WO2012103214A2 - Pointe de pipette magnétique - Google Patents

Pointe de pipette magnétique Download PDF

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Publication number
WO2012103214A2
WO2012103214A2 PCT/US2012/022545 US2012022545W WO2012103214A2 WO 2012103214 A2 WO2012103214 A2 WO 2012103214A2 US 2012022545 W US2012022545 W US 2012022545W WO 2012103214 A2 WO2012103214 A2 WO 2012103214A2
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WO
WIPO (PCT)
Prior art keywords
pipette tip
magnetic
lumen
housing
magnet
Prior art date
Application number
PCT/US2012/022545
Other languages
English (en)
Other versions
WO2012103214A3 (fr
Inventor
Gerard Albert LAWTHER
Steven Patrick MOSELEY
Lydia Wu
Chris LE
Original Assignee
Molecular Bioproducts, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Molecular Bioproducts, Inc. filed Critical Molecular Bioproducts, Inc.
Publication of WO2012103214A2 publication Critical patent/WO2012103214A2/fr
Publication of WO2012103214A3 publication Critical patent/WO2012103214A3/fr

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Classifications

    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1009—Characterised by arrangements for controlling the aspiration or dispense of liquids
    • 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
    • B01L2300/0609—Holders integrated in container to position an object
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N2035/1027—General features of the devices
    • G01N2035/1048—General features of the devices using the transfer device for another function
    • G01N2035/1053—General features of the devices using the transfer device for another function for separating part of the liquid, e.g. filters, extraction phase

Definitions

  • the present invention relates generally to the isolation of biological molecules and, more particularly, to the magnetic isolation of biological molecules.
  • Bio molecules such as DNA, RNA, proteins, and genomic DNA, may be conventionally isolated and purified from a sample using magnetic particles having an affinity to the biological molecules and a larger magnet that is positioned external to a vessel containing the sample.
  • the process of isolating and purifying the biological molecule begins with the lysis of cells, which are cultured to produce a desired biological molecule.
  • samples may include serum, cell culture supernatant, and PCR reactions as well.
  • Magnetic particles having a small magnetic or paramagnetic core coated with one or more coating materials are added into the lysed sample and the mixture is incubated under conditions that allow the desired biological molecule to reversibly bind to the outer coating of the magnetic particle.
  • Various coating materials are known and may include one or more polymers, biological molecules, or functional groups to facilitate the capture of the target molecule.
  • the biological molecule reversibly binds to the coating material.
  • a magnet is positioned adjacent to and external of the vessel containing the sample, thereby attracting the biological molecule bound magnetic particles to the internal surface of the vessel that is adjacent to the magnet.
  • the biological sample is separated from the lysis solution (i.e., the matrix).
  • the binding of the biological molecule to the coating material then may be disrupted by altering one or more properties of the sample containing the biological sample, releasing the biological material from the vessel while the magnetic particles are retained at the internal surface of the vessel that is adjacent to the magnet.
  • the present invention overcomes the foregoing problems and other shortcomings, drawbacks, and challenges of known conventional processes for isolating desired biological molecules using magnetic particles. While the invention will be described in connection with certain embodiments, it will be understood that the invention is not limited to these embodiments. To the contrary, this invention includes all alternatives, modifications, and equivalents as may be included within the spirit and scope of the present invention.
  • a magnetic pipette tip includes a pipette tip housing having proximal and distal ends. A lumen within the pipette tip housing extends between the proximal and distal ends. A first magnet is located within the lumen of the pipette tip housing between the proximal and distal ends.
  • a pipetting system in accordance with another illustrative embodiment, includes the magnetic pipette tip and a pipetter.
  • the pipetter has a pipetter housing, a shaft extending from the pipetter housing configured to receive the proximal end of the pipette tip housing, and a fluid aspirator within the housing.
  • the fluid aspirator is configured to aspirate a fluid into the lumen of the pipette tip housing after the magnetic pipette tip is received by the shaft.
  • the invention is directed to a magnetic pipette tip assay system that includes the magnetic pipette tip and a plurality of magnetic beads.
  • Each of the magnetic beads has a magnetic core and a coating.
  • the coating is configured to reversibly bind a biological molecule to the magnetic beads.
  • Each of the magnetic beads may pass through the distal end of the pipette tip housing, into the lumen, and be located proximate the first magnet.
  • the invention is directed to a method of isolating a desired biological molecule.
  • the method includes introducing the plurality of magnetic beads into a sample that contains the desired biological molecule.
  • the desired biological molecule reversibly binds to a binding portion of the plurality of magnetic beads within the sample.
  • the sample with the desired biological molecule reversibly bound to the plurality of the magnetic beads is aspirated into a magnetic pipette tip having a pipette tip housing, a lumen extending through the pipette tip housing, and a magnet located within the lumen.
  • the biological molecule is released from the plurality of magnetic beads and expelled from the magnetic pipette tip while the magnet and the plurality of magnetic beads remain within the lumen.
