusion, inside the opening of the mini-column capable of contributing, together with the upper part of the corresponding droplet collector, to keep the filter element in place. The junctions of the transition zone with the hole and the widened hole can be of any suitable type. For example, such splices could take the form of a smooth curve. Furthermore, the transition zone itself between said joints can be of any type, that is, flat, curved, stepped, or any combination of the same as long as only a suitable zone with the reduced diameter is provided in the opening of the mini-tube. column to make contact with the upper edge area of ​​the filter element.

In the preferred embodiment illustrated in Figure 4, the transition zone between the hole 12a and the widened hole 12b, delimits an internal annular projection indicated as 12d. In this embodiment, each of the joints of the projection 12d with the hole 12a and the widened hole 12b delimits a sharp angle or a corner. Between said joints, the projection 12d takes the form of an annular wall having a practically constant taper, with a decreasing circumference along the direction from the widened hole 12b to the hole 12a. In the longitudinal direction, the surface of the projection 12d is oblique with respect to the surfaces of the hole 12a and the widened hole 12b. Preferably, the surface of the boss 12d forms an acute angle with a plane perpendicular to the central axis of the mini-column and extends through the junction of the boss 12d with the widened hole 12b. In one embodiment, this angle, indicated with "α" in Figure 4, is within a range of 30-85 degrees; and preferably between 6085 degrees.

The drop collector 16 is configured to facilitate the elution of a mobile phase from the well by channeling it to a lower opening. In the embodiment of Figure 4, the drop collector 16 comprises (i) an annular ridge or edge 16a arranged above the plane of the upper surface of the drop collector plate 14, (ii) convergent side walls inclined 16b, and (iii) a lower conduit or outlet opening 16c disposed below the plane of the lower surface of the drip collector plate 14. The interior surface of the downwardly sloping converging walls 16b between the edge 16a and the outlet opening 16c delimits a conical and / or horn-shaped gap at the bottom of the well opening.

As mentioned above, the upper part of the droplet collector 16 provides a support structure adapted to abut against the lower peripheral area of ​​the edge of the filter element. In the embodiment of Figure 4, said structure takes the form of an annular upper edge 16a. The surface area of ​​the uppermost part of edge 16a (that is, the part of edge 16a that faces directly the lower peripheral zone of the filter element and is arranged to support it) can vary. In a preferred embodiment, the highest area of ​​edge 16a delimits a narrow circular strip. In this embodiment the contact between edge 16a and filter element 8a is tangential in nature. That is, the area of ​​contact between the edge 16a and the filter element 8a defines a very narrow circular band. Edge 16a contacts no more than 15% and preferably less, about 10%, and more preferably less than 5% of the lower surface area of ​​filter element 8a.

In the illustrated embodiment, the peripheral edge area of ​​the filter element 8a is preferably pinched or compressed between the protrusion 12d and the edge 16a in an effective manner to fix the filter element in place and press its circular lateral edge against the edge. inner surface of the column opening. This arrangement prevents upward or downward movement of the filter element and prevents leakage around its edges.

Figure 5 is a partial sectional view showing a microfiltration well manufactured according to a preferred embodiment of the invention. The filter element 8a is compressed between the edge of the droplet collector 16a and the shoulder 12d of the minicolumn, such that the membrane is held firmly in place. Furthermore, the compression fit causes the edge of the circular lateral area of ​​the filter element to be pressed against the inner face of the column opening in an effective way to prevent any drift of the liquid around the edges of the filter element. The projection 12d extends into the mini-column opening at an angle α of about 45 degrees. Furthermore, the surface area of ​​the upper part of the edge 16a is minimal, approaching that of a circular stripe, so that only the outermost perimeter of the lower surface of the filter element is in contact with it.

Continuing with reference to Figure 5, both compression and dead volume have been estimated for a filter element of this type in a microfiltration well, using the computer-aided engineering program "Pro / ENGINEER" (version 18) of Parametric Technology Corporation (Waltham, MA). Membrane compression for a QM-B filter element (Whatman, Inc. Tewskbury, MA) of 950 μιιι thick that has a diameter of 6.88 mm is estimated to be only 2.6 μΐ (area 52 of Figure 5) and the dead volume for said filter element is estimated at only 3 μΐ (area 54 of Figure 5).

Below the filter element 8a, the inner surface of the converging side walls 16b of the droplet collector 16 delimit a cavity. The cavity is configured to expose most of the bottom surface of the filter element to open space or open air. By having this space

ES 2 212 652 T3 free below the filter element 8a (that is, the volume between the convergent side walls 16b of the droplet collector and the lower surface of the filter element), preferential flow paths are avoided.

