WO2020256695A1 - Procédé et appareil employant des perles magnétiques pour des analyses de liaison par ligands d'échantillons biologiques - Google Patents

Procédé et appareil employant des perles magnétiques pour des analyses de liaison par ligands d'échantillons biologiques Download PDF

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Publication number
WO2020256695A1
WO2020256695A1 PCT/US2019/037628 US2019037628W WO2020256695A1 WO 2020256695 A1 WO2020256695 A1 WO 2020256695A1 US 2019037628 W US2019037628 W US 2019037628W WO 2020256695 A1 WO2020256695 A1 WO 2020256695A1
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Prior art keywords
sample
beads
ligand binding
magnetic beads
binding assay
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Application number
PCT/US2019/037628
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English (en)
Inventor
Marvin L. Vestal
Original Assignee
Virgin Instruments Corporation
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.)
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Publication date
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Priority to PCT/US2019/037628 priority Critical patent/WO2020256695A1/fr
Publication of WO2020256695A1 publication Critical patent/WO2020256695A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry

Definitions

  • the present application is related to U.S. Patent Application No. 15/861,265, entitled“Ligand Binding Assays Using MALDI-TOF Mass Spectrometry” filed January 3, 2018, which claims priority to U.S. Provisional Patent Application Serial No. 62/442,512, entitled “Ligand Binding Assays Using MALDI-TOF Mass Spectrometry” filed on January 5, 2017.
  • the present application is also related to U.S. Patent Application No. 15/079,900, entitled“MALDI- TOF MS Method And Apparatus For Assaying An Analyte In A Bodily Fluid From A Subject”, which claims priority to U.S. Provisional Patent Application Serial No.
  • ELISA Immunosorbent Assays
  • Ligand binding assays have been used to measure the interactions that occur between two molecules, such as protein-bindings, as well as the degree of affinity for which the reactants bind together. More specifically, ligand binding assays are used to test for the presence of target molecules in a sample that is known to bind to the receptor. Various detection methods have been used to determine the presence and extent of the ligand-receptor complexes formed. For example, known methods include electrochemical detection through various fluorescence detection methods.
  • FIG. 1 illustrates a workflow diagram for an embodiment of an apparatus and method for ligand binding assays according to the present teaching.
  • FIG. 2A illustrates a front cut-away view of an embodiment of a magnetic bead picker of the present teaching.
  • FIG. 2B illustrates a side-view of the embodiment of the magnetic bead picker of
  • FIG. 2A is a diagrammatic representation of FIG. 2A.
  • FIG. 3 A illustrates an expanded view of a lower section of an embodiment of a well that is part of a plate preparation apparatus that uses a magnetic bead picker of the present teaching.
  • FIG. 3B illustrates a table that provides approximate bead capture volume dimensions for various embodiments of the magnetic bead picker according to the present teaching.
  • FIG. 4A illustrates an embodiment of part of a plate preparation apparatus of the present teaching at different points during processing.
  • FIG. 4B illustrates the steps of an embodiment of a method that is associated with the apparatus of FIG. 4 A.
  • FIG. 5 illustrates a schematic diagram of an embodiment of a bead
  • FIG. 6A illustrates an embodiment of part of a plate preparation apparatus of the present teaching at different points during processing from bead pick-up through preparation for washing and incubation.
  • FIG. 6B illustrates the steps of an embodiment of a method that is associated with the apparatus of FIG. 6 A.
  • FIG. 7A illustrates an embodiment of portion of a plate preparation apparatus of the present teaching at different points during processing for incubating and washing the mixture of beads plus sample.
  • FIG. 7B illustrates the steps of an embodiment a method associated with the apparatus of FIG. 7 A.
  • FIG. 8A illustrates an embodiment of portion of a plate preparation apparatus of the present teaching at different points during processing for picking up beads through applying a MALDI matrix.
  • FIG. 8B illustrates the steps of an embodiment of a method associated with the apparatus of FIG. 8 A.
  • FIG. 9A illustrates another embodiment of a portion of a plate preparation apparatus of the present teaching at different points during processing for picking up beads through applying a MALDI matrix.
  • FIG. 9B illustrates the steps of an embodiment of a method associated with the apparatus of FIG. 9 A.
  • FIG. 10 illustrates an embodiment of a high-volume automated MALDI plate preparation apparatus of the present teaching.
  • FIG. 1 1 illustrates an embodiment of a MALDI sample plate of the present teaching.
  • FIG. 12 illustrates an embodiment of a gasket of the present teaching.
  • FIG. 13 A illustrates a diagram of a well layout for a microwell plate of the present teaching that accommodates ⁇ 0.04-mm-diameter beads.
  • FIG. 13B shows a detailed view of one particular hexagon in the hexagonal array layout shown in FIG. 13 A.
  • FIG. 14 illustrates a table that presents the probability of missing one of n distinguishable beads in a collection of m for various values of m/n and n as applies to an embodiment of the system of the present teaching.
  • FIG. 15A illustrates a front- view of an embodiment of a MALDI sample plate that can accommodate a large number of beads of the present teaching.
  • FIG. 15B illustrates a side-view of a section of the MALDI sample plate of FIG.
  • FIG. 16A illustrates a top-view of an embodiment of a cassette comprising four
  • MALDI sample plates that can accommodate a large number of beads according to the present teaching.
  • FIG. 16B illustrates an end-view of the cassette comprising four MALDI sample plates of FIG. 16 A.
  • MS Mass spectrometry
  • MS has significantly advanced human protein biomarkers research.
  • MS has yet to make a significant inroad into clinical laboratories and find use its use in protein biomarker diagnostic applications.
