U.S. patent application number 13/711807 was filed with the patent office on 2013-07-11 for methods and apparatus for treating samples with acoustic energy.
This patent application is currently assigned to Covaris, Inc.. The applicant listed for this patent is Covaris, Inc.. Invention is credited to Brevard S. Garrison, James A. Laugharn, JR., Douglas A. Yates.
Application Number | 20130177922 13/711807 |
Document ID | / |
Family ID | 38997723 |
Filed Date | 2013-07-11 |
United States Patent
Application |
20130177922 |
Kind Code |
A1 |
Laugharn, JR.; James A. ; et
al. |
July 11, 2013 |
METHODS AND APPARATUS FOR TREATING SAMPLES WITH ACOUSTIC ENERGY
Abstract
This invention relates to systems and methods for applying
acoustic energy to a sample. According to one aspect of the
invention, a system comprises a housing, a chamber for receiving
the sample, an acoustic energy source for providing a focused
acoustic field to the sample according to a treatment protocol, a
processor for determining the treatment protocol, a sensor for
detecting information about the sample, and a user interface for
communicating with a user.
Inventors: |
Laugharn, JR.; James A.;
(Winchester, MA) ; Garrison; Brevard S.; (Reading,
MA) ; Yates; Douglas A.; (North Andover, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Covaris, Inc.; |
Woburn |
MA |
US |
|
|
Assignee: |
Covaris, Inc.
Woburn
MA
|
Family ID: |
38997723 |
Appl. No.: |
13/711807 |
Filed: |
December 12, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11888708 |
Aug 1, 2007 |
8353619 |
|
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13711807 |
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60923335 |
Apr 13, 2007 |
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60834979 |
Aug 1, 2006 |
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Current U.S.
Class: |
435/7.1 ;
422/128; 422/20; 436/174 |
Current CPC
Class: |
C01B 25/327 20130101;
B01F 11/0283 20130101; C12Q 1/6806 20130101; G01N 2001/2866
20130101; B01L 99/00 20130101; G01N 1/286 20130101; G01N 1/28
20130101; B01F 2215/0454 20130101; B01J 2219/00486 20130101; G01N
2035/00554 20130101; Y10T 436/25 20150115 |
Class at
Publication: |
435/7.1 ; 422/20;
422/128; 436/174 |
International
Class: |
G01N 1/28 20060101
G01N001/28 |
Claims
1-34. (canceled)
35. A method of processing a sample held within a vessel, the
method comprising: subjecting the sample to pressurization such
that a pressure within the vessel is greater than a pressure
outside of the vessel; providing focused acoustic energy to the
sample held within the vessel, the focused acoustic energy having a
frequency of between about 100 kilohertz and about 100 megahertz
and a focal zone having a width of less than about 2 centimeters,
and the focused acoustic energy originating from an acoustic
transducer exterior to the vessel; and inducing cavitation within
the vessel at a location proximate to the sample held within the
vessel.
36. The method of claim 35, wherein the pressure within the vessel
is greater than standard atmospheric pressure.
37. The method of claim 35, wherein pressurization of the sample
lowers a threshold of acoustic energy required for cavitation
within the vessel to occur.
38. The method of claim 35, wherein subjecting the sample to
pressurization comprises adding at least one of compressed air,
nitrogen, argon, helium and inert gas to a space within the
vessel.
39. The method of claim 35, wherein subjecting the sample to
pressurization comprises sealing the vessel with a cover, piercing
the cover with a needle, and delivering at least one fluid through
the needle into the vessel.
40. The method of claim 35, wherein subjecting the sample to
pressurization comprises reducing a volume of the vessel.
41. The method of claim 40, wherein subjecting the sample to
pressurization comprises sealing the vessel with a plunger and
moving the plunger in a direction that reduces the volume of the
vessel.
42. The method of claim 40, wherein the vessel comprises a
deformable container and subjecting the sample to pressurization
comprises sealing the vessel with a clip and moving the clip in a
direction that reduces the volume of the vessel.
43. The method of claim 35, wherein providing focused acoustic
energy to the sample sterilizes the sample.
44. An apparatus for treating a sample using acoustic energy, the
apparatus comprising: a vessel constructed and arranged to hold the
sample and to subject the sample to pressurization such that a
pressure within the vessel is greater than a pressure outside of
the vessel; and an acoustic energy source comprising a transducer
that is spaced from and exterior to the vessel, the transducer
being adapted to generate acoustic energy for forming at least one
focused acoustic field having a frequency of between about 100
kilohertz and about 100 megahertz and a focal zone having a width
of less than about 2 centimeters.
