WO2014026008A1

WO2014026008A1 - Disposable functional pipette tips for the isolation of nucleic acids

Info

Publication number: WO2014026008A1
Application number: PCT/US2013/054149
Authority: WO
Inventors: Jeffrey L. Helfer; George E. Diaz; Evan C. BURLEY
Original assignee: Diffinity Genomics, Inc.
Priority date: 2012-08-08
Filing date: 2013-08-08
Publication date: 2014-02-13

Abstract

The present invention preferably relates to disposable functional pipette tips, particularly disposable functional pipette tips for the purification of nucleic acids. The present invention also preferably relates to methods of making these tips, methods of using these tips, and the structures of these tips. Although the present invention is preferably directed to disposable functional pipette tips, the invention explicitly also contemplates other disposable and non-disposable functionalized formats including, but not limited to, functionalized wells, functionalized columns, functionalized capillaries, functionalized cartridges, etc.

Description

DISPOSABLE FUNCTIONAL PIPETTE TIPS FOR

THE ISOLATION OF NUCLEIC ACIDS

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent Serial No.

61/680,877, filed August 8, 2012. The contents of this application are herein incorporated in their entirety by reference.

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

As stated in Applicants co-pending application Ser. No. 13/201 ,415, the contents of which are herein incorporated in its entirety by reference, chemical reactions and analysis involving fluids often use pipette tips to manipulate the fluids during the course of the reaction or analysis. To help improve the efficiency and reduce the cost of related workflows, manufacturers often integrate elements of the reaction or analysis into the pipette tip itself— what are typically referred to a "functional" pipette tips. These chemical reactions and analysis often include the use of specially treated particles within the pipette tip to improve the interaction between the sample and the chemistry provided by the particles.

In co-pending application Ser. No. 13/201 ,415, Applicants provided a number of designs for functional pipette tips. The present invention is directed to other disposable (and non-disposable) functional pipette tips, with various embodiments of these tips provided in the specification below. Although the present invention is preferably directed to disposable functional pipette tips, the invention explicitly also contemplates other disposable and non-disposable functionalized formats including, but not limited to, functionalized wells, functionalized columns, functionalized capillaries, functionalized cartridges, etc. SUMMARY OF THE INVENTION

The present invention preferably relates to disposable functional pipette tips, particularly disposable functional pipette tips for the purification of nucleic acids. The present invention also preferably relates to methods of making these tips, methods of using these tips, and the structures of these tips. Although the present invention is preferably directed to disposable functional pipette tips, the invention explicitly also contemplates other disposable and non-disposable formats including, but not limited to, wells, columns, capillaries, cartridges, etc.

In embodiment 1 , the present invention is directed to a disposable functional pipette tip comprising an external distal membrane.

In embodiment 2, the present invention is directed to the disposable functional pipette tip of embodiment 1 , where the disposable functional pipette tip is optimized for the isolation of nucleic acids.

In embodiment 3, the present invention is directed to the disposable functional pipette tip of embodiment 1 , wherein the optimization comprises one or more of: optimal membrane design; increased membrane wettability and uniformity of wettability; means to minimize or eliminate optical interference effects; use of low static charge polymer resins; multi-chamber pipette tip; non-plugging pipette tip; optimum nozzle and membrane dimensions (e.g. wide bore); perforated nozzle; use of desiccant within a pipette tip; multi-chamber reaction column; and, multi-reaction column.

In embodiment 4, the present invention is directed to the disposable functional pipette tip of embodiment 1 , where the external distal membrane thickness is between 0.001 inches and 0.008 inches.

In embodiment 5, the present invention is directed to the disposable functional pipette tip of embodiment 1 , where the external distal membrane diameter-to- thickness ratio is between 2.5:1 and 5:1 .

In embodiment 6, the present invention is directed to the disposable functional pipette tip of embodiment 5, where the external distal membrane diameter is 5-20 times the membrane pore size.

In embodiment 7, the present invention is directed to the disposable functional pipette tip of embodiment 1 , where the disposable functional pipette tip is optimized to have a high fluid flow rate.

In embodiment 8, the present invention is directed to a disposable well or column comprising an external distal membrane.

In embodiment 9, the present invention is directed to the disposable well or column of embodiment 8, where the disposable well or column is optimized for the isolation of nucleic acids.

In embodiment 10, the present invention is directed to the disposable well or column of embodiment 8, wherein the optimization comprises one or more of:

optimal membrane design; increased membrane wettability and uniformity of wettability; means to minimize or eliminate optical interference effects; use of low static charge polymer resins; multi-chamber configuration; multi-reaction

configuration; non-plugging configuration; optimum membrane dimensions (e.g. wide bore); use of desiccant within the lumen of the well or column.

In embodiment 1 1 , the present invention is directed to the disposable well or column of embodiment 8, where the external distal membrane thickness is between 0.001 inches and 0.008 inches.

In embodiment 12, the present invention is directed to the disposable well or column of embodiment 8, where the external distal membrane diameter-to-thickness ratio is between 2.5:1 and 5:1 .

In embodiment 13, the present invention is directed to the disposable well or column of embodiment 12, where the external distal membrane diameter is 5-20 times the membrane pore size.

In embodiment 14, the present invention is directed to the disposable well or column of embodiment 8, where the disposable well or column tip is optimized to have a high fluid flow rate.

In embodiment 15, the present invention is directed to a coated capillary comprising an external distal membrane.

In embodiment 16, the present invention is directed to the coated capillary of embodiment 15, where the coated capillary is optimized for the isolation of nucleic acids.