  • the present teaching relates to simpler, faster, and cheaper sample preparation workflows that will facilitate clinical MS protein tests adoption.
  • One aspect of the present teaching is that the apparatus and methods of the present teaching can transform cancer research by enabling fast and cost-effective screening of protein biomarkers and clinically relevant proteoforms that may have significant implications in cancer diagnostics and therapy monitoring.
  • the solution described in connection with the present teaching includes bead- based immunoaffmity capture, with the straightforward MS detection offered by matrix assisted laser desorption ionization (MALDI) based analysis.
  • matrix assisted laser desorption ionization time-of-flight mass spectrometry MALDI-TOF MS
  • Some steps in this workflow are the transfer of beads to a MALDI sample plate and the release of the captured proteins and, in an efficient, non-dilutive, and sample-loss minimizing fashion that results in millimeter-size sample spots on the MALDI target.
  • the apparatus and methods of the present teaching can be used to quickly determine the concentration of biomarkers with concentrations below the current detection threshold of many state-of-the-art MALDI instruments.
  • concentration of the target marker(s) can be increased by conventional methods known in the art. Such methods include one or more of drying, evaporation, centrifugation, sedimentation, precipitation, differential mobility or retention, ion exchange and amplification.
  • a particularly powerful method of enrichment employs an appropriate antibody to capture a specific component of interest.
  • the antibody may be covalently bound to the bead.
  • An example of a targeted analyte that requires concentration is the biomarker and diagnostic substance known as troponin, which is commonly used for the diagnosis of various heart disorders.
  • Functional or healthy troponin occurs as a complex of three subunits that are distinguished by name as troponin C, troponin I, and troponin T.
  • the troponin complex is involved in the contraction of cardiac and skeletal muscle diseases. More specifically, measurement and quantification of troponin subtypes T and I in blood are used as indicators of damage to heart muscle. These measurements are diagnostic, and are used to differentiate between unstable angina and myocardial infarction (heart attack) in people with chest pain or acute coronary syndrome.
  • non-thrombotic cardiac conditions myocardial contusion, infiltrative myocardial diseases
  • non-thrombotic diagnoses sepsis, pulmonary embolism, stroke, renal failure
  • the current prognostic threshold for troponin T in blood is 0.01 ug/L or ⁇ 1 pmol /
  • the present teaching describes a system and method for ligand binding assay of biological samples.
  • the system uses a plurality of magnetic beads with bait molecules attached to each bead.
  • the beads are positioned within a sample well that is part of a sample plate.
  • a magnetic bead picker picks magnetic beads from a bead well in a predetermined volume. The magnetic bead picker then releases the magnetic beads into a sample well that contains samples of interest.
  • the samples may be obtained from bodily fluids that include, but are not restricted to, blood and blood products (serum, plasma, platelets), ascites fluid, breast milk, cerebrospinal fluid, lymph fluid, saliva, urine, gastric and digestive fluid, tears, stool, semen (and semen- derived fluids such as aspermic semen), prostatic fluid, vaginal fluid, amniotic fluid, and interstitial fluids derived from tissue.
  • the samples of interest and the magnetic beads are already positioned together within a well of a sample plate, and, as such, a magnetic bead picker is not used to move the predetermined volume of magnetic beads to a well that includes a sample.
  • the magnetic bead picker may pick up a predetermined volume of beads plus sample, and transfer these beads plus sample to another test plate for analysis, as required by the sample preparation process.
  • the magnetic bead picker is used to pick a predetermined volume of beads plus sample and place the beads plus sample into a well positioned on a sample plate that is suitable for washing and incubation.
  • washing and incubation binds the bait molecule to the sample molecule, and washes away any weakly bound molecules.
  • washing includes washing with a Tris pH 7.3 buffer solution.
  • the washed and incubated beads are then loaded using a magnetic bead picker onto a MALDI sample plate containing wells.
  • the wells are sized to allow only one bead per well.
  • multiple beads are placed in a single well.
  • a multiplexing analysis technique is employed to be able to provide ligand binding assay on multiple analytes of interest simultaneously or nearly simultaneously.
  • a MALDI matrix solution can be added to the loaded beads plus sample. Then, a matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer receives the loaded sample plate and performs time-of-flight mass spectrometry to generate mass spectra. A computer executes an algorithm using the mass spectra generated by the MALDI- TOF mass spectrometer to produce a ligand binding assay.
  • MALDI-TOF matrix assisted laser desorption ionization time-of-flight
  • FIG. 1 illustrates a workflow diagram 100 for an embodiment of an apparatus and method for producing ligand binding assays according to the present teaching.
  • a first step 102 of the method 100 one or more affinity capture media are attached to a plurality of beads.
  • the affinity capture media may include any of the known media used to capture biological and/or chemical molecules, including proteins.
  • the plurality of beads with one or more capture media are mixed in specified proportion. In some embodiments, equal proportions of beads of each different capture media are mixed.
  • the capture media is a bait molecule.
  • a sample of the bead mixture generated in step two 104 is extracted.
  • the sample extracted in step three 106 is then deposited in a target test sample in a fourth step 108 of method 100.
  • the test sample is incubated in the fifth step 110 to bind analytes to the beads.
  • the test sample incubated in step five 110 is then washed in a sixth step 112 of the method 100 to remove weakly bound analytes.
  • the washed beads are deposited onto a sample plate and a MALDI matrix is applied to produce a MALDI-TOF sample plate.
  • a MALDI-TOF scan is performed to produce a TOF mass spectrum of the analytes that are bound to the beads.