  • a magnetic pipette tip that includes a pipette tip housing having proximal and distal ends. A lumen within the pipette tip housing extends between the proximal and distal ends. A magnetic portion is supported by the pipette tip housing and is located between the proximal and distal ends.
  • FIG. 1 is a flowchart of an exemplary method of isolating a desired biological molecule in accordance with one embodiment of the present invention.
  • FIGS. 2A-2C are diagrammatic views illustrating the process of the exemplary method of FIG. 1 .
  • FIG. 3 is a side elevational view illustrating a magnetic pipette tip in accordance with one embodiment of the present invention.
  • FIG. 3A is an enlarged elevational view of the magnetic pipette tip of FIG. 3.
  • FIG. 4 is a side elevational view illustrating a magnetic pipette tip in accordance with another embodiment of the present invention.
  • FIG. 4A is an enlarged elevational view of the magnetic pipette tip of FIG. 4.
  • FIG. 5 is a side elevational view illustrating a magnetic pipette tip in accordance with another embodiment of the present invention.
  • FIG. 5A is an enlarged elevational view of the magnetic pipette tip of FIG. 5.
  • FIG. 6 is a side elevational view illustrating a magnetic pipette tip in accordance with another embodiment of the present invention.
  • FIG. 6A is an enlarged elevational view of the magnetic pipette tip of FIG. 6.
  • FIG. 7 is a side elevational view illustrating a magnetic pipette tip in accordance with another embodiment of the present invention and one exemplary method of using the same.
  • FIGS. 8A-8D are side elevational views illustrating additional embodiments of magnetic pipette tips.
  • FIGS. 8E-8G are side elevational views illustrating magnets having adaptors in accordance with embodiments of the present invention.
  • FIG. 9 illustrates one embodiment of a conventional pipetter.
  • FIGS. 10A, 10B, and 1 0D are photographs of electrophoretic gels exemplifying the use of a magnetic pipette tip in accordance with one embodiment of the present invention.
  • FIG. 10C is a graph illustrating PCR sequencing data of a base pair sequence purified by using a magnetic pipette tip in accordance with one
  • FIG. 1 is a flowchart 1 0 illustrating one method of isolating and purifying a desired biological molecule ("biomolecule"), which is described in further detail with reference to FIGS. 2A-2C.
  • a cell culture 12 is raised or cultured according to a known manner to achieve expression of the biomolecule 14. It is understood that the methods and apparatii described herein may also be employed to isolate biomolecules from samples derived from non-cultured cells, such as tissue homogenates, biological fluids, environmental samples, whole organisms, PCR reactions, and so forth.
  • the biomolecule 14 may include DNA, RNA, genomic DNA, proteins, or other biological molecules of interest such as steroids, growth factors, hormones, cytokines, chemokines, amino acids, fatty acids, carbohydrates, biomarkers, and so forth.
  • the cells comprising the cell culture 12 are lysed with a lysis solution 18 (block 16).
  • This solution in the case of an alkaline lysis, may contain a strong base (such as sodium hydroxide, "NaOH”) in addition to a detergent (such as sodium dodecyl sulfate, "SDS”) and usually has a pH that is greater than about 8.
  • sample is then placed into a suitable labware 20, such as sample wells, reservoirs, tubes, vials, vessels, etc. While the labware 20 is illustrated herein as being the same container throughout the method, it would be understood that multiple types of labwares or containers may be used with this method.
  • a plurality of magnetic beads 24 then may be introduced into the sample (block 22).
  • the sample may be centrifuged and the resultant precipitate (not shown) containing the biomolecule 14 may be washed and re-suspended in another buffer prior to introducing the plurality of magnetic beads 24.
  • the magnetic beads 24 may comprise less than about 10% of the total bead solution, and more specifically may range from about 5% to about 1 0%; however, other concentrations may be used if necessary or desired by a particular isolation assay.
  • the magnetic beads 24, also known as magnetic particles, may be constructed in a known manner, which generally includes a magnetic or
  • the paramagnetic cores may be comprised of any suitably magnetic material, for example, iron oxide, that is capable of being magnetically attracted to a magnetic field yet is chemically stable with the desired coating.
  • the coating may include one or more materials having a particular chemistry to confer a generalized or specific affinity for the biomolecule 14. Exemplary coatings may include carboxyl, silica, proteins, metals, peptides, and oligonucleotides. Some examples of commercially-available magnetic beads may include those that are manufactured by Thermo Fisher Scientific (Fremont, CA), such as magnetic beads available under the SERA-MAG tradename.
  • a suitable diameter may range from about 10 nm to about 100 ⁇ .
  • the three-dimensional structure of the magnetic beads 24 may also vary, including shapes (such as cylinders, spheres, or cones) and irregular shapes. Any size or shape of magnetic bead may be used that has an outer dimension that is sufficiently small to be used with one or more magnetic pipette tips as described in detail below.
  • the biomolecule 14 includes a surface charge that is dependent on several factors: the particular functional groups comprising the molecular structure of the biomolecule 14; the tertiary folding of the biomolecule 14, which positions certain functional groups at the external surface; and the matrix (i.e., the solvent, lysis solution, etc.) containing the biomolecule 14.