In another embodiment, to prevent the filter element from sinking or shifting into the well, the invention provides a structure to support the central points or zones of each filter element. For example, a supporting buttress may be provided within the well of the droplet collector 16 to provide a fulcrum, ridge, or surface for one or more centrally located areas on the bottom surface of the filter element. In this case, the expression "central" refers to those parts of the filter element that are located radially towards the inside of the peripheral edges of the filter element, and especially to those parts that are not supported or pinched between a diametral zone compressed between a mini-column and the highest part of the drip collector. In a preferred embodiment, the highest area of ​​said support structure is practically coplanar with the highest part of the edge of the drop collector. It should be taken into account that said structure prevents the subsidence or downward movement of the filter element inside the cavity. This is especially advantageous in relation to filter elements that lack mechanical strength and / or substantial rigidity.

In a preferred embodiment, which appears in the exploded view of Figure 6, said support structure takes the form of three fin-shaped support buttresses, indicated by numbers 58a-58c, located radially and equally spaced inside the drip collector cavity 16 over the central outlet opening 16c. It should be noted that any other reasonable number of supporting buttresses could be used, ie 4 or 6, instead of three. Small parts of the filter element 8a rest on the highest part of the elongated and narrow surfaces or edges of the support buttresses 58a-58c. Preferably, the support buttresses 58a-58c are configured to support the filter element with virtually no dead volume or preferential flow into the system. In this regard, the upper part of each supporting buttress, close to the filter element, can have a curved, arcuate or angular shape, so that the contact area between the filter element 8a and the buttress is practically along a line (or tangent by nature). Furthermore, the profile of each supporting buttress is narrow and slender in the direction of fluid flow.

In the illustrated embodiment, the support buttresses 58a-58c are integrally formed with the droplet collector 16. Alternatively, a plurality of independent support buttress devices (not shown), formed separately from the drop collectors, removably positioned or permanently fixed inside the respective drop collectors.

Advantageously, the invention also provides a very efficient and low-cost method for manufacturing the apparatus described herein. According to one embodiment, a sheet of filter material is placed between a first plate that has a mini-column formed therein, inside which a sample can be placed, and a second plate that has an evacuation conduit, or collector. drops, with an outlet through which the sample can exit. The plates are positioned in such a way that the mini-column is axially aligned with the droplet collector. The plates are then pressed together so that the upwardly projecting part of the drip collector fits snugly inside the opening at the bottom of the mini-column. During this last operation, a path is formed for the flow, which is directed from the interior of the minicolumn to the outlet of the drop collector. Likewise, during the compression stage, a piece of the filter medium is cut from the sheet that is located on a part of the flow path inside the mini-column.

The process of the invention is especially advantageous for the manufacture of a multi-well microfiltration apparatus as mentioned above. Accordingly, the method of the invention will now be described with reference to the illustrated apparatus. The filter sheet 8 is sandwiched between the surfaces of the column plate 10 and the plate of the drip collector 14 that are facing each other, as shown in Figure 2. Plates 10 and 14 are arranged so that each minicolumn 12 is axially aligned with the corresponding drop collector 16. Plates 10 and 14 are then pressed together to achieve a configuration basically like that shown in Figure 3. During In the compression stage, the upper annular edge 16a of each droplet collector 16 acts as a die to punch out a piece of the filter medium 8a (that is, in the form of a disk) from the filter sheet. On the other hand, compressing the droplet collector 16 against the mini-column 12 fixes the filter element in place, inside the opening of the mini-column. As a result, an outer and peripheral edge of the filter element 8a is pinched between an annular upper edge 16a of the droplet collector 16 and an annular projection 12d of the minicolumn 12. The droplet collector 16 and the mini-column 12 are clamped. then by any suitable means. For example, an ultrasonic weld or tab device within a slot can hold the mini-columns 12 and the droplet collectors 16 together, as discussed above.

A further aspect of the present invention corresponds to a multi-well microfiltration device that provides the filtration flow rate for each well, while preventing cross-contamination due to aerosols and splashes.

As indicated above, the arrangement of the collection wells corresponds to the arrangement of the drop collectors, each drop collector being located directly above a receiving or collection well. In turn, the collection well plate is adapted to fit within an open reservoir of the lower vacuum chamber, with its collection wells extending downward toward the reservoir. Once an adequate vacuum has been established in the lower chamber,

ES 2 212 652 T3 the filtrate will flow from each microfiltration well into the corresponding collection well. In accordance with this aspect of the invention, means are provided to prevent aerosols related to filtrates and residues present in any of the wells from traveling, and potentially contaminating neighboring wells. Such means may include, for example, a cross flow limiter, also called aerosol protection, made of a material practically impervious to aerosols, sandwiched between the area between the upper surface of the collection plate and the lower surface of the drip collector plate. Although it prevents the passage of aerosols and debris associated with the filtrates, the cross flow limiter is adapted to allow a vacuum to be drawn through it.