  • the method includes steps for assaying an analyte in a bodily fluid from a subject. See, for example, U.S. Patent Application No. 15/079,900, entitled “MALDI-TOF MS Method And Apparatus for Assaying an Analyte in a Bodily Fluid From a Subject”, which is assigned to the present assignee, and which has been incorporated herein by reference.
  • the method may include saving only mass spectra that exceed a predetermined intensity level, and/or determining the mass-to-charge ratios from the saved spectra, and/or analyzing the mass-to-charge ratios to interpret a resulting mass spectrum.
  • Ionizing light pulses from the MALDI-TOF spectrometry may be scanned over a predetermined area of the sample plate, and/or saved spectra may be averaged over a sample spot.
  • Mass spectrometry may be performed by irradiating a spot on the sample plate with a plurality of light pulses and/or the number of the plurality of light pulses may be chosen to reduce noise, and/or achieve a desired level of reproducibility.
  • the magnetic beads are Sepharose beads with a magnetic core.
  • Bait molecules can be attached to the magnetic beads by variety of techniques.
  • a mass tag molecule may be attached to assist in identifying these beads by mass spectrometry.
  • the mass tag molecules and bait molecules may be biotinylated and bound to the beads by the Streptavidin-biotin interaction.
  • the bait molecules may comprise biotinylated aptamers or biotinylated peptides.
  • Biotinylation is a process of covalently attaching biotin to a protein, nucleic acid, or other molecule.
  • Suitable beads are commercially available from a variety of sources including GE Healthcare and Cube Biotech.
  • the beads are available in a range of sizes from about 1 pm up to 1 mm.
  • the beads used are nominally 40 pm in diameter.
  • the beads used are nominally 400 pm in diameter and typically range in size from 350 pm to 450 pm.
  • Known methods of processing magnetic beads use magnets to keep beads in a volume while liquid is introduced and removed to incubate or wash the beads.
  • the present teaching expands the use of magnetic beads to provide a more efficient, controlled sample preparation apparatus and method that is suitable for high-volume manufacture.
  • FIG. 2A illustrates a front cut-away view of an embodiment of a magnetic bead picker 200 of the present teaching.
  • FIG. 2B illustrates a side-view of the embodiment of the magnetic bead picker 200 of FIG. 2 A.
  • the bead picker 200 uses a magnet 202 that is inserted in the direction 204 shown to pick up beads and removed along the direction 204 shown to release beads.
  • the magnet 202 may be an electromagnet that is activated by a current to pick up beads and deactivated by turning off a current to release beads (not shown).
  • the magnetic bead picker 200 comprises a bead capture volume 206 and a pair of arms formed from soft magnetic material 208 to concentrate the magnetic field in the bead capture volume 206.
  • the beads are thus captured by the concentrated magnetic field from a liquid suspension (not shown) into the bead capture volume 206.
  • the arms of soft magnetic material 208 are confined using an internal plastic spacer 210.
  • a plastic shield 212 surrounds the outside of the magnetic bead picker 200.
  • a dimension 214, x, of the plastic shield is approximately six millimeters or less.
  • the size of the bead capture volume 206 may be chosen to accommodate a
  • FIG. 3 A illustrates an expanded-view of a lower section of an embodiment of a well that is part of a plate preparation apparatus 300 that uses a magnetic bead picker 302 of the present teaching.
  • FIG. 3 A illustrates beads 304 moving to occupy the bead capture volume 306.
  • the bead capture volume 306 has dimensions chosen to capture a specified number of beads.
  • FIG. 3B illustrates a table 350 that provides approximate bead capture volume dimensions for various embodiments of the magnetic bead picker of the present teaching.
  • FIG. 3A illustrates beads 304 occupying the bead volume 306. The beads 304 are attracted by the magnetic field generated by magnet (not shown) and directed by the arms formed of a soft magnetic material 308. The beads 304 are retained on filter 310 at the bottom of a well 312.
  • the plastic shield 314 surrounds the arms of soft magnetic material 308.
  • the magnetic material may be iron or an iron-containing compound.
  • a lower manifold 316 has a diaphragm 318 with an aperture 320 positioned under the bead capture volume 306.
  • An o-ring 322 is positioned under the well 312 and on top of the diaphragm 318.
  • the aperture 320 is on the order of 1 mm in diameter or smaller and located at the top of the lower manifold 316 so that the beads are concentrated near the center as liquid flows from the sample well into a lower chamber 324 of the lower manifold 316.
  • the actual number of beads in a bead volume is determined empirically, rather than prescribed by specific dimensions of the volume.
  • the actual bead numbers captured will be determined empirically in the volumes and adjusted as required.
  • one or more magnetic bead pickers can be used in multiple steps of a process of preparing a MALDI sample plate for MALDI-TOF analysis.
  • samples are provided in a plate that includes one or more wells with filters at the bottom of each well that retains the beads and allows liquid to flow through.
  • Beads with bait molecules attached may be supplied in separate plates containing wells, where each well may contain a large number of beads that may represent a number of different bait molecules.
  • the sample plate contains 96 separate wells. Other embodiments use sample plates with different numbers and sizes of wells.
  • FIG. 4A illustrates an embodiment of part of a plate preparation apparatus 400 of the present teaching at different points during processing.
  • the plate preparation apparatus 400 uses a magnetic bead picker 402, 402’, 402”, 402’” to prepare a MALDI sample plate 404.
  • FIG. 4B illustrates the steps of an embodiment of a method 430 that is associated with the apparatus of FIG. 4A.
  • a mixture 408 containing a large number of beads is supplied in a well 410.
  • the mixture contains a large number of beads with different antibodies attached.
  • the number of 0.4 mm beads in 1 mL of bead slurry is approximately 10,000 beads.
