Patent Publication Number: US-2021162358-A1

Title: Device and method for mixing and bubble removal

Description:
PRIORITY CLAIM 
     This application claims priority from U.S. Provisional Patent Application No. 62/213,669 filed on Sep. 3, 2015, which is hereby incorporated by reference in its entirety in the present application. 
    
    
     BACKGROUND 
     Technical Field 
     The present disclosure relates to the field of mixing devices and mixing methods, and in particular, to a magnetic mixing device and method for using a magnetic spin bar to circulate fluid and remove air bubbles within a reaction chamber. 
     Background 
     The mixing of solutions is routinely used in many industrial processes and is often essential in some chemical and biological reactions. Mixing is beneficial in any chemical or biological reaction where an equal and homogeneous concentration is needed throughout the solution. 
     In biotechnology, mixing is generally performed by methods such as pipette mixing or vortexing. There are advantages and disadvantages of each method, for example, mixing may yield variability between samples. 
     Biotechnology companies use extensive amounts of culture media, buffers, and reagents. Such materials originally come in powdered form and must be rehydrated prior to use. Rehydrating the reaction components reduces reaction time and improves consistency between reactions. 
     In one area of biotechnology where rehydration of reaction components is done for nucleic acid amplification, the reaction components may be rehydrated by pipette mixing or vortexing, or thermal mixing before the reaction begins. Pipette mixing requires both specialized laboratory skills and specialized laboratory equipment. Vortexing requires specialized laboratory equipment that is not typically battery operated or portable. Both vortexing and pipette mixing are variable, depending on the manner in which the operator performs the process. For example, there may be variability in the number of times the fluid is cycled through the micropipette or the duration of vortexing. Thermal mixing occurs during the temperature cycling, however optimal amplification may be delayed a few cycles until the solution is properly mixed. Thermal mixing is time-consuming, often taking approximately 10 minutes for the reaction solution to become thoroughly mixed. When using the invention, all parameters of the process are controlled, for example motor ramp up rate, motor revolutions per minute, motor duration at maximum revolutions per minute, and motor ramp down rate. 
     Air bubble(s) may get trapped within or beneath the fluid. Traditionally, the air bubble is removed by methods such as a centrifugation or pipette mixing. Another common method of removing air bubbles is tapping the reaction plate or the tube. In a magnetic mixer where the stir bar rotates at the bottom of the reaction chamber, the air bubble may not be disrupted and may remain at the bottom of the reaction chamber. An air bubble can cause inconsistent results for a number of reasons including reducing the effective volume of the reaction, preventing the reaction from achieving the appropriate reaction temperatures, interfering with a detector, and preventing the complete mixing of reaction components. 
     A device that quickly rehydrates dried reaction components, produces evenly distributed mixing throughout the reaction volume, and removes air bubbles in a reaction chamber is highly desirable. It is also advantageous if this device is designed so any operator can use it, so it does not require a trained technician. Therefore, it is desirable that the device be configured to alert a user if the device is not operating properly. 
     SUMMARY 
     The present disclosure is directed to a magnetic mixer that can be used for mixing in any chemical or biological application. It is to be understood that the term “mix” in this disclosure refers to any movement that creates a uniform solution, e.g. mix, stir, blend, agitate, etc. 
     It is to be understood that the term “holder” in this disclosure refers to any mechanical expedient, e.g. support, spindle, bracket, prop, etc. 
     Consistent with a disclosed embodiment, a device is disclosed that quickly mixes solutions and removes air bubbles that may be present in the reaction chamber. One application of the device and method of the present disclosure is the quick rehydration of dried reaction components by a magnetic mixer. In a nucleic acid amplification assay, for example, the dried down reaction components must be rehydrated to re-suspend reagents, reduce reaction time and improve consistency between reactions. Dried down reaction components may include, but are not limited to, polymerase chain reaction (PCR) primers, PCR probes, nucleotides, taq polymerase, magnesium chloride, Bovine Serum Albumin, trehalose, and PCR buffer. Dried reaction components may also include, but not be limited to, NASBA, RPA, HDA, LAMP, RCA, ICAN, SMART, SDA, and LDR reaction components. 
     The geometry of a reaction chamber could cause air bubble(s) to be trapped within or beneath the fluid. The present disclosure describes devices and methods that address both challenges of quick mixing and removing trapped air bubbles. 
     Consistent with an exemplary embodiment of the present disclosure, a device is disclosed that detects when a magnetic stir bar is moving within a reaction chamber. A control unit detects a change in current to a motor due to the presence of a magnetic stir bar and a feedback loop provides data to a magnetic mixing device or a master instrument. A lack of change in current to a motor represents the absence of a magnetic stir bar within a reaction chamber. The feedback loop to a master instrument can alert an untrained user to the absence of the magnetic stir bar and any other potential problems with the magnetic mixing device. 
     Additionally, a device is disclosed that contains at least two magnetic stir bars of a shape that promotes a grinding-type action to breakdown a sample. 
     The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate various embodiments and together with the description, serve to explain the principles of the various aspects of the embodiments. Other embodiments of this disclosure are disclosed in the accompanying drawings, description, and claims. Thus, this summary is exemplary only, and is not to be considered restrictive. 
    
