Abstract:
This invention is directed to a sample preparation apparatus for grinding or homogenizing test samples. More specifically, but without restriction to the particular embodiments hereinafter described in accordance with the best mode of practice, this invention relates to a reciprocating apparatus based upon a slider-crank mechanism for grinding or homogenizing of a sample within a sample vial attached to an oscillating connecting linkage that has an amplitude of oscillatory motion equal to or greater than the length of the sample processing chamber.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 61/816,094 entitled “Mechanism for Grinding of Biological Samples” filed Apr. 25, 2013, the disclosure of which application, including the specification, claims and drawings, are specifically incorporated herein by reference in their entireties without disclaimer. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention is directed to hand held and table top grinders and homogenizers such as those used to release proteins and nucleic acids from biological samples and those that grind geological samples for composition analysis. Samples may include, for example, seeds, soil, bones, teeth, tissues, spores, yeasts, and gram positive microbes. Certain embodiments, described below, result in more efficient and faster processing of said samples. 
     The most commonly used homogenizers are of the bead mill type (bead-beater), in which a biological or geological sample is added to a container such as a sample vial or tube that is preloaded with grinding media such as, but not limited to, for example, ceramic, glass or metal beads. A special buffer designed to dissolve proteins, nucleic acids, minerals and/or metals may also be added, followed by sealing of the tube typically with a screw cap. The sealed tube is then placed into the homogenizer, which mechanically agitates it in an oscillatory manner, causing the media (hard beads) within the tube to impact and break the sample. Typical rates of oscillation are in the range of 4,000 to 5,000 cycles per minute. This grinding or homogenization of the sample facilitates the extraction of proteins, nucleic acids, metals and/or minerals for use in downstream processes such as amplification, analysis of DNA and/or analysis of metal/mineral composition. 
     The biggest drawback with most bead mill homogenizers is that their range of oscillatory motion is not high enough to cause the beads to traverse the whole length of the sample processing chamber such as a sample vial or tube. Furthermore, if the sample tube is oriented vertically in the homogenizer, gravity tends to cause the beads to localize at the bottom of the tube during homogenization. At the same time, the biological sample, which is less dense than the beads will tend to stay closer to the top of the tube. As a result, the time to achieve complete homogenization is long, usually in the range from 5 to 60 seconds; perhaps longer if the sample is also hard (e.g. dry corn kernel, bone, etc.). Another undesired effect from this inefficient homogenization scheme is the generation of heat proportional to the length of time of homogenization due to internal friction within the tube. This heat is problematic if the desired nucleic acids are RNAs, which quickly degrade at elevated temperatures. 
     The previously mentioned drawbacks could be mitigated if the amplitude of oscillatory motion were to be extended to be at least equal to the length of the tube being used to process a biological sample such as that in the apparatus of the present invention. Doing so would cause the milling beads within the tube to traverse the whole length of the tube, enhancing the incidence of encounters between the beads and the sample. This more efficient design of the apparatus of the present invention in turn results in shorter homogenization times and thus, less heat generation. The preferred embodiment of the present invention has substantially improved grinding/homogenization efficiency, leading to shorter homogenization times, and less heat generation compared to those in the prior art. 
     2. Description of Related Art 
     There are a number of bead mill homogenizers (bead-beaters) on the market including that disclosed in U.S. Pat. No. 5,567,050, entitled “Apparatus and method for rapidly oscillating specimen vessels”, which describes an apparatus and method for rapidly oscillating specimen containing vessels such as those used in an RNA recovery operation wherein small sized glass sized beads in the vessel are employed to disrupt the cell walls of an RNA component to release the RNA, includes a specimen vessel holder provided as a disc in which the containers are received. The disc is operably connected with oscillatory motion producing means that in operation oscillates the disc rapidly in an oscillatory movement up and down symmetrically of a fixed vertical axis. The disc is haltered so it cannot rotate about the fixed axis. Locking means in the form of a locking plate locks the vessels on the vessel holder and applies a clamping force thereto to prevent relative movement between the vessels and the holder to prevent generation of heat that could be of deleterious effect to the specimen material or the vessels holding same. 
     Another example of a bead beater is described in U.S. Patent Application Publication No. 20120263010, entitled “Device for the Quick Vibration of Tubes Containing, In Particular, Biological Samples” which discloses an appliance for rapidly vibrating test tubes containing samples to be ground up, the appliance comprising an electric motor for driving rotation of a disk that is provided with an eccentric pin, the appliance being characterized in that the test tubes are perpendicular to the eccentric pin and are held by a clamp mounted on a support that is substantially parallel to the eccentric pin, being connected to a fixed baseplate via a Cardan type hinge having two mutually perpendicular axes of rotation (X and Y), one of which axes (Y) is substantially parallel to the eccentric pin and connects the support to the other axis (X) of the hinge that prevents the support from moving in rotation about a perpendicular axis (Z). The support is connected to the eccentric pin by a link. 
     Both of the above devices produce a Figure 8 motion, with respective capacities of 12×2 mL and 3×2 mL sample tubes/vials, and peak to peak amplitudes of ⅝ and ¾ inch. Since the sample tubes/vials in both examples are 1.5 inches tall, this means that the sample and hard matrices do not have the opportunity to travel throughout the whole length of the sample vial and they stay mostly at the bottom of the sample vial. In contrast, the apparatus of the present invention produces an elliptical motion/path and has a peak to peak amplitude equal to the length of the sample vial (1.5 inches, for example) allowing the matrices to fully interact with the sample throughout the sample tube/vial (sample processing chamber), making it much more efficient, achieving the same results, as the devices above, in about 1/10th the time of the other bead mill/bead-beater homogenizers. 
     Table 1 below outlines characteristics of commonly used sample grinders and homogenizers, for example, including a preferred embodiment of the apparatus of the present invention showing the peak to peak amplitude advantage of the present invention. The peak to peak amplitude of the present invention is not limited to 1.5 inches but is shown only as an example. Peak to peak amplitude can be adjusted in the apparatus of the present invention to match the sample processing chamber (sample vial) length by varying the diameter of its crank to match the length of the sample processing chamber. 
     