With special reference to the embodiment of Figures 2 and 3, the sheet-shaped cross flow restrictor 30 is provided with an arrangement of passages 32 complementary to the arrangements of the collection wells and of the droplet collectors, which allows the filtrate to pass from each microfiltration well 18 to the corresponding collection well 26. Except for such passages, the cross flow restrictor 30 substantially fills the area between the facing faces of the plates of the drop collector and the collection well (14, 24). In this way, the movement of the aerosols from well to well above the collection plate 24 is practically prevented. Consequently, the risk of cross contamination posed by the movement of the aerosols is substantially reduced. In addition, aerosols formed in any collection wells that may inadvertently pass through the cross-flow restrictor (that is, those that are not effectively blocked or trapped) will be drawn by the vacuum source through the adjacent opening. 28 down into the area of ​​the bottom plate 24 without passing over the openings of the neighboring collection wells, as will be described in greater detail below.

The embodiments of the present invention contemplate the coupling of the cross flow restrictor to the upper face of the collection well plate 24 or to the lower face of the drop collector plate 14. Said coupling can be done by any suitable means, that is, using fixing elements, welding and / or one or more adhesives, such as adhesive tapes, rubbers, cements, pastes or glues. Instead of attaching the aerosol shield to a plate, the aerosol shield may simply be sandwiched between the facing surfaces of the plates and held in place, for example by friction and / or compression forces.

The protection against aerosols can be formed as a single sheet, that is, with a thickness between 0.10 "and 0.15" or, as a variant, it can be made up of two or more sheets, for example, each one of them 0.060 "to 0.065" thick, layered. In a preferred embodiment, the single-layer aerosol shield made of a porous hydrophilic polymer having suitable characteristics, such as ethyl vinyl acetate (EVA) or the like, is attached to the underside of the plate. drip collectors using a pressure sensitive adhesive. Another embodiment contemplates a multilayer construction, including: (i) a shaping layer comprising a foam pad, about 0.062 "thick having a pressure sensitive adhesive on both sides, and (ii) a A layer of a porous UHMW (ultra high molecular weight) polymer, about 0.062 "thick, permeable to air but practically impervious to aerosols. In the latter embodiment, the shaping layer is bonded to the underside of the drip collector plate and then the UHMW polymer layer is bonded to the shaping layer.

Other materials (i.e., hydrophobic, non-polymer, etc.) may be used to form the aerosol protection of the present invention as long as the material (s) only limit the passage of the aerosols, but allow the propagation of the vacuum. through them.

In another embodiment, the means for preventing cross contamination due to movement of aerosols from one well to another includes vents or openings 28 that extend through the surface of the collection plate 24. In one form of preferred embodiment, at least one of said openings is located near each collection well. It should be noted that a reduced pressure applied from below the plate will propagate through the openings to the microfiltration wells.

Any number and any spatial configuration of the openings can be used, as long as only the area between the outlet of each drop collector and the corresponding collection well is arranged in fluid communication (that is, allowing a vacuum) with the area below. of the collection plate following a path that does not pass over the openings of the neighboring wells. For example, a centered aperture can be arranged within a group of four wells, said wells being arranged at the corners of a quadrilateral. If 24 such 4-well groups are provided, each standard 96-well arrangement may be provided with a vent or an opening adjacent thereto. As a variant, the number of openings can be equal to or greater than the number of collection wells, each well having one or more associated openings in close proximity thereto. For example, a 96-well collection plate could be provided with at least 96 apertures arranged so that each well has at least one aperture in close proximity. In this regard, the openings may be provided for example in a regular 12 x 8, or 13 x 9 rectangular arrangement.

As described above, the openings 28 allow fluid communication between the areas above and below the collection plate 24. Once the vacuum has been made in the lower chamber 29, a vacuum will be established that will reach from the outlet opening 51 to the area between each microfiltration well and the corresponding collection well. In particular, the vacuum will perform aspiration following the paths that extend from each microfiltration well 18 to the interconnection zone between the facing surfaces of the drop collector plate 14 and the collection well plate 24. The path of the The flow of the vacuum rotates downward through the surface of the collection plate 25, via the respective vents 28, to the open reservoir of the chamber 29. Here, it flowed through them19

ES 2 212 652 T3 two of the vacuum flow will continue following the lower chamber until reaching the outlet opening 51. The large black arrows in Figure 3 illustrate an example of the path that the vacuum follows. Advantageously, aerosols and filtrate residues that are entrained by the vacuum flow are largely directed away from the area of ​​each collection well and out of the system, without passing over nearby collection wells. Also, it should be noted that the vacuum paths are directed in such a way as to drive a flow rate that is basically laminar in nature and is directed downward. Cross flows, and consequently turbulence, are kept to a minimum when compared to standard arrangements.