45. The apparatus of claim 44, wherein the vessel comprises a cover
configured to form an air-tight seal between a space within the
vessel and outside of the vessel.
46. The apparatus of claim 45, wherein the cover comprises a
pierceable septum that allows for delivery of at least one fluid
into the space within the vessel for pressurization of the
sample.
47. The apparatus of claim 45, wherein the cover comprises a
plunger movable in a direction that reduces a volume of the vessel
for pressurization of the sample.
48. The apparatus of claim 44, wherein the vessel comprises a
deformable container.
49. The apparatus of claim 48, further comprising a clip
constructed and arranged to seal the vessel and to move in a
direction that reduces a volume of the vessel.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/888,708 filed Aug. 1, 2007 entitled
"Methods and Apparatus for Treating Samples with Acoustic Energy,"
which claims the benefit of priority from U.S. Provisional Patent
Application No. 60/834,979 filed Aug. 1, 2006 entitled "Methods and
Apparatus for Treating Samples with Acoustic Energy" and U.S.
Provisional Patent Application No. 60/923,335 filed Apr. 13, 2007
entitled "Methods and Apparatus for Focused Ultrasonic Sample
Processing under High Pressure." The disclosures of each of the
foregoing applications is incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The invention generally relates to the field of controlled
acoustic energy-emitting devices for treating biological and/or
chemical material, and more particularly to performing such
treatment in a laboratory or benchtop setting.
BACKGROUND
[0003] Ultrasonics have been utilized for many years in a variety
of diagnostic, therapeutic, and research purposes. Some uses of
ultrasonic or acoustic energy in materials processing include
breaking up and/or mixing of fluid suspensions of materials.
Additional uses are in solubilizing or otherwise ensuring that all
or substantially all of the constituents of a sample are in
solution and/or in suspension. Regardless of the particular use,
sample materials are typically contained in a plastic or glass
enclosure, such as vials, tubes, culture plates/well, or
micro-titer plates, with an acoustic transducer coupled to the
sample by way of a coupling medium, such as water. Typically,
systems in which acoustic energy is precisely controlled and
transferred to a sample in a vessel are relatively low power.
Examples of low power systems include high-frequency, low-intensity
focused acoustic dispensing systems, which transfer droplets of
sample from a fluid-air interface through an air gap to a receiving
vessel, and high-frequency focused interrogation systems commonly
used in non-destructive testing of materials. Alternatively,
acoustic transducers can be directly immersed in the material to be
treated. This type of system, in which an acoustic transducer
directly contacts the sample, is capable of relatively high power;
however, it is typically of lower frequency. A distinct
disadvantage of lower frequency systems is the lack of control
inherent with long wavelength acoustics. For example, the
low-frequency probe-type sonicator typically used in biological and
chemical laboratories is operated at approximately 15 KHz, which
results in wavelengths in aqueous media measuring several
centimeters. Other systems can implement both high-power and
high-control processing of diverse samples. However, there exists a
need for a system with both high-power and high-control which is
easy to use on a routine basis with minimal a priori sample
preparation, process optimization, or operator training.
[0004] The foregoing arrangements have been used for a number of
applications, including large-scale batch processing, yet there is
still a need for acoustic systems and methods that are more
flexible, convenient, and effective, in particular for on-demand
uses, such as for automated processing of small quantities of
samples, for example, in laboratory or benchtop settings.
SUMMARY
[0005] The invention provides methods and systems for selectively
exposing a sample or samples to acoustic energy in a benchtop or
laboratory setting for the purpose of, for example, heating,
fluidizing, mixing, stirring, disrupting, comminuting, sterilizing,
or solubilizing the sample, or for enhancing a reaction in the
sample. The foregoing applications are merely illustrative, and one
skilled in the art will recognize other uses for the application of
focused acoustic energy. Altering the sample in a controlled
manner, especially biological and chemical samples, allows
manipulation of the sample while preserving the viability, chemical
and/or biological activity of the material. Samples may comprise
one or more constituents such as, for example, solvents, reagents,
nucleic acids, proteins, small organic or inorganic molecules,
chemical compounds, or pharmaceutical or biopharmaceutical agents.
Non-clinical samples may also advantageously be treated by acoustic
energy. A sample to be processed with acoustic energy may be
physically isolated in a vessel from the surrounding environment
and an acoustic energy source (e.g., transducer) which applies
acoustic energy to the sample. The acoustic energy may be applied
to the sample through a coupling medium such as water.