In embodiment 17, the present invention is directed to a non-disposable functional pipette tip comprising an external distal membrane.

In embodiment 18, the present invention is directed to a non-disposable functionalized well or wells, column, capillary or cartridge comprising one of the features provided in the specification.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings provided in the present invention are provided solely to better illustrate particular embodiments of the present invention, and specifically do not provide an exhaustive or limiting set of embodiments of the present invention.

Figures 1-2 provide an embodiment of the present invention in which the distal end of the pipette tip (bottom end in the figure) is covered with an attached membrane (see expanded view in Figure 1 ); the upper (top) end of the pipette tip is referred to as the "proximal" end.

Figure 3 provide an alternate embodiment of the present invention in which the membrane at the distal end of the functional pipette tip is integrally formed (see Figure 4 provides data on treatments used to minimize UV absorption.

Figures 5-12 provide alternate embodiments of the present invention.

Figure 13 provides stability testing data on absorbance changes over time. Figures 14-15 provide alternate embodiments of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Note that in the present invention, "a" or "an" are explicitly not limited to the singular form; instead, "a" and "an" are explicitly intended to be synonymous with "at least - but not limited to - one instance of the term being referenced.

"Functional Pipette Tips"

The present invention preferably relates preferably to disposable functional pipette tips, particularly disposable functional pipette tips for the purification of nucleic acids, and to methods of making these tips, methods of using these tips, and the structures of these tips. Although the present invention is preferably directed to disposable functional pipette tips, the invention explicitly also contemplates other disposable formats including, but not limited to, wells, columns, capillaries, cartridges, etc. Thus whenever the term "functional pipette tip" or "pipette tip" is used in the present application, it is understood that, while this is one preferred embodiment, functionalized wells, cartridges, columns, capillaries, etc. are also contemplated. Also, while disposable embodiments are preferred, the present invention explicitly contemplates non-disposable embodiments as well. Use Of Flush/External Membrane

Conventional design functional pipette tips (e.g. Thermo-Fisher Aspire™ or Millipore ZipTip™) utilize porous frits to immobilize the active materials (e.g.

purification resins) used in these tips. Referring to Figure 1 , one aspect of the present invention is the use of a membrane permanently attached to the distal face/external surface of a pipette tip to contain active materials in the pipette tip, i.e., an "external distal membrane." The lower flow resistance of the thinner membrane, relative to the much thicker porous frits, enables much faster sample fluid flow rates into and out of the pipette tip for given sample aspiration and dispensing pressures, which in turn enables much faster sample aspirate and dispense cycle times. Other benefits also include minimum sample volume retention and ease of adapting standard pipette tips to a "functional" design configuration, since such adaption is dependent only upon attaching a membrane to the distal face/external surface of the pipette tip and not the use of, for example, internal components such as porous frits which are custom designed to fit the unique internal physical dimensions (lumen) of a specific pipette tip.

Thus Figure 1 provides an embodiment of the present invention in which the distal end of the pipette tip (bottom end in the figure) is covered with an attached membrane (see expanded view in Figure 1 ); the upper (top) end of the pipette tip is referred to as the "proximal" end. This terminology is in keeping with that of copending application Ser. No. 13/201 ,415 (e.g., Figure 2 in this co-pending application). Applicants note that "attached" refers to any method of joining that ensures the membrane does not separate from the distal end of the pipette tip within the intended lifetime of the functional pipette tip.

In functional terms, "attached" refers to a state in which preferably fluid is unable to enter the lumen of the pipette tip except by flowing through the membrane, although an alternate/additional definition refers to attached such that resin particles inside the pipette tip are not able to escape the interior of the tip but fluid is able to flow.

"Attached" as contemplated herein refers to any appropriate method of joining that accomplishes this functional endpoint, including but not limited to heat sealing, welding, cementing, brazing, etc. Applicants note that a "permanently attached" membrane refers to attachment of sufficient longevity to prevent leakage into the lumen of the pipette tip (i.e., fluid flow through a path other than the membrane) during the intended lifetime of the functional pipette tip.

Applicants note that the membrane in this embodiment is joined to what is referred to as the "distal face/external surface" of the pipette tip; this terminology refers generally to any joining configuration of the membrane to the pipette tip in which the membrane is not contained within the lumen (inner surface) of the pipette tip. Thus the flush joining of the membrane to the distal end of the pipette tip shown in Figure 1 is an example of the contemplated configuration; so too would be, e.g., a membrane cap over the end of the pipette tip, etc.

Optimal Membrane Design

Yet another aspect of the present invention is the use of an optimally designed membrane positioned on the nozzle-end of a pipette tip. The primary purpose of the membrane is to allow the free passage of fluid (e.g. liquid samples) while retaining pre-selected materials (e.g. particles of a specific size) within the pipette tip. However, the ability to effectively perform this function, while

simultaneously providing other important product performance benefits, represents complex and competing membrane design tradeoffs that are not known in the art. Therefore, knowing what these optimal tradeoffs are and knowing how to simultaneously satisfy all of them in a single product to achieve a superior performing "system" represents novelty.

More specifically, the membranes used in the present functionalized pipette tip product concept must satisfy the following product performance requirements: a) particle retention (e.g. membrane design and effective pore size); b) wettability (e.g. natural or as treated hydrophillicity); c) manufacturability (e.g. weldability, ease of perforation, physical integrity following perforation); d) bio-inertness (e.g. low molecular adsorption, low surface energy); e) fluid volume recovery (e.g. thin, low void volume); f) uniformity (e.g. spatial consistency of pores); and, g) high flow rate (e.g. membrane thickness and pore size, self-cleaning flow).

