Integrated magnetic bead assay processing method and apparatus

Abstract

An integrated magnetic microbead processing apparatus includes a microwell plate having magnetic microbeads in microwells. And the bottom of the micro-pore plate comprises cavities among the micro-pores. A heating / cooling plate supports the base of the microplate and has a plurality of pores. A vertically movable magnet support plate includes a plurality of magnetic pins, each of which may protrude through one of the plurality of apertures of the heating plate and into the cavity of the microwell plate. The height of the magnetic pin is adjustable. A vibrator is operably connected to the microwell plate and is configured to vibrate the microwell plate, the heating / cooling plate, the magnetic support plate.

Description

Technical Field

[0001] The present invention generally relates to assay beads and methods of use thereof for conducting protein, nucleic acid, and other specific binding bioassays utilizing magnetic beads, and more particularly to an integrated, compact, multifunctional bioassay processing device for magnetic beads that includes washing, separation, mixing, and reaction incubation with minimal bead loss. Background Art

[0002] Magnetic microbeads used in bioprocessing are typically paramagnetic, meaning they possess magnetic properties when placed in a magnetic field but retain no residual magnetism when removed from the field. This paramagnetic property allows for magnetic collection of the beads and their resuspension when the magnetic field is removed. The collection and resuspension of the magnetic microbeads can be easily and rapidly repeated any number of times. Consequently, magnetic microbeads are widely used for target enrichment or capture in bioprocessing. In protein assays (such as sandwich immunoassays), unbound or nonspecific antibodies or antigens can be removed after the magnetic microbead-antibody-antigen reaction. Secondary antibodies are coupled to the antibody-antigen complex, and optical detection is then performed using fluorescent or chemiluminescent labels. Magnetic microbeads allow for washing unbound molecules from the beads, adding buffer solutions, or removing any contaminants from the solution. In nucleic acid-specific binding assays (such as DNA or RNA assays), unbound or nonspecific nucleotides can be removed after hybridization. Consequently, magnetic microbead bioprocessing typically requires a series of biochemical reactions and extensive washing. These processes are typically performed in 96-well, 384-well, or 1536-well microplates with automated robotic systems and fixtures for high-throughput assays.

[0003] Over the past 20 years or so, barcoded magnetic microbeads or digital magnetic microbeads have been developed and used for multiplexed or syndromic bioassays. Multiplexed assays not only expand the range from a single test per sample to multiple tests per sample, providing comprehensive test results, but also allow a panel of tests to be performed with minimal sample volume. Each micron-sized magnetic microbead, either color-coded or digitally encoded, can be immobilized with a specific protein or molecular probe to capture a specific desired target in the sample. The probe-target complex is then labeled with a labeled agent (such as a fluorophore) for optical detection. Thus, barcoded magnetic microbeads are used for both target identification and fluorescent detection. Barcoded magnetic microbeads offer many advantages, but they are more expensive than non-barcoded magnetic microbeads. For multiplexed assays, it is crucial to have a sufficient and consistent number of magnetic microbeads with a specific barcode in each well for each assay and each sample. For example, for a 20-plex assay, each of the 96 wells is required to have magnetic beads with 20 barcodes, and each barcode needs to have a minimum number of beads, such as 20-30 beads or 200-300 beads, to ensure data accuracy.

[0004] Magnetic bead-based washers are available on the market. For example, BioTek's ELx405™ Magnetic Bead Washer provides full microplate washing with magnetic microspheres. The microplate rests on a magnetic plate with an array of magnetic blocks, so that each microwell is positioned on top of a magnetic block. Peristaltic and syringe pump dispenser modules feature autoclavable fluid paths for automating wash / dispense / aspiration steps on a single platform.

[0005] Microplate vibrating and heating devices are currently available on the market. For example, Thermo Scientific's microplate shaker can vibrate, heat, or both microplates. It provides oscillations of 250-1,200 rpm / 2 mm amplitude. Heat is transferred to the sample from the platform and heated lid. The temperature can be adjusted from 25°C or room temperature to 60°C. Mixing, heating, and cooling modes can be used simultaneously or independently, meaning the device functions as both a vibrator and a thermostat.