[0006] The term "acoustic energy" used herein refers to acoustic
energy, acoustic waves, acoustic pulses, including forms of
ultrasonic energy and/or shock waves. As used herein, sonic
energy/acoustic energy refers to the focused, high frequency (e.g.,
typically 100 kHz-100 MHz; greater than 500 kHz; greater than or
approximately equal to 1 MHz; etc), short wavelength (e.g.,
approximately 1-1.5 mm), acoustic energy. As used herein, focal
zone or focal point means an area where sonic energy converges
and/or impinges on a target, although that area of convergence is
not necessarily a single focused point. According to one aspect,
the present invention provides an acoustic energy source that
provides a focused acoustic field. The acoustic energy source can
be a focused transducer having a focal length, which generates an
ellipsoidal focal zone. The focused transducer may be spherical,
ellipsoidal, cylindrical, or any other suitable shape. The acoustic
focal length of the focused transducer may be any suitable length,
e.g., from 0.1-65 mm or more in diameter. The focal zone resulting
from the focused transducer may be between 0.1 millimeter and 2
centimeters in diameter, e.g., between 0.1 mm and 100 mm, or
between 0.1 nun and 10 mm, and the axial length of the focal zone
may be between 0.1 millimeter and 6 centimeters, for example,
depending on the size of the sample vessel.
[0007] In one aspect, the present invention provides a benchtop
apparatus that can treat a sample effectively with little input
from a user. In certain embodiments, the apparatus may also offer
the user varying degrees of control over the treatment applied to
the sample. In certain embodiments, an apparatus of the invention
may feature one or more components such as a user interface for
communicating with the user or an easily accessed chamber for
holding the sample.
[0008] In certain embodiments, an apparatus of the invention may
include an interchangeable memory component for storing treatment
protocols. Interchangeable memory components can include memory
cards, flash drives, CDs, DVDs, CD-ROMs, diskettes, chips, and any
other suitable memory storage device. Treatment protocols may be
preprogrammed, adjust to inputs from the user, adjust to measured
changes in the sample during the treatment process, be based on
initial conditions or characteristics of the sample, and/or be
configured manually by the user. Operation of the apparatus can be
at least partially automated. Steps that may be automated include
selecting treatment parameters, selecting a treatment protocol,
initiating acoustic treatment, and monitoring of sample parameters
during treatment.
[0009] For example, the acoustic energy delivered to the sample may
be adjusted by the controller according to the volume of the
sample, the sample temperature, and/or based on the type or
concentration of particulate matter in the sample, for the purpose
of, for example, comminuting the particles. The sensors may include
temperature sensors, pressure sensors, optical sensors, such as
infrared sensor, microscopes and/or video cameras, lasers, acoustic
sensors such as electromagnetic or piezoelectric sensors, or a
combination of such sensors. The sensors may be arranged coaxially
or at an angle to each other.
[0010] The sensors may be employed for measuring a physical
characteristic of one or more samples before, during and/or
following acoustic treatment of the samples. The results of the
measured characteristic can be stored for use in subsequent
processing steps or to compile a treatment history for the
sample(s). For example, samples may be selected for further
processing or interchanged for other samples based on their
previously measured characteristics, or samples may be grouped
and/or classified based on treatment history. Similarly, a
characteristic measured post-treatment can be assessed by itself or
can be compared to the characteristic measured pre-treatment and
used to determine whether a desired condition of the sample has
been reached and/or to assign a subsequent treatment or processing
step for the sample.
[0011] The samples may be coupled to the acoustic energy source by
a liquid, semi-solid or solid medium. For example, the acoustic
transducer may be placed in a tray surrounded by a fluid with a
high transmissivity for the acoustic energy, and the semi-solid or
solid layer may be placed between the fluid and the sample to
prevent direct contact between the sample and the fluid. The
semi-solid or solid layer may be made of silicone gel, elastomeric
polyurethane, thermoplastic elastomer and the like, and may also
have an additional cover layer to further protect the sample from
contamination.
[0012] According to the systems and methods disclosed herein,
pressure may be applied to the sample or to the medium transmitting
the acoustic energy, for example, by pressurizing the fluid, to
improve acoustic coupling between the acoustic energy source and
the sample. In another embodiment, the isolated sample inside a
vessel may be pressurized relative to standard atmospheric pressure
(e.g., to 2, 3, 4, or more .cndot.atmospheres of pressure) to
improve sample processing. When focused acoustic energy is
subsequently applied to the sample, the desired result may be
obtained in a shorter time period and/or, in some applications, may
also result in improved sample processing and output quality (e.g.,
a narrower size distribution in a sheared DNA strand
population).