    
     
       BRIEF DESCRIPTION OF DRAWING(S) 
       The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate the disclosed embodiments and together with the description, serve to explain the principles of the various aspects of the disclosed embodiments. In the drawings: 
         FIG. 1  illustrates a cross-sectional view of an exemplary embodiment of an exemplary magnetic mixer of the present disclosure; 
         FIGS. 2A and 2B  illustrate the complete mixing of the dried components within the reaction chamber before and after mixing; 
         FIGS. 3A and 3B  illustrate an air bubble trapped by the introduction of a liquid, represented by hatched lines, to the reaction chamber and the removal of the air bubble after mixing; 
         FIG. 4  illustrates the logic of a master instrument controlling the magnetic mixing device, in accordance with an exemplary embodiment of the present disclosure. 
     
    
    
     It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims. 
     DESCRIPTION OF EXEMPLARY EMBODIMENTS 
     Reference will now be made in detail to certain embodiments consistent with the present disclosure, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts. 
     The present disclosure describes a magnetic mixing device. Exemplary embodiments may include a rare earth magnet that attracts and/or repels a paramagnetic stir bar. 
     The present disclosure further describes a device that removes trapped air bubbles beneath or within a fluid. 
       FIG. 1  illustrates an exemplary magnetic mixing device  10  comprising a source of motive power  20 , a holder  30  containing an embedded magnet  40 , a reaction chamber  50 , stir bar(s)  60 , housed in an outer housing  70 . The stir bar(s)  60  is is co-located within the reaction chamber  50 . 
     In some embodiments, source of motive power  20  may be an electric motor, which in an illustrative embodiment is configured to generate a circular motion. In alternative embodiments, source of motive power  20  may be an electric linear actuator configured to generate linear motion. In other embodiments, the movement of magnet  40  may be performed without a motor or a linear actuator, for example, by hand or other manual means. The source of motive power  20  can also be mixed-mode, relying on more than one motor, actuator, manual motion, etc. 
     Another aspect of the present disclosure is holder  30  that contains one or more embedded magnet  40 . In an exemplary embodiment, holder  30  is embedded with one rare earth magnet  40 . In alternative embodiments, holder  30  has more than one rare earth magnet  40  embedded in holder  30 . In alternative embodiments, magnet  40  is another type of permanent magnet. In alternative embodiments, magnet  40  is an electromagnet. In alternative embodiments, holder  30  itself is a permanent magnet or an electromagnet. In alternative embodiments, magnet  40  is moved relative to reaction chamber  50  without being embedded in a holder. 
     Reaction chamber  50  may be configured for various functionalities. In an exemplary embodiment, reaction chamber  50  may be optimized to perform a nucleic acid amplification reaction. In another exemplary embodiment, reaction chamber  50  may be contained within cartridge  90 . 
     Another aspect of the present disclosure is one or more magnetic stir bar  60 . The motion of stir bar  60  is driven by the movement of magnet  40 . Exemplary shapes of stir bar  60  may include disc, rod, cross, ring, and any other shape or construction capable of mixing. The size of stir bar  60  should be coupled to the size of reaction chamber  50  to allow stir bar  60  to move unrestricted. 
     In exemplary embodiments, stir bar  60  may be stainless steel, though stir bar  60  may be any magnetic or paramagnetic material. The magnetic material of stir bar  60  may be coated or uncoated. In exemplary embodiments, stir bar  60  is coated with a material that does not react with or contaminate the reaction components. In an exemplary embodiment, stir bar  60  may be coated with parylene. In alternative embodiments, the coating may be any number of coatings other than parylene. In such embodiments, a preferred coating is one that has been FDA approved for use in food, drug, and cosmetic applications. 
     Another aspect of the present disclosure is housing  70 , which is used to align source of motive power  20  and reaction chamber  50 . 
     Holder  30  orientation relative to reaction chamber  50  may be in any manner that allows for movement of stir bar  60  within reaction chamber  50 . In one embodiment, holder  30  is rotated parallel relative to reaction chamber  50 . In another embodiment, holder  30  is rotated perpendicularly relative to reaction chamber  50 , along the longitudinal axis of reaction chamber  50 . In another embodiment, mixing device  10  is configured to allow reaction chamber  50  to sit at the center and holder  30  moves magnet  40  around reaction chamber  50 . In an exemplary embodiment, holder  30  may be orientated to rotate alongside of reaction chamber  50  in a manner so that stir bar  60  moves vertically within reaction chamber  50 . 
