Abstract:
An ultrasonic sonication device includes a velocity transformer or probe which, when coupled to a vibrating transducer of the piezoelectric or magnetostrictive type, resonates in sympathy with the transducer and either increases or decreases the magnitude of the transducer&#39;s vibration. A shallow cup assembly is attached to the distal end of the probe. The cup assembly holds a microtiter tray in a suitable orientation and contains an amount of liquid which provides efficient acoustic coupling between a transverse end face of the probe and the microtiter tray.

Description:
BACKGROUND OF THE INVENTION  
         [0001]    This invention relates to ultrasonic vibration probes. More particularly, this invention relates to such an ultrasonic probe or horn assembly which is particularly useful in the simultaneous sonication of biological and cellular materials disposed in multiple wells of a tray.  
           [0002]    It has been well known for decades that a probe which vibrates at ultrasonic frequencies (i.e. frequencies greater than 16,000 Hz) and has its distal end submerged under fluids will create cavitation bubbles if the amplitude of vibration is above a certain threshold. Many devices have been commercialized which take advantage of this phenomenon. An example of such an ultrasonic cellular disrupter is disclosed in the Sonicator™ sales catalog of Misonix Incorporated of Farmingdale, N.Y. In general, devices of this type include an electronic generator for producing electrical signals with frequencies ranging from 16 to approximately 100 KHz, a piezoelectric or magnetostrictive transducer to convert the signal to mechanical vibrations and a probe (a.k.a. horn or velocity transformer) which amplifies the motion of the transducer to usable levels and projects or removes the operating face away from the transducer itself. The design and implementation of these components are well known to the art.  
           [0003]    The cavitation bubbles produced by such ultrasonic vibration devices can be utilized to effect changes in the fluid or upon particles suspended therein. Such changes include biological cell disruption, deagglomeration of clumped particles, emulsification of immiscible liquids and removal of entrained or dissolved gases, among many others.  
           [0004]    Cell disruption has been a particularly good application for probe type devices, in that the cells may be disrupted without the heat or cellular changes which prevent further analysis by conventional methodology. Many scientific protocols have been written which name the Sonicator™ (or similar devices) as the instrument of choice for the procedure.  
           [0005]    One characteristic of the probe type ultrasonic vibration devices which limit their use is the fact that the standard probes must be inserted directly into the fluid. Because the probe occupies volume as it is submersed, very small samples cannot be processed. In addition, the probe becomes contaminated with the fluid since the probe is in direct contact with the fluid. If the probe is subsequently dipped into another sample, contamination of that sample may occur. In some cases, this cross contamination renders the second sample unusable for analysis.  
           [0006]    One way to mitigate these deficiencies is to have the probe tip separated from the sample by a membrane or other solid surface. If liquid is present on both sides of the membrane or surface, the acoustic waves will propagate through the membrane and transfer the cavitation forces to the second liquid volume without having the probe in direct contact with that second liquid volume. This membrane does not have to be elastic. In fact, experience shows that glass or hard plastic is an acceptable material. Consequently, glass and plastic test tubes and beakers are routinely used in this service. Misonix Inc. produces and sells a device called the Cup Horn™ which uses this method of acoustic wave transfer to allow the researcher to segregate the probe from the sample.  
           [0007]    One requirement for use of the Cup Horn is that the beaker or test tube diameter be significantly smaller than the distal diameter of the Cup Horn probe itself. This allows the acoustic energy to be relatively uniform across the diameter of the sample container. In addition, liquid is forced to surround the entire probe end in order to provide the transfer fluid for the acoustic wave. FIG. 1 shows the relationship of the Cup Horn probe  12 , transfer fluid  14  and sample test tube. A cup  16  having a cylindrical sidewall  18 , an inwardly extending annular flange  20  and a cylindrical sleeve  22  is mounted to the horn or probe  12  via a coupling sleeve  24  and a pair of O-rings  26  disposed in a region about a node of ultrasonic vibration of the probe. The transfer fluid not only covers a transverse end face  28  of probe  12 , but also surrounds a substantial portion of the cylindrical distal surface  30  of the probe.  