By way of example, difficulties arise due to the following types of competing conditions:

1 . Particle retention versus manufacturability. Separation of molecules from liquids in the present invention works most effectively with particles whose size is approximately 50-250 microns, which is close in size to pipette tip nozzle diameters, which typically range from 250 to 500 microns in diameter. Membranes suitable for filtering such particles are often many times this thickness to achieve the proper effective pore size. Referring to Figure 2, thick membranes are difficult to attach to pipette tips with small nozzle diameters which can be less than the thickness of the membrane. One such example of an attachment process involves welding membranes to the nozzle of a pipette tip. Referring again to Figure 2, the welding process requires that heat be applied from outside the pipette tip and transferred through the membrane to soften and melt the pipette tip at the tip-membrane interface. The thicker membrane shown in Figure 2 makes such heat transfer much more difficult and can often result in thermally damaging the membrane.

Accordingly, there is an optimum membrane thickness to pipette tip nozzle diameter that enables effective welding. We have found that pipette tip nozzle diameters of 0.005 to 0.060 inches work best with welded membranes whose thickness ranges from 0.001 to 0.008 inches, or a diameter-to-thickness ratio of 2.5:1 to 5: 1 or greater. Knowing the composition of a membrane able to filter 50-250 micron particles and still be thin enough to be easily and reliably attached to a small diameter pipette tip represents novel insight.

2. Membrane pore size versus cross-sectional area. The choice of optimum pore size for membranes used to retain particles in functional pipette tips requires membranes large enough in cross-sectional area and pore size to permit reasonably high fluid flow rates through the membrane at the very low pressures available from pipettors yet small enough in cross-sectional area to fit commonly available pipette tips while maintaining small pore size to retain functionalized particles.

Membranes small enough to fit commonly available pipette tip nozzles and remain physically stable with uniform physical properties (e.g. uniform pore size across their surface) and chemical properties (e.g. uniform wettability across their surface) typically have very small pore sizes, typically less than one micron, making them unsuitable for functional pipette tips due to their very high and often variable liquid intrusion pressures and low fluid flow rates associated with them.

Accordingly, yet another aspect of the present invention is the use of a membrane that has large pore size relative to the overall cross-sectional dimensions of the membrane and is simultaneously very thin. Ideally, the membrane diameter is 5 to 20 times the membrane pore size.

3. Particle retention versus fluid volume recovery. Likewise, thicker membranes retain larger amounts of liquid in their void volume, thereby increasing the amount of liquid retained by the membrane and reducing the amount of sample fluid returned by the functional pipette tip. Knowing the composition of a membrane able to filter larger particles and simultaneously minimize retained fluid volume represents novel insight.

4. Particle retention and wettability. Membranes need to have an effective pore size smaller than the size of the particles they need to filter. However, deceasing pore size increases the pressure required to initiate flow through the membrane, what is referred to as water intrusion pressure, due to reduced capillary pressure. Since most pipettors typically deliver only a few Kilopascals of operating pressure, knowing the composition of a membrane able to filter smaller particles and still have very low fluid intrusion pressure represents novel insight.

5. Bio-inertness and wettability. Popular membrane materials can adsorb nucleic acids, proteins and other materials to varying degrees, which is undesirable. Bio-inertness, or the ability to resist adsorbing biological materials, is typically the result of low surface energies (e.g. PTFE is a very low surface energy material and is also very bio-inert). However, low surface energy produces surfaces are very difficult to wet, which results in higher water intrusion pressures. Knowing the composition of a membrane that is bio-inert (i.e. membranes that adsorb minimal or negligible amounts of desirable biological materials passing through the membrane) while still enabling flow through at low pipettor operating pressures represents novel insight.

There are materials, typically referred to as surfactants, known to increase the wettability of a surface. However, these materials often interact adversely with biological materials. One example is the adverse impact that certain surfactants have on polymerase chain reaction ("PCR") processes. Therefore, the means to enhance membrane wettability without adversely interfering with the biological materials processed by the functional pipette tip represents novel insight. Referring to Table 1 below, we see the results of membrane wetting and bio-inertness experiments performed in which a melt-blown polypropylene membrane (Pall Corporation product number S80904 70 micron HDC media) when treated with a representative surfactant (Sigma-Aldrich Docusate Sodium product number D1685). By inspection we see that i) treating the membrane surface with surfactant increases membrane wettability, and ii) surfactant concentrations of 0.2% or greater result in degradation of ds-DNA sequencing performance. Accordingly, there exists an optimal surfactant concentration range (0% < concentration < 0.1 %) that eliminates undesirable ds-DNA interaction while maintaining membrane wettability:

Table 1

Note: For purposes of comparison, a sequencing read length difference of 50 bp or less is considered to be equivalent.

6. Membrane pore size uniformity versus membrane wettability, flow rate and particle retention. Membrane materials are typically produced in large sheets or discs using membrane manufacturing processes (e.g. "melt blown" membrane fabrication process) that can demonstrate very significant spatial variability in effective pore size, wettability and flow rate. This variability can be on a physical scale that is comparable to the very small dimensions of membranes used to seal pipette tip nozzles (i.e. 0.10 millimeter to 0.50 millimeter membrane diameters), resulting in significant tip-to-tip variation in pipette tip particle retention, wettability and as a result liquid intrusion and extrusion pressure (i.e. the pressure required to initiate fluid flow into and out of the membrane by the pipette tip), and sample flow rate. Therefore, the means to achieve uniform membrane properties, including pore size uniformity, particle retention, wettability, and flow rate on such a very small scale represents novel insight.