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                   
                   
                   
                 Sample 
                 Frequency 
                 Amplitude 
               
               
                 Product 
                 Type 
                 Action 
                 throughout 
                 Range (cpm) 
                 (inches) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 PRESENT INVENTION 
                 Bead-Beater 
                 crank-slider (combination of circular  
                 1 
                   750-4,400 
                 1.5 
               
               
                   
                   
                 and linear motion) 
                   
                   
                   
               
               
                 Xpedition 
                 Bead-Beater 
                 Vertical Linear impaction 
                 1 
                 3,600 
                 0.25 
               
               
                 Tissue-Tearor 
                 Rotor-stator 
                 Probe Rotor-stator 
                 1 
                  5,000-35,000 
                 not applicable 
               
               
                 Shredder SG3 
                 Pressure 
                 Hydraulic forcing of sample through  
                 1 
                 200 
                 not applicable 
               
               
                   
                   
                 a sieve with simultaneous rotational  
                   
                   
                   
               
               
                   
                   
                 mechanical grinding 
                   
                   
                   
               
               
                 Disruptor Genie 
                 Bead-Beater 
                 horizontal orbital motion 
                 12 
                 1,000-3,000 
                 0.5 
               
               
                 Bead-Bug 
                 Bead-Beater 
                 Arc-shaped trajectory (~2 inch radius) 
                 3 
                 2,700-4,000 
                 0.75 
               
               
                 Mini-Bead beater-1 
                 Bead-Beater 
                 Arc-shaped trajectory (~2 inch radius) 
                 1 
                 2,500-4,800 
                 0.75 
               
               
                 Bullet Blender (BBX6F) 
                 Bead-Beater 
                 Horizontal Linear impaction 
                 24 
                   100-1,200 
                 0.125 
               
               
                 TissueLyser LT 
                 Bead-Beater 
                 Vertical linear motion 
                 12 
                   900-3,000 
                 0.75 
               
               
                 PRECELLYS ® 24 
                 Bead-Beater 
                 Arc-shaped trajectory (~3 inch radius) 
                 24 
                 6,500 
                 0.315 
               
               
                 MINILYS 
                 Bead-Beater 
                 Arc-shaped trajectory (~3 inch radius) 
                 3 
                 3000, 4000, 5000 
                 0.63 
               
               
                   
               
             
          
         
       
     