           [0008]    The requirements of (a) the relative sizes of the probe  12  and the test tube and (b) the surrounding of the probe end surface  30  by the transfer fluid  14  give rise to at least two problems. First, the size of the vessel is limited to that of the surface area of the probe  12  and second, the liquid  14  surrounding the probe  12  places a great load upon the probe. The power required to overcome this load is many times that needed for acoustic coupling into the small sample. In some cases, as the probe has been made larger to accommodate larger samples, the energy required has become greater than the power capability of the electronic generators currently available. In such cases, system overloads have occurred.  
           [0009]    These limitations become especially apparent when the sample vessel takes the form of a multi-well microtiter plate or tray. Such a plate is typically made from clear hard plastic such as polystyrene, polyvinylchloride or acrylics. The tray is fairly shallow and may contain up to approximately 96 depressions (wells) into which the samples or specimens are placed. Each depression may contain only a few microliters of sample. In most cases, the insertion of a probe device is problematic since each sample must be isolated from the others, the wells are too small and the total processing time would be an unacceptable multiple of the processing time of one cell. Therefore, most researchers would prefer a device which would isolate the samples from the ultrasound probe and process all cells simultaneously.  
           [0010]    It would be obvious to most persons skilled in the art to simply enlarge the diameter of the probe to allow the entire tray to be covered. However, as previously stated, the probe becomes very large, leading to non uniformity in the vibrational amplitude of the distal surface, very high power requirements and high cost of manufacture. In the past, probes of smaller square section were made which allow a quarter of the tray to be processed at a time, which decreased processing time substantially. However, most researchers required a further reduction in time in order to process their entire workload in one day. Also, the outer edges of the trays received irregular ultrasonic energy and therefore inconsistent cell breakdown in successive samples.  
         OBJECTS OF THE INVENTION  
         [0011]    An object of the present invention is to provide an ultrasonic device which could treat a full microtiter tray simultaneously.  
           [0012]    Another object of the present invention is to provide such an ultrasonic device which increases the degree of uniformity of acoustic intensity across the cells of the microtiter tray.  
           [0013]    A further object of the present invention is to provide such an ultrasonic device which does not heat the fluid or the sample liquids, and which require minimum energy to operate, thereby allowing the use of the device on existing laboratory scale ultrasonic processors.  
           [0014]    These and other objects of the present invention will be apparent from the drawings and descriptions herein.  
         BRIEF DESCRIPTION OF THE INVENTION  
         [0015]    The present invention is directed to an ultrasonic sonication device which includes two basic components, namely, (1) a velocity transformer (or probe) which, when coupled to a vibrating transducer of the piezoelectric or magnetostrictive type, resonates in sympathy with the transducer and either increases or decreases the magnitude of the transducer&#39;s vibration and 2) a shallow cup assembly which holds a microtiter tray in a suitable orientation and contains an amount of liquid which provides efficient acoustic coupling.  
           [0016]    An ultrasonic horn assembly comprises, in accordance with the present invention, an ultrasonic horn or probe having an axis and a distal end with an end face oriented substantially transversely to the axis. The end face of the probe is disposed at least approximately at an antinode of ultrasonic vibration of the horn or probe. A cup member is attached to the horn or probe at least approximately at the antinode so as to define a liquid reservoir covering the end face of the horn or probe. This attachment of the cup member at, or approximately at, the antinode at the distal end of the probe enables the formation of the reservoir as a shallow reservoir covering essentially only the end face of the probe. A small or marginal circumferential surface of the probe, contiguous with the end face thereof, may be submerged in the coupling liquid, as well.  
           [0017]    In an ultrasonic horn assembly in accordance with the present invention, the load placed upon the probe is decreased owing to the reduction in the area of contact between the coupling fluid and the probe. The power requirements are accordingly reduced for a probe end face of a given area.  