[0006] U.S. Patent No. 5,779,907, issued on July 14, 1998 (the entire contents of which are incorporated herein by reference), discloses an apparatus for using a 96-well microplate and including a mechanism for supporting the microplate in a relatively fixed position. A magnetic microplate assembly containing a plurality of cylindrical magnets is positioned in a 4x6 array for insertion from the bottom of the microplate into the spaces between the wells of the microplate. A means for moving the magnet microplate assembly relative to the microplate allows for the selective separation of magnetic components within the wells of the microplate. Magnets, preferably cylindrical in configuration, are positioned between each group of four wells in the microplate. The apparatus is intended for manual use, and the height of the magnets is fixed.

[0007] U.S. Patent No. 6,645,431, issued November 11, 2003 (the entire contents of which are incorporated herein by reference), discloses an apparatus for automated magnetic separation of materials in laboratory trays. The apparatus includes a frame on whose upper surface a multi-well laboratory tray can be placed. A base plate is mounted with a plurality of upright magnets positioned below the upper surface, and a flexible bladder is used to elevate the base plate to insert the upright magnets into the spaces between the wells of the laboratory tray.

[0008] U.S. Patent No. 8,512,558, issued on August 20, 2013, discloses a magnetic separation system for use in methods employing magnetic particles. The system includes a magnetic separation plate having a support plate and magnetic pins arranged in a predetermined geometric arrangement. The magnetic pins have a fastening portion, an intermediate portion, and a separating portion, and are secured to the support plate at the fastening portion via a retainer comprising one or more flexible elements or O-rings. The magnetic pins are individually movable laterally at the separating portion. Summary of the Invention

[0009] Problems can arise when processing bioassays using two separate magnetic washers and heating / cooling / vibration units. For example, once the plate is removed from the heating / cooling / vibration unit, the magnetic beads no longer remain suspended and sink, randomly adhering to the bottom of the microwells through static forces. Furthermore, once the magnetic beads are randomly distributed and adhere to the surface, some are farther from the tip of the magnetic pin (approximately 6.8 mm microwell diameter) than others. This can cause low and uneven paramagnetic forces, leading to high bead loss and inconsistency.

[0010] An integrated magnetic microbead handling device, as described herein, includes a microplate having magnetic microbeads in microwells. The bottom of the microplate includes cavities between the microwells. A heating / cooling plate supports the base of the microplate and has a plurality of holes. A vertically movable magnet support plate includes a plurality of magnetic pins, each of which can protrude through one of the plurality of holes in the heating plate and into the cavity of the microplate. The magnetic pins are height-adjustable. A vibrator is operably connected to the microplate and is configured to vibrate the microplate, the heating / cooling plate, and the magnetic support plate.

[0011] In one embodiment, the shaker can have an adjustable orbital rotation speed between 0 rpm and 1200 rpm. The orbital rotation provides mixing for the reaction and also provides a circular force to move the magnetic microbeads to the sides of the microwells and closer to the magnet pins for efficient bead attraction.

[0012] In another embodiment, the height of the magnetic pins protruding above the heating plate can be adjusted according to the liquid level in the microwells.

[0013] In another embodiment, the height of the magnetic pins protruding above the heating plate can be adjusted between 1.5 mm and 4 mm.

[0014] In another embodiment, the heating / cooling plate is configured to provide a temperature between 25°C or room temperature and 60°C.

[0015] In another embodiment, the vibrator has a home position mark that ensures that the track stops rotating at the same designated position.