  • the magnetic bead picker 402 is used to pick up beads from a well 410.
  • the well 410 may be in a plate (not shown) containing 96 wells that each contains beads and/or bead mixtures. Only one well is shown in the apparatus illustrated in FIG. 4 A.
  • the beads are deposited by the magnetic bead picker 402’ into a well 414.
  • This well 414 may reside in another 96-well plate containing samples.
  • the well 414 has a filter 416 at the bottom.
  • the plate (not shown) containing samples and beads in well 414 is then moved to the bead incubator or washer for the next step 418 where the beads and sample in well 414’ with filter 416’ are incubated and washed.
  • the bead washer/incubator comprises an upper chamber whereby liquid can flow in or out of the well through a filter and air can also flow in or out through the filter.
  • the bead washer/incubator also includes a bottom chamber with a volume at least equal to the volume of the well. The bottom chamber allows both liquid and air to flow in or out of the chamber.
  • the upper chamber also includes a liquid metering pump and a valve to direct flow either to the well or to a waste area.
  • the upper chamber also includes an air pump and a valve to direct flow either to the well or to the vent.
  • the lower chamber comprises similar pumps and valves as does the upper chamber.
  • FIG. 5 illustrates a schematic diagram of an embodiment of a bead
  • washer/incubator 500 of the present teaching.
  • the upper manifold 502 connects to a two-way valve 508 that connects to a vent and air supply. The input air passes through an air filter 510 and an air pump 512.
  • the upper manifold 502 also connects to a two-way valve 514 that sends liquids to a syringe pump 516 from a liquid reservoir 518 to refill the syringe pump 516 or to direct flow from the syringe pump 516 to upper manifold 502.
  • the well 504 contains a buffer 520 and sample plus beads 522.
  • the lower manifold 524 forms a chamber that contains a buffer 526. There is a filter 528 at the bottom of well 504 where the well 504 connects to the lower manifold 524.
  • the bottom of the lower manifold 524 is connected to a two-way valve 530 that goes to liquid waste 532 and a syringe pump 534.
  • FIG. 6A illustrates an embodiment of part of a plate preparation apparatus 600 of the present teaching at different points during processing from bead pick-up through preparation for washing and incubation.
  • FIG. 6B illustrates the steps of an embodiment of the method 602 that is associated with the apparatus of FIG. 6A.
  • the magnetic bead picker 606 picks-up a selected number or volume of beads from a mixture 610 that includes a large number of beads with different antibodies attached and then deposits them into a bead capture volume 608.
  • the large number of beads may be contained in a first well 612.
  • the bead picker 606’ then moves to the sample well 616 and releases the beads from the bead capture volume 608’ into the sample 618.
  • the sample well 616 includes a filter 620 at the bottom.
  • the sample well 616 may be located on a sample plate (not shown).
  • the plate containing the sample well 616’ with sample and beads 619 and filter 620’ is moved to a bead washer/incubator.
  • a buffer 626 is added to the sample well 616” that contains the sample plus beads 619’ and the air is expelled to fill sample well 616” with buffer 626 up to a top filter 628 in the upper chamber. Buffer 630 is also added to the lower chamber 632 and the air is expelled.
  • the process then moves onto other steps of the method, such as illustrated in FIG. 7B.
  • FIG. 7A illustrates an embodiment of a portion of a plate preparation apparatus
  • FIG. 7B illustrates the steps of an embodiment of a method 702 associated with the apparatus 700 of FIG. 7A.
  • buffer 706 is added to well 726 containing sample plus beads 708 through the top chamber 710.
  • Buffer 712 is also added to the bottom chamber 714 and air is expelled.
  • a filter 716 at the bottom of well 726 sits atop the buffer 712 in lower chamber 714. The process then moves to incubation 718.
  • a second step 720 associated with incubation 718 buffer 722 is added through the upper chamber 710’ to force the sample through the filter 716’ into the lower chamber 714’ with the beads 724 being retained in the well 726’ by the filter 716’ at the bottom of the well 726’ .
  • the buffer flow is reversed in a next step 728 to force the sample back through the filter 716” and to re-suspend the beads in the sample to produce beads plus sample 730 in the well 726”.
  • the method proceeds to a washing step 732.
  • the buffer flows from the top chamber through the bottom chamber and a valve in the liquid flow at the bottom directs the flow to the waste container (not shown).
  • Multiple wash cycles can then be initiated by forcing buffer from the bottom chamber through the filter re-suspending the beads in the buffer.
  • the flow can be reversed to direct the buffer flow back through the filter 716’” with the beads 734 being retained on the filter with the flow directed to the waste container.
  • This cycle in the washing step 732 can be repeated as many times as necessary to thoroughly wash the beads.
  • the last step 736 in the cycle is to flow air into the top chamber to push the buffer through the filter with the beads 734’ retained on the filter 716”” in a small volume of buffer.
  • FIG. 8A illustrates an embodiment of portion of a plate preparation apparatus 800 of the present teaching at different points during processing for picking up beads through applying a MALDI matrix.
  • FIG. 8B illustrates the steps of an embodiment of the method 802 associated with the apparatus 800 of FIG. 8 A.
  • a well 804 with the beads in a small volume 806 adjacent to the filter 808 is moved from the incubator/washer.
  • a magnetic bead picker 812 picks up the beads into a bead capture volume 814.
  • the magnetic beads are pulled into the bead capture volume 814 by application of a magnet 816 to a soft magnetic core 818 that forms arms and concentrates the magnetic field to the bead capture volume 814.
  • the magnetic bead picker 812’ deposits the beads onto a MALDI plate 822.