       FIG. 2A  illustrates the introduction of dried components  110  and fluid  100  within a reaction chamber  50 .  FIG. 2B  illustrates the resulting uniform solution  120  after mixing in a magnetic mixing device of the present disclosure. 
     An air bubble  80  may be trapped at the bottom of reaction chamber  50  when fluid is introduced, as shown in  FIG. 1  and  FIG. 3A . In an orientation where stir bar  60  rotates at the bottom of reaction chamber  50 , trapped air bubble  80  may not be removed by motion of stir bar  60 . In an orientation that moves stir bar  60  vertically within reaction chamber  50 , air bubble  80  trapped at the bottom of a narrow reaction chamber  50  can be disrupted and/or air bubble  80  can be forced to the top of reaction chamber  50 . 
       FIG. 3A  illustrates air bubble  80  trapped in a solution caused by the introduction of a liquid, represented by hatched lines, to reaction chamber  50  within cartridge  90 .  FIG. 3B  illustrates the removal of the air bubble after mixing using a magnetic mixing device of the present disclosure. 
     In an embodiment where the fluid has a viscosity high enough that air bubbles  80  may be trapped within the fluid, such as a gel or cream, an orientation that moves stir bar  60  vertically within reaction chamber  50  may similarly disrupt air bubbles  80 . 
     Further, an orientation that moves stir bar  60  vertically within reaction chamber  50  may result in a turbulent flow, rather than a predictable vortex. The vertical motion advantageously creates an evenly distributed mixing throughout the reaction volume. 
     It will be apparent to those skilled in the art that in embodiments where stir bar  60  is of a paramagnetic material, the distance between magnet  40  and stir bar  60  must be within range to effectively attract or repel stir bar  60  to cause the desired movement of stir bar  60  within reaction chamber  50 . 
     In an embodiment where there is more than one reaction chamber  50 , corresponding stir bar(s)  60  must be in each reaction chamber  50 . They must be positioned adjacent to magnet  40  within a range to effectively attract or repel stir bars  60  to cause the desired movement within respective reaction chambers  50 . 
     In an alternative embodiment, magnet  40  may be stationary without the use of a holder, and reaction chamber  50  may be moved relative to magnet  40  by source of motive power  20  in the form of an electric motor or other manual means. 
     The present disclosure may contain more than one magnetic stir bar  60 , within reaction chamber  50 , of a shape to optimize a grinding-type action to break down a sample. 
     In an alternative embodiment, a microprocessor controls the ramp rate and speed of the source of motive power  20 , for instance a motor, and therefore stir bar  60 . In an exemplary embodiment a master control unit controls magnetic mixing device  10  and selects a mixing protocol. The control unit may be part of a larger master instrument. 
     In such an embodiment, a control unit is configured to detect if magnetic stir bar  60  is within reaction chamber  50 . A current necessary to run a motor serving as the source of motive power  20  without the presence of magnetic stir bar  60  is known. A control unit detects a difference in that current when magnetic stir bar  60  is present and moving within reaction chamber  50 . A lack of a change in current may represent an absence of magnetic stir bar  60  within reaction chamber  50 . A feedback loop provides input to magnetic mixing device  10  or a master instrument. An alert may be provided to a user regarding the absence of magnetic stir bar  60  and of any other potential problems with the magnetic mixing device. 
     In an alternative embodiment, a master device controls magnetic mixing device  10  as a slave device. In an exemplary embodiment, the master device is a master instrument that controls magnetic mixing device  10 .  