Methods and devices for simplified DNA extraction

Abstract

The present invention discloses a DNA extraction pipette tip that contains a proximal end and a distal end. An aspiration device opening is located at the proximal end and constructed to receive an aspiration device, such as a pump or a pipetter (manual or automatic). A distal end opening is located at the distal end, and a central lumen fluidly connects the aspiration device opening to the distal end opening. The central lumen defines an internal wall. A filter matrix is disposed in the central lumen and positioned closer to the distal end opening than the aspiration device opening. The filter matrix may be a silica gel membrane matrix or a plastic and silica bead matrix. Methods of manufacture are also disclosed, as are method of DNA extraction.

Description

[0001] METHODS AND DEVICES FOR SIMPLIFIED DNA EXTRACTION

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003]

[0001] This application claims priority to US 63 / 608,797 filed on December 11, 2023 entitled “Methods and Devices for Simplified DNA Extraction” and to US 63 / 567,753 filed on March 20, 2024 entitled “Methods and Devices for Simplified DNA Extraction”. The entire contents of these applications are incorporated herein by reference.

[0004] FIELD OF THE INVENTION

[0005]

[0002] This invention relates to the design, materials, and methods for the extraction of DNA.

[0006] BACKGROUND OF THE INVENTION

[0007]

[0003] An efficient DNA extraction is critical to studying the genetic causes of diseases and developing diagnostics and drugs. It is also essential in forensic science, detecting bacteria and viruses, determining paternity, genome sequencing, etc. Current DNA extraction techniques consists of several steps and requires a great deal of plastic consumables. The current DNA extraction technique is shown in FIG. 5 and can be described as follows:

[0008] • Step 1: Use a first conventional pipette tip 5-1 to aspirate the sample from the sample collection tube 10.

[0009] • Step 2: Insert a spin column 15 into a first microcentrifuge collection tube 20-1.

[0010] • Step 3: Dispense the sample into the first microcentrifuge collection tube 20-1 / spin column 15.

[0011] • Step 4: Insert the first microcentrifuge collection tube 20-1 / spin column 15 into a centrifuge and spin it for at least 5 minutes.

[0012] • Step 5: Removed spin column 15 from first microcentrifuge collection tube 20-1 and place spin column 15 into a second microcentrifuge collection tube 20-2; dispose of first microcentrifuge collection tube 20-1 and its fluid contents.

[0013] • Step 6: Use a second conventional pipette tip 5-2 to aspirate washing solution 25; dispose of first conventional pipette tip 5-1.

[0014] • Step 7 : Dispense washing solution into the second microcentrifuge collection tube 20- 2 / spin column 15. • Step 8: Insert the second microcentrifuge collection tube 20-2 / spin column 15 into a centrifuge and spin it for at least 5 minutes.

[0015] • Step 9: Removed spin column 15 from second microcentrifuge collection tube 20-2 and place the spin column 15 into a third microcentrifuge collection tube 20-3; dispose of second microcentrifuge collection tube 20-2 and its fluid contents.

[0016] • Step 10: Use a third conventional pipette tip 5-3 to aspirate elution solution 30; dispose of second conventional pipette tip 5-2.

[0017] • Step 11 : Dispense elution solution into the third microcentrifuge collection tube 20- 3 / spin column 15.

[0018] • Step 12: Insert the third microcentrifuge collection tube 20-3 / spin column 15 into a centrifuge and spin it for at least 5 minutes.

[0019]

[0004] For this extraction technique the following is used and consumed: three microcentrifuge collection tubes, three pipette tips, and one spin column. Not only does the current extraction method waste materials, it is also very time consuming. Specifically, three steps (steps 4, 8 and 12) require 5-10 minutes in a centrifuge. The entire process therefore can take 30-45 minutes.

[0020]

[0005] What is needed are methods and devices that both speed up the extraction process and produce less waste.

[0021] SUMMARY OF THE INVENTION

[0022]

[0006] Disclosed herein is a DNA extraction pipette tip that contains a proximal end and a distal end. An aspiration device opening is located at the proximal end and constructed to receive an aspiration device, such as a pump or a pipetter (manual or automatic). A distal end opening is located at the distal end, and a central lumen fluidly connects the aspiration device opening to the distal end opening. The central lumen defines an internal wall. A filter matrix is disposed in the central lumen and positioned closer to the distal end opening than the aspiration device opening. The filter matrix may be a silica gel membrane matrix or a plastic and silica bead matrix. The aspiration device opening may be constructed to receive a pipetter or a pump.

