WO2014026008A1 - Embouts de pipette fonctionnels jetables pour l'isolement d'acides nucléiques - Google Patents

Embouts de pipette fonctionnels jetables pour l'isolement d'acides nucléiques Download PDF

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Publication number
WO2014026008A1
WO2014026008A1 PCT/US2013/054149 US2013054149W WO2014026008A1 WO 2014026008 A1 WO2014026008 A1 WO 2014026008A1 US 2013054149 W US2013054149 W US 2013054149W WO 2014026008 A1 WO2014026008 A1 WO 2014026008A1
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Prior art keywords
membrane
pipette tip
disposable
column
well
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PCT/US2013/054149
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English (en)
Inventor
Jeffrey L. Helfer
George E. Diaz
Evan C. BURLEY
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Diffinity Genomics, Inc.
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Publication of WO2014026008A1 publication Critical patent/WO2014026008A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/02—Burettes; Pipettes
    • B01L3/0275—Interchangeable or disposable dispensing tips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5025—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
    • B01L3/50255—Multi-well filtration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/12—Specific details about manufacturing devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/14—Process control and prevention of errors
    • B01L2200/141—Preventing contamination, tampering
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0832—Geometry, shape and general structure cylindrical, tube shaped
    • B01L2300/0838—Capillaries

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention is directed to a disposable functional pipette tip comprising an external distal membrane.
  • 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.
  • 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.
  • 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
  • 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.
  • 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 .
  • 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.
  • 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.
  • the present invention is directed to a disposable well or column comprising an external distal membrane.
  • 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.
  • 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
  • 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.
  • 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 .
  • 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.
  • 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.
  • the present invention is directed to a coated capillary comprising an external distal membrane.
  • the present invention is directed to the coated capillary of embodiment 15, where the coated capillary is optimized for the isolation of nucleic acids.
  • the present invention is directed to a non-disposable functional pipette tip comprising an external distal membrane.
  • 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.
  • 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.
  • FIG 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.
  • FIGS 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.
  • 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.
  • disposable functional pipette tips the invention explicitly also contemplates other disposable formats including, but not limited to, wells, columns, capillaries, cartridges, etc.
  • 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.
  • disposable embodiments are preferred, the present invention explicitly contemplates non-disposable embodiments as well. Use Of Flush/External Membrane
  • 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.”
  • 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.
  • 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.
  • 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.
  • 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 refers to any appropriate method of joining that accomplishes this functional endpoint, including but not limited to heat sealing, welding, cementing, brazing, etc.
  • 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.
  • 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.
  • 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.
  • 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.
  • pre-selected materials e.g. particles of a specific size
  • 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).
  • particle retention e.g. membrane design and effective pore size
  • b) wettability e.g. natural or as treated hydrophillicity
  • manufacturability e.g. weldability, ease of perforation, physical integrity following perforation
  • bio-inertness e.
  • 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.
  • 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.
  • 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.
  • the membrane diameter is 5 to 20 times the membrane pore size.
  • 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.
  • 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.
  • bio-inert i.e. membranes that adsorb minimal or negligible amounts of desirable biological materials passing through the membrane
  • 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.
  • membrane manufacturing processes e.g. "melt blown" membrane fabrication process
  • 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:
  • 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 provide unanticipated design flexibility in terms of membrane construction, pore size to thickness properties, wettability, bio-inertness, etc.
  • 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.
  • 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.
  • the membrane melt temperature would be 90 or more degrees Centigrade higher than that of the pipette tip;
  • 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
  • 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.
  • 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 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.
  • 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.
  • solute e.g. polypropylene
  • 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.
  • 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.
  • 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.
  • chromatographic grade silica gel e.g. the ability of the silanol groups to interact with an adsorbent
  • 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.
  • 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.
  • 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.
  • 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.
  • a reaction vessel such as a tube or a well closed at one end
  • the functionalized materials are permanently attached to the sample-contacting walls of the tube or well.
  • 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.
  • 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.
  • 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.
  • 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.
  • sample purification is achieved by aspirating sample into one end of the fiber and dispensing it from the other end of the fiber.
  • 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.
  • yet another aspect of the present invention is a pipette tip with two reaction chambers, located in series.
  • 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.
  • 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.
  • nozzle diameters 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.
  • d nozzle diameters
  • 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.
  • 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.
  • 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.
  • 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.
  • the desiccant material could include a suitably sized desiccant paper such as the desiccant paper produced by Sorbent Systems (e.g. Product number DP50SG0812A).
  • Sorbent Systems e.g. Product number DP50SG0812A.
  • One advantage provided by Diffinity's functionalized materials is purification in a single exposure (i.e. single-pass) to a sample.
  • yet another aspect of the present invention is a multi-chamber reaction vessel that takes unique advantage of this single-pass purification capability.
  • 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.
  • 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.
  • FIG. 15a-c 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.
  • 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.
  • 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.