     SUMMARY OF THE INVENTION 
     The present invention is an apparatus that generates a reciprocating motion for the purpose of grinding, or homogenizing (if liquid is present), biological or geological samples. A motor-driven crank is linked to a linearly-confined carriage via a connecting linkage. The linkage contains a holder for a sample tube or vial. The sample vial is preferably cylindrical or rectangular in shape. As the crank rotates, the sample vial in the holder experiences an elliptical oscillatory reciprocating motion that causes the sample and grinding media, such as, for example ceramic beads, within the sample vial to collide with each other as they traverse the length of the sample vial. The sample breaks apart as a result of the collisions with the beads. The invention allows for substantial reduction of process time down to the range of 1 to 5 seconds versus 5 to 60 seconds for bead-beaters commonly used by laboratories, using the same reciprocation rates of about 4,000 to 5,000 cycles per minute. 
     Compared to the above-mentioned bead-beaters, the apparatus of the present invention stands apart because it is more efficient and therefore about 1 order of magnitude shorter processing times than other bead-beaters. Its mechanism consists of a rotating crank connected to a slider via a connecting linkage (linkage). A holder for a sample vial or tube is attached to middle of the connecting linkage, which is approximately 7.3 inches long in a preferred embodiment of the present invention but can be longer or shorter depending on the desired elliptical path and the number of holders attached thereto, i.e., the apparatus of the present invention may include multiple holders. The diameter of the crank is equal to or greater than the axial length of the sample vial (if the sample vial is 1.5 inches then the crank diameter will be 1.5 inches or greater), so the crank diameter preferably corresponds to the axial length of the sample vial or tube. For example, when a 1.5 inch sample vial is inserted into the holder, it will undergo an elliptical oscillatory motion (elliptical path) with peak to peak amplitude of 1.5 inches, causing the grinding matrices and sample within the sample vial or tube to traverse the whole length of the sample vial or tube thereby maximizing cascade impaction and shearing forces. With the other previously described bead-beaters, the matrices (beads) and samples tend to stay localized at the bottoms of the sample vial or tube, strongly influenced by gravity, which tends to separate the heavy matrices from the lighter matrices into a density gradient. 
     Another important characteristic that gives the apparatus of the present invention an advantage over the other bead-beaters is that the sample vial or tube in the apparatus of the present invention is rigidly held at the top and bottom, making the collisions (during change of axial direction) at those extremities mostly inelastic. This means that most energy will go into cascade collisions instead of moving the tube or the tube holder. The impaction-based bead-beaters experience inelastic collisions mostly at the time of impaction. The rest of the time, the matrices and samples are colliding elastically within the tube. This makes processing with those bead-beaters much more inefficient and time-consuming. 
     The present invention is specifically directed to a mechanical reciprocating apparatus for grinding of samples comprising one or more sample vials or tubes of predetermined length, a grinding media in the sample vial, a holder to hold the sample vial, a connecting linkage where said holder is attached to, said connecting linkage having a distal and proximal end with pivot points at each end, a crank pivot connected to the proximal end of said connecting linkage, a slide pivot connected to the distal end of said connecting linkage, a frame top and a bottom frame wherein the various components of the apparatus are attached, a sliding carriage on a rail that is affixed to the frame top, said sliding carriage is connected to the slide pivot, a crank having a diameter equal to or greater than the length of the sample vial, said crank attached to the crank pivot; and a motor operatively connected to the crank. The connecting linkage may also accommodate multiple holders and the holders may be placed at various distances (adjustable) from the ends of the connecting linkage to optimize its elliptical path of samples in the sample vial. In addition to varying the location of the holder/tube on the connecting linkage, the length of the connecting linkage and the diameter of the crank may also be varied and optimized to achieve an optimal sample/sample vial and/or media path to allow efficient grinding of the samples. The rail of the apparatus of the present invention may alternatively be attached to the connecting linkage and the sliding carriage may be fixed to a pivot point on the frame top of the apparatus instead of the sliding carriage. Many types of commercially available sample vials may be used in the present invention including, for example, but not limited to, cryogenic tubes, multi-well plates, plastic, metal or glass vials, and other sample processing tubes/vials such as those listed in Table 2, below. Different types of motors may also be used for the present invention including, for example; electrically, pneumatically, or hydraulically driven motors. The apparatus of the present invention may be configured as a hand held device or a table top device depending on its capacity for holding multiple sample vials or tubes. 
     
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                   
                   
                 Capacity 
                   
                   
               
               
                 Model # 
                 Description 
                 (mL) 
                 Skirt 
                 Vendor 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 72.693 
                 Screw cap tube 
                 2 
                 no 
                 Sarstedt 
               
               
                 72.694 
                 Screw cap tube 
                 2 
                 yes 
                 Sarstedt 
               
               
                 72.730.406 
                 Screw cap tube 
                 0.5 
                 yes 
                 Sarstedt 
               
               
                 72.703.406 
                 Screw cap tube 
                 1.5 
                 yes 
                 Sarstedt 
               
               
                 330TX 
                 Screw cap tube with  
                 2 
                 no 
                 Biospec 
               
               
                   
                 extra thick walls 
                   
                   
                   
               
               
                 2007 
                 Stainless Steel tubes 
                 2 
                 no 
                 Biospec 
               
               
                 S6003-50 
                 Lysis Tubes prefilled  
                 2 
                 yes 
                 Zymo  
               