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Features and advantages of the invention will be apparent
upon consideration of the following detailed description, taken in
conjunction with the accompanying drawings, in which like reference
characters refer to like parts and in which:
[0014] FIG. 1A depicts an exterior view of a benchtop apparatus for
processing a sample according to an embodiment of the
invention;
[0015] FIG. 1B depicts a cross-sectional view of the interior of a
benchtop apparatus for processing a single sample according to an
embodiment of the invention;
[0016] FIG. 2 is a schematic illustration of one embodiment of the
apparatus according to an embodiment of the invention;
[0017] FIG. 3 is a schematic illustration of one embodiment of a
control system according to an embodiment of the invention;
[0018] FIGS. 4A-4D depict an illustrative process for treating a
pressurized sample with acoustic energy according to an embodiment
of the invention;
[0019] FIGS. 5A-5D depict an illustrative process for treating a
pressurized sample with acoustic energy according to an embodiment
of the invention;
[0020] FIGS. 6A-6C depict an illustrative process for treating a
pressurized sample with acoustic energy according to an embodiment
of the invention;
[0021] FIG. 7 depicts an illustrative pressurizing device according
to an embodiment of the invention;
[0022] FIG. 8 depicts an illustrative vessel according to an
embodiment of the invention;
[0023] FIG. 9 depicts an illustrative vessel according to an
embodiment of the invention; and
[0024] FIG. 10 depicts a graph of the absorbance of yeast samples
after treatment at various durations, pressures, and acoustic
intensities.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS AND EXAMPLES
[0025] FIGS. 1A and 1B depict, respectively, an exterior view and
an interior cross-sectional view of a benchtop ultrasonicator 10
for processing a sample 14 according to an embodiment of the
invention. The ultrasonicator 10 is adapted for use by a single
operator, and is generally sized to fit on and be supported by a
table or bench in a laboratory setting. The ultrasonicator 10 can
have a user interface 16 disposed on an outer housing 18, which can
include a cover 12, or other access mechanism, for accessing the
interior of the ultrasonicator 10. In particular, the access
mechanism may allow access to a chamber within the outer housing
18.
[0026] The operator can access the ultrasonicator 10 by opening the
cover 12. The cover 12 may open by detaching from the outer housing
18, by tilting on a hinge that attaches one side of the cover 12 to
the outer housing 18, by sliding laterally along grooves that
engage the cover 12 on the outer housing 18, by rotating in a
lateral direction along a joint that attaches a corner of the cover
12 to the outer housing 18, or any other suitable mechanism. The
mechanism may include one or more buttons, tabs, handles, latches,
or catches that may be depressed, pulled, toggled,
engaged/disengaged, or rotated to open and/or close the cover 12.
Alternatively, the ultrasonicator 10 may be remotely controlled.
For example, a USB or RS232 connector may link the ultrasonicator
10 to a robotic system that may, for example, use ActiveX control
or any other suitable control protocol. In embodiments where the
cover 12 slides laterally along grooves on the outer housing 18,
the cover 12 may, for example, be disposed on the outer surface of
the housing, or may be disposed in a recess within the housing 18.
The mechanisms may be at least partially controlled electronically,
such that the operator pushes a button on the user interface 16 or
elsewhere on the outer housing 18 to open and/or close the cover
12. Although ultrasonicator 10 is depicted with the cover 12 being
disposed on its top surface, one of skill in the art will recognize
that other embodiments may additionally or instead have a door or
cover 12 on a side of the outer housing 18, a drawer that slides
laterally from the outer housing 18, or any other suitable
mechanism for accessing the interior of the ultrasonicator 10. In
certain embodiments, the ultrasonicator 10 may include a latch or
other means for securing the cover 12 in a closed position during
operation of the ultrasonicator 10. In certain embodiments, the
ultrasonicator 10 may include a safety mechanism such that the
device will not operate when the cover 12 is in an open position.
In certain embodiments, the cover 12 in a closed position forms an
airtight seal with the outer housing 18, to permit pressurization
of the treatment chamber relative to the surrounding
atmosphere.
[0027] The cover 12 may be made of the same material as the outer
housing 18, which can be made of any suitable material, such as
plastic, glass, metal, etc. The interior of the cover may be lined
with soundproofing material capable of dampening acoustic energy so
as to reduce danger, disruption, or annoyance to the operator,
e.g., to allow the device to operate quietly or silently in the
perception of the user. The soundproofing material may be
configured to absorb any acoustic energy that reaches it, or to
reflect the acoustic energy back towards the sample 14. The outer
housing 18 can be between about 5 centimeters and about 30
centimeters in width, height, or length.