[0016] In another embodiment, an integrated magnetic microbead processing method includes: providing a microplate having magnetic microbeads in microwells, the bottom of the microplate having cavities between the microwells; a heating / cooling plate supporting the microplate, the heating / cooling plate having a plurality of wells; a vertically movable magnetic support plate containing a plurality of magnetic pins, each of the plurality of magnetic pins being able to protrude through one of the plurality of wells and into the cavity of the microplate, the magnetic pins being height-adjustable; and a vibrator configured to vibrate the microplate, the heating / cooling plate, and the magnetic support plate to enhance separation of the magnetic microbeads. The vibrator is activated using appropriate orbital rotation, and then the magnetic pins are raised to increase microbead capture efficiency.

[0017] In another embodiment, the method includes the vibrator having an adjustable orbital rotation speed between 0-1200 rpm.

[0018] In another embodiment, the method further comprises the steps of rotating the magnetic microbeads when the magnetic pins are downward, and attracting the magnetic microbeads toward the sides of the microwells when the magnetic pins are upward.

[0019] In another embodiment, a microplate plastic lid and heated lip assembly is provided to enhance uniform temperature distribution and automation of liquid handling robotic systems.

[0020] In another embodiment, the microplate has a flat and optically clear bottom.

[0021] In another embodiment, the magnetic microbeads comprise at least some barcoded magnetic microbeads.

[0022] In another embodiment, a heated cover and spring loaded pins help prevent the micro plastic cover from sticking to the heated cover.

[0023] In another embodiment, to improve the efficiency of magnet capture of magnetic microbeads, a gentle shaking speed of <500 RPM for a short period of time (<30 seconds) can very efficiently move the magnetic microbeads into proximity with the magnet to achieve a high yield of magnetic microbead capture.

[0024] Unless otherwise stated, in other, non-described embodiments, any feature described in the Summary above or Detailed Description below may be combined with any other feature of the integrated magnetic microbead handling apparatus or method. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In the following drawings, like reference numerals refer to like or similar parts throughout the drawings.

[0026] 1 illustrates a perspective view of magnetic microbead loss in a prior art magnetic bead handling system having an external magnet block on the bottom of a microplate, as is common in prior art magnetic bead handling systems having base-mounted magnets.

[0027] 2 illustrates a perspective view of magnetic bead loss in a prior art magnetic bead handling system having external cylindrical magnetic pins on the sides of microwells or in the cavities between microwells.

[0028] FIG. 3 a shows a perspective view of one embodiment of the present invention, wherein magnetic beads are suspended in orbital rotation when the magnetic pins are facing downward.

[0029] 3b illustrates a perspective view of the embodiment of FIG. 3a , wherein the magnetic pin is raised with appropriate adjustable height and vibration speed to attract the magnetic microbeads toward the magnetic pin and thus toward the sides of the microwell as the microbeads rotate.

[0030] Figure 4 A perspective view of the apparatus of the present invention is shown having a heating / cooling plate, a collapsible and adjustable plurality of magnetic pins, and a vibrating mechanism.

[0031] Figure 5 is a perspective view of the integrated device with the adjustable plurality of magnetic pins in a downward position.

[0032] Figure 6 is a perspective view of the track vibrating element of the apparatus with adjustable vibration speed.

[0033] Figure 7 is a perspective view of the heated cover assembly. DETAILED DESCRIPTION

[0034] The magnetic microbead bioassay process can be lengthy, involving more than 10 steps. The process includes a probe-target reaction, multiple washes, a secondary antibody reaction, multiple washes, a fluorescent labeling reaction, and multiple washes; and the plate is transferred back and forth between a heater / cooler / shaker and a magnetic washer. The heater / cooler / shaker serves as an incubator for performing antigen-antibody immunoassays, sandwich assay chemistries, nucleic acid hybridization for molecular assays, and labeling chemistries. All reactions require temperature control and mixing of the magnetic microbeads. To mix the magnetic microbeads, a shaking mechanism is implemented. After each reaction, the plate is transferred to a magnetic microbead washer. The magnetic bead washer uses external magnets at the bottom or side of the microwells to separate the magnetic microbeads from the liquid and prevent the magnetic microbeads from being vacuumed out of the wells during liquid aspiration. The purpose of washing is to maintain the reaction on the magnetic microbeads and wash any unbound chemicals, such as free fluorophores, contaminants, sample matrix material, or residual buffer solution, out of the wells.