               
                   
                 with 2.0 mm 
                   
                   
                 Research 
               
               
                   
                 diameter ceramic balls 
                   
                   
                   
               
               
                 S6002-50 
                 Lysis Tubes prefilled  
                 2 
                 yes 
                 Zymo  
               
               
                   
                 with 0.5 mm 
                   
                   
                 Research 
               
               
                   
                 diameter ceramic balls 
               
               
                   
               
             
          
         
       
     
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Further objects of the present invention together with additional features contributing thereto and advantages accruing therefrom will be apparent from the following description of the preferred embodiments of the invention which are shown in the accompanying drawing figures with like reference numerals indicating like components throughout wherein: 
         FIG. 1  is blow-up illustration of the apparatus of the present invention showing the individual components thereof; 
         FIG. 2  is an isometric view of the apparatus of the present invention; and 
         FIGS. 3A, 3B, 3C and 3D  are a top views of the apparatus of the present invention showing four different angular positions of the crank and corresponding locations of grinding media within a sample vial. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The components of the preferred embodiment of the apparatus of the present invention are shown in the blown-up illustration of  FIG. 1 . The individual components of the apparatus, with like reference numbers corresponding to the drawing of  FIG. 1 , are listed below in Table 3: 
     
       
         
               
               
             
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                 Ref No. 
                 Description 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 10 
                 crank 
               
               
                 11 
                 crank pivot 
               
               
                 12 
                 sliding carriage/ 
               
               
                   
                 carriage/slide 
               
               
                 13 
                 slide pivot 
               
               
                 14 
                 rail 
               
               
                 15 
                 rail screws 
               
               
                 16 
                 linkage or  
               
               
                   
                 connecting linkage 
               
               
                 18 
                 holder 
               
               
                 20 
                 sample vial or tube 
               
               
                 21 
                 frame top 
               
               
                 22 
                 ceramic bead 
               
               
                 26 
                 motor 
               
               
                 28 
                 drive belt 
               
               
                 30 
                 small pulley 
               
               
                 32 
                 big pulley 
               
               
                 34 
                 spacer 
               
               
                 36 
                 spacer 
               
               
                 38 
                 big pulley shaft 
               
               
                 40 
                 small pulley shaft 
               
               
                 42 
                 frame bottom 
               
               
                 44 
                 threaded adapter 
               
               
                 46 
                 frame screws 
               
               
                 48 
                 adapter screws 
               
               
                 50 
                 crank pivot bolt 
               
               
                 52 
                 slide pivot bolt 
               
               
                 54 
                 slide pivot nut 
               
               
                 56 
                 top frame screw 
               
               
                 58 
                 slide pivot  
               
               
                   
                 carriage screw 
               
               
                 60 
                 slide pivot bearing 
               
               
                 62 
                 crank pivot bearing 
               
               
                 100 
                 reference line 
               
               
                   
               
             
          
         
       
     