The illustrated embodiments feature a cross flow restrictor 30 used in combination with a vented collection well plate 24, such as the one just described. It should be noted that the cross flow restrictor 30 covers the openings 28, so that the path of the vacuum that extends from the area between each microfiltration well 18 and the corresponding collection well 26 to the area below the plate of the collection wells 24, through the nearby aperture 28, must pass through the cross flow restrictor 30. Since the cross-flow limiter 30 allows a vacuum to be created through it, but prevents the passage of aerosols, the aerosols associated with the filtering are practically separated (that is, filtered by the cross-flow limiter) by the vacuum. aspirated and thus the potential movement of the aerosols from well to well above the surface of the collection plate 25 is further reduced.

Instead of using a single cross-flow restrictor for the plurality of drop collectors and collection wells (i.e., a sheet having a plurality of circular perforations that are distributed throughout), as described above and As shown in the accompanying drawings, an alternative embodiment contemplates a plurality of individual cross-flow restrictors in the form of a neck or skirt. In a horizontal section, such individual cross flow limiters can take any suitable shape, viz. Annular, elliptical, elongated, etc. In one embodiment, each individual cross-flow restrictor coaxially and laterally surrounds the area between the drop collector and the corresponding collection well. Such cross-flow restrictors can be made of a substantially rigid material, i.e., like that of the drip collector plate, or they can be made of a suitable porous and hydrophilic material, i.e., a polymer such as ethyl acetate. vinyl (EVA) or similar. In one embodiment, a plurality of substantially rigid limiters, annular or elliptical in shape, are integrally molded with one of the trays, that is, depending on the lower surface of the drip collector plate and extending downward, towards the plate in the collection wells, near the respective drop collectors. Furthermore, each of these rigid cross-flow limiters is configured in such a way as to allow the vacuum drawn from under the collection plate, located under the plate of the drip collector, to be drawn in to extend it to the area close to the drip collector. that is surrounding. In this regard, each cross-flow restrictor can be configured to encompass, in addition to the corresponding collection well, an adjacent opening leading to the area below the collection plate. That is, the cross-flow limiter can extend both around the corresponding collection well and the adjacent opening. In an alternative embodiment, the cross-flow restrictor extends only around the corresponding collection well. That is, the cross flow limiter does not further span the adjacent opening. Rather, in this embodiment, a small through-hole formed in the cross-flow restrictor, close to the opening, allows fluid communication between the opening and the area near the drop collector. It should be noted that, like the sheet-shaped cross-flow restrictor 30 described above, individual cross-flow limiters protect against filtrate splashes and against unwanted movements of aerosols above the surface of the tube. plate from collection wells that could cause cross contamination.

As mentioned above, it should be noted that in all the embodiments described herein, the vacuum flow paths established between the areas above and below the collection plate are directed in a manner that encourages downward and mostly laminar flow. (including entrained gases and / or aerosols). Compared to conventional arrangements, horizontal flow over the upper surface of the plate from the collection wells is minimized. Not only is this the case in the areas near the microfiltration and collection wells, but it is also the case in the peripheral areas of the edges of the plates. In this regard, and with particular reference to the embodiment of Figure 3, the contact between the fin 44a of the stepped joint 44 extending inwardly, and the top of the rim 48 of the well plate collection 24 is such that the air circulation between the two is obstructed or partitioned. In this way, once the vacuum has been made in the lower chamber 29, the gases located above the stepped joint 44, in the area indicated by the arrow 46, will be drawn into the lower vacuum chamber through the vent 28. On the other hand, the gases comprised in the space located below the lower surface of the stepped joint 44, indicated globally by the arrow 47, will be drawn into the lower vacuum chamber through a space 49 arranged between the plate of the collection wells and surface 50 around vacuum chamber 29. By thus limiting the amount of air flow above the collection well plate, cross flow turbulence along the periphery of the device is minimized.

Additional means to avoid cross contamination due to the movement of aerosols from one well to the other, as well as to splashing the filtrate, refer to the positioning of the openings of each drop collector with respect to the upper edge, or lip, of the corresponding collection well. According to this characteristic, the outlet orifice 16c of each of the drop collectors 16 extends towards

ES 2 212 652 T3 down from the plate of the drop collector 14, so that it is inserted into the corresponding collection well 26. In this regard, the lower part of each of the drop collectors 16 has a diameter that allows to do so exactly match the open top of the corresponding collection well 26 inside the collection plate 24. As illustrated in the embodiment of Figure 3, the outlet port 16c of each of the drop collectors 16 is located below the upper edge or lip of the corresponding collection well 26. By locating the outlet port 16c in an area that is laterally surrounded by the inner side walls of the collection well 26, most of the aerosol generated during filtration will impact the collection well walls, which are opposed to lateral displacement until stand on top of a nearby collection well. As an additional advantage, such placement of the outlets of the drip collectors helps to reduce splashing of the filtrate.