  • the pH of the aqueous matrix may affect the protonation of some functional groups and thereby alter the surface charge.
  • the coating for the magnetic beads 24 may be selected to possess a surface charge that opposes the surface charge of the biomolecule 14 and thereby electrostatically interacts with the biomolecule 14 and form the weak reversible bond therewith. This interrelationship also can be modulated through use of different buffers, with variations in pH, ionic strength, detergents, and so on.
  • the biomolecules 14 reversibly bind to the coating of the magnetic beads 24 and form a "biomolecule-magnetic bead complex.”
  • the sample then may be fully aspirated into a magnetic pipette tip 32 (block 30), a first embodiment of which is shown in greater detail in FIGS. 3 and 3A.
  • the magnetic pipette tip 32 includes an elongated housing 34 that may be constructed from a molded, inert material, generally a plastic material, such as polypropylene, or a combination of suitable materials.
  • the housing 34 has a distal end 36 that is tapered, a proximal end 38 that is enlarged to form a hub configured to be received by a shaft 92 (FIG. 9) of a pipetter 86 (FIG. 9), and a lumen 40 extending therebetween.
  • the distal end 36 of the illustrated embodiment of the magnetic pipette tip 32 is molded to include a sharp taper for retaining a magnet 42 that is located within the lumen 40, as described in greater detail below.
  • the sharp taper of the distal end 36 may include an abrupt change in the diameter, i.e., from a gentle decreasing diameter to a narrow diameter fluid port 44 that extends distally away from the distal end 36.
  • the narrow diameter fluid port 44 is configured to provide fluid movement into and out of the lumen 40 of the magnetic pipette tip 32.
  • the magnet 42 within the lumen 40 may be constructed from any inert magnetic or paramagnetic material that may be structured to reside within the lumen 40 of the magnetic pipette tip 32.
  • the magnet 42 should have dimensions that are suitable for residing within the lumen 40 while maximizing the surface area of the magnet 42 for capturing a majority of the magnetic beads 24 from the sample.
  • Some suitable diameters may range from about 1/16 th inch (1 .58 mm) in diameter and about 1 /16 th inch (1 .58 mm) in thickness, and may vary in three- dimensional structure, including, for example, cylinders, spheres, cones, rings, or other shape/structure as appropriate.
  • One suitable magnet is a neodymium cylinder magnet from K&J Magnetics, Inc. of Jamison, PA.
  • the magnetic field strength generated by the magnet 42 must be sufficiently large to magnetically attract and retain the magnetic beads 24; for example, the 6619 Gauss surface magnetic field strength of the commercially-available neodymium cylinder magnet may be considered to be suitable but not limiting.
  • the sample When the sample is aspirated into the pipette tip 32 (block 30), the sample passes through the fluid port 44, past the sharp taper of the distal end 36, and into the lumen 40 of the housing 34. Once within the lumen 40, the magnetic beads 24 are positioned within the magnetic field of the magnet 42 and thus magnetically attracted and bound to the magnet 42. Because the biomolecule 14 is reversibly bound to the coating of the magnetic beads 24, the magnetic attraction of the magnetic bead 24 to the magnet 42 effectively immobilizes and retains the biomolecule 14 within the magnetic pipette tip 32.
  • the lysis solution 1 8 may be expelled, with contaminants, out of the magnetic pipette tip 32 and into the labware 20 (FIG. 2A) (block 46).
  • the biomolecule-magnetic bead complex, the magnet 42, and lumen 40 may be washed (block 48). Washing of the magnetic beads 24 with the bound biomolecule 14 may include aspirating and dispensing a wash buffer 50, one or more times, between the magnetic pipette tip 32 and the labware 20, to further remove contaminants or lysis solution 18 (FIG. 2A) from the magnetic pipette tip 32.
  • the use of the wash buffer 50 may be optionally excluded or repeated one or more times, as necessary or desired, and in accordance with the particular isolation procedure. Once the washing is complete, the wash buffer 50 is expelled from the magnetic pipette tip 32 and disposed in an appropriate manner.
  • wash buffers may be used in the repeated washings.
  • a suitable wash buffer may be a 70% ethanol solution (pH of about 7.4).
  • a 5 M sodium chloride (“NaCI”) solution followed by a solution comprised of 25 mM Tris Acetate (pH of about 7.8), 1 00 mM potassium acetate (“KOAc”), 10 mM magnesium acetate (“Mg 2 OAc”), and 1 mM dithiothreitol (“DTT”) may be used.
  • the biomolecule 14 may be eluted from the magnetic pipette tip 32 into a clean buffer for further analysis or processing.
  • an elution buffer 56 is aspirated into the magnetic pipette tip 32 to release the biomolecule 14 (block 54).
  • the elution buffer 56 differs from the previous lysis solution 1 8 and wash buffer 50 in at least one chemical property that is configured to sufficiently disrupt the reversible bond between the biomolecule 14 and the coating of the magnetic bead 24.