     As illustrated in  FIG. 1 , a sample tube or vial ( 20 ) fits inside a holder ( 18 ). The holder ( 18 ) is attached to a connecting linkage or linkage ( 16 ). The linkage ( 16 ) sits on top of bearing pivots; the crank pivot ( 11 ) and slide pivot ( 13 ). The proximal end of the linkage ( 16 ) is attached to the crank pivot ( 11 ) via the top frame screws ( 56 ) while its distal end is attached to the slider pivot bearing ( 60 ) in the slider pivot ( 13 ) and is held in place via the slide pivot bolt ( 52 ) and slide pivot nut ( 54 ). The slide pivot ( 13 ) is connected to the sliding carriage or slide ( 12 ) via slide pivot carriage screws ( 58 ). The slide ( 12 ) sits on top of the rail ( 14 ) which is attached to the frame top ( 21 ) via slide screws ( 15 ). The crank pivot ( 11 ) is attached to the crank ( 10 ) via a crank pivot bearing ( 62 ). The crank ( 10 ) is connected to the big pulley ( 32 ) via the big pulley shaft ( 38 ). The big pulley ( 32 ) is driven or turned via a small pulley ( 30 ) through a drive belt ( 28 ). The small pulley ( 30 ) is connected to a small pulley shaft ( 40 ) which connects directly to the motor ( 26 ). The big pulley ( 32 ), big pulley shaft ( 38 ), drive belt ( 28 ) and the small pulley ( 30 ) are held in place between the frame top ( 21 ) and frame bottom ( 42 ) via frame screws ( 46 ) and spacers ( 34  and  36 ). The frame bottom ( 42 ) is attached to a threaded motor adapter ( 44 ) via adapter screws ( 48 ). The threaded motor adapter ( 44 ) allows for the attachment of the device of the present invention to the motor ( 26 ) which drives the small pulley ( 30 ) thereby moving the sample vial ( 20 ) in an elliptical path at a predetermined rate. In an alternate embodiment of the present invention, the sliding carriage ( 12 ) and rail ( 14 ) is made longer so that the holder ( 18 ) could be placed directly on the sliding carriage ( 12 ). The motion of the sample vial ( 20 ) is linear in this alternative embodiment. 
     With reference now to  FIG. 2 , there is depicted an isometric view of a fully assembled apparatus of the present invention with the sample vial ( 20 ) in vial holder ( 18 ) which is in turn mounted on the connecting linkage ( 16 ), which has a pivot point at each end for connecting it to sliding carriage ( 12 ) at the distal end and crank  10  at the proximal end as shown in  FIG. 2 . Slide/carriage ( 12 ) slides on rail ( 14 ), which is fixed to the frame top ( 21 ) of the apparatus as discussed above. Motor ( 26 ) is linked to crank ( 10 ) via small pulley ( 30 ), drive belt ( 28 ), and big pulley ( 32 ). As crank ( 10 ) is driven to rotate by motor ( 26 ), its rotational motion is converted to linear motion as carriage ( 12 ) slides on rail ( 14 ). Since holder ( 18 ) is placed approximately halfway between the crank ( 10 ) and carriage ( 12 ), it experiences a combination of linear and rotational motion, resulting in an elliptical trajectory of sample vial/tube ( 20 ). 
     The next figures,  FIGS. 3A to 3D , shows a top view of the apparatus at four angular positions of the cycle of crank ( 10 ), as it rotates in a counterclockwise direction. Also illustrated is a ceramic bead ( 22 ) that acts as pestle in grinding samples placed in the sample vial ( 20 ). Since the ceramic bead ( 22 ) has a high finite inertia, it will tend to stay in place at the level of reference line ( 100 ) while vial ( 20 ) surrounding it reciprocates in an elliptical path. 
     Specifically,  FIG. 3A  depicts the ceramic bead ( 22 ) at midway the length of the vial ( 20 ), pressed against its left wall as it travels towards the top.  FIG. 3B  depicts the bead ( 22 ) impacting the top end of the tube.  FIG. 3C  depicts the ceramic bead ( 22 ) again midway the length of the tube, pressed against the right wall as it travels towards the bottom of the tube. Finally, the ceramic bead ( 22 ) impacts the bottom of the tube in  FIG. 3D . 
     OPERATION: 
     Example 1 
     
         
         
           
             1. A sample to be ground or homogenized, if liquid is present, is inserted into a sample vial ( 20 ) that has preloaded hard grinding matrices [ceramic beads ( 22 )] inside. 
             2. The vial ( 20 ) is sealed and inserted into the holder ( 18 ) of the device. 
             3. The device is turned on for a set period of time (usually 5 seconds or less, for example) at about 4,000 to 5,000 cycles per minute to cause the sample to be ground or homogenized. 
             4. The tube is removed from the holder and unsealed to remove the ground sample for analysis. 
           
         
       
    
     Example 2 
     
         
         
           
             1. The user determines the optimal location for the holder on the connecting linkage. 
             2. The holder is attached onto the connecting linkage at the pre-determined optimal location (distance from the crank pivot) to allow optimal grinding or homogenization of a sample. 
             3. A sample to be ground or homogenized, if liquid is present, is inserted into a sample vial ( 20 ) that has preloaded hard grinding matrices [ceramic beads ( 22 )] inside. 
             4. The vial ( 20 ) is sealed and inserted into the holder ( 18 ) of the device. 
             5. The device is turned on for a set period of time (usually 5 seconds or less, for example) at about 4,000 to 5,000 cycles per minute to cause the sample to be ground or homogenized. 
             6. The tube is removed from the holder and unsealed to remove the ground sample for analysis. 
           
         
       
    
     CONCLUDING STATEMENT 
     All patents, provisional applications, patent applications and other publications mentioned in this specification are herein incorporated by reference. 
     While this invention has been described in detail with reference to certain preferred embodiments, it should be appreciated that the present invention is not limited to those precise embodiments. Rather, in view of the present disclosure, which describes the current best mode for practicing the invention, many modifications and variations would present themselves to those of skill in the art without departing from the scope and spirit of this invention. The scope of the invention is, therefore, indicated by the following claims rather than by the foregoing description. All changes, modifications, and variations coming within the meaning and range of equivalency of the claims are to be considered within their scope. 
     Furthermore, those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.