In a related aspect, the present invention provides a method of preventing cross-contamination due to movement of aerosols from one well to another in a multi-well microfiltration system. According to one embodiment, the method includes the following steps:

(i) providing a series of microfiltration wells (containing fluid samples) above a collection well tray that supports the corresponding series of collection wells;

(ii) draw the vacuum along the flow paths that extend: (a) from each microfiltration well, (b) down through a plane defined by the upper surface of the collection tray at a point near or, next to the corresponding collection well, and (c) to an area located below the collection tray, thereby achieving that the filtrate flows from each of the microfiltration wells to the corresponding collection wells; and (iii) preventing the aerosols formed by the filtrate in any of the microfiltration wells from moving over the upper surface of the collection tray to a collection well that does not correspond to it, thereby limiting cross-contamination.

It should be understood that the apparatus described above is particularly suitable for carrying out this procedure. For example, a vacuum chamber, such as chamber 29 shown in Figure 3, may be connected to a low pressure source, such as a vacuum pump (not shown), to establish a pressure differential across the filter elements 8a , 8b arranged inside the microfiltration wells 18. The reduced pressure will then cause the filtrate to emanate from the droplet collectors 16. The aerosol shields 30 provide means to prevent the aerosols formed, associated with the filtrate in any of the microfiltration wells 18, from traveling along from the top surface 25 of the collection well plate 24 to a nearby collection well.

Apertures 28, which pass through the surface 25 of the collection plate 24, allow the vacuum to propagate between each microfiltration well and the area below the plate of collection wells 24 without having to pass over it. from neighboring collection well openings.

When drawing a vacuum in the lower chamber, it is advantageous to slowly decrease the pressure (pressure ramp) to the desired value, combining it with the use of very low pressures (that is, less than 2 psi and preferably less, up to 1 psi), to further reduce the possibility of cross-contamination from aerosols. For example, to go from ambient pressure to 0.75 to 2 psi, a ramp period of 2 to 3 seconds is used.

Another aspect of the present invention corresponds to a multi-well microfiltration device that provides a flow rate of filtration from each well, while avoiding cross-contamination due to hanging droplets that can adhere to the droplet collectors of the various wells. microfiltration. As mentioned above, such hanging droplets can fall into neighboring collection wells when the drop collector plate is moved over the collection well plate.

According to one of the embodiments, the vacuum or evacuation in a microfiltration well is carried out in the direction of its upper opening, thereby sucking up any drop of fluid that may hang from its drop collector and returning it back to the well. To perform evacuation, a pressure control source, i.e. a vacuum pump, can be connected in communication with the top of the mini-column to empty the mini-column in the direction that it extends from the manifold. drops to the top opening.

Another embodiment provides the "wringing contact" of the tips of the drip collectors, to remove hanging filtrate droplets, which could sag from the drip collectors. To this end, the outlets of all the droplet collectors of the microfiltration wells are simultaneously brought into contact with the inner walls of the corresponding collection wells.

Means are provided for relative movement between the plate of the drop collectors and the plate of the collection wells to simultaneously move the evacuation conduits to contact the internal walls of the respective collection wells and separate them at a time. continuation. In one embodiment, said means can be activated to move the plate from the collection wells following a plane practically orthogonal to the longitudinal axes of the microfiltration wells, while the microfiltration wells themselves are kept in a practically fixed position. In another embodiment, the means for performing relative movement can be activated to move the microfiltration wells along a plane substantially orthogonal to the longitudinal axes of the collection wells, while the collection wells are held in one direction. practically fixed position.

An example of an arrangement for performing this relative movement is illustrated in Figures 7 to 10. Referring initially to Figures 7

ES 2 212 652 T3 and 8, an L-shaped carriage, indicated by reference number 60, is provided with a central opening 62 configured to house and support a multi-well microfiltration assembly, indicated globally by reference number 6 in the previous figures. Below carriage 60, a collection plate 24 having a series of collection wells 26 is supported within a lower vacuum chamber (not shown).