[0023]

[0007] When a plastic and silica bead matrix is employed, the beads are fused together to form a porous matrix that permits a fluid connection between the aspiration device opening to the distal end opening. The ratio by volume of plastic beads to silica beads may range from 85: 15 to 50:50. The ratio of the diameter of the plastic beads to silica beads may range from 1 :3 to 3 : 1. The silica beads may range from 50 to 150 microns in diameter; and the plastic beads may range from 60 to 150 microns.

[0024]

[0008] The filter matrix may form an interference / press fit with the internal wall. To this end, the filter matrix may be constructed to be inserted into the central lumen, has a diameter that is larger than the diameter of the central lumen. The tip may also have a retainer constructed to retain the filter matrix within the central lumen. The filter matrix may be positioned adjacent to the distal end opening, preferably is position to reduce dead space. The filter matrix may have a length of 0.20”-1.50”.

[0025]

[0009] A method of DNA extraction is also disclosed that includes: (a) loading a DNA extraction pipette tip onto an aspiration device; (b) aspirating a DNA-containing sample into the pipette tip; (c) dispensing the sample from the pipette tip; (d) trapping DNA in the filter matrix in step (b) or step (c) or both; (e) aspirating a washing solution into the pipette tip; (f) dispensing the washing solution from the pipette tip; (g) aspirating an elution solution into the pipette tip; (h) dispensing elution solution (30) from the pipette tip into a collection tube (55); and (i) dislodging DNA within the filter matrix (105) in step (g) or step (h) or both, such that the dispensed elution solution (30) contains DNA.

[0026]

[0010] A method of manufacturing a DNA extraction pipette tip is also disclosed that includes: mixing silica beads and plastic beads into a homogenous mixture; pouring the homogenous mixture into a mold; heating the homogenous mixture to a temperature between 280°F and 340°F; forming a porous filter matrix using a sintering process (melting the outer surface of the plastic beads and adhering the plastic beads to each other) such that the silica beads get trapped between the plastic beads; removing the filter matrix from the mold; and inserting the filter matrix into a central lumen of a pipette tip.

[0027]

[0011] Additional aspects, alternatives and variations as would be apparent to persons of skill in the art are also disclosed herein and are specifically contemplated as included as part of the invention. The invention is set forth only in the claims as allowed by the patent office in this or related applications, and the following summary descriptions of certain examples are not in any way to limit, define or otherwise establish the scope of legal protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028]

[0012] The invention can be better understood with reference to the following figures. The components within the figures are not necessarily to scale, emphasis instead being placed on clearly illustrating example aspects of the invention. In the figures, like reference numerals designate corresponding parts throughout the different views and / or embodiments. Furthermore, various features of different disclosed embodiments can be combined to form additional embodiments, which are part of this disclosure. It will be understood that certain components and details may not appear in the figures to assist in more clearly describing the invention.

[0029]

[0013] FIG. 1 illustrates a novel DNA extraction process using a novel DNA extraction pipette tip.

[0014] FIG. 2A illustrates a novel DNA extraction pipette tip.

[0030]

[0015] FIG. 2B is an enlarged view of the DNA extraction pipette tip, showing the matrix filter in greater detail.

[0031]

[0016] FIG. 2C is an enlarged view of the distal end of the DNA extraction pipette tip, showing the matrix filter interference fit.

[0032]

[0017] FIG. 3 illustrates the silica / plastic bead matrix.

[0033]

[0018] FIG. 4A is an electrophoresis graph of test results of DNA extraction pipette tips made according to the techniques described herein.

[0034]

[0019] FIG. 4B is an electrophoresis graph of test results of DNA extraction pipette tips made according to the techniques described herein.

[0035]

[0020] FIG. 5 illustrates the conventional spin column DNA extraction process.

[0036] DETAILED DESCRIPTION OF THE INVENTION

[0037]

[0021] Reference is made herein to some specific examples of the present invention, including any best modes contemplated by the inventor for carrying out the invention. Examples of these specific embodiments are illustrated in the accompanying figures. While the invention is described in conjunction with these specific embodiments, it will be understood that they are not intended to limit the invention to the described or illustrated embodiments. To the contrary, they are intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims.