7. High fluid flow rate versus membrane pore size and particle retention. Membranes able to retain particles as small as 50 microns typically do not produce high liquid flow rates at the operating pressures available in commonly used pipetting systems (e.g. 2-4 KPa), particularly through such small membranes (e.g. 0.10 millimeter to 0.50 millimeter diameters). Therefore, the means to obtain high flow rates through such small membranes represents novel insight.

In light of points 1 -7 above, one aspect of the present invention is a membrane design that simultaneously meets all of the requirements outlined above for using a functional pipette tip for separating bio-molecules from a fluid, specifically a non-obvious optimization of particle retention, wettability, manufacturability, bio- inertness, fluid volume recovery, uniformity and high flow rate. This performance is achieved by optimizing the following parameters:

A. Surface filtration membranes which do not rely upon membrane thickness to achieve an effective pore size (e.g. mesh design);

B. Self-cleaning fluid handling protocol wherein flow through the membrane is periodically reversed to forcibly remove any particles that might become trapped in the membrane and therefore serve to increase the pressure required to maintain a specific rate of fluid flow through the membrane;

C. Treatment of the membrane surface to increase surface energy and wettability, including the use of specially optimized plasma treatment and surfactants;

D. Membrane materials that are not known to be easily attachable to widely used pipette materials. In the present invention, it is desirable to utilize membrane materials that can be permanently attached to the distal end of a pipette tip, such as by welding. Conventional wisdom holds that only like-materials can be welded to each other (e.g. polypropylene membranes to polypropylene pipette tips).

Membrane materials that can be attached to pipette tip materials (e.g. membrane materials with higher melting points able to "wet" and "mechanically bind" to polypropylene) provide unanticipated design flexibility in terms of membrane construction, pore size to thickness properties, wettability, bio-inertness, etc.

Therefore, knowledge of such materials represents novel insight. Referring to Table 1 , we show the results of membrane-to-pipette tip welding experiments. This data clearly shows the ability to weld a polypropylene membrane to a polypropylene pipette tip, as expected, but also shows the ability to weld dissimilar materials to a polypropylene pipette tip, in this example nylon and polyester. Observation confirms the ability of the nylon and polyester materials to "wet" the polypropylene enabling it to bond with the dissimilar material by direct adhesion and by flow of the lower melting point polypropylene material into the openings in the dissimilar membrane material, while the dissimilar nylon and polyester materials maintain their necessary structural integrity due to the much higher melting points.

The structural integrity of the welded membrane is very important, since the very thin nature of preferred membrane designs creates the risk that excessive heat applied during the welding process can weaken or even sever the membrane at the very thin point of attachment to the pipette tip. To avoid this potential problem, it is desirable to use membrane materials that have slightly higher temperatures than the pipette tip to which they are being attached, thereby helping to ensure that plastic from the lower melting point pipette tip flows into and around the membrane, rather than vice versa, which would endanger the physical integrity and strength of the welded membrane.

Accordingly, yet another aspect of the present invention is the use of membrane materials that "wet" typical (polypropylene) pipette tip materials and have melt temperatures that are higher than that of the pipette tip to which they are being attached. Ideally, the membrane melt temperature would be 90 or more degrees Centigrade higher than that of the pipette tip;

E. Ease and stability of perforation as evidenced by sharply defined membrane cut edges that do not unravel or fray so as to enable reliable attachment to the very small wall thickness at the nozzle of a pipette tip;

F. Low surface energy to minimize bio-adsorption;

G. Small membrane area to sample volume ratio that minimizes the total amount of bio-adsorption that might take place for any sample passing through the membrane;

H. Thin with low void volume so as to minimize the volume of sample retained by the membrane; and,

I. Thin with small area that improves membrane weldability. Increased Membrane Wettability And Uniformity Of Wettability

Membrane materials are typically provided in sheets or discs that can demonstrate non-uniformity in wettability (i.e. surface energy) that can vary by position within the sheet or disc. This non-uniformity can be on a scale that is comparable to the very small dimensions of a pipette tip nozzle that the membrane is to seal. Therefore, cutting such a small piece of a membrane from a larger sheet or disc that contains such non-uniformity can result in significant tip-to-tip variability in the wettability of any membrane attached to the nozzle of a pipette tip. This in turn can result in significant tip-to-tip variability in pipette tip flow parameters such as liquid intrusion and extrusion pressures and fluid flow rate.

Accordingly, another aspect of the present invention is the use of membranes that are specially chosen or prepared, prior to assembly, to increase their wettability and the uniformity of wettability across the sheet or disc. Specific means for doing so include plasma treatment and surfactant treatment.

Plasma treatment. Plasma treatment enables customized molecular re- engineering of materials to impart unique surface properties, without affecting the bulk properties. The effect of plasma on a material is determined by the chemistry of the reactions between the surface and the reactive species present in the process gas employed. A multitude of gases can be used. Each gas produces a unique plasma composition resulting in different surface properties. Referring to Table 2, we see that proprietary plasma treatment processes resulted in approximately 50% reduction in membrane intrusion pressures, relative to an untreated sample.