Carriage 60 is mounted on a pair of longitudinal and parallel transport rails for reciprocating linear movement along a substantially horizontal first axis. In the illustrated embodiment, one of the transport rails is a rail with a linear bearing, indicated as 64, which supports the carriage 60 by means of an insert composed of a linear bearing 65 attached to the lower surface of the carriage 60 towards one from the side edges. The other transport rail is a U-shaped bearing guide, indicated as 66, which houses a wheel 68 of a bearing, which extends laterally outwards from the other edge of the carriage 60, inside a track or elongated slot 66a.

The carriage 60 is moved along the rails 64, 66 by means of a set of a belt constituted by a flexible belt 70 that has its ends joined to each of the longitudinal ends of a U-shaped support 74 that is part of a spring-loaded motion control mechanism 72, which will be described in detail later. Belt 70 passes around a driven roller 76 and idler roller 78, arranged longitudinally close to opposite ends of the transport rail device. To prevent slippage, the belt may be provided with teeth 70a suitable for engaging sets of complementary teeth 76a, 78a located on the rollers.

The driven roller 76 is mechanically connected to a motor, such as 82, by an assembly of a power transmission system, indicated as a whole by the reference numeral 84. When the motor 82 is driven, the belt 70 moves, making that the carriage 60 slides along the rails 64, 66, the direction of movement depending on the direction of rotation of the drive shaft 86 exiting the motor 82. The motor 82 can be of any known type, that is, a stepper motor, a servo motor, or a similar device.

A preferred embodiment of the present invention contemplates the use of a stepper motor to move the belt. As a reminder, a stepper motor is a motor of a special type that moves in individual steps. Unlike servo motors, the position of a stepper motor can be determined without the need for expensive encoders to check its position. Stepper motors are much cheaper than servo systems due to their simplified control and drive circuitry. In a stepper motor there are no brushes to replace, thus reducing the maintenance frequency. Because of their ease of use and relatively low cost, stepper motors are often preferred over servo motors in many modern computer motion control systems.

According to this embodiment of the invention, a control system for driving the stepper motor is provided in the desired form. For example, a microcontroller, such as the Motorola 68332, can be used to control the motor using standard techniques.

As noted above, by advancing the stepper motor 82, it causes the belt 70 to move on the rollers 76, 78, the direction of movement depending on the direction of rotation of the motor shaft 86. The movement of the belt 70 it in turn causes carriage 60 to slide along guide rails 64, 66, thereby laterally displacing the series of drop collectors 16 relative to the series of collection wells 26. If the drop collectors 16 are positioned to extend into the respective collection wells 26, a few steps in a given direction will be sufficient for the drop collectors 16 to engage the upper internal surfaces of the collection wells 26, as illustrated in the sectional views of Figures 9 (A) -9 (C). In this way, the filtrate droplets hanging from the droplet collectors 16 come into "wringing contact" with the bottom surfaces of the respective collection wells 26. Similarly, by reversing the direction of the steps, the droplet collectors can be moved to contact the upper internal surfaces on the opposite side of the collection wells 26 to further ensure effective contact for draining of the drops. drops that are hanging.

As mentioned above, alternative embodiments of the invention contemplate the use of a servo motor to move the belt. In one of these embodiments, means are provided for providing position feedback, such as an encoder (not shown) in order to track the position of the servomotor.

The carriage further supports means for moving and positioning the microfiltration device 6 along a second axis, normally vertical. With particular reference to the embodiment of Figure 7, on the upper surface of the carriage, along each of the lateral sides of the microfiltration device, a vertical positioning mechanism is arranged. Each of the vertical positioning mechanisms includes: (i) lifting springs, such as 92, which provide a continuous upward force that tends to raise the microfiltration device 6 to a higher position in which the droplet collectors 16 pass completely over the upper edges the collection wells 26, and (ii) the fluid cylinders, such as 94, which can be actuated to lower the microfiltration device 6 by overcoming the force of the lifting springs 92, up to a seating position in which each droplet collector 16 extends towards the upper area of ​​the respective collection well 26. In the fully seated (lowered) position the microfiltration device 6 forms a seal with the lower vacuum chamber (not represented).

Both the springs 92 and the fluid cylinders 94 are coupled at their upper ends to handles, indicated 96, which extend upward and outward from each of the lateral sides of the support frame 38 of the microfiltration device. In one embodiment, the spring / cylinder devices can be actuated to hold the microfilm device13

ES 2 212 652 T3 traction in any one of three positions: (i) an upper or displacement position, (ii) a contact position for wringing, and (iii) a lower or sealing position.

The wringing contact operation can be carried out with the microfiltration device 6 arranged in any of the positions, along the second (vertical) axis, provided that the droplet collectors 16 penetrate at least in part into the wells of pickup 26. In one embodiment, the wringing contact of the drop collectors 16 towards the inner side walls of the collection wells 26 is made with the microfiltration device 6 slightly raised above its fully seated position so that the lower areas of the drop collectors 16, close to their outlets 16c, abut against the lower surfaces of the collection wells 26.