  • One exemplary method of disrupting the reversible bond is to use an elution buffer 56 having a pH that differs from the pH of the previous buffers 18, 50.
  • Altering the pH alters the concentration of protons in the solution and may resultantly affect the degree of protonation of some functional groups (i.e., acidic buffers will protonate anionic functional groups while alkaline buffers remove protons from cationic functional groups with a by-product of water).
  • Affecting the protonation of the functional groups of at least one of the biomolecule 14 or the magnetic bead coating may alter the static surface charge of the biomolecule 14 or the coating, respectively.
  • elution buffers 56 are known, and may alternatively include, for example, varying the salt concentration and/or including detergents.
  • the elution buffer 56 does not affect the magnetic attraction between the magnet 42 and the magnetic beads 24; therefore, the magnetic beads 24 remain within the lumen 40 and magnetically attracted to the magnet 42 even after expelling the elution buffer 56 from the magnetic pipette tip 32.
  • the released biomolecule 14 is free to be expelled with the elution buffer 56 from the magnetic pipette tip 32 and into the labware 20.
  • the magnet 42 and the magnetic beads 24 remain within the lumen 40 of the magnetic pipette tip 32.
  • the isolated biomolecule 14 then may be studied in accordance with an assay or other biotechnique that is known to those of ordinary skill in the art.
  • FIGS. 4-7 illustrate other magnetic pipette tips in accordance with other embodiments of the present invention.
  • One such magnetic pipette tip 60 is shown in FIG. 4A.
  • the magnetic pipette tip 60 includes a molded housing 62 having a gentle taper from a proximal end hub 66 to a distal tip 64 and a lumen 70 therebetween.
  • the magnetic pipette tip 60 further includes a porous member 72 within the lumen 70 and proximate the distal tip 64.
  • the porous member 72 spans a cross-sectional dimension of the lumen 70, which as shown may have a diameter ranging from about 3 mm to about 5 mm, in order to retain a magnet 74 within the lumen 70 of the magnetic pipette tip 60 while permitting passage of the magnetic beads 24. While the magnet 74 of the particular embodiment is shown to include a spherical shape, it would be understood that the cubic magnet 42 of FIG. 3 or another shape may alternatively be used.
  • the porous member 72 may be a porous frit, constructed from polyethylene or ceramic materials with a porosity ranging from about 10 nm to about 100 ⁇ , or larger, as necessary to permit passage of the magnetic beads 24.
  • Suitable porous members 72 may include, for example, those that are commercially- available from Porex Technologies (Fairburn, GA). Other frits may include those that are described in detail in U.S. Patent No. 7,482,1 69, entitled “LOW DEAD VOLUME EXTRACTION COLUMN DEVICE,” issued to Gjerde et al. on January 27, 2009, and U.S. Patent No. 6,566,145, entitled “DISPOSABLE PIPETTE EXTRACTION,” issued to Brewer on May 20, 2003, the disclosures of both incorporated herein by reference, in their entireties. Briefly, these porous members 72 include sintered glass plugs, glass wool plugs, porous polymer plugs, or metal screens. Alternatively, the porous member may be a membrane or a filter, such as those that are constructed from nylon, polyester, polyamide, polycarbonate, cellulose, polyethylene, nitrocellulose, cellulose acetate, or polypropylene.
  • the porous member 72 may include a functionalized structure or coating that is similar to those described in PCT
  • the functionalized structures and coatings as disclosed by Diffinity Genomics include a target rejection chemistry, i.e., having a hydrophobicity, charge, and/or affinity that is specialized to absorb undesired molecules from the lysis solution 1 8 (FIG. 2A) and not the biomolecules 14 (FIG. 2A).
  • the functionalized coatings taught by Diffinity Genomics may be included as a coating on at least a portion of an inner surface of the lumen 70 of the magnetic pipette tip 80.
  • the magnetic pipette tip 60 functions in a manner that is similar to the magnetic pipette tip 32 of FIG. 3.
  • the porosity of the porous member 72 is selected such that the lysis solution 1 8 (FIG. 2) and the biomolecule-magnetic bead complex may traverse the porous member 72 while the magnet 74 does not.
  • the biomolecule-magnetic bead complex enters the lumen 70 of the magnetic pipette tip 60 and is attracted and retained by the magnetic field.
  • the process of releasing the biomolecule 14 from the magnetic beads 24 and expelling the biomolecule 14 then may proceed in a manner that is similar to the method described in detail above.
  • FIG. 4 further illustrates an optional barrier member 75 that is positioned distal to the proximal end hub 66 for the purpose of retaining the magnet 74 within the lumen 70 in the event that the magnetic pipette tip 60 is inverted.
  • the barrier member 75 may be constructed in a manner that is similar to any porous member described herein or any membrane, plug, frit, or other structure that permits a displacement of air, and thus functioning of, the magnetic pipette tip 60.
  • the position of the barrier member 75 may be just distal to a "nose cone" 77 of the magnetic pipette tip 60, which is the proximal portion of the tip 60 that is tapered to couple to the shaft 92 (FIG. 9) of the pipetter 86 (FIG. 9) by frictional fit.