[0035] The embodiments described herein advantageously integrate magnetic bead washing with vibration and heating / cooling into a single device, while providing efficient magnetic bead processing with minimal bead loss. Conventional heating / cooling / vibration devices and magnetic bead washer / vibrators are separate units. Each unit is equipped with a vibration mechanism. After the chemical reaction in the heating / cooling / vibration device, the microplate must be transferred to the magnetic vibrator manually or via a robotic system.

[0036] Two prior art magnetic bead washer configurations are known in the art. The first configuration features an external magnetic block at the bottom of the microplate, as shown in Figure 1 (prior art). Once the microplate 11 is placed on the washer's magnetic block 12, beads 16 are immediately attracted to the bottom of the microwells 13. When the pipette tip 15 is lowered to vacuum out the liquid, the meniscus 14 drops. The challenge is to aspirate as much liquid as possible while retaining as many magnetic beads as possible in the well. As shown in the figure, the pipette tip can easily vacuum out the beads. The second configuration, as shown in Figure 2 (prior art), features external magnetic pins or rods on the sides of the microwells. Magnetic pins 23 are affixed to a base plate 22. When the microplate 11 is placed on the magnetic base 22, as shown in the figure, beads are attracted to the sides of the microwells 13. In this second configuration, the height of the magnetic pins 23 is critical. The tip of the pin has the strongest magnetic force. If the pins are too high or too low, the efficiency of bead attraction will vary, depending on the liquid level in the microwells. The height of the magnetic pins is not adjustable. In addition, the positioning of the magnetic pins is very sensitive. The tolerance of the pin positioning and the distance 25 of the pre-adapted cavity often leads to an uneven paramagnetic field distribution 26 between four adjacent wells. Finally, once the microplate is removed from the heating / vibration device, the magnetic microbeads 16 will begin to fall toward the bottom of the microwells and be randomly distributed at the bottom due to gravity. Once the microbeads settle on the bottom surface, the distance between each surface magnetic microbead 27 and the tip of the magnetic pin will vary significantly, for example when the microwell diameter is about 6.8 mm. Therefore, the magnetic force will also vary significantly. In addition, weak electrostatic forces cause the magnetic microbeads to stick to the bottom surface, which leads to the problem of distant microbeads being attracted to the magnetic pins. All of these factors will lead to inconsistency and low bead attraction efficiency.

[0037] Figure 3(a) illustrates one embodiment of a microwell 13 according to the present disclosure, wherein the microwell is positioned atop a heating / cooling plate 31 and has rotating beads 36 within the microwell 13 prior to engaging a magnetic pin 33. The purpose of this continuous orbital rotation 35 is to evenly distribute the beads 36 within the liquid and to move the magnetic beads 36 closer to or further from the magnetic pin 33. Thus, once the magnetic pin 33 is raised, all beads 36 have an opportunity to be attracted to the pin 33. A vibrator with an orbital motion (not shown in Figure 3a) induces a circular or orbital motion of the beads 36, thereby creating a more uniform distribution of the beads 36 within the microwell 13. Other vibration mechanisms, such as oscillating, reciprocating, or periodic shaking, are unable to move the beads 36 in a circular motion within the microwell 13.

[0038] As microbeads 36 circulate within microwells 13, external magnetic pins 33 on the magnet substrate 32 are raised relative to the heating / cooling plate 31 to protrude through the heating / cooling plate 13, as shown in Figure 3(b). Depending on the rotation speed, the orbital rotation force can be controlled to be smaller than the magnetic force; otherwise, the orbital rotation force would rotate the microbeads 36 out of the magnetic field. Multiple slow rotation speeds (e.g., 500 rpm, then 300 rpm) can be used to allow microbeads 36 to circulate freely and gently within microwells 13. After the magnetic pins 33 are raised, the microbeads 36 are captured by the magnetic field of the magnetic pins 33. Through 10-30 seconds of continuous pre-vibration or rotation, the majority of microbeads 36 will be attracted to the magnetic pins 33.