The area of ​​each droplet collector 16 closest to its outlet may have an angled or chamfered shape around its lower perimeter, to favor the location of any filtrate droplets that remain hanging in certain areas of the drop collector 16 and to make better contact between said areas and the inner side wall of the corresponding collection well 26 during contact for wringing. The upper zone of each collection well 26 can also be shaped in a similar way, that is, in a complementary way (that is, fitting) with the shape of the drop collector 16, so that adequate contact is made between them. elements during contact for wringing so that the drip collector 16 is practically free of any filtrate droplets that could be left hanging. In a preferred embodiment, as seen in Figures 9A-C, the top of each collection well is formed with an outwardly sloping inner side wall that engages an inwardly sloping outer surface along the lower zone of the corresponding drop collector, thereby providing a surface that practically abuts between these elements during the contact operation for wringing.

As described above, the differentiated magnitude of angular rotation imparted to shaft 86 each time stepper motor 82 advances is eventually converted to a predetermined length of linear travel by support 74. For example, by advancing a few steps to the motor 82, the bracket 74 can be made to move! 4 "(of an inch) in a certain direction. It should be noted that the minimum number of steps required for the stepper motor 82 to make a wringing contact may cause the droplet collectors 16 to travel beyond what is necessary. That is, the drop collectors 16 can be displaced into contact with the interior walls of the collection wells 26, exerting continuous pressure to move beyond the interior walls. As will be described below, such excess linear momentum can be an advantage as it can aid in the removal of hanging droplets. It should be noted that it is desirable to move the droplet collectors to a suitable position against the side walls of the collection well (that is, making a secure stop against the side walls) in order to effectively fix the removal of debris. earring drops. By providing a considerable excess of momentum in the drift collector side travel, such positioning can be ensured (that is, the drip collectors will not miss the side walls), despite some small inherent positioning inaccuracies. to device. In this way, by providing a reasonable amount of excess linear impulse, it is the side walls themselves that determine the final position of the droplet collectors. On the other hand, it is desirable to absorb or compensate for some of the excess linear impulse to avoid overstressing the drop collectors 16 and / or the collection wells 26.

In this regard, an embodiment of the invention contemplates the use of a spring tensioned movement control mechanism 72 in the mechanical connection system between motor 82 and carriage 60. The displacement control mechanism 72 ensures proper positioning of the droplet collectors that abut against the side walls, while absorbing excess linear displacement beyond the magnitude required to move the droplet collectors 16 until they make contact. with the inner side walls of the collection wells 26. As a further advantage, the travel control mechanism 72 provides a damping of resistance to sliding movement of the carriage 60 along the rails 64, 66.

In one embodiment, the travel control mechanism includes a spring arranged so that movement of the carriage in any direction along the first axis puts the spring in tension. With particular reference to the partially schematic top plan views of Figures 10 (A) - (C), the U-shaped bracket 74 that forms part of the strap assembly is rigidly connected to a housing 101 that it contains large and small holes, respectively indicated together as 102 and 108. The hole 102 has a large diameter portion 102a and a small diameter portion 102b, separated by a radial step 102c. A shaft of a stepped diameter, indicated as a whole as 104, and having a large diameter portion 104a and a small diameter portion 104b, separated by a radial step 104c, passes through hole 102 and is joined together. rigid at the end of the large diameter, with the elongated portion 60a of the carriage arm 60 in an L shape. A guide pin 106, which helps to maintain the substantially horizontal orientation of the carriage 60, is rigidly attached to the elongated portion 60a of the carriage arm 60 at one end and is housed in the small hole 108 at the other end. . Inside the large diameter portion 102a of the hole 102, a spring 110 is concentrically mounted on the small diameter portion 104b of the shaft 104 between a pair of spaced apart washers, indicated 112 and 116. The two washers 112, 116 are mounted concentrically so that they can slideably move along the small diameter portion 104b of the stepped shaft 104. The spring 110 biases the two washers 112, 116 against opposite ends of the small diameter portion 104b of shaft 104. A washer 114 is seated in a fixed position within a circular groove (not

ES 2 212 652 T3 shown) formed in the small diameter portion 104b of the shaft 104 near its free end.

As belt 70 moves U-shaped bracket 74 in the direction indicated by arrow "A" in Figure 10B, hole 102 slides along axis 104 in the direction of elongated arm 60a of carriage 60. As a result, an annular lip 120 extending radially inward at the end of hole 102 acts against the peripheral annular portion of washer 112, causing washer 112 to slide along small diameter portion 104b. of stepped shaft 104, thereby compressing spring 110. When the compression force exceeds the pre-tensioned holding force, carriage 60 will then move in the same direction ("A" direction).