  • the use of the barrier member 75 is not necessary and should not be considered to be limiting.
  • FIGS. 5 and 5A illustrate a magnetic pipette tip 80 in accordance with another embodiment of the present invention.
  • the magnetic pipette tip 80 is constructed substantially similar to the magnetic pipette tip 60 of FIG. 4; however, a porous member 81 replaces the barrier member 75 and is spaced farther from the distal end 64 as compared with the barrier member 75.
  • the magnetic pipette tip 80 further includes a plurality of magnets 74n (shown with three magnets 74a, 74b, 74c) located within the lumen 70. Inclusion of the plurality of magnets 74n within the lumen 70 increases the magnetic field strength and/or the volume covered by the magnetic field, which results in the capture and retention of more magnetic beads 24.
  • the plurality of magnets 74n increases the surface area of coating material available for binding the biomolecule 14 (FIG. 2A) to the magnetic beads 24. In any event, a larger percentage of biomolecule-magnetic bead complexes may be retained by the magnetic pipette tip 80 and larger concentrations of biomolecule 14 (FIG. 2A) isolated.
  • FIGS. 6 and 6A illustrate a magnetic pipette tip 82 in accordance with yet another embodiment of the present invention.
  • the magnetic pipette tip 82 is generally constructed in a manner that is similar to the magnetic pipette tips 60, 80 of FIGS. 4 and 5, respectively.
  • the magnetic pipette tip 82 of FIG. 6 includes a plurality of porous members 84n (shown with three porous members 84a, 84b, 84c) separating each of the plurality of magnets 74a, 74b, 74c.
  • this embodiment of the magnetic pipette tip 82 with the plurality of magnets 74n allows for the capture and isolation of larger amounts, or concentrations, of the biomolecules 14 (FIG. 2A).
  • the plurality of porous members 84n may be constructed with varying degrees of porosity. As a result, various diameters of magnetic beads could be used, each having a separate coating for different biologies, for isolating more than one biologic material of interest, thereby allowing multiplex assay formats.
  • FIG. 7 a magnetic pipette tip 100 in accordance with still another embodiment of the present invention is shown with greater detail.
  • the magnetic pipette tip 100 may be constructed in a manner that is similar to the magnetic pipette tip 32 of FIG.
  • a cylindrical magnet 102 having a cylindrical lumen 104 extending lengthwise therethrough is located within the lumen 40 and resides on a surface created by the abrupt change between the distal end 36 and the fluid port 44.
  • the magnetic poles of the cylindrical magnet 102 may correspond with the top and bottom surfaces of the cylindrical magnet 102. While the specific embodiment of the cylindrical magnet 1 02 shown in FIG. 7 has a conical shape that generally corresponds with the taper of the distal end 36, this is not necessary. Indeed, the cylindrical magnet 1 02 needs only have a diameter that is sufficiently small to allow the cylindrical magnet 102 to reside within the lumen 40.
  • the cylindrical lumen 1 04 has a diameter that is similar to the diameter of the fluid port 44; however, this dimension is not necessary.
  • the diameter of the cylindrical lumen 1 04 should meet or exceed the diameter of the fluid port 44 so as to not hinder fluid motion into the lumen 40 when the sample is aspirated.
  • the lumen 104 may have a diameter of about 1/16 th inch (about 1 .59 mm).
  • the magnetic beads 24 are introduced into the lysis solution 1 8.
  • the lysis solution 18 may be incubated to permit binding of the biomolecules 14 to the magnetic beads 24.
  • the lysis solution 1 8 then may be aspirated from the labware 20 into the magnetic pipette tip 1 00, where the lysis solution 18 passes through the fluid port 44, the cylindrical lumen 104, and into the lumen 40 of the magnetic pipette tip 100.
  • the biomolecule-magnetic bead complexes are retained at the cylindrical magnet 102 while the lysis solution 18 may be dispensed back into the labware 20. Release of the biomolecules 14 from the magnetic beads 24 then may proceed as described previously.
  • FIGS. 8A-8D illustrate additional embodiments of magnetic pipette tips in accordance with the present invention.
  • the magnetic pipette tips 1 10, 1 12, 1 14, 1 15 of FIGS. 8A, 8B, 8C, and 8D respectively may be constructed in a manner that is similar to the magnetic pipette tip 60 of FIG. 4; however, in FIG. 8A the magnetic pipette tip 1 10 includes a ring-shaped magnet 1 16 that is molded into the housing 62, and the magnetic pipette tip 1 1 2 of FIG. 8B includes a plurality of rectangular magnetic strips 1 18 that is molded into the housing 62.
  • a porous member 72 FIG.
  • the thickness of the molded housing 62 at the position of the magnets 1 1 6, 1 18 may be optimized to reduce interference of the magnetic field strength experienced by the magnetic beads 24 within the lumen 70 of the housing 62.
  • the magnetic pipette tip of FIG. 8C includes a magnetic coating 120 applied to an inner wall surface 1 19 of the housing 62 and within the lumen 70.