           [0018]    The cup member is attached to the horn or probe via a flexible coupling element such as an O-ring or an annular elastomeric membrane. Where the cup member includes a sidewall and a lower wall or flange extending inwardly from the sidewall, the lower wall is provided with at least one port for feeding liquid to the reservoir. Preferably, the port is one of at least a pair of ports disposed on substantially opposite sides of the cup member. The feeding of the coupling liquid through a lower wall of the cup member has advantages detailed below.  
           [0019]    The end face of the probe is disposed in a first plane and an upper surface of the flange is disposed in a second plane spaced a first predetermined distance from the first plane, so that a lower surface of a specimen-containing tray resting on the upper surface of the flange is spaced a second predetermined distance from the probe end face. This spacing optimizes the acoustic effects of the ultrasonic energy on specimens contained in wells of a microtiter tray. To enable an optimal spacing, the probe end face is provided with a plurality of grooves for receiving peripheral lower edges of the tray so that contact between the tray and the vibrating probe is prevented.  
           [0020]    Where the end face of the probe is circular, the end face has a diameter larger than a largest dimension of the portion of the tray containing the sample wells. Thus, all of the sample wells are located over the end face of the probe.  
           [0021]    In accordance with another feature of the present invention, the probe is provided at the distal end, proximately to the end face, with an annular concavity for providing or enhancing uniformity of the ultrasonic wave field generated in the coupling fluid reservoir.  
           [0022]    An ultrasonic sonication device in accordance with the present invention is an effective apparatus to acoustically treat or disrupt samples within a multiwell microtiter tray. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0023]    [0023]FIG. 1 is a cross-sectional view, taken along an axial plane, of an ultrasonic sonication device in accordance with the prior art.  
         [0024]    [0024]FIG. 2 is a cross-sectional view, taken along an axial plane, of an ultrasonic sonication device in accordance with the present invention.  
         [0025]    [0025]FIG. 3 is a cross-sectional view, taken along an axial plane, of another ultrasonic sonication device in accordance with the present invention.  
         [0026]    [0026]FIG. 4 is a top plan view of the ultrasonic sonication device of FIG. 2, showing a microtiter tray in place on the probe.  
         [0027]    [0027]FIG. 5 is a partial cross-sectional view taken along line V-V in FIG. 4.  
         [0028]    [0028]FIG. 6 is a detail, on a larger scale, of a portion VI of FIG. 5.  
         [0029]    [0029]FIG. 7 is an enlarged top plan view similar to FIG. 4, showing flow paths for a transfer fluid. 
     
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0030]    As illustrated in FIG. 2, an ultrasonic sonication device comprises a horn or probe  32  having an axis  34  defining a direction of ultrasonic standing wave propagation. Probe  32  has a distal end portion  36  formed with an active end face  38  oriented transversely to axis  34  and provided with at least one pair of parallel grooves  40  and  42 . Distal end portion  36  of probe  32  is further formed with an annular groove  44  receiving an elastomeric O-ring seal  46 .  
         [0031]    The ultrasonic sonication device of FIG. 2 additionally comprises a cup member  48  having a vertical cylindrical sidewall  50  and a horizontal annular flange  52  extending inwardly from a lower end of the sidewall. An inner periphery of flange  52  is in fluid tight contact with an outer periphery of distal horn portion  36 , through or over O-ring seal  46 . Flange  52  is provided on opposite sides with a pair of liquid ports or fittings  54  and  56  for the continuous introduction and removal, respectively, of a pressure-wave transfer fluid  58  from a reservoir defined in part by probe end face  38  and cup member  48 .  
         [0032]    As depicted in FIG. 3, a modified ultrasonic sonication device comprises a cup member  60  having a sidewall  50 ′ with a larger diameter than sidewall  50  of cup member  48 . An inner periphery of an annular flange  52 ′ is spaced from and connected to the outer periphery of distal horn portion  36  by an annular elastomeric membrane  62 . Membrane  62  is sealingly fixed along an inner side to distal horn portion  36  and along an outer side to flange  52 ′.  