[0039] Tables 1 and 2 show the percentage loss of beads 36 after six wash cycles for a 96-well plate containing 1,000-2,000 magnetic beads 36. As Table 1 demonstrates, without pre-vibration, bead 36 loss is high and highly uneven across the plate. While the average loss is approximately 32%, the loss can be quite uneven. Some wells experience losses as high as 70%-80%, and there is significant variability (see Table 1). High bead loss is a major concern. However, with pre-vibration at 500 rpm for 30 seconds with the magnetic pins facing downward and no magnetic field, bead 36 loss is significantly lower, averaging approximately 12% and being relatively uniform across the plate (see Table 2). After six washes, the maximum loss across all 96 wells is 25%, an excellent result and an unexpected improvement over prior art vibrators and washers.

[0040] The height of the magnetic pin 33 can be adjusted based on the liquid level in the microwell 13 and the positioning of the pipette tip 15. By appropriately adjusting the height of the magnetic pin 33, the pipette tip 15 can be lowered near the bottom of the microwell 13 to aspirate a higher percentage of the liquid because the beads 36 move laterally to the sides of the microwell 13, which significantly reduces bead 36 loss (see Table 3). The pipette tip 15 provides a vacuum force to aspirate liquid from the microwell 13 without descending below the liquid surface to avoid contamination of the beads 36. The microbead washing system described herein advantageously suspends the magnetic beads 36 in orbital rotation through vibration and raises the magnetic pin 33 to an adjustable height to attract the magnetic beads 36 to the walls of the microwell 13 and away from the center of the microwell 13, where the pipette tip 15 is positioned.

[0041] Table 1. Percentage of bead loss without vibration

[0042]

[0043] Table 2. Percentage of bead loss under vibration conditions

[0044]

[0045] Table 3. Bead loss relative to magnetic pin height

[0046]

[0047] Figure 4 One embodiment of a highly efficient integrated magnetic microbead assay processing device according to the present disclosure is presented. Figure 4 A perspective view of the apparatus is shown, including a heating / cooling plate 103, which serves as a support base for the microplate holding the microwells 13 shown in Figures 3a and 3b. The heating / cooling plate 103 has multiple holes 104 for receiving the magnetic pins 33 shown in Figures 3a and 3b. A vertically movable magnetic support plate 101 includes multiple magnetic pins 102, which can protrude through the holes 104 in the heating / cooling plate 103 and into the cavities of the microplate. The height of the vertically movable magnetic support plate 101 is adjustable, allowing the magnetic pins 102 to be adjusted relative to the microwells 13 (Figures 3a and 3b). An orbital shaker 105 is used to vibrate the microplate, heating / cooling plate 103, and magnet support plate 101. The multiple holes 104 in the heating / cooling plate 103 are positioned according to the positioning of the magnetic pins 102. The magnetic support plate 101, which is not inherently magnetic, includes multiple magnetic cylindrical pins 102 permanently mounted on the magnetic support plate 101. In one embodiment, each magnetic pin 102 is a cylindrical rod with a diameter of approximately 2 mm and a length of 10 mm. In other embodiments, other diameters, shapes, and lengths are possible. In one embodiment, when the magnet pins 102 are in the fully raised position, the length of the exposed magnetic pins is > 4.5 mm above the heating / cooling plate 103. In other embodiments, other heights are possible. The up and down movement of the magnet support plate 101 is controlled by a stepper motor actuator. The microplate, heating / cooling plate 103, and magnet support plate 101 are placed on an orbital shaker 105. In one embodiment, the heating / cooling plate 103 provides a temperature of 0°C to 60°C for the entire microplate with an accuracy of + / - 1°C. In other embodiments, other temperatures are possible and can be selected by one of ordinary skill in the art.