[0028] In certain embodiments, a ultrasonicator 10 may include a
sensor 20 on the outer housing 18 that triggers the cover 12 to
open. The sensor 20 may be placed on the upper portion 22 of the
outer housing 18, or any other suitable location, preferably
selected to avoid accidental activation. The sensor 20 may include
a motion detector configured to detect motion within a
predetermined range, such as about 5 inches, so that the operator
may move his/her hand or a sample 14 over the sensor 20 to trigger
the cover 12, but motion further from the ultrasonicator 10 would
not trigger the cover 12.
[0029] In certain embodiments, the sensor 20 may be configured to
detect information about the sample 14. For example, the sample 14
could be labeled with a radio frequency identification (RFID) tag,
text, a barcode, a symbol, or any other type of identifying mark,
which sensor 20 could recognize using an RFID interrogator, optical
recognition, acoustic recognition, or any other suitable means. In
certain embodiments, the sample 14 could be marked using a special
ink, a reflective material, or other distinguishing features that
could be recognized using sensor 20.
[0030] Upon accessing the ultrasonicator 10, the operator may place
the sample 14 within a sample holder 24 adapted to hold a sample
vessel 26 containing the sample 14. Suitable sample vessels 26
include tubes, vials, aerosol vials, flasks, jars, bottles, wells,
arrays, blister packs, ampoules, pouches, bags, envelopes, and
other containers that are manipulable by the operator and capable
of containing a sample under sonication conditions. In certain
embodiments, the sample holder 24 can be a concavity or depression
having a shape similar to an outer surface of the sample vessel 26,
such that the sample vessel 26 can nest within the concavity or
depression. The sample holder 24 can include a clamp, clip, or any
other suitable fastener capable of holding the sample vessel 26 in
place, e.g., around the neck of a flask or bottle, or around the
body of a jar or tube. The sample holder 24 may also be configured
to detect information about the sample 14. For instance, an
adjustable clamp can encircle a test tube to both hold the test
tube in place and measure the circumference of the test tube. A
concavity or depression can be equipped with a scale to weigh the
sample vessel 26 and sample 14. The ultrasonicator 10 may have
other sensors or interrogation systems capable of detecting
characteristics of the sample 14. For instance, the ultrasonicator
10 may detect an identifying mark, the location of the sample
vessel 26, the level of fluid in the sample vessel 26, or any other
initial characteristics of the sample 14. Other suitable
characteristics and sensors are discussed in more detail below in
reference to FIG. 2. The ultrasonicator 10 may also include a
safety mechanism for determining that the sample 14 has been
appropriately and securely positioned in the sample holder 24, such
that the ultrasonicator 10 will not operate unless the sample 14 is
appropriately and securely positioned.
[0031] In certain embodiments, the user may close the cover 12
manually. Alternatively, the ultrasonicator 10 may automatically
close the cover 12, e.g., when the user activates the
ultrasonicator 10 and/or when the ultrasonicator 10 determines that
the sample is ready for sonication. The ultrasonicator 10 may also
automatically devise and then execute a treatment protocol for the
sample 14, or may prompt the operator to select or devise a
treatment protocol using the user interface 16. The ultrasonicator
10 may also signal when the treatment protocol is finished, for
example, by emitting an audio signal, turning an indicator light on
or off, flashing or displaying a message on the user interface 16,
or re-opening the cover 12.
[0032] The user, or operator, can use the user interface 16 to
communicate with a control system of the ultrasonicator 10 which
controls the operation of the ultrasonicator 10. Generally, the
user interface 16 can query the user for input that may be
communicated to a processor of the control system. The user
interface 16 can include a display 16a to communicate information
such as which treatment process options are available, the value of
a particular setting, or data detected by any sensors the
ultrasonicator 10 may have. The user interface 16 may also have
buttons, dials, touchpads, knobs, sliders, or any other suitable
control interfaces 16b with which an operator may indicate
preferences, instructions, or parameters to the ultrasonicator 10.