FIG. 4  illustrates an exemplary logic of a master instrument in controlling a magnetic mixing device  10 . By way of background, when an assay is developed, an optimal thermal protocol, mixing protocol, and results interpretation methodology is prescribed. These pieces of information can be advantageously encoded onto an information carrier, else contained in a database that can be referenced by indicia. The information carrier can be in an illustrative embodiment a bar code, such as a 2-D barcode, which can contain, or provide the location of such information, for example a thermal protocol ID, a mixing protocol ID, a results interpretation ID, the manufacturing lot number of the assay, the catalog number of the assay, etc. At step  130 , the illustrative master instrument reads a bar code. At step  140 , the master instrument chooses a mixing protocol. This can be accomplished, for instance, by reference to an online or offline database and/or filesystem to retrieve the information referred to by the ID number. At step  150 , the master instrument sends commands to magnetic mixing device  10 . At step  160 , magnetic mixing device  10  sends a reply to the master instrument. In exemplary embodiments, the instrument may communicate with the bar code module over USB, by wired or by wireless communications. The command structure in an exemplary embodiment may be modified as necessary to communicate with the bar code module employed. However any other communication protocol could be used, for instance serial, RS232, RS485, SPI, I2C, WiFi direct, Bluetooth, etc. Imaging systems such as cameras are also envisioned for capturing data labels, for example QR codes. RFID or other electromagnetic-based information tags can also be used to encode the information described above in place of an optical system. 
     Another embodiment of the present disclosure provides a kit for amplifying DNA from dried reaction components. The kit may comprise a magnetic mixing device as described above, dried down reaction components, and a thermocycler for DNA amplification. 
     An exemplary method of mixing by magnetic mixing device  10  will now be described. In describing the exemplary method, it will be assumed that a user is operating magnetic mixing device  10  shown in  FIG. 1 . However, it should be understood that an automated, semi-automated, or manually operated machine could also operate device  10  in a similar manner. 
     A user may fill reaction chamber  50  with the desired substances to be mixed, provided there is at least one substance that is a fluid. At least one magnetic stir bar  60  must be inserted into reaction chamber  50 , co-located with the substances to be mixed. Through movement of magnet  40  relative to a stationary reaction chamber  50  or movement of reaction chamber  50  relative to a stationary magnet  40 , magnetic stir bar  60  moves within reaction chamber  50  to create turbulent flow and uniform mixing throughout. Alternatively, a user may receive reaction chamber  50  containing at least one of the desired substances to be mixed, and/or magnetic stir bar  60 . Reaction chamber  50  may be contained within cartridge  90 . In an embodiment where magnetic stir bar  60  moves vertically throughout reaction chamber  50 , any air bubbles  80  present within or beneath the fluid are disrupted and/or moved to the top of reaction chamber  50 . 
     In an exemplary embodiment, a microprocessor may control the ramp rate and the speed of a motor serving as the source of motive power  20 . 
     In an exemplary embodiment where magnetic mixing device  10  is used for nucleic acid amplification, a user fills reaction chamber  50  with dried reaction components  110  and a liquid. Through the use of magnetic mixing device  10 , the dried reaction components  110  are rehydrated in the liquid. Alternatively, the user may receive reaction chamber  50  with dried reaction components  110  and magnetic stir bar  60  already within reaction chamber  50 . Reaction chamber  50  may be contained within cartridge  90 . 
     In an exemplary embodiment, magnetic mixing device  10  may be used before the reaction chamber is placed in a master instrument. 
     In an alternative embodiment, magnetic mixing device  10  may be used during the run on a master instrument. At a specified time, the user may remove the reaction chamber from the master instrument, place the reaction chamber in magnetic mixing device  10 , run a mixing protocol, and place the reaction chamber back in the master instrument. 
     In an alternative embodiment, one or more magnetic mixing devices may be incorporated as part of a master instrument, rather than as an accessory that communicates with a master instrument. 
     Table 1 presents data from a set of experiments where the magnetic mixing device was used for nucleic acid amplification. The data indicates the cycle threshold comparison between magnetic mixing, thermal mixing, and pipette mixing. The cycle threshold is the number of cycles of amplification required to cross a threshold value. The data shows magnetic mixing is equal to or better than conventional thermal mixing or pipette mixing, with the added advantages of being faster and allowing an unskilled user to operate the magnetic mixing device. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Comparison between Magnetic Mixing, 
               