  • the magnetic coating 120 may be unitary, as shown, or may be partitioned within the lumen 70 and having any shape, including both regular and irregular shapes. Again, the coating 120 may be located at any position along the length of the housing 62.
  • the coating 1 20 need not be limited to the inner wall surface 1 19 of the housing 62 but may be a material that is included within the moldable material during the molding process or may be applied to an outer wall surface 1 17 of the housing 62 after the molding process.
  • the thickness of the applied coating 120 may vary and depends on a thickness necessary to provide sufficient magnetic field strength to capture the magnetic beads 24 once the magnetic beads 24 are within the lumen 70 of the magnetic pipette tip 1 14.
  • a small magnet may be inserted the lumen into a non-magnetic, conventional pipette tip.
  • a width dimension of the magnet (which may be, for example, a diameter of approximately 2 mm) is less than a cross-sectional dimension of the lumen, then the magnet may move freely within the lumen.
  • the magnet may be constructed with an outer surface matching a shape of an inner wall of the lumen of the magnetic pipette tip (for example, as described with reference to FIG. 7), the manufacture of particularly- shaped magnets increases manufacturing costs. Therefore, it may be beneficial to support a standard magnet within the lumen of the magnetic pipette tip so as to reduce tumbling of the magnetic and maintain the desired orientation of the magnet.
  • the magnetic pipette tip 1 15 includes a magnet 1 22 (shown as cylindrical in shape and having a lumen 123 extending therethrough) supported within the lumen 70 of the magnetic pipette tip 1 15.
  • the magnet 122 is supported by an adaptor 124, which may be constructed from any non-magnetic, semi-compliant, and moldable material, including, for example, generally plastics and specifically polypropylene, polystyrene, and polyethylene. Because the inner surface 126 lumen 70 of the magnetic pipette tip 1 15 is generally circular in cross-section, the adaptor 124 may be molded to include a generally cylindrical outer surface 1 28.
  • the outer surface 128 may have a dimension (“D1 ”) that approximates the cross-sectional dimension (“D2") of the lumen 70.
  • an adaptor 130 may be molded to correspond to the particular shape of the lumen 70.
  • the magnetic pipette tip 1 15 may taper from the proximal end hub 66 to the distal tip 64 such that the inner surface 126 of the lumen forms an angle, a, with a vertical plane 132. Therefore, an outer surface 134 of the adaptor 130 may be similarly angled, ⁇ , with the vertical plane 132, wherein ⁇ is substantially similar to a.
  • the adaptor 130 may better conform to the lumen 70 of the magnetic pipette tip 1 15 while maintaining a friction fit with the same.
  • each adaptor 124, 130 may further include an inner lumen 136, 138 sized and shaped to receive the magnet 122.
  • the inner lumen 136, 138 is generally cylindrical to match the generally cylindrical outer surface of the magnet 1 22; however, the shape is not so limited.
  • a cubic-shaped magnet 140 having a lumen 141 extending therethrough, is positioned within a square lumen 142 of an adaptor 144.
  • the square lumen 142 is molded to have a size and shape similar to the cubic-shaped magnet 140 while an outer surface 146 of the adaptor 144 retains a cylindrical shaped similar to the adaptor 1 24 of FIG. 8E.
  • the magnet 122 may be placed within the adaptor 124 and retained by friction fit or an adhesive.
  • the adaptor 1 24, with the magnet 1 22 positioned therein, may then be inserted into the lumen 70 of the magnetic pipette tip 1 15 to a desired, final position, and retained by friction fit or adhesive.
  • the lysis solution 1 8 (FIG. 2A), wash buffer 50 (FIG. 2B), and elution buffer 56 (FIG. 2C) may be aspirated into and expelled from the magnetic pipette tip 1 15 via the lumen 123 extending through the magnet 121 .
  • FIG. 9 illustrates one embodiment of a conventional pipetter 86 suitable for use with a magnetic pipette tip in accordance with any one embodiment of the present invention; however, the present invention should not be limited to use with the particular pipetter 86 shown.
  • the pipetter 86 is described in detail in U.S. Patent No. 7,690,274, entitled “PIPETTE WITH A TIP REMOVING MECHANISM,” issued to Thermo Fisher Scientific (Vantaa, Finland) on April 6, 2010, the disclosure of which is incorporated herein by reference in its entirety.
  • the pipetter 86 includes a housing 88 having a finger rest 90 and a shaft 92 extending from the housing 88.
  • An activator shown here as a plunger 94, is operably associated with the aspirating mechanism (not shown) located within the housing 88.
  • the particular embodiment also includes a tip removal mechanism 96. Further details of the aspirating mechanism and the tip removal mechanism 96 are provided in the incorporated disclosure.
  • FIGS. 10A-10D illustrate results of the use of the invention for particular applications.
  • Example 1 illustrated in FIG. 10A, demonstrates the results of a plasmid DNA purification using a magnetic pipette tip was constructed in a manner that is similar to the magnetic pipette tip 60 of FIG. 4.