Table 2 Integral "Formed" Membrane

One popular method for creating membranes in sheet form is create a saturated solution of the membrane substrate material (e.g. polypropylene) and allow the solvent to evaporate away, leaving holes in the then-dried membrane, which collectively then create an effective membrane pore size. Referring to Figure 3, yet another aspect of the present invention is to integrally form a membrane at the nozzle-end of a pipette tip. Unlike many filtration pipette tip designs, in this invention the membrane is an integral part of the pipette tip. This is achieved by aspirating a very small volume of a mixture of solute (e.g. polypropylene) and solvent into the pipette tip and allowing it to evaporate from the tip, leaving behind a porous membrane that is then integral to the pipette tip. Means To Minimize Or Eliminate Optical Interference Effects

All DNA absorbs maximally at 260 nm. DNA concentration in a solution is proportional to absorbance at 260 nm according to the following relationships: ds- DNA concentration (ng/uL) = 260 nm absorbance * 50 and ss-DNA concentration (ng/uL) = 260 nm absorbance ^* 33.

Researchers commonly use 260 nm absorbance measurements to determine DNA concentration and consider 10% variability acceptable. Interfering substances in the DNA-bearing sample can skew the 260 nm absorbance measurement making it difficult to determine the true concentration of DNA in the sample. Pipette tips and other plastic laboratory disposables are known to release compounds that absorb light in the ultraviolet region (e.g. 260 nanometers) as described by Lewis/et.al. This phenomenon can be unpredictable, as some tips show a significant problem while others show little or no effect at all. This phenomenon is exacerbated with functional pipette tips that containing absorbing or adsorbing materials (e.g. silica gel) which can absorb or adsorb the interfering substances released by the laboratory disposable from the time the functional pipette tip is manufactured until the time it is used. The absorbed or adsorbed material can then wash off the functionalized material when the functional pipette tip is used with aqueous solutions.

Representative data showing UV-spectrum of pH 8.5 PCR-buffer and water with an absorption peak at A_max = 264 nm is shown in Figure 4 - "Control, No Treatment". As a result, the UV absorbing materials released by laboratory disposables prevent accurate quantitative measurement of the concentration of nucleic acids in solution. Accordingly, yet another aspect of the present invention is the ability to eliminate this interference effect and enable accurate measurement of nucleic acids processed in plastic laboratory disposables. This performance is achieved in several, non-obvious ways:

A. Use of specially prepared molding resins. Most polypropylene (PP) pipette tips are molded from propylene homopolymers with melt flow rate (MFR) of about 35 g/10 min. These are generally "vise-broken" (viscosity broken) resins, i.e. they are produced as lower MFR (higher molecular weight) polymers in the polymerization reactor and then chemically degraded by organic peroxides to the higher final MFR (lower molecular weight) during melt extrusion with additives to give the

commercially sold pelletized form. Vise-breaking not only raises the MFR but also narrows the molecular weight distribution of the resin making it more suitable for injection molding. The peroxides are used at around 0.1 % of the weight of polymer and are essentially completely destroyed in the high temperature extrusion process. The exact amount of peroxide used depends upon the amount of molecular weight change intended and the stabilization additives used in the formulation. We have shown that the peroxide reaction products (e.g. acetone and t-butanol) which originate from the hydrolysis or photolysis of a by-product of vise-breaking, have UV absorption peaks throughout the 200-300 nm range and have reasonable volatility which allows them to transfer from the pipette tip to samples processed by the pipette tip. The specific mechanisms of transfer are most commonly migration to the surface and either evaporation or extraction. The less compatible the component is with PP, the more likely the migration and the greater extent to which the relevant partition coefficient will favor transfer out of the polymer. Accordingly, yet another aspect of the present invention is the use of polymer resins prepared without the "vise-breaking" process in the processing of samples containing nucleic acids.

B. The use of sacrificial (interferent-absorbinq) materials. Yet another aspect of the present invention is the use of materials in a functional pipette tip that preferentially absorb or adsorb the interfering substances released by the pipette tip. One example of such a material is non-functionalized materials whose surfaces have not been treated to repel interfering substances, used in the presence of

functionalized materials, wherein the non-functionalized materials preferentially adsorb or absorb the interfering materials.

C. Heat curing. Yet another aspect of the present invention describes a solution to the unwanted absorption by heat treating the pipette tip prior to assembly. Per the above, the source of the UV absorbance problem is the tip itself and that the UV absorbing material originates from the hydrolysis or photolysis of a by-product of vis-breaking process. This secondary by-product has reasonable volatility and can be removed by either heating the tips at elevated temperatures, typically 120-130°C , at atmospheric pressure, or at lower temperatures and high vacuum conditions, e.g. 65°C and 10 ³ Torr. Experiments have shown that the treatment under high vacuum results in more efficient removal and a cleaner tip. For our purposes and equipment availability, heating at atmospheric pressure has been shown to be the more practical solution. Referring to Figure 4, the curve labeled "Old treatment ( 20°C)" shows the absence of the 264 nanometer peak associated with the untreated pipette tip control sample - the curve labeled "Control, no treatment".

D. UV irradiation. Yet another aspect of the present invention describes a solution to the unwanted absorption by optically irradiating the pipette tip prior to assembly. Per the above, the source of the UV absorbance problem is the tip itself and that the UV absorbing material originates from the hydrolysis or photolysis of a by-product of vis-breaking process. This secondary by-product can be rendered benign by irradiating the pipette tips with UV radiation. Referring to Figure 4, the curve labeled "New treatment (hv)" shows the absence of the 264 nanometer peak associated with the untreated pipette tip control sample - the curve labeled "Control, no treatment". The main spectral line from our UV-light source emitted at 254 nm. However, not all materials that absorb in and around 254 nm, undergo a photochemical reaction with resultant chemical decomposition. The material causing the unwanted absorption at 264 nm in polypropylene pipette tips unexpectedly decomposed after absorbing the light." Therefore, the use of UV radiation to eliminate the UV absorbance problem represents novel insight.