In one possible mode of operation of the ultrasonicator 10, the
control system automatically devises and executes a treatment
protocol upon detecting the presence of a sample 14 properly
disposed within the ultrasonicator 10 and/or other sample
characteristics, including any identifying marks. In another
possible mode of operation, the user interface 16 prompts the
operator to input information that the control system can use to
select a treatment protocol. In one embodiment, the operator can
manually configure a treatment protocol, for instance by selecting
which waveform(s) to use, the duty cycle, the total energy, the
relative positioning of the sample 14 to a acoustic energy source,
and/or any other treatment parameters. In another embodiment, the
operator can select a treatment protocol from a set of
preprogrammed treatment protocols. The preprogrammed treatment
protocols can be configured to each achieve a different objective
or desired result, such as sterilization, mixing, reaction
enhancement, and any other application of acoustic treatment. Each
preprogrammed treatment protocol can also be configured to
correspond to a particular sample, where the control system uses
the characteristics of a particular sample either detected by
sensors of the ultrasonicator 10 or from information entered by the
operator via the user interface 16. In yet another embodiment, the
operator can input information about the sample, such as the sample
size, sample vessel, and desired objective or result, and then the
control system automatically selects and executes a treatment
protocol based on the information from the operator. The operator
may also input acceptable ranges for any measured sample
characteristics or parameters, such as temperature and pressure,
which can help guide the control system's selection of a treatment
protocol. In yet another embodiment, the operator can adjust the
treatment protocol during the treatment process, for instance by
lowering or raising the duty cycle, modifying the waveform, and/or
switching to a different treatment protocol. The operator may also
designate whether or not the treatment protocol should feedback
information about the sample from sensors during the treatment
process to adjust the treatment protocol. The control system can
make feedback adjustments according to instructions from a
preprogrammed treatment protocol and/or input from the operator.
Further information about the control system is described below in
reference to FIG. 3.
[0033] The user interface 16 can have an input mechanism that when
activated initiates application of a focused acoustic field
provided by the acoustic energy source to the sample 14. For
example, the user interface 16 can have a pulse button that
initiates sonication when the pulse button is pressed and stops
sonication, for example, when the pulse button is released or
pressed a second time. The parameters of the acoustic treatment can
be preset by either the control system or the operator to a default
treatment process, or may be selected by the operator at each use.
The user interface 16 can have multiple pulse buttons, each
corresponding to a different application, sample size, and/or
treatment protocol.
[0034] In addition to allowing the operator to indicate his/her
selections to the control system of the ultrasonicator 10, the user
interface 16 can Impart information to the operator, such as which
treatment protocols and/or operating modes (e.g., feedback mode,
fully automatic mode, etc.) are available to the operator. The user
interface 16 can also display information measured, detected, or
recognized by any sensors of the ultrasonicator 10, such as a
sample ID from an identifying mark, sample temperature, or sample
size. Information from sensors can be continuously displayed and
updated during a treatment process to allow the operator to monitor
the progression of the process. The control system may save the
monitored data for later use or review by the operator. The saved
data may be stored on a removable memory component compatible with
a computer or other processing device. The ultrasonicator 10 may
also be configured to produce hard copies of the data, such as a
paper printout. The user interface 16 can also alert the operator,
either through an audio or visual indicator such as a beeping
sound, flashing light, or message within the display 16a, if the
sample approaches or exceeds any boundaries defining an acceptable
range for a sample parameter or characteristic. This combined
monitoring/alert feature can be advantageous in embodiments or
operating modes that do not use feedback to adjust the treatment
protocol during treatment.
[0035] Treatment protocols and similar instructions for treating
samples may be stored on a memory component 30 of the control
system. In some cases it may be advantageous to store treatment
protocols that are relatively specialized to specific uses, sample
types, or objectives. The memory component may be fixed to the
ultrasonicator 10, such as a silicon chip or other hardware
component, or may be configured to be readily removable and
exchanged for other memory components, e.g., such that the operator
can select a memory component comprising treatment protocols
pertinent to the needs of the operator. For example, if the
ultrasonicator 10 is reallocated to a different use, a different
memory programmed with protocols or other programs appropriate may
be used in place of the original memory component. Similarly, as
improved protocols are developed, the operator can also acquire
newer memories including these improved protocols. The
interchangeable memory component can be a memory card that slides
through a memory card slot 28 on the outer housing 18.
Alternatively, the memory card can be stored exterior to the outer
housing 18, similar to a flash drive. Other suitable
interchangeable memory components include compact discs (CDs),
compact discs with read-only memory (CD-ROMs), digital versatile
discs (DVD), diskettes, flash drives, and memory chips. In one
embodiment, the ultrasonicator 10 can download treatments protocols
from the internet, either onto an interchangeable memory component
separable from the ultrasonicator 10, directly to a memory
component built into the ultrasonicator 10, or to an ActiveX or
other controller.
[0036] FIG. 1B depicts a cross-sectional view of the interior 44 of
the benchtop ultrasonicator 10 according to an embodiment of the
invention. The interior of the ultrasonicator 10 can have a cavity
32 specially shaped to fit around and hold a suitable sample vessel
26, such as a tube, that contains a sample 14. The cavity 32, or
any other suitable sample holder, can be substantially surrounded
by a first fluid bath 34 that is contained within a sample tank 36.