  • E. coli XL10 Gold (Strategene, San Diego, CA)
  • pBluescript (pBSK) (Strategene, San Diego, CA) plasmid transformants were cultured overnight in a suspension culture under antibiotic selection. The cells were divided into 2 ml_ aliquots and pelleted by centrifugation at 13,000 rpm for 10 minutes. All cell pellets were stored at 20 °C until use. The cells were lysed using alkaline lysis conditions.
  • this method involves resuspension of the cells in a solution comprised of 50 mM glucose, 25 mM Tris-HCI (pH of about 8), 10 mM ethylenediaminetetraacetic acid ("EDTA”), and 1 00 ⁇ g/mL RNAse. This was followed by the actual lysis of the cells in a solution of 0.2 N NaOH with 1 % SDS. Finally, the entire lysis is neutralized by the addition of 3 M KOAc. Precipitated cellular proteins were pelleted by centrifugation, and clarified lysis solution samples containing plasmid DNA were processed.
  • the plasmid DNA isolated using embodiments of the invention was analyzed using a 0.8% agarose gel prepared with Tris-acetate-EDTA ("TAE”) buffer and 0.5 ⁇ g/mL ethidium bromide. After loading the samples, the gel was run at 100 V for about 1 hour. The results were visualized using a UV light box and are displayed in FIG. 10A.
  • TAE Tris-acetate-EDTA
  • Lane “L” shows a DNA marker, Fisher BioReagents exACTGeneTM DNA Ladder (Thermo Fisher Scientific, Fair Lawn, NJ). Lanes “1 " and “2” show plasmid DNA that was eluted after processing and using 1 ⁇ functionalized magnetic particles. Lanes “3” and “4" show plasmid DNA that was eluted after processing and using 3 ⁇ functionalized magnetic particles; and Lanes “5" and “6” show plasmid DNA that was eluted after processing and using 5 ⁇ functionalized magnetic particles. All samples were eluted in Tris-EDTA ("TE”) buffer by aspiration/dispensing for five cycles.
  • TE Tris-EDTA
  • Example 2 illustrated in FIG. 10B, compares the results of a plasmid DNA purification using a conventional spin column protocol with the use of a magnetic pipette tip constructed in a manner that is similar to the magnetic pipette tip 60 of FIG. 4.
  • E. coli XL10 Gold (Strategene, San Diego, CA)
  • pBluescript (pBSK) (Strategene, San Diego, CA) plasmid transformants were cultured overnight in a suspension culture under antibiotic selection. The cells were divided into 2 mL aliquots and pelleted by centrifugation at 13,000 rpm for 10 minutes. All cell pellets were stored at -20 °C until use. The cells were thawed to room temperature and lysed using alkaline lysis conditions.
  • this method involves resuspension of the cells in a solution comprised of 50 mM glucose, 25 mM Tris-HCI (pH of about 8), 10 mM EDTA, and 100 ⁇ g/mL RNAse. This was followed by the actual lysis of the cells in a solution of 0.2 N NaOH with 1 % SDS. Finally, the entire lysis is neutralized by the addition of 3 M KOAc. Precipitated cellular proteins were pelleted by centrifugation, and clarified lysis solution samples containing plasmid DNA were processed.
  • the plasmid DNA isolated using embodiments of the invention was analyzed using a 0.8% agarose gel prepared with TAE buffer and 0.5 ⁇ g/mL ethidium bromide. After loading the samples, the gel was run at 100 V for about
  • Lane “L” shows a DNA marker, Fisher BioReagents exACTGeneTM DNA Ladder (Thermo Fisher Scientific, Fair Lawn, NJ).
  • Lane “C” shows plasmid DNA that was isolated using a conventional spin column purification protocol.
  • Lanes “1 " and “2” show plasmid DNA that was purified using a ring-shaped magnet (4 mm x
  • Lanes "3" and “4" show plasmid DNA that was purified using a ring-shaped magnet (4 mm x 2 mm x 1 mm) that was magnetized across the diameter of the magnet.
  • Lanes "5" and “6” show plasmid DNA that was purified using a ring-shaped magnet (2 mm x 1 mm x 1 mm) that was magnetized across the diameter of the magnet.
  • Lanes "7” and “8” show plasmid DNA that was isolated using a ring-shaped magnet (3 mm x 1 mm x 1 mm) that was magnetized across the diameter of the magnet.
  • Example 3 illustrated in FIG. 10C, demonstrates the results of a polymerase chain reaction ("PCR") product purification using a magnetic pipette tip that was constructed in a manner that is similar to the magnetic pipette tip 60 of FIG. 4.
  • PCR polymerase chain reaction
  • a 526-base pair sequence was amplified using pBluescript (pBSK) (Stratagene, San Diego, CA) plasmid as a template, purified using the
  • the PCR reaction mixture included 1 ⁇ _ of both forward and reverse primers (25 ⁇ ), 1 ⁇ _ of pBSK template (1 0 ng/mL), 5 mL of 10X PCR buffer, 1 ⁇ of MgCI 2 (100 mM), 1 ⁇ of dNTP mixture (10 mM), 0.25 ⁇ Taq Polymerase (5 ⁇ / ⁇ ), and 39.75 ⁇ of HPLC water.