E. The activity of chromatographic grade silica gel (e.g. the ability of the silanol groups to interact with an adsorbent) can be controlled by regulating the amount of residual water adsorbed on its surface. Conformation of this fact was observed from our long term PCR stability testing data shown in Figure 13, which was obtained under 10%, 58% and 100% relative humidity environments. The resulting PCR-material was found to have little, if any, 264 nm absorption when washed. Thus indicating that under these conditions, we had produced materials ineffective at picking up the UV-absorbing polypropylene by-product from the tips. So by carefully controlling the drying of our fresh PCR-material, i.e. leaving a certain level of water adsorbed on its surface, that we should be able to eliminate, or at least significantly reduce, the silanol interactions and thus prevent the removal of tip impurities by PCR materials. The advantages of this design include eliminating the need for post-manufacturing pipette tip treatments and ease of incorporation into the current manufacturing process of our materials.

F. Use of a blank with functional pipette tips. To reduce false A260 readings, it is also possible to utilize a "blank" UV-Vis absorbance spectrophotometer measurement using the same functional pipette tip and buffer or solution used to dilute DNA samples. Blanking ensures that the A260 signal is not coming from components in the buffer but rather from the DNA solution. To correct for this unwanted absorption component, the data are subjected to a pre-determined correction factor, based upon the level of UV absorbance in the blank reading.

Accordingly, yet another aspect of the present invention is a method of correcting for UV absorption in samples processed in the present functional pipette tip, that involves running a blank reading to correct for UV absorbance.

Use Of Low Static Charge Polymer Resins

At the very low level of functionalized materials (i.e. small particles) used in the pipette tip of the present invention, static charge can result in a significant mass of functionalized material clinging to the upper inside wall of the pipette tip and not locating near the nozzle where they need to be located in order to effectively interact with the fluids aspirated into the pipette tip. Accordingly, yet another aspect of the present invention is he use of anti-static agents in the pipette tip molding resin, such as glycerol monostearate, which enable the particles to more effectively settle into the lower portion of the pipette tip where they can more effectively interact with the sample.

Coated Wells (Tubes And Plates)

Referring to Figure 5, yet another aspect of the present invention involves the use of a reaction vessel, such as a tube or a well closed at one end, in which the functionalized materials are permanently attached to the sample-contacting walls of the tube or well. By placing the functionalized materials in the reaction vessel and not the pipette tip, standard design non-functionalized pipette tips can be used in the sample purification process. Interaction between the functionalized particles and sample is achieved by cycling sample in and out of the coated tube or well. Rapid reaction times (e.g. short purification time) is achieved by maintaining a narrow gap between the pipette tip and wall of the tube or well while the sample is dispensed into and aspirated from the well, thereby creating high shear forces at the sample- well interface, which minimize the thickness of the chemical boundary layer - the region of depleted reactants, that exists at the sample-well interface and serves to reduce the rate at which reactants are transported to and adsorbed by the functionalized materials bound to the surface of the reaction well. The chemical boundary layer is further minimized by aspirating into and dispensing sample to and from the well at high aspirate and dispense rates, as well as by using multiple sample fluid aspirate and dispense cycles to minimize the time the sample is stagnant within the tube or well. Optimum sample handling parameters include tip- to-well separation of 1.0 millimeter or less, sample flow rates of 50 micro-liters per second or greater, and multiple aspirate and dispense cycles that begin and end once fluid flow comes to rest (i.e. the aspirate cycle begins at the moment the dispense cycle ends. A significant advantage of this approach is that immobilization of particles in the tube or well (versus a pipette tip) dramatically simplifies the design of the pipette tip by eliminating the need for discrete components in the pipette tip for retaining loose particles, thereby reducing tip cost and enabling the use of very small size pipette tips and sample volumes, while the aforementioned high agitation means maintains rapid purification times not generally associated with reaction kinetics of liquids in wells.

Coated Capillaries

Referring to Figure 6, yet another aspect of the present invention involves the use of a small-bore tube, such as a capillary tube or very small pipette tip, in which the functionalized materials are permanently attached to the inner (preferably) or outer wall of the tube. Interaction between the functionalized particles and sample is achieved by cycling sample into and out of the coated tube. Rapid reaction times (e.g. short purification time) are achieved due to the small distance between the inner wall of the tube and the sample it contains, which minimizes the maximize distance materials in the sample must diffuse to reach the immobilized functionalized materials on the wall of the tube, as well as by the high fluid shear forces that are created at the sample-wall interface as sample is aspirated into and dispensed from the tube, which minimize the thickness of the chemical boundary layer of depleted reactants that exists at the sample-particle interface which serves to reduce the rate at which reactants are transported to and adsorbed by the functionalized materials bound to the surface of the reaction well.

In the present invention, the chemical boundary layer is further minimized by aspirating into and dispensing sample to and from the well at high aspirate and dispense rates, as well as by using multiple sample fluid aspirate and dispense cycles to minimize the time the sample is stagnant within the coated tube or pipette tip. Optimum sample handling parameters include capillary orifice diameter of 2.0 millimeters or less, sample flow rates of 50 micro-liters per second or greater, and multiple sample aspirate and dispense cycles that begin and end once fluid flow comes to rest (i.e. the aspirate cycle begins at the moment the dispense cycle ends. A significant advantage of this approach is that immobilization of particles in the tube dramatically simplifies the design of the fluid transfer device by eliminating the need for discrete components to retain loose particles, thereby reducing transfer device cost, as well as enabling the use of very small size transfer devices and sample volumes, while simultaneously maintaining the aforementioned high agitation means and rapid purification times generally associated with loosely defined particles. Yet another advantage is higher sample fluid flow rates enabled by replacing solid frits of functionalized materials with a flow through design.