  • the magnetic beads are released from the bead capture volume 814’ by removing the magnet 816’, causing the magnetic beads to drop from the bead capture volume 814’.
  • MALDI matrix solution is applied to the plate 822’.
  • a MALDI matrix solution is sprayed onto the MALDI plate 822’ and allowed to dry.
  • other known methods of applying a MALDI matrix solution to a plate containing samples are used. As the matrix solution dries, analytes non-covalently bound to capture media on the beads are released from the capture media and are incorporated into matrix crystals.
  • FIG. 9A illustrates another embodiment of a portion of a plate preparation apparatus 900 of the present teaching at different points during processing for picking up beads through applying a MALDI matrix.
  • FIG. 9B illustrates the steps of an embodiment of the method 902 associated with the apparatus 900 of FIG. 9A.
  • a MALDI matrix solution is added to the well 906 containing the beads so that the buffer is displaced and the beads are left in a slurry 908 of MALDI matrix solution on top of the filter 910 at the bottom of the well 906.
  • the beads saturated with matrix solution are then picked up from the well 906’ with the filter 910’ by a magnetic bead picker 912 in a second step 914.
  • the beads saturated with MALDI matrix solution are pulled into the bead capture volume 916 by energizing the magnetic bead picker 912 by inserting the magnet 918.
  • the beads saturated with the MALDI matrix solution are then deposited in a third step 920 on a MALDI plate 922 and allowed to dry.
  • the beads saturated with matrix solution are released from the bead capture volume 916’ by removing the magnet 918’ from the magnetic bead picker 912’.
  • an electro-magnet that is energize by application of an electric current may be used in place of the permanent magnet 918, 918’ that is inserted and removed.
  • analytes non-covalently bound to capture media on the beads are released from the capture media and are incorporated into matrix crystals.
  • One feature of the present teaching is that it can be used to prepare samples in select individual wells of a multiple well plate. Samples can also be prepared in columns and/or rows and/or various shapes of two-dimensional arrays of a multiple well plate.
  • a single bead picker can be employed and the incubator/washer can accommodate one well at a time.
  • the incubator/washer accommodates one column of eight wells from a 96-well plate.
  • One feature of the present teaching is that it is possible to provide higher volume and a greater degree of automation by ganging multiple magnetic bead pickers together.
  • ninety-six magnetic bead pickers are assembled in an 8 x 12 array that matches a standard 96-well plate.
  • the incubator/washer is also configured to accommodate the same 8 x 12 array. This arrangement can be automated and can provide relatively high throughput, but is rather inflexible in that all 96 samples are analyzed together.
  • FIG. 10 illustrates an embodiment of a high-volume automated MALDI plate preparation apparatus 1000 according to the present teaching.
  • Twenty-four magnetic bead pickers 1002 are assembled in an 8 x 3 array 1004 that corresponds to one quarter of a 96-well plate 1006.
  • Twenty-four pipettes 1008 are assembled in an 8X3 array 1010.
  • three 8X3 arrays 1004, 1012, 1014 of magnetic bead pickers 1002 are used.
  • Two 8X3 arrays 1004, 1012 are used for picking and placing beads.
  • a third, optional, 8X3 array 1014 of magnetic bead pickers 1002 is used to remove beads.
  • Multiple 96-well plates 1006, 1016, 1018, 1020 are arranged along three parallel tracks 1022, 1024, 1026.
  • a fourth track 1028 holds MALDI plates 1030 that are one quarter of the size of a 96 well plate. Four of these MALDI plates 1030 are ganged together along the fourth track 1028 so they are approximately equal to a size that corresponds to the 96-well plates 1018, 1020 on track three 1026. Some embodiments include one or more additional tracks (not shown) to accommodate additional plates and additional processing steps. Each track 1022, 1024, 1026, 1028 of plates supports a different processing step.
  • the first track 1022 uses the 8X3 array 1010 of pipettes 1008 to apply the MALDI matrix.
  • the MALDI matrix is extracted from plate 1016 and the array 1010 is moved to track four 1028 to deposit the matrix.
  • the plate 1006 on the second track 1024 supplies magnetic beads, that are picked up by the 3X8 array 1004 of pickers 1002 and subsequently deposited on the first quarter of plate 1020 on the third track 1026 that supports plates 1018, 1020 that contain samples. This is accomplished using a y-directed motion of the array 1004.
  • a bead washer/incubator 1032 is sized to accommodate one quarter of a 96 well plate, such as plate 1020, and the washer/incubator 1032 is positioned over the third track 1026.
  • the sample plate 1020 After receiving the deposited beads, the sample plate 1020 then moves the first quarter over in the x-direction into the bead washer/incubator 1032 where the incubation and washing process occur.
  • the first magnetic bead picker array 1004 then picks up beads from the plate 1006 containing beads and deposits them into ninety-six wells that are in the adjacent quarter of the sample plate on track three 1026, so that these beads can be moved into the washer/incubator on the next cycle.
  • the adjacent quarter of the sample plate that was just loaded with beads is introduced into the bead incubator/washer 1032 by x-directed motion along the track 1026, and the incubation washing cycle proceeds on the adjacent quarter of the sample plate.
  • a second magnetic bead picker array 1012 picks up the beads from the first quarter of the sample plate 1020 and deposits them to the first MALDI plate 1034 located on the fourth track 1028.
  • the second picker array 1012 picks and deposits beads to the fourth track 1028 simultaneously with the first magnetic bead picker array 1004 picking up beads from the plate 1006 containing beads and depositing them into the next adjacent quarter of the sample plate.