         [0033]    [0033]FIGS. 4, 5, and  6  depict the use of the sonication device of FIG. 2 with a microtiter tray or plate  64  having a plurality of specimen-receiving wells or cells  66  disposed in a rectangular array. Four corners  68  of tray  64  rest on flange  52  so that a bottom surface  70  (FIG. 6) of the tray is disposed in a plane P 1  spaced a predetermined distance D from a plane P 2  in which the vibrating end face  38  of probe  32  is located. This distance D is selected to optimize the transmission of ultrasonic wave energy from end face  38  through fluid  58  and into tray  64 .  
         [0034]    Tray  64  is conventionally configured to have a peripheral lower rim  72  (FIG. 6) which extends below the plane P 1  of bottom tray surface  70 . This rim  72  is in contact with an upper surface  76  (FIGS.  4 - 6 ) of flange  52  and is spaced from horn or probe  32  by virtue of grooves  40 ,  42 , etc., provided in end face  38 .  
         [0035]    Probe  32  functions in part as a velocity transformer which amplifies the motion of a piezoelectric or magnetostrictive transducer (not shown) to usable levels. Probe  32  can be designed and constructed using standard techniques known to the art. However, several important operating characteristics must be obtained for probe  32  to be useful in this device. First, distal end face  38  of probe  32  must be large enough to cover the entire area of bottom surface  70  of microtiter tray  64 . In the embodiment described herein, distal end face  38  is circular and has a diameter of 5.25 in., but other diameters or geometric shapes may be employed as well. One important aspect regarding size is that microtiter tray wells  66  must not be less than 0.125 inches from an outer edge  74  of probe end face  38 . If a tray cell  66  is located at edge  74  or within 0.125 inches of that edge, acoustic input to the well will be decreased due to ultrasonic edge effects. Second is that it is advantageous if a uniform amplitude of vibration is generated across the entire end face  38  of probe  32 . If significantly non-uniform vibrations are present, then non-uniformity of processing in the microtiter wells  66  will result. In order to obtain this uniform vibration for the size of probe discussed herein, the shape of probe  32  must be as that shown in FIG. 2. It should be noted that the dimensions given describe a probe  32  which has a fundamental resonant frequency of approximately 20 kc. Other frequencies of operation may be employed without deviating from the scope of this disclosure.  
         [0036]    Grooves or reliefs  40 ,  42 , etc., are machined or otherwise formed in probe end face  38  (FIG. 6) to allow microtiter tray edge or rim  72  to sit in these recesses. In this way, the bottom surface  70  of microtiter tray  64  sits within 0.100 inches (preferably between about 0.001 and 0.100 inches) of the vibrating probe end face  38 . Controlling this distance D is of paramount importance if enough acoustic energy is to be transmitted through the wall of tray  64  to the samples contained in wells or cells  66  thereof. The geometry of probe end face  38  is particularly shown in FIGS.  4 - 6 . Of course, probe  32  must be manufactured from an acoustically efficient material such as aluminum, titanium, certain stainless steels and certain ceramics. These materials are all known to the art. Harder materials such as titanium or ceramics will yield a device which does not wear quickly due to cavitation erosion. Connection to the transducer (not shown) can be accomplished by a threaded stud (not shown) or other techniques well known to the art.  
         [0037]    The seal provided by O-ring  46  or membrane  62  is elastomeric to provide a compliant joint between cup member  48  or  60  and probe  32 . This seal is liquid tight and yet isolates cup member  48  or  60  from the vibrations transmitted by probe  32 . This isolation prevents loading and possible detuning of probe  32  while keeping acoustic power from being absorbed by cup member  48  or  60 , preventing melting thereof if the cup member is manufactured from thermoplastics. It is to be noted that O-ring  46  and membrane  62  are placed at or near an anti-node (point of maximum displacement) of probe operation as opposed to being placed at a node (point of no displacement) as is generally practiced by the art. Since the node point is found approximately at the midpoint of probe  12  (see FIG. 1), placing the seal at the node would mean that half of the probe would be submerged under cooling/coupling fluid  14 . Prior art, as shown in FIG. 1, uses the node point sealing method, with all of the inherent problems as described above. Moving the seal position near the antinode (and thus near probe end face  28 ) greatly reduces the power loading and energy consumption of the device.  