Coated Filtration Fibers

Yet another aspect of the present invention involves the use of the coated small-bore tubes mentioned above, but with much longer tube lengths. In this particular embodiment, sample purification is achieved by aspirating sample into one end of the fiber and dispensing it from the other end of the fiber. As before, high agitation and rapid purification times are achieved by using small bore and high fluid transfer velocities. Optimum sample handling parameters include capillary orifice of 2.0 millimeters or less and sample flow rates of 50 micro-liters per second or greater. A significant advantage of this aspect of the invention is rapid filtration capability enabled by high agitation, and applicability to larger sample volume, continuous flow purification processes.

Multi-Chamber Pipette Tip

Referring to Figure 7, yet another aspect of the present invention is a pipette tip with two reaction chambers, located in series. In this embodiment, a particle retainer is positioned between a distally-positioned membrane and proximally- positioned aerosol barrier/retainer to create two independent reaction chambers Each chamber may or may not contain loosely-contained functionalized materials. By varying sample aspirate and dispense volumes, sample can be processed, sequentially, in the lower reaction chamber and upper reaction chamber, enabling multiple reactions to be performed on a sample with a single pipette tip.

Non-Plugging Pipette Tip

Referring to Figure 8, yet another aspect of the present invention is a pipette tip that is produced with a slight angle to the surface of the pipette tip nozzle.

Standard design pipette tips have nozzle faces that are perpendicular to their long axis and as a result, can become plugged against the bottom of the vessel containing the sample fluid to be aspirated. The angled pipette tip avoids this condition by maintaining a slight gap between the pipette tip and bottom of the vessel containing the sample, even if the pipette tip is in direct contact with the vessel.

Optimum Nozzle And Membrane Dimensions (E.G. Wide Bore)

Laboratory disposable designs are currently trending towards the use of smaller and smaller sample volumes so as to minimize the cost of the reagents required to process the sample. This in turn requires the use of smaller and smaller sized pipette tips for transferring the smaller and smaller sample and reagent volumes. However, smaller pipette tips increase the difficulty of attaching membranes to their much smaller nozzle diameters. Referring to Figure 9, yet another aspect of the present invention is the use of small-sized pipette tips with disproportionately larger sized nozzle dimensions. More specifically, we claim the use of nozzle diameters ("D") in small-sized pipette tips, typically pipette tips sized to handle 2-50 micro-liters, that are 0.010 to 0.060 inches in diameter, which is much larger, relative to the typical 0.005 to 0.020 inch nozzle diameters ("d") used in the larger-sized pipette tips used to handle 00-1 ,000 micro-liter samples. The advantages of this design include improved manufacturability of small volume pipette tips.

Perforated Nozzle

Referring to Figure 10, yet another aspect of the present invention is pipette tip which is produced with an integral distal particle retainer that is formed by perforating the pipette tip with orifices to enable fluid flow into and out of the pipette tip. The orifices are small enough to contain particles confined in the pipette tip while large enough to enable higher sample fluid flow rates. The orifices may be circular holes and/or slits that are formed during the pipette tip molding process or added afterwards.

Disposable that Enable Direct Measurement of Sample Optical Properties (e.g., DNA Concentration)

Samples processed by functional pipette tips must often be characterized before further processing of the sample. One example is quantification of DNA concentration prior to subsequent DNA sequencing. Yet another aspect of the present invention is the ability to characterize the optical properties of the processed sample without needing to dispense the sample from the pipette tip. Referring to Figure 1 1 , a pipette tip is described with an optical inspection path, which is positioned above the position required for the functionalized materials to settle in the pipette tip. By inspecting the sample above the functionalized materials, there is no possibility that the impurities removed by the functionalized materials (i.e. the impurities bound to the settled particles) will interfere with the optical signal produced by the desired materials remaining in the solution above the settled particles.

Use Of Desiccant Within A Pipette Tip

Referring to Figure 12, yet another aspect of the present invention is the use of a desiccant material positioned within the pipette tip, versus for example externally positioned in the box or carton containing one or more pipette tips, as is typical practice. By way of example, the desiccant material could include a suitably sized desiccant paper such as the desiccant paper produced by Sorbent Systems (e.g. Product number DP50SG0812A). The advantage of this approach includes a desiccant material in closer proximity with the protected species (i.e. the

functionalized materials located within the pipette tip) and reduced moisture absorption requirements of the desiccant due to the resistance to the transmission of water vapor into the pipette tip caused by the pipette tips distal and proximal retainers, which act as barriers to the transmission of water vapor and thereby reduce the amount of moisture that must be absorbed by the desiccant material.

Multi-Chamber Reaction Column

One advantage provided by Diffinity's functionalized materials is purification in a single exposure (i.e. single-pass) to a sample. Accordingly, yet another aspect of the present invention is a multi-chamber reaction vessel that takes unique advantage of this single-pass purification capability. Referring to Figure 14, we see a reaction vessel positioned in an outer collection vessel, wherein the inner reaction vessel contains an upper and lower reaction chamber, each of which contain functionalized materials. Sample is added to the upper sample reaction/addition chamber and progressively moved through the device under the aid of centrifugation or vacuum applied to the lower collection chamber (not shown), enabling the sample to sequentially interact with the particles in the upper and lower reaction chambers. The particles in each reaction chamber may be rigidly or loosely confined by the indicated fluid-permeable membranes.