  • the automated plate preparation apparatus 1000 efficiently and completely loads each of the plates 1020, 1018 on track three 1026 and also the plates 1030,
  • FIG. 10 also helps to illustrate how an embodiment of a complete plate preparation apparatus 1000 can carry out the various movements under computer control.
  • a bottom deck 1036 accommodates at least four parallel tracks 1022, 1024, 1026, 1028 that allow plates to be moved as required in the x-direction. Some embodiments use five or more tracks.
  • the fourth track 1028 includes one for the MALDI plates 1030, 1034 that are each one quarter of the 96 well plate size and are ganged in sets of four with the same dimensions as the 96-well plates used for the other tracks 1022, 1024, 1026.
  • the configuration of MALDI plates illustrated in FIGS. 16A and 16B are described further below. Tracks are provided for sample plates 1018, 1020, the plates 1006 containing beads, and plates 1016 containing MALDI matrices. In some embodiments, plates may also be included with enzymes, such as tripsin for cleaving proteins into peptides (not shown). An upper deck (not shown explicitly in FIG.
  • the system includes y- and z- directed motion control for the other elements, including the magnetic bead picker arrays 1004, 1012, an 8 x 3 array 1010 of pipettes for transferring MALDI matrix to the MALDI sample plates. It also may include a magnetic bead remover array 1014 for cleaning the dried beads from the MALDI process.
  • the system also includes z-directed motion for opening and closing the incubator/washer chamber 1032 and for moving the permanent magnets (not shown) from the magnetic bead picker arrays 1006, 1012, 1014.
  • the motion control elements required may be summarized as follows. There are up to five x-directed motion controls for moving MALDI plates 1016, sample plates 1018, 1020, bead plates 1006, matrix plates 1030, 1034 and enzyme plates (not shown). There are five y-z- directed motion controllers for the three bead picker arrays 1004, 1012, 1014, the matrix pipette array 1010, and optional enzyme pipette array (not shown). There are two z-directed motion controllers for the incubator washer 1032 that open and close the incubator washer 1032 and that move magnets (not shown). There are also three syringe pumps and three valves. This embodiment of the apparatus, therefore, requires seventeen motors, five syringe pumps, three valves, and two solenoids or motors for moving magnets in and out.
  • FIG. 11 illustrates an embodiment of a MALDI sample plate 1100 of the present teaching.
  • the dimensions of this plate correspond to one quarter of a 96- well plate.
  • a region 1102 that includes an array of wells (wells not shown) suitable for use with the beads nominally 0.4 mm in diameter are shown.
  • the plate 1100 has a width A 1104 and a length B 1106. In some embodiments, the width A is approximately 85 mm, and length B is approximately 27 mm.
  • the region 1102 that contains wells is of length B 1106 and width C 1108. In some embodiments, B 1106 is approximately 27 mm, and C 1108 is approximately 75 mm.
  • the region 1102 comprises a gasket (details not shown) that is 0.5 mm thick with 0.5-mm-diameter holes.
  • the wells are 0.5-mm diameter and 0.5-mm deep and are spaced at intervals of 0.75 mm. This provides an array that is 36 spots wide and 96 wells long, for a total of 3456 wells.
  • the wells are formed in a silicone gasket that can be removed after the beads are dried. Note that four of the plates 1100 are equals in size to one known 384-well microtiter plate.
  • a barcode may be used to tag information associated with the plate.
  • the wells are grouped into spots that are formed in a second silicone gasket that can be removed after the beads are dried.
  • FIG. 12 illustrates an embodiment of a second silicone gasket 1200 of the present teaching. This is a layout that accommodates 400 micrometer diameter beads, one bead per well.
  • the gasket 1200 has spots 1202 arranged in a 3X8 array of spots.
  • each spot 1202 comprises a 10X10 array of wells, so the gasket 1200 includes 2400 total wells. Individual wells are not shown in FIG. 12.
  • Some embodiments of a gasket include ninety-six spots that are
  • Each spot comprises a 5X5 array of wells, for 2400 total wells.
  • Some embodiments of a gasket include 384 square spots of 1.5 X 1.5 mm dimension on 2.25 mm centers, each with a 2X2 array of wells for 1536 total wells.
  • FIGS. 11 and 12 support nominally one bead per well with a 400-micrometer bead. However, smaller bead sizes can be employed using large wells using multiplexing. For example, in FIG. 12 each of the 24 spots accommodates about 25,000 beads of 0.04 mm diameter and so the total number of beads is up to 600,000 beads. In various methods that use larger beads, a smaller number of beads is required for a given assay, but these methods to some extent limit the degree of multiplexing that can be done. With the smaller beads, i.e.
  • each spot accommodates about 1000 of the small beads. This could be used, for example, to do 50-fold multiplexing, 20 beads with each bait molecule, for a total of 1000 beads in each spot.
  • the plurality of magnetic beads comprises at least two sets of a plurality of beads, wherein each of the at least two sets comprises a mass tag and a bait molecule that are unique to that set.
  • microwell plates that accommodate small beads in a nominally single-bead-per-well configuration can be used.
  • particular beads that are only 0.04 mm in diameter can be used with a micro well plate that has a hexagonal array of 0.04 mm in diameter wells that are up to 0.04 mm deep.
  • FIG. 13A illustrates a diagram of a well layout 1300 for a microwell plate of the present teaching that accommodates ⁇ 0.04-mm-diameter beads.
  • This hexagonal well layout 1300 provides approximately 250 times as many wells as compared to the designs in each of the spots described above in connections with FIGS. 11 and 12.
  • the microwells 1302 are 40 pm in diameter and are arranged in a regular hexagonal array 1304 with individual hexagons 1306 having a 50 pm height.