[0048] Figure 5is a perspective view of the integrated device with the magnetic pins 102 in a downward position. The heating / cooling plate 202, which serves as a support for the microplate, has multiple heating elements below the heating plate and has 24 holes for the magnetic pins 102 to move up and down through. The heating element has a dual-cartridge heater 201 that can quickly heat the heating plate 202 with uniform heat distribution. In one embodiment, the heater can control the temperature of the microplate between 30°C and 60°C. A thermal cutoff 203 cuts off power to the heater in the event of thermal overshoot. At the edge of the heating plate, a two-piece Delrin insert 204 is used to secure the microplate, a plastic cover (not shown), and a heating cover (not shown), which are stacked on top of each other.

[0049] Figure 6 is a perspective view of the integrated device with the magnetic pin 102 in an upward position. The system allows for control of the speed and duration of shaking of the mixed magnetic microbeads. In one embodiment, a motor drives an eccentric shaft 301 to rotate the microplate at 0-1200 rpm on a 0.08" or 2 mm diameter orbital path. In other embodiments, other orbital paths are possible. A stack 302 of flexible rubber mounts at the four corners allows for sufficient freedom of motion for orbital rotation of the microplate. The speed of the orbital shaker table is adjustable between 0-1200 rpm. Typical frequencies for reactions are 800-1200 rpm and frequencies for moving the magnetic beads into proximity of the magnetic pin are 300-800 rpm for 10 to 30 seconds. The processor protocol provides the user with the ability to input the shaker rpm and duration.

[0050] The home position marker 303 advantageously ensures that orbital rotation stops at the same designated point, enabling reliable and precise positioning. The orbital shaker stage returns to its exact home position, allowing pipette tips to be reproducibly aligned with each of the 96 wells. Even after vibration, the exact position of the pipette tip in the microwell remains the same for all 96 wells. This precise position relative to the magnetic pin and microwell ensures consistent liquid aspiration, thus preventing fluctuations in bead counts.

[0051] The height of the magnetic pins is advantageously adjustable depending on the relative positioning of the liquid level and the pipette tip. Each magnetic pin in the magnet plate provides the same magnetic strength. In one embodiment, the magnetic strength is in the range of 0.65-0.9 lb, but in other embodiments, other magnetic strengths are possible. It is known that the magnetic field distribution is strongest near the top of the pin. Therefore, the magnetic microbeads will be attracted to the wall of the hole near the top pin position. If the pin height is raised too low, the microbeads will be sucked up when the aspiration tip approaches the bottom of the hole. In one embodiment, the optimal positioning is to raise the magnetic pins above the bottom of the hole, such as > 2 mm, but not exceed the height of the liquid solution, such as < 6 mm, otherwise the microbeads cannot be captured.

[0052] The vibrator provides a moving mechanism to mix the magnetic microbeads in a homogenous medium. Not only does the vibrator ensure uniform distribution of the magnetic microbeads in the solution, it also accelerates the reaction between the probes attached to the magnetic microbeads and the target molecules in the solution. The device is an integrated, compact, and versatile module that can be incorporated into liquid handling robotic systems.

[0053] All biochemistry requires a series of reactions. Probe and target reactions, antigen-antibody reactions, nucleic acid hybridization reactions, and fluorescent labeling reactions at varying temperatures (30-65°C) require a magnetic microbead incubator. The barcoded magnetic microbead handling device described herein is advantageously designed to be integrated into a robotic system. Three common problems with incubators are: 1) difficulty controlling the temperature across all 96 wells to avoid temperature gradients; 2) difficulty preventing evaporation of liquids when heating them; and 3) preventing condensation under any plastic lids or covers.