As belt 70 moves U-shaped bracket 74 in the direction indicated by arrow "B" in Figure 10C, hole 102 slides along axis 104 in a direction away from elongated arm 60a of the carriage 60. As a result, radial step 102c of hole 102 acts against the peripheral annular area of ​​washer 116 causing washer 116 to slide along small diameter portion 104b of stepped shaft 104, thereby compressing spring 110. When the compression force overcomes the pre-tensioned holding force, the carriage 60 will then travel in the same direction ("B" direction).

In one embodiment, spring 110 provides a pre-tension force of about 1 pound. In this manner, the force provided by the stepper motor 82 will not be sufficient to move the carriage 60 until the threshold of about 1 pound is exceeded. Advantageously, the device provides: (i) a constant center hold mode, or neutral position, and (ii) a constant force mode to make contact for wringing. Spring 110 is system compliant, that is, it allows the wringing contact to start at 1 pound and end at 1.2 pounds.

With reference to the apparatus as described above, a preferred embodiment of the present invention contemplates the following steps:

(i) the microfiltration device 6 is loaded onto the carriage 60 and fixed in position;

(ii) carriage 60 is centered over lower vacuum chamber 29;

(iii) the microfiltration device 6 is lowered and settles in its seating position (that is, retracting the fluid cylinders 94), effecting the sealing on the lower vacuum chamber 29;

(iv) a robot (not shown) lowers the upper vacuum chamber 20 to the top of the microfiltration device 6 and optionally applies a downward force of about 5 pounds to the stacked device;

(v) the lower vacuum chamber 29 is evacuated (ie 0.5-3 psi) to carry out elution / purification;

(vi) carriage 60 is slightly raised from its fully seated position to a wringing contact height in which only the lowest areas of the drop collectors extend below the upper edges of the collection wells 26;

(vii) motor 82 advances a few steps in the forward direction until droplet collector 16 touches one of the side walls of collection wells 26;

(viii) motor 82 moves back a few steps in a rearward direction until droplet collector 16 touches the opposite inner side wall of collection wells 26;

(ix) the forward and reverse steps of the motor 82 are repeated to repeat once more each of the steps of the wringing contact; the carriage 60 is again centered on the lower vacuum chamber 29;

(x) the microfiltration device 6 is lowered into its seating position and is sealed on the lower vacuum chamber 29;

(xi) optionally, the robot can apply a downward force of about 5 pounds to the stacked device;

(xii) the upper chamber 20 is evacuated to achieve a suction effect of the pending drops (0.1 to 0.3 psi);

(xiii) the microfiltration device 6 is raised to its fully elevated position such that the droplet collectors 16 fully release the collection wells 26; and then (xiv) the carriage 60 moves to the next station.

Figure 11 illustrates a high throughput automated workstation 202 for sample preparation, including, by way of example, a microfiltration apparatus, cross-contamination control devices, as well as the well cap assemblies. collecting and heat sealing (described below), and associated components and reagents, according to the explanations of the present invention. As illustrated, several collection trays can be arranged in adjacent vacuum chambers placed side by side adjacent to one end of the work station. For example, a closed bottom collection tray, such as tray 24, may be located in each of the two furthest vacuum chambers, while open bottom collection trays can be located in the two central vacuum chambers. . The carriage 60 can successively transfer a microfiltration device 6 from one vacuum chamber to the next. For example, an initial collection of filtrate may be performed in the vacuum chamber that supports the closed bottom collection plate 24 adjacent to the front of the workstation. Successive washes can then be carried out in each of the two central vacuum chambers in which the open-bottom collecting plates are placed. Subsequently, a final collection of filtrate can be carried out in the vacuum chamber arranged next to the back of the work station, where another collection tray with a closed bottom is located.

Regarding the orientation in space, it should be noted that the various components (that is, the upper chamber, the mini-column plate, the filter element, the droplet collection plate, the frame,

ES 2 212 652 T3 the cross-flow limiter, the collection well plate and the lower chamber) are illustrated and described herein as stacked vertically, with the upper vacuum chamber being the uppermost component. Furthermore, the microfiltration well is described as having a central axis arranged in a substantially vertical manner, with a flow path leading down the length of the well. However, it should be noted that these guidelines may be adopted merely for convenience in developing the detailed description and to facilitate understanding of the invention. In practice, the invention contemplates that the components and the wells can be arranged in any orientation.

Those skilled in the art will now appreciate from the foregoing description that the extensive explanations of the present invention can be implemented in a wide variety of ways.