  • FIG. 13 A shows the size of a 25-pm pixel 1308 and a 10-pm pixel 1310 in relation to the size of an individual hexagon 1306 and the regular hexagon array 1304.
  • FIG. 13B shows a detailed view of one particular hexagon in the hexagonal array
  • the hexagon layout has two hexagonal dimensions,“a” 1312 and“b” 1314.
  • the laser beam in MALDI-TOF mass spectrometer is raster scanned over the surface of a microwell plate comprising the layout 1300 illustrated in FIG. 13A.
  • the raster scanning is performed at intervals of 10 pm with a 10-pm-diameter laser beam using laser repetition rate of 5 kHz, a scanning speed of 2 mm/s with 24 shots per pixel. This provides 22 total pixels per cell with 58% on the well and no significant crossover between wells.
  • the total number of laser shots on well is 300 with a time per cell of 0.12 seconds with 462 cells per square millimeter.
  • the raster scanning is performed at intervals of 25 pm with a 10-pm diameter laser beam using laser repetition rate of 5 kHz, a scanning speed of 2.5 mm/s, and summing of 50 laser shots per pixel to produce 25 pm long pixels.
  • the total pixels/cell ratio is about 3.5 with about half on the well and about half with significant contribution from adjacent wells.
  • Total number of laser shots on each well is 100, with the laser irradiation time per cell equal to about 0.035s.
  • the raster scanning is performed over the surface of microwell plate comprising the layout 1300 illustrated in FIG. 13A at intervals of 10 pm with a 10-pm diameter laser beam using a laser repetition rate of 5 kHz, scanning speed of 1 mm/s, and summing of 50 laser shots per pixel to produce 10 pm long pixels.
  • the total pixels/cell ratio is about 25 with about 58% on the well having no significant contribution from adjacent wells.
  • Total number of laser shots on the well is 600, and the laser irradiation time per cell is 0.25 seconds.
  • the raster scanning is performed over the surface of microwell plate comprising the configuration 1300 illustrated in FIG. 13A at intervals of 12.5 pm with a 10-pm diameter laser beam using laser repetition rate of 5 kHz, scanning speed of 1.25 mm/s, and summing of 50 laser shots per pixel to produce 12.5 pm long pixels.
  • the total pixels/cell ratio is about 16 with about 58% on the well and with no significant contribution from adjacent wells.
  • Total number of laser shots on each well is 400 with the laser irradiation time equal to 0.16 seconds.
  • FIG. 14 illustrates a table 1400 that presents the probability of missing one of n distinguishable beads in a collection of m beads for various values of m/n and n as applies to an embodiment of the system of the present teaching. As shown by the calculation and the table 1400 in FIG. 14, the probability that one of the n beads is missed is significant unless the total number m is large compared to the number of distinguishable beads.
  • the mixture of beads required for a particular multiplexed assay is prepared off-line and provided in one or more wells of the bead plate.
  • the magnetic bead picker described herein allows a predetermined number of beads to be accurately collected. For example, a single bead can be picked from a large collection of beads. This allows the mixture of beads required for a particular multiplexed assay to be mixed directly on the sample plate.
  • the first quarter of the bead plate includes of a large number of beads with a particular bait molecule attached.
  • the next quarter includes a large number of beads with a second bait molecule.
  • the third quarter includes a large number of beads with a third bait molecule.
  • the fourth quarter includes a large number of beads with a fourth bait molecule.
  • the sample plate might contain a different sample in each well. By sequential use of the bead picker, the four different beads can be introduced into each well.
  • each with four wells in a 2X2 array allows fourfold multiplexing of 384 samples on a single small MALDI plate.
  • the time to produce and process the data from one of these plates is less than two hours and can be as little as one hour or less. This provides the ability to analyze up to 400 samples per hour for four different analytes.
  • the throughput is limited only by the time required to produce and prepare the samples. This means a system operating 24/7 could produce samples up to 9600 per day for four different analytes. Higher multiplexing with a smaller number of samples is also possible with very high throughput.
  • the capacity is approximately 100 pmol per bead.
  • a single bead may be adequate for many applications. For some applications, multiplexing is not required, for example a competing ELISA assay. For these applications, a single bead can be introduced to each sample well. After washing, the bead can be transferred to a predetermined well on the MALDI sample plate. This allows up to 3456 samples to be analyzed from a single MALDI plate. This corresponds to 36 plates each with 96 wells and provides very high throughput and low cost for these applications.
  • FIG. 15A illustrates a front- view of an embodiment of a MALDI sample plate 1500 that can accommodate a large number of beads of the present teaching.
  • FIG. 15B illustrates a partial side-view of the MALDI sample plate 1500 of FIG. 15 A. Referring to both FIG. 15A and B, in some embodiments, the dimensions of this plate 1500 correspond to one quarter of a 96-well plate.
  • a region 1502 that comprises an array of wells suitable for use with the beads nominally 0.04 mm in diameter and smaller is illustrated.
  • the plate 1500 is of length A 1504 and width B 1506. In some embodiments, A is approximately 27 mm, and B is approximately 85.5 mm.
  • the region 1502 that contains wells is of length A 1504 and width C 1508.
  • a 1504 is approximately 27 mm
  • C 1508 is approximately 72 mm
  • the array of wells comprise wells with diameter D 1510 of 0.6 mm, and a depth E of 0.05 mm. As such, the wells are 0.6 mm in diameter and 0.05 mm deep and are spaced at intervals of dimension F 1514 of 0.75 mm.
  • the plate 1500 has a thickness of dimension G 1516 of 0.3 mm. This provides an array that is 36 spots wide and 96 wells long, which includes a total of 3456 wells.