         [0038]    Cup members  48  and  60  are fabricated alternatively from clear acrylic and clear polyvinylchloride. However, other materials such as thermoplastics, metals, ceramics or thermosets may be used with equal results.  
         [0039]    Several features of cup member  48  and  60  are important to the operation of the device. First, cup members  48  and  60  must have an internal diameter just slightly greater than the diagonal dimension of the microtiter tray  64 . This centers the tray  64  with respect to the end face  38  of probe  32 , as shown particularly in FIG. 4. Upper surface  76  of flange  52 ,  52 ′ must be designed in conjunction with the dimensions of microtiter tray  64  in order to hold the tray off the probe end face  38  by the proper distance D. To that end, a plane P 3  in which surface  76  is disposed is located at a predetermined distance D 2  (FIG. 6) from the plane P 2  of probe end face  38 . Microtiter tray  64  sits on cup surface  76  and does not contact probe  32  at any point. If tray  64  is allowed to touch end face  38  of the probe, melting of the tray will result.  
         [0040]    Next, cup member  48  or  60  must incorporate liquid fittings or ports  54  and  56 , to allow coupling fluid  58  to be pumped in and out of the cup member. If fluid transport is not provided, then heating of the fluid will result with extended use. The temperatures generated may exceed the cytocoagulation temperature of the biological samples in wells  66 , effectively cooking the specimens. A constant flow of fresh or cooled fluid obviates this eventuality. Although the necessity for cooling is well known to the art, an improvement disclosed herein is to place the fittings  54  and  56  so that the coupling fluid or liquid  58  is introduced and removed from under the microtiter tray  64 . FIG. 7 shows general paths  78  of fluid flow under microtiter tray  64  from one port or fitting  54  to the other port  56 . When the ports or fittings  54 ,  56  are disposed on opposite sides of cup member  48 ,  60  and along flanges  52 ,  52 ′ thereof, the coupling fluid  58  has maximum cooling effect and reduces or eliminates splashing onto the top of the tray  64 , thereby preventing contamination of the samples. Another benefit is extremely important in that the liquid flow as illustrated in FIG. 7 will purge or flush trapped air from the underside or bottom surface  70  of tray  64 . Air bubbles, if present between the probe end face  38  and the bottom surface  70  of the tray  64 , will not allow acoustic coupling to the tray wells  66  and no processing will result. Therefore, bubbles or air entrapment must be eliminated, something which this embodiment accomplishes. In the disclosed embodiment, port elements  54 ,  56  are standard liquid tubular fittings provided on the lower surface of the cup member  48 ,  60 . The coupling fluid or liquid can be plain tap water, saline, distilled water or, if sub freezing temperatures are desired, a solution of glycol and water may be employed.  
         [0041]    In operation, a thin plastic film (not shown) should be applied to the top of microtiter tray  64  in a fashion known to the art. This thin film prevents loss of samples from the tray wells  66  during acoustic processing, from either bubbling or atomization. In addition, cross contamination of samples is eliminated. Although when using non-ultrasonic techniques of sample preparation, this film is optional, the film is deemed essential in use of the ultrasonic sonication devices disclosed herein.  
         [0042]    Cup member  48 ,  60  must incorporate features such as a counterbore to prevent slippage of the cup relative to probe  32 . This prevents the cup from lowering with respect to the probe end face  38  and maintains the clearance between the bottom surface  70  of microtiter tray  64  and the probe end face.  
         [0043]    Although the invention has been described in terms of particular embodiments and applications, one of ordinary skill in the art, in light of this teaching, can generate additional embodiments and modifications without departing from the spirit of or exceeding the scope of the claimed invention. Accordingly, it is to be understood that the drawings and descriptions herein are proffered by way of example to facilitate comprehension of the invention and should not be construed to limit the scope thereof.