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  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Hematology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La présente invention concerne de préférence des embouts de pipette fonctionnels jetables, en particulier des embouts de pipette fonctionnels jetables pour la purification d'acides nucléiques. La présente invention concerne également de préférence des procédés de fabrication de ces embouts, des procédés d'utilisation de ces embouts, et les structures de ces embouts. Bien que la présente invention concerne de préférence des embouts de pipette fonctionnels jetables, l'invention concerne également de façon explicite d'autres formats fonctionnalisés jetables et non jetables comprenant, mais sans y être limités, des puits fonctionnalisés, des colonnes fonctionnalisés, des capillaires fonctionnalisés, des cartouches fonctionnalisés, etc.
PCT/US2013/054149 2012-08-08 2013-08-08 Embouts de pipette fonctionnels jetables pour l'isolement d'acides nucléiques WO2014026008A1 (fr)

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US201261680877P 2012-08-08 2012-08-08
US61/680,877 2012-08-08

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WO2014026008A1 true WO2014026008A1 (fr) 2014-02-13

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018165635A1 (fr) * 2017-03-10 2018-09-13 Hitachi Chemical Co., Ltd. Dispositif de filtrage, dispositif de capture et leurs utilisations
WO2020180497A1 (fr) * 2019-03-06 2020-09-10 Smulevitch Sergey Pointe de pipette contenant une ou plusieurs barrières
EP3940052A4 (fr) * 2019-03-12 2023-01-25 Hui Chen Appareil d'extraction d'acide nucléique

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6048457A (en) * 1997-02-26 2000-04-11 Millipore Corporation Cast membrane structures for sample preparation
US20100081209A1 (en) * 2007-02-21 2010-04-01 William Brewer Pipette tips for extraction, sample collection and sample cleanup and methods for their use
US20110107855A1 (en) * 2008-03-28 2011-05-12 Pelican Group Holdings, Inc. Sample preparation devices and methods for processing analytes
US8053247B2 (en) * 2006-10-11 2011-11-08 Phynexus, Inc. Method and device for preparing an analyte for analysis by mass spectrometry
US20120071643A1 (en) * 2009-02-14 2012-03-22 Diffinity Genomics, Inc. System and methods for purifying biological materials

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6048457A (en) * 1997-02-26 2000-04-11 Millipore Corporation Cast membrane structures for sample preparation
US8053247B2 (en) * 2006-10-11 2011-11-08 Phynexus, Inc. Method and device for preparing an analyte for analysis by mass spectrometry
US20100081209A1 (en) * 2007-02-21 2010-04-01 William Brewer Pipette tips for extraction, sample collection and sample cleanup and methods for their use
US20110107855A1 (en) * 2008-03-28 2011-05-12 Pelican Group Holdings, Inc. Sample preparation devices and methods for processing analytes
US20120071643A1 (en) * 2009-02-14 2012-03-22 Diffinity Genomics, Inc. System and methods for purifying biological materials

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018165635A1 (fr) * 2017-03-10 2018-09-13 Hitachi Chemical Co., Ltd. Dispositif de filtrage, dispositif de capture et leurs utilisations
WO2020180497A1 (fr) * 2019-03-06 2020-09-10 Smulevitch Sergey Pointe de pipette contenant une ou plusieurs barrières
US11701650B2 (en) 2019-03-06 2023-07-18 Sergey Smulevitch Pipette tip containing one or more barriers
EP3940052A4 (fr) * 2019-03-12 2023-01-25 Hui Chen Appareil d'extraction d'acide nucléique

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