The sample tank 36 can be suspended within a coupling medium, such
as a second fluid bath 38, through which an acoustic energy source
40 may transmit acoustic energy to the sample 14. The second fluid
bath 38 can be contained within an interior tank 42 of the
ultrasonicator 10. The sample tank 36 and acoustic energy source 40
are capable of moving, and their positions may each be controlled
by the control system of the ultrasonicator 10. At least a portion
of the sample tank 36 can be made of a thin film material having
low acoustic absorption and an acoustic impedance similar to the
fluid within the second fluid bath 38. This portion of the sample
tank 36 may be arranged so that it is aligned with the acoustic
energy source 40.
[0037] In other embodiments, non-fluidic coupling means may be
used. The acoustic energy can be transmitted through a viscous
(semi-solid) layer 34 of, for example, silicone gel or rubber, or
other material with a gel consistency or rubber consistency, which
may optionally be sealed by an impervious membrane such as, for
example, a plastic sheet or film, to form a laminate. Exemplary
suitable sound-transmitting media are listed in Table 1. This list,
however, should not be viewed as comprehensive and exhaustive, and
other acoustic coupling media with adequate sound transmission
properties may be used instead. In this arrangement, the sample
vessel 26 may be pressed against the layer 34 for more efficient
transfer of acoustic energy. In one embodiment, the cover 12 may be
configured to provide this pressure by having an interior portion
of the cover 12 disposed in contact with the sample vessel 26 when
the cover 12 is closed, such that the sample vessel is pressed
against the layer 34. To adjust for different sample vessel sizes
while still applying pressure, the interior portion of the cover 12
may be coupled to a spring, bellows-like structure, or any other
structure which can be compressed but also resists being
compressed. Layer 34 may be free-floating on the fluid surface of
fluid bath 38 or may be suitably supported in other ways, such as
by making the membrane of the laminate of layer 34 in contact with
fluid bath 38 more rigid, by a lattice frame (not shown) or the
like.
[0038] Table 1 below lists the relative acoustic transmission of
various materials relative to water (100%):
TABLE-US-00001 TABLE 1 Transmission at 1 MHz Material Thickness (in
mm) (in % relative to water) No material (water) 100 Acetate 0.13
80 Latex 0.10 50 PET (Mylar) 0.13 90 Silicone 0.13 95 PET (Mylar)
0.05 >95
[0039] FIG. 2 depicts an electronically controlled ultrasonic
processing apparatus 100 that includes an ultrasound treatment
system and associated electronics 200, a positioning system 300 for
the sample target 800 being treated, and a control system 400 which
controls, generates, and modulates the ultrasound signal and
controls the positioning system 300 in a predetermined manner that
may or may not include a feedback mechanism. The source of acoustic
energy 230 and the target 800 being treated are arranged in a fluid
bath 600, such as water, such that the source of acoustic energy
230 is oriented towards the target 800. The target 800 may be
positioned proximate the surface of the fluid bath 600, above the
source of acoustic energy 230, all being contained within a sample
processing vessel 500. Any of a multitude of sensors 700 for
measuring processing parameters and/or recognizing sample
characteristics can be arranged in or proximate to the fluid bath
600. A temperature control unit 610 may be used to control the
temperature of the fluid in the fluid bath 610. An overpressure
system 900 can control, for example, cavitation, by maintaining a
positive pressure on the target 800 and may be adjusted, in a
predetermined manner that may or may not include feedback
processing, by a target pressure controller 910 that is connected
to the control system 400.
[0040] Target 800 may be a sample, multiple samples, or other
device, and may be contained in a variety of sample vessels. Sample
vessels are sized and shaped as appropriate for the material to be
treated, and can be any of a variety of shapes. For instance, a
sample vessel can be an ampoule, vial, pouch, bag, or envelope.
These and other sample vessels can be formed from such materials as
polyethylene, polypropylene, poly(ethylene terephthalate) (PET),
polystyrene, acetate, silicone, polyvinyl chloride (PVC), phenolic,
glasses and other inorganic materials, metals such as aluminum and
magnesium, and laminates such as polyethylene/aluminum and
polyethylene/polyester. Certain configurations of a sample vessel
can be made by vacuum forming, injection molding, casting, and
other thermal and non-thermal processes.
[0041] An ultrasound acoustic field 240 can be generated by the
acoustic energy source 230, for example, a focused piezoelectric
ultrasound transducer, into the fluid bath 600. According to one
embodiment, the acoustic energy source 230 can be a 70 mm diameter
spherically focused transducer having a focal length of 63 mm,
which generates an ellipsoidal focal zone approximately 2 mm in
diameter and 6 mm in axial length when operated at a frequency of
about 1 MHz. The acoustic energy source 230 is positioned so that
the focal zone is proximate the surface of the fluid bath 600. The
acoustic energy source 230 can be driven by an alternating voltage
electrical signal generated electronically by the control system
400.