               
                 Thermal Mixing, and Pipette Mixing 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Magnetic 
                 Thermal 
                 Pipette 
               
               
                   
                 Assay 
                 Mixing 
                 Mixing 
                 Mixing 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Type 1 
                 29.4 
                 30.1 
                 30.4 
               
               
                   
                 Type 1 
                 30.3 
                 30.4 
                 30.1 
               
               
                   
                 Type 2 
                 33.8 
                 36 
                 not tested 
               
               
                   
                 Type 2 
                 34.2 
                 36.1 
                 not tested 
               
               
                   
                 Type 1 
                 31.4 
                 33.7 
                 not tested 
               
               
                   
                 Type 1 
                 30.8 
                 33.8 
                 not tested 
               
               
                   
                 Type 1 
                 31.4 
                 35 
                 not tested 
               
               
                   
                 Type 1 
                 31.3 
                 33 
                 not tested 
               
               
                   
                 Type 1 
                 29.9 
                 30.7 
                 not tested 
               
               
                   
                 Type 2 
                 37.5 
                 not tested 
                 36.8 
               
               
                   
                 Type 2 
                 37.4 
                 not tested 
                 37.2 
               
               
                   
                 Type 2 
                 37.7 
                 not tested 
                 37.9 
               
               
                   
                 Type 2 
                 37.3 
                 not tested 
                 37.9 
               
               
                   
                 Type 2 
                 35.5 
                 not tested 
                 38.4 
               
               
                   
                 Type 2 
                 38.3 
                 not tested 
                 37.8 
               
               
                   
                 Type 2 
                 37.2 
                 not tested 
                 38.2 
               
               
                   
                 Type 2 
                 37.9 
                 not tested 
                 38.5 
               
               
                   
                   
               
            
           
         
       
     
     The foregoing description has been presented for purposes of illustration. It is not exhaustive and is not limited to the precise forms or embodiments disclosed. Exemplary embodiments have been presented as being used for nucleic acid amplification, this disclosure is not limited to nucleic acid amplification and can be used for mixing in any chemical or biological application. Other embodiments will be apparent to those skilled in the art from consideration of the specification and practice of the embodiments disclosed herein. 
     Moreover, while illustrative embodiments have been described herein, the scope of any and all embodiments include equivalent elements, modifications, omissions, combinations (e.g., of aspects across various embodiments), adaptations and/or alterations as would be appreciated by those skilled in the art based on the present disclosure. The limitations in the claims are to be interpreted broadly based on the language employed in the claims and not limited to examples described in the present specification or during the prosecution of the application. The examples are to be construed as non-exclusive. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the embodiments being indicated by the following claims and their full scope of equivalents.