  • the well volume is 25 nL.
  • a bead volume of a monolayer for 2.7 micrometer beads is 0.8 nL.
  • the volume of matrix solution is approximately 50 nL/well.
  • a barcode 1511 may be used to identify various information.
  • the barcode 1511 can be used to identify information associated with the plate and/or associated samples and/or processing steps or other instructions.
  • FIG 16A illustrates a top-view of an embodiment of a cassette 1600 comprising four MALDI sample plates that can accommodate a large number of beads of the present teaching wherein each of the four sample plates may be loaded into a MALDI mass spectrometer either manually or under automated or semi-automated control.
  • FIG 16B illustrates an end-view of a cassette 1600 comprising four MALDI sample plates of FIG. 16A.
  • the cassette 1600 is of length A 1604 and width B 1606. In some embodiments, A is approximately 127 mm, and B is approximately 85 mm.
  • MALDI sample plates 1500 can be formed, for example, by photo etching the array of wells in a stainless steel plate. In these cases, a monolayer of beads may be spread uniformly over the bottom of the well. MALDI matrix material may be added to each well to release biomolecules from these beads. As the matrix solution dries, the biomolecules are released from the beads incorporated into matrix crystals in the well. The MALDI plate is then transferred to the mass spectrometer and the mass spectra acquired and processed. [0064] Referring to FIGS.
  • the MALDI sample plates 1500 can be formed, for example, by photo etching the array of wells in a magnetic stainless steel plate. These sample plates are held by magnets on a sled that is compatible for loading into a specified MALDI-TOF mass spectrometer.
  • a second magnetic stainless steel plate with through holes in the same pattern as MALDI plate 1500 is used. But this second plate is substantially thicker. In one example, this second plate is 0.6 mm thick and is held in place by the magnets with the holes in the second plate being aligned with the wells in sample plate 1500. This allows larger volumes of matrix solution to be added to each well. After the matrix dries, the second plate is removed before the plate 1500 is introduced into the MALDI- TOF mass spectrometer.
  • the mass spectra acquired by laser raster scanning over a well are summed to produce an average spectrum. If each well contains beads with a number of different bait molecules attached, than the average spectrum will represent the sum of the spectra from all of the beads in the well.
  • the spectra of the captured analytes are generated. With a mixture of beads having different bait molecules, the average spectrum includes a sum of the spectra for each analyte with the relative intensities determined by the concentration of the biomolecules in the sample.
  • spectra from each well can be deconvoluted to determine the concentration of each biomolecule.

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Abstract

L'invention concerne un appareil destiné à lier par ligands des échantillons biologiques qui inclut un puits de perles servant à confiner une pluralité de perles magnétiques. Un puits d'échantillon comprend un fond de filtre servant à contenir des échantillons d'intérêt. Un premier sélecteur de perles magnétiques capture des perles magnétiques depuis le puits de perles et libère les perles magnétiques capturées dans le puits d'échantillon. Un incubateur incube les perles magnétiques dans le puits d'échantillon, liant les molécules appâts à des molécules d'échantillon contenues dans l'échantillon d'intérêt. Un appareil de lavage lave les perles magnétiques incubées, retirant les molécules d'échantillon faiblement liées tout en retenant les perles magnétiques comprenant des molécules d'échantillon fortement liées. Un deuxième sélecteur de perles magnétiques capture les perles magnétiques comprenant des molécules d'échantillon fortement liées depuis le puits d'échantillon et libère les perles magnétiques capturées comprenant des molécules d'échantillon fortement liées sur une plaque d'échantillon. Un applicateur de matériau de matrice dépose un matériau de matrice MALDI sur une surface de la plaque d'échantillon. Un spectromètre de masse MALDI-TOF reçoit la plaque d'échantillon avec le matériau de matrice MALDI déposé et procède à une spectrométrie de masse de temps de vol sur les molécules d'échantillon fortement liées, générant ainsi des spectres de masse de l'échantillon. Un ordinateur exécute un algorithme au moyen des spectres de masse générés par le spectromètre de masse MALDI-TOF pour produire une analyse de liaison par ligands.
PCT/US2019/037628 2019-06-18 2019-06-18 Procédé et appareil employant des perles magnétiques pour des analyses de liaison par ligands d'échantillons biologiques WO2020256695A1 (fr)

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WO2012109460A1 (fr) * 2011-02-09 2012-08-16 Adeptrix Corp. Dispositifs et procédés d'obtention et d'analyse de microréseaux
WO2016160511A1 (fr) * 2015-03-30 2016-10-06 Virgin Instruments Corporation Procédé et appareil de spectrométrie de masse à temps de vol à désorption-ionisation par impact laser assistée par matrice (maldi-tof ms) pour doser un analyte dans un fluide corporel provenant d'un sujet
US20180188241A1 (en) * 2017-01-05 2018-07-05 Virgin Instruments Corporation Ligand Binding Assays Using MALDI-TOF Mass Spectrometry
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WO2012109460A1 (fr) * 2011-02-09 2012-08-16 Adeptrix Corp. Dispositifs et procédés d'obtention et d'analyse de microréseaux
WO2016160511A1 (fr) * 2015-03-30 2016-10-06 Virgin Instruments Corporation Procédé et appareil de spectrométrie de masse à temps de vol à désorption-ionisation par impact laser assistée par matrice (maldi-tof ms) pour doser un analyte dans un fluide corporel provenant d'un sujet
US20180188241A1 (en) * 2017-01-05 2018-07-05 Virgin Instruments Corporation Ligand Binding Assays Using MALDI-TOF Mass Spectrometry
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