[0042] The positioning system 300 can include at least one
motorized linear stage 330 that allows the target to be positioned
according to a Cartesian coordinate system. The positioning system
300 may position and move the target 800 relative to the source 230
in three dimensions (x, y, z) and may optionally move either or
both of the target 800 and the acoustic energy source 230. The
positioning system 300 can move target 800 during and as part of
the treatment process and between processes, as when multiple
samples or devices within the target 800 are to be processed in an
automated or high-throughput format. The positioning system 300 may
position or move the target 800 in a plane transverse to the focal
axis of the acoustic energy source 230 (x and y axes). The
positioning system 300 can position and move the target 800 along
the focal axis of the acoustic energy source 230 and lift or lower
the target 800 from or into the fluid bath 600 (z axis).
[0043] The positioning system 300 can also position the acoustic
energy source 230 and any or all of the sensors 700 in the fluid
bath 600 along the focal axis of the acoustic energy source 230, if
the sensors 700 are not affixed in the water bath 600, as well as
lift, lower, or otherwise move the acoustic energy source 230. The
positioning system 300 also can be used to move other devices and
equipment such as detection devices and heat exchange devices from
or into the fluid bath 600 (z axis). The linear stages of the
positioning mechanism 330 can be actuated by stepper motors (not
shown), which are driven and controlled by electrical signals
generated by the control system 400, or other apparatus known to
those skilled in the art.
[0044] Sensors 700 can be used prior to, during, or after the
acoustic treatment to analyze the samples and/or detect certain
physical properties of the sample, for example, by measuring
responses to electromagnetic stimulation, such as optical
spectroscopy, energy dispersion, scattering absorption, and/or
fluorescence emission. Other measurable variables can include
electromagnetic properties, such as electrical conductivity,
capacitance or inductivity, as well as other physical parameters,
such as sample uniformity or pattern analysis. Exemplary sensors
may include an additional ultrasonic acoustic transducer suitable
to transmit and/or receive an acoustic probe interrogation beam
which can be used to assess one or more characteristics, such as
the fill level, temperature, cavitation, homogeneity (e.g.,
presence of absence of particulate matter in the solvent, and/or
the size of such particles), volume, etc., of the sample located
within the sample vessel. It will be understood by those skilled in
the art that the roles of the acoustic energy transducer 230 and
the sensor transducer can be reversed in that the sensor transducer
may operate to emit the acoustic processing beam while the
transducer 230 performs sensing function. The system may include
other types of sensors as well, such as an infrared (IR)
temperature sensor to measure the sample temperature.
[0045] Interfaces, such as an interface between air and water,
cause reflection of an incident ultrasound field. While reflection
should be minimized for transmitting acoustic energy to the sample,
a signal emitted from the acoustic energy source 230 or from a
separate sensor and reflected by an interface, such as the meniscus
of the sample within the sample vessel, can be used to quantify the
height and therefore also the volume of the sample. In one
embodiment, the sensor may be implemented as an acoustic transducer
and emit a short burst of acoustic energy with a duration of 1 ms
or less for interrogating the sample. Such short burst is also
referred to as a "ping." As mentioned above, the interrogation
burst can be focused on the sample. Due to reflection at the
various interfaces encountered by the propagating interrogation
sound wave, the sensor receives a return signal after a transit
time proportional to the distance between the sensor and the
respective interface. For example, it takes a sound wave
approximately 10 ms to travel a distance of 1 cm, which is easily
resolved by a detection system. The height location of the meniscus
of the sample can then be determined from the arrival time
difference between the sound wave reflected at the bottom of the
sample, and the reflection at the liquid-air interface at the
meniscus. The volume of the sample can be taken into consideration
when applying acoustic energy for treatment of the sample.
[0046] Likewise, air bubbles and particulates can also block or
reflect energy transmission through the sample volume. The same
principle described above for determining the position of the
meniscus can therefore also be used to evaluate the sample volume
for the presence or absence of particulates, and/or the size and/or
amount of such particles.
[0047] The control system 400 can include a computer 410, or other
processor or microprocessor, and a user input/output device or
devices 420 such as a keyboard, display, printer, etc. The control
system is linked with the ultrasound treatment system 200 to drive
the acoustic energy source 230, with the positioning system 300 to
drive the stepper motors described above, with one or more sensors
700 to detect and measure process parameters and/or sample
characteristics, and with one or more controllers, such as the
target pressure controller 