Referring to Figure 14a, we see yet another embodiment of the multi-chamber reaction vessel. In this specific embodiment, the upper membrane has a liquid intrusion pressure that is lower than the intrusion pressure of the lower membrane. Sample is first forced through the upper membrane by centrifuging the reaction vessel at a lower G-force sufficient to create a high enough pressure to force liquid through the upper membrane but not sufficient to force liquid through the lower membrane, thereby enabling the sample to collect and remain in the space between the two membranes, which may or may not have functionalized materials and/or reagents located in it. Sample is then forced through the lower membrane by centrifuging the reaction vessel at a higher G-force sufficient to create a high enough pressure to force liquid through the lower membrane, thereby enabling the sample to exit the reaction vessel into the collection vessel.

Multi-Reaction Column

Yet another aspect of the present invention is a column containing Diffinity's functionalized materials which enable multiple reactions to take place in a single reaction column. Referring to Figures 15a-c and by way of example: A sample containing molecular species to be removed from solution is added to the top of the column and drawn through the indicated particles by either centrifugation or vacuum into the lower portion of an external sample collection vessel. In this example, the lower sample collection vessel and sample are then discarded. Referring to Figure 15d: The column of particles containing the molecular species bound in the vessel of Figure 15a (Note: Columns shown in Figures 15b or 15c apply here as well) is placed onto a clean sample collection vessel. A release agent is then added to the reaction column and drawn through the particles containing the bound molecular species, thereby releasing the species into the clean collection column where it is available for further processing.

Referring to Figure 15e: The column of particles containing the molecular species bound in the vessel of Figure 15a (Note: Columns shown in Figures 15b or 15c apply here as well) is placed onto a clean sample collection vessel. A release agent is then added to the reaction column and drawn through the particles containing the bound molecular species, enabling the released molecular species to interact with a second set of particles contained in the upper portion of the sample collection vessel before passing into the lower portion of the clean collection column where it is available for further processing. The advantages of this approach, relative to the previous approach, include simplifying the composition of the release agent and/or enabling additional processing of the sample, due to the additional interaction provided by the second set of particles.

Referring to Figure 15f: The column of particles containing the molecular species bound in the vessel of Figure 15a (Note: Columns shown in Figures 15b or 15c apply here as well) is placed onto a clean sample collection vessel. A release agent is then added to the reaction column and drawn through the particles containing the bound molecular species, enabling the released molecular species to pass into the new collection vessel and interact with the second set of particles contained in the lower portion of the sample collection vessel. A further refinement of this aspect of the present invention is to use the pipette tip design shown in Figure 1 to aspirate the purified sample from the sample collection vessel without aspirating the second set of particles into the pipette tip. The sample solution can then be dispensed from the pipette tip free of particles.

The following claims provide a non-limiting list of some of the embodiments of the present invention. Other embodiments are presented elsewhere herein.

Claims

What is claimed is:

1. A disposable functional pipette tip comprising an external distal membrane.

2. The disposable functional pipette tip of claim 1 , where the disposable

functional pipette tip is optimized for the isolation of nucleic acids.

3. The disposable functional pipette tip of claim 1 , wherein the optimization

comprises one or more of: optimal membrane design; increased membrane wettability and uniformity of wettability; means to minimize or eliminate optical interference effects; use of low static charge polymer resins; multi-chamber pipette tip; non-plugging pipette tip; optimum nozzle and membrane dimensions (e.g. wide bore); perforated nozzle; use of desiccant within a pipette tip; multi-chamber reaction column; and, multi-reaction column.

4. The disposable functional pipette tip of claim 1 , where the external distal membrane thickness is between 0.001 inches and 0.008 inches.

5. The disposable functional pipette tip of claim 1 , where the external distal membrane diameter-to-thickness ratio is between 2.5:1 and 5: 1.

6. The disposable functional pipette tip of claim 5, where the external distal membrane diameter is 5-20 times the membrane pore size.

7. The disposable functional pipette tip of claim 1 , where the disposable functional pipette tip is optimized to have a high fluid flow rate.

8. A disposable well or column comprising an external distal membrane.

9. The disposable well or column of claim 8, where the disposable well or

column is optimized for the isolation of nucleic acids.

10. The disposable well or column of claim 8, wherein the optimization comprises one or more of: optimal membrane design; increased membrane wettability and uniformity of wettability; means to minimize or eliminate optical interference effects; use of low static charge polymer resins; multi-chamber configuration; multi-reaction configuration; non-plugging configuration;

optimum membrane dimensions (e.g. wide bore); use of desiccant within the lumen of the well or column.

1 1 . The disposable well or column of claim 8, where the external distal membrane thickness is between 0.001 inches and 0.008 inches.

12. The disposable well or column of claim 8, where the external distal membrane diameter-to-thickness ratio is between 2.5:1 and 5:1 .

13. The disposable well or column of claim 12, where the external distal membrane diameter is 5-20 times the membrane pore size.

14. The disposable well or column of claim 8, where the disposable well or column tip is optimized to have a high fluid flow rate.

15. A coated capillary comprising an external distal membrane.

16. The coated capillary of claim 15, where the coated capillary is optimized for the isolation of nucleic acids.

17. A non-disposable functional pipette tip comprising an external distal

membrane.

18. A non-disposable functionalized well or wells, column, capillary or cartridge comprising one of the features provided in the specification.