[0054] To address these issues, the incubator described herein can also be equipped with a microplate plastic cover (not shown) and a heated lid assembly (e.g. Figure 7 (as shown). The microplate plastic lid is used to prevent liquid evaporation, and the heated lid assembly located on top of the microplate lid is used to prevent temperature gradients. Both the microplate lid and the heated lid assembly are picked up by a robot, which moves and releases the heated lid on top of the microplate lid from the docking station. The heated lid assembly 400 includes a heating metal block 401, a cable 402, a thermistor 403, and a latch mechanism 404 for robotic pickup. Once the steady-state set point is reached, the heated lid provides a temperature accuracy within ±1.5°C. The heated lid should have a thermal cutoff to prevent heat loss. The heating plate and heated lid enclose the microplate, maintaining the same temperature for all 96 wells. All reactions can be incubated in a uniform environment. The heated lid includes a thermistor and a spring-loaded "spring pin" 405, which helps prevent the microplate plastic lid from sticking to the heated lid. Four spring pins are mounted underneath the heated lid. The arrangement is advantageous for incorporation into a liquid handling robotic system.

[0055] In view of the foregoing description of its presently preferred embodiments, it is expected that those skilled in the art will effect numerous modifications and alterations in the practice of the invention. Therefore, the only limitations to the scope of the invention are those that appear in the appended claims.

Claims

1. An integrated magnetic microbead processing device comprising: A microplate having a plurality of magnetic microbeads in a plurality of microwells, and a bottom of the microplate having a plurality of cavities between the microwells; a heating / cooling plate supporting the microplate, the heating / cooling plate having a plurality of wells; a vertically movable magnet support plate, the vertically movable magnet support plate comprising a plurality of magnetic pins capable of protruding through the plurality of holes of the heating / cooling plate and entering the cavities of the microplate, the plurality of magnetic pins having adjustable heights; as well as A vibrator is used to vibrate the microplate, the heating / cooling plate and the magnetic support plate.

2. The integrated magnetic microbead handling device of claim 1, wherein the vibrator has an adjustable orbital rotation speed between 0-1200 rpm. 3 . The integrated magnetic microbead processing device according to claim 1 , wherein the height of the magnetic pins protruding above the heating plate can be adjusted according to the liquid level in the microwells.

4. The integrated magnetic microbead handling apparatus of claim 1, wherein the height of the magnetic pins protruding above the heating plate is adjustable between 1.5 mm and 4 mm.

5. The integrated magnetic microbead handling device of claim 1, wherein the heating / cooling plate is configured to generate a temperature of 25°C or room temperature to 60°C.

6. The integrated magnetic microbead handling apparatus according to claim 1, wherein the vibrator has a home position mark to ensure that the orbital rotation stops at the same designated position.

7. The integrated magnetic bead handling device of claim 1, wherein the device has a microplate plastic lid and heated lip assembly that is automated by a liquid handling robotic system.

8. The integrated magnetic bead handling device of claim 1, wherein the microplate has a flat and optically transparent bottom.

9. The integrated magnetic bead handling device according to claim 1, wherein the magnetic microbeads are barcoded magnetic microbeads.

10. An integrated magnetic microbead processing method comprising: Providing a microplate having a plurality of magnetic microbeads in a plurality of microwells, the bottom of the microplate having a plurality of cavities between the microwells; and a heating / cooling plate supporting the microplate, the heating / cooling plate having a plurality of wells; a vertically movable magnet support plate, the vertically movable magnet support plate comprising a plurality of magnetic pins, the magnetic pins being capable of protruding through the plurality of holes of the heating plate into the plurality of cavities of the microplate, the height of the magnetic pins being adjustable; and a vibrator for vibrating the microplate, the heating / cooling plate, and the magnetic support plate to enhance the separation of the magnetic microbeads; activating the vibrator to uniformly suspend the magnetic microbeads using orbital rotation; as well as The magnetic pins were then raised to increase bead capture efficiency.

11. The integrated magnetic bead processing method according to claim 10, wherein the vibrator has an adjustable orbital rotation speed between 0-1200 rpm. 12 . The integrated magnetic microbead processing method according to claim 10 , further comprising the steps of rotating the magnetic microbeads when the magnetic pins are downward, and attracting the magnetic microbeads toward the magnetic pins when the magnetic pins are upward.

Images