  • the 50 ⁇ samples were placed in a thermal cycler and cycled for 30 repetitions (95 °C for 30 seconds followed by 52 °C for 30 seconds and finally 72 °C for 30 seconds).
  • Example 4 illustrated in FIG. 10D, demonstrates the results of
  • Glutathione S-Transferase GST
  • GST Glutathione S-Transferase
  • E. coli BL21 (DE3) (Novagen, Darmstadt, Germany) plasmid
  • transformants expressing the recombinant GST-tagged Green Fluorescent Protein were cultured and induced in a suspension culture under antibiotic selection. The cells were divided into 2 ml_ aliquots and pelleted by centrifugation at
  • the proteins isolated by the magnetic pipette tip were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis ("SDS PAGE") gel. A 15 ⁇ _ aliquot of each sample was mixed with 5 ⁇ _ reducing sample buffer and heated at 95 °C for 5 minutes. Samples then were loaded onto an 8-20%
  • polyacrylamide gel and run at 100 V for approximately 1 hour. The gel then was stained using a Coomassie Blue based stain and photographed on a light box.
  • Lane “L” shows a protein ladder (Fisher BioReagents EZ-RunTM

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Abstract

La présente invention a trait à une pointe de pipette magnétique (32) qui inclut un logement de pointe de pipette (34) qui est doté d'extrémités proximale et distale (38, 36) et d'un lumen (40) qui s'étend entre les extrémités proximale et distale (38, 36). Au moins un aimant (42) est placé à l'intérieur du lumen (40) du logement de pointe de pipette (34) entre les extrémités proximale et distale (38, 36). La pointe de pipette magnétique (32) est configurée de manière à être utilisée dans l'isolation magnétique de molécules biologiques.
PCT/US2012/022545 2011-01-26 2012-01-25 Pointe de pipette magnétique WO2012103214A2 (fr)

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US8900533B2 (en) 2012-06-29 2014-12-02 Molecular Bioproducts, Inc. Welded blister pack for tips
WO2015018937A1 (fr) * 2013-08-09 2015-02-12 Novacyt Procédé et dispositif de lavage d'un dispositif de pipetage-distribution
WO2015048663A1 (fr) * 2013-09-27 2015-04-02 The Johns Hopkins University Extraction en phase solide de peptides, glycopeptides et glycanes globaux en utilisant l'immobilisation chimique dans une pointe de pipette
CN104911103A (zh) * 2015-05-25 2015-09-16 广东省人民医院 一种用于悬浮细胞快速换液的移液枪头
WO2018026886A1 (fr) * 2016-08-02 2018-02-08 DPX Technologies, LLC Précipitation de protéines et/ou extraction en phase solide dispersive automatisées au moyen d'embouts filtrants
CN117000324A (zh) * 2023-06-30 2023-11-07 上海金鑫生物科技有限公司 一种移液元件

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US7690274B2 (en) 2003-11-19 2010-04-06 Thermo Fisher Scientific Oy Pipette with a tip removing mechanism
WO2010093998A2 (fr) 2009-02-14 2010-08-19 Diffinity Genomics, Inc. Système et procédés destinés à purifier des substances biologiques

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8900533B2 (en) 2012-06-29 2014-12-02 Molecular Bioproducts, Inc. Welded blister pack for tips
WO2015018937A1 (fr) * 2013-08-09 2015-02-12 Novacyt Procédé et dispositif de lavage d'un dispositif de pipetage-distribution
FR3009622A1 (fr) * 2013-08-09 2015-02-13 Novacyt Procede et dispositif de lavage d'un dispositif de pipetage-distribution
CN105518466A (zh) * 2013-08-09 2016-04-20 诺维茨公司 用于清洗吸取-分配设备的方法与设备
US20160195565A1 (en) * 2013-08-09 2016-07-07 Novacyt Method and device for washing a pipetting-dispensing device
WO2015048663A1 (fr) * 2013-09-27 2015-04-02 The Johns Hopkins University Extraction en phase solide de peptides, glycopeptides et glycanes globaux en utilisant l'immobilisation chimique dans une pointe de pipette
CN104911103A (zh) * 2015-05-25 2015-09-16 广东省人民医院 一种用于悬浮细胞快速换液的移液枪头
WO2018026886A1 (fr) * 2016-08-02 2018-02-08 DPX Technologies, LLC Précipitation de protéines et/ou extraction en phase solide dispersive automatisées au moyen d'embouts filtrants
US11193930B2 (en) 2016-08-02 2021-12-07 DPX Technologies, LLC Automated protein precipitation and/or dispersive solid phase extraction using filter tips
US11567067B2 (en) 2016-08-02 2023-01-31 Dpx Technologies, Inc. Automated solid phase extraction using filter tips
CN117000324A (zh) * 2023-06-30 2023-11-07 上海金鑫生物科技有限公司 一种移液元件
CN117000324B (zh) * 2023-06-30 2024-08-13 上海金鑫生物科技有限公司 一种移液元件

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