[0038]

[0022] In the following description, numerous specific details are set forth to provide a thorough understanding of the present invention. Example embodiments of the present invention may be implemented without some or all these specific details. In other instances, process operations well- known to persons of skill in the art have not been described in detail so as not to obscure unnecessarily the present invention. Various techniques and mechanisms of the present invention will sometimes be described in singular form for clarity. However, it should be noted that some embodiments include multiple iterations of a technique or multiple mechanisms, unless noted otherwise. Similarly, various steps of the methods shown and described herein are not necessarily performed in the order indicated, or performed at all, in certain embodiments. Accordingly, some implementations of the methods discussed herein may include more or fewer steps than those shown or described. Further, the techniques and mechanisms of the present invention will sometimes describe a connection, relationship or communication between two or more entities. It should be noted that a connection or relationship between entities does not necessarily mean a direct, unimpeded connection, as a variety of other entities or processes may reside or occur between any two entities. Consequently, an indicated connection does not necessarily mean a direct, unimpeded connection, unless otherwise noted.

[0039]

[0023] The following list of example features corresponds with the attached figures and is provided for ease of reference, where like reference numerals designate corresponding features throughout the specification and figures:

[0040]

[0024] Conventional Pipette Tip 5-1, 5-2, 5-3

[0041]

[0025] DNA-Containing Sample 6

[0042]

[0026] Sample Collection Tube 10

[0043]

[0027] Spin Column 15

[0044]

[0028] Microcentrifuge Collection Tube 20-1, 20-2, 20-3

[0045]

[0029] Washing Solution 25

[0046]

[0030] Elution Solution 30

[0047]

[0031] DNA Extraction Pipette Tip 50

[0048]

[0032] Aspiration Device Opening 51

[0049]

[0033] Proximal Pipette Tip End 52

[0050]

[0034] Distal Pipette Tip End 53

[0051]

[0035] Distal End Opening 54

[0052]

[0036] Central Lumen 54.1

[0053]

[0037] Internal Wall 54.2

[0038] Microcentrifuge Collection Tube 55-1 , 55-2

[0054]

[0039] Filter Matrix 105

[0055]

[0040] Pipette Tip 110

[0056]

[0041] Retainer 115

[0057]

[0042] Interference / Press Fit 118

[0058]

[0043] Dead Space 119

[0059]

[0044] Silica Beads 120

[0060]

[0045] Plastic Beads 125

[0061]

[0046] FIG. 1 illustrates the DNA extraction method that uses a novel DNA extraction pipette tip described in detail below. The steps of this novel method include:

[0062] • Step 1 : Use a DNA extraction pipette tip 50 to aspirate the DNA-containing sample 6 from the sample collection tube 10.

[0063] • Step 2: Dispense the sample into a first microcentrifuge collection tube 55-1.

[0064] • Step 3 : Using the DNA extraction pipette tip 50, aspirate washing solution 25. A nonlimiting example of a washing solution is 80% ethanol, 20 mM NaCl, 2 mM Tris-HCl, pH 7.5.

[0065] • Step 4: Dispense washing solution into the first microcentrifuge collection tube 55-1; dispose of the first microcentrifuge collection tube 55-1 and its fluid contents.

[0066] • Step 5: Using the DNA extraction pipette tip 50, aspirate elution solution 30. Two non-limiting examples of elution solution are: (1) 10 mM Tris, pH 8.5, 0.1 mM EDTA; and (2) 10 mM Tris-Cl pH 8.3, 0.1 mM EDTA and 0.04% NaN3 (Sodium-azide).

[0067] • Step 6: Dispense elution solution into a second microcentrifuge collection tube 55.

[0047] This novel extraction process is far more simple, less wasteful and faster than the previous method. The novel process is only six steps, as opposed to twelve, and takes less than five minutes because the centrifuge steps are not needed. This method also uses only a single DNA extraction pipette tip and two microcentrifuge collection tubes. Fewer steps and less consumables yields workflow automation opportunities and more throughput.

[0068]

[0048] The DNA extraction pipette tip offers excellent advantages over traditional methods because the tip fuses the fluid dispensing capabilities of a pipette tip with the DNA binding properties of silica, and the same tip is used in the binding, washing, and elution procedures.

[0049] The DNA extraction pipette tip 50 (FIGS. 2A-2C) consists of two main elements: the filter matrix 105 and a pipette tip 110. The filter matrix 105 can be created by different methods. Two preferred methos are (a) silica gel membrane (or frits) matrix, such as those found on DNA extraction spin columns; and (b) plastic and silica beads matrix. a. Silica gel membrane matrix: The filter matrix 105 may consist of silica membranes inserted inside the pipette tip 110 towards the distal end (see FIG. 2B) and held in place by a plastic retainer 115 that interacts with the pipette tip’s 110 internal walls, forming an interference fit (see FIG. 2C) that keeps the membranes in place during pipetting. The number of membranes or frits will depend on the application or type of DNA that we want to bind. Typically, the number of membranes is between three to eight. b. Plastic and silica beads matrix; The filter matrix 105 may consist of a mixture of etched silica beads ranging from 50 to 150 microns in diameter and a plastic bonding agent, such as an ultra-high molecular weight polyethylene with a particle size ranging between 60 to 150 microns. For the filter matrix to be effective, the particle size ratios between silica and plastic beads should be controlled. Both the preferred silica / plastic and plastic / silica ratios should be approximately within the range of 0.9 to 3.0, meaning the size of the silica can be three times greater or three times smaller than the size of the plastic. Experimentation has shown that outside of these ratios, the overall performance of the matrix may suffer. For example, a silica size that is four times smaller than the size of the plastic will cause the silica to become loose and flow out of the filter matrix as the DNA solution passes through or the loose silica will accumulate in some areas of the matrix clogging the filter and preventing proper DNA solution flow. On the other hand, if the silica is too big, the structural integrity will be compromised, making the filter very fragile and causing it to collapse during assembly or manipulation. The silica-to-plastic mix ratios are also important and may depend on the application. For proper DNA extraction, a range between 15% to 50% silica and 85% to 50% plastic by volume is preferred. The blend is mixed using mechanical methods to achieve a homogeneous mix. The mixture is then poured into a mold with the desired shape of the filter matrix. Inside each mold cavity, the silica beads are surrounded by plastic beads, creating a porous matrix. The mold is then heated to a temperature range between 280°F and 340°F to allow the outer layer of the plastic beads to soften enough to adhere to the surrounding plastic beads. This plastic bead structure traps and locks the silica beads between the plastic beads, forming the fdter matrix. FIG.3 illustrates the plastic 125 and silica 120 matrix. The mold may be constructed to form a fdter matrix that is slightly larger than the central lumen of the pipette tip, creating a tight interference fit.

[0069]

[0050] In either the silica gel membrane matrix or the plastic and silica beads matrix implementation, the filter matrix should be installed as close as possible to the distal end of the tip to minimize the “dead space” 119 (i.e., the space between the distal end of the pipette tip and the distal end of the filter matrix). This installation is done to ensure that most, if not all, of the liquid containing the DNA will pass through the filter matrix.

[0070]

[0051] Show in FIGS. 2A through 2C is a DNA extraction pipette tip 50 is detailed, and contains a proximal end 52 and a distal end 53. An aspiration device opening 51 is located at the proximal end 52 and constructed to receive an aspiration device, such as a pump or a pipetter (manual or automatic). A distal end opening 54 is located at the distal end 53, and a central lumen 54.1 fluidly connects the aspiration device opening 51 to the distal end opening 54. The central lumen 54.1 defines an internal wall 54.2. A filter matrix 105 is disposed in the central lumen 54.1 and positioned closer to the distal end opening 54 than the aspiration device opening 51. The filter matrix 105 may be a silica gel membrane matrix or a plastic and silica bead matrix, discussed above.

[0071]

[0052] FIG. 2B shows how the filter matrix 105 shape follows the pipette tip 110 contour, particularly on the draft angles. A tight interference fit (shown as dashed line 118 in FIG. 2C) results in an even seal between the pipette tip 110 and the filter matrix 105, ensuring that the fluid is always in contact with the filter matrix 105. The diameter of the filter matrix should be approximately .002” to .010” bigger than the pipette tip to allow for a tight interference / press fit. If the filter matrix 105 is not tight against the pipette tip’s 110 internal walls 54.2, then liquid will flow through the potential gaps between the filter matrix 105 and the internal walls 54.2 of the pipette tip 110.

[0072]

[0053] The matrix filter length also affects performance. A long filter will allow for more silica to be embedded into the matrix, and will give the target DNA more opportunities to bind to the filer matrix. A preferred filter length will be in the range of 0.20-1 .50 inches. The length depends on the amount of DNA that is to be trapped.

[0073]

[0054] The DNA extraction pipette tip 50 eliminates the need for excessive force to make the DNA solution pass through the filter matrix 105. This unique filter matrix 105 allows a regular positive displacement hand-held pipettor to make the DNA solution flow through the filter matrix 105. In comparison, the traditional DNA extraction method requires high centrifuge forces (e.g., 10,000 to 14,000 X g) to push the DNA solution through the spin column membranes, sometimes breaking the DNA strings.

[0074]

[0055] Table 1 presents test results of DNA extraction pipette tips made according to the techniques described herein. Tip A has a weight ratio of 70% GHR (Plastic) / 30% silica, while tip B has a weight ratio of 65% GHR (Plastic) / 35% silica. Both tips were tested using a low molecular weight DNA (<500bp), with two aspiration methods: handheld pipetter or pump. The electrophoresis graph shown in FIG. 4A confirms that both tips A and B had a sufficient capability of binding low molecular weight DNA.

[0075] Table 1- Low Molecular Weight DNA (<500bp)

[0076]

[0056] Table 2 presents test results of DNA extraction pipette tips made according to the techniques described herein. Again, tip A has a weight ratio of 70% GHR (Plastic) / 30% silica, while tip B has a weight ratio of 65% GHR (Plastic) / 35% silica. Both tips were tested using a medium molecular weight DNA (>1000bp), with two aspiration methods: handheld pipetter or pump. The electrophoresis graph shown in FIG. 4B confirms that both tips A and B had a sufficient capability of binding medium molecular weight DNA. Interestingly, the handheld pipetter aspiration showed a noticeably better recovery of DNA (see graph wells 2-4) as compared to the pump aspiration (see graph wells 6-9). Table 2- Medium Molecular Weight DNA (>1OOObp)

[0077] 057] The above description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles described herein can be applied to other embodiments without departing from the spirit or scope of the invention. Thus, it is to be understood that the description and drawings presented herein represent a presently preferred embodiment of the invention and are therefore representative of the subject matter that is broadly contemplated by the present invention. It is further understood that the scope of the present invention fully encompasses other embodiments that may become obvious to those skilled in the art, and that nothing accordingly limits the scope of the present invention other than the appended claims.

Claims

CLAIMS1. A DNA extraction pipette tip (50) comprising: a proximal end (52) and a distal end (53); an aspiration device opening (51) located at the proximal end (52) and constructed to receive an aspiration device; a distal end opening (54) located at the distal end (53); a central lumen (54.1) fluidly connecting the aspiration device opening (51) to the distal end opening (54), the central lumen defining an internal wall (54.2); a filter matrix (105) disposed in the central lumen (54.1) and positioned closer to the distal end opening (54) than the aspiration device opening (51), the filter matrix (105) comprising: plastic beads (125) and silica beads (120) fused together in a porous matrix that permits a fluid connection between the aspiration device opening (51) to the distal end opening (54).

2. The pipette tip (50) of claim 1, wherein the ratio by volume of plastic beads (125) to silica beads (120) ranges from 85:15 to 50:50.

3. The pipette tip (50) of any of the claims 1 or 2, wherein: the silica beads (120) range from 50 to 150 microns in diameter; and the plastic beads (125) range from 60 to 150 microns.

4. The pipette tip (50) of any of the claims 1 through 3, wherein the ratio of the diameter of the plastic beads (125) to silica beads (120) ranges from 1 :3 to 3: 1.

5. The pipette tip (50) of any of the claims 1 through 4, wherein the filter matrix (105) forms an interference / press fit (118) with the internal wall (54.2).

6. The pipette tip (50) of any of the claims 1 through 5, further comprising a retainer (115) constructed to retain the filter matrix (105) within the central lumen (54.1).

7. The pipette tip (50) of any of the claims 1 through 6, wherein the filter matrix (105) is positioned adjacent to the distal end opening (54).

8. The pipette tip (50) of any of the claims 1 through 7, wherein the filter matrix (105) is positioned to reduce dead space (119).

9. The pipette tip (50) of any of the claims 1 through 8, wherein: the filter matrix (105) is constructed to be inserted into the central lumen (54.1); the filter matrix (105) comprises a first diameter, and the central lumen (54.1) comprises a second diameter at the location where in the filter matrix (105) is disposed; and the first diameter is .002” to .010” larger than the second diameter prior to insertion into the central lumen (54.1).

10. The pipette tip (50) of any of the claims 1 through 9, wherein the filter matrix (105) comprises a length of 0.20”-1.50”.

11. The pipette tip (50) of any of the claims 1 through 10, wherein the aspiration device opening (51) is constructed to receive a pipetter or a pump.

12. A DNA extraction pipette tip (50) comprising: a proximal end (52) and a distal end (53); an aspiration device opening (51) located at the proximal end (52) and constructed to receive an aspiration device; a distal end opening (54) located at the distal end (53); a central lumen (54.1) fluidly connecting the aspiration device opening (51) to the distal end opening (54), the central lumen defining an internal wall (54.2); a filter matrix (105) disposed in the central lumen (54.1) and located closer to the distal end opening (54) than the aspiration device opening (51), the filter matrix (105) comprising a silica gel membrane.

13. The pipette tip (50) of claim 12, wherein the filter matrix (105) forms an interference / press fit (118) with the internal wall (54.2).

14. The pipette tip (50) of any of the claims 12 or 13, further comprising a retainer (115) constructed to retain the filter matrix (105) within the central lumen (54.1).

15. The pipette tip (50) of any of the claims 12 through 14, wherein the filter matrix (105) is positioned adjacent to the distal end opening (54).

16. The pipette tip (50) of any of the claims 12 through 15, wherein the filter matrix (105) is positioned to reduce dead space (119).

17. The pipette tip (50) of any of the claims 12 through 16, wherein: the filter matrix (105) is constructed to be inserted into the central lumen (54.1); the filter matrix (105) comprises a first diameter, and the central lumen (54.1) comprises a second diameter at the location where in the filter is disposed; and the first diameter is .002” to .010” larger than the second diameter prior to insertion into the central lumen (54.1).

18. The pipette tip (50) of any of the claims 12 through 17, wherein the filter matrix (105) comprises a length of 0.20”-1.50”.

19. The pipette tip (50) of any of the claims 1 through 18, wherein the aspiration device opening (51) is constructed to receive a pipetter or a pump.

20. A method of DNA extraction comprising: a. loading a DNA extraction pipette tip (50) of any of the previous claims onto an aspiration device; b. aspirating a DNA-containing sample (6) into the pipette tip (50); c. dispensing the sample (6) from the pipette tip (50); d. trapping DNA in the filter matrix (105) in step (b) or step (c) or both;e. aspirating a washing solution (25) into the pipette tip (50); f. dispensing the washing solution (50) from the pipette tip (50); g. aspirating an elution solution (30) into the pipette tip (50); h. dispensing elution solution (30) from the pipette tip (50) into a collection tube (55); and i. dislodging DNA within the filter matrix (105) in step (g) or step (h) or both, such that the dispensed elution solution (30) contains DNA.

21. A method of manufacturing a DNA extraction pipette tip (50) comprising: mixing silica beads (120) and plastic beads (125) into a homogenous mixture; pouring the homogenous mixture into a mold; heating the homogenous mixture to a temperature between 280°F and 340°F; forming a porous filter matrix (105) by softening and adhering the plastic beads (125) to each other during heating; removing the filter matrix (105) from the mold; and inserting the filter matrix (105) into a central lumen (54.1) of a pipette tip (110).

22. The method of claim 21, wherein the ratio by volume of plastic beads (125) to silica beads (120) ranges from 85: 15 to 50:50.

23. The method of any of the claims 21 or 22, wherein: the silica beads (120) range from 50 to 150 microns in diameter; and the plastic beads (125) range from 60 to 150 microns.

24. The method of any of the claims 21 through 23, wherein the ratio of the diameter of the plastic beads (125) to silica beads (120) ranges from 1 :3 to 3: 1.

25. The method of any of the claims 21 through 24, wherein a central lumen (54.1) defines an internal wall (54.2), and the filter matrix (105) forms an interference / press fit (118) with the internal wall (54.2).

6. The method of any of the claims 21 through 25, wherein: the filter matrix (105) comprises a first diameter prior to insertion; the filter matrix (105) is disposed in the central lumen (54.1) at a position during insertion; the central lumen (54.1) comprises a second diameter at the position; and the first diameter is larger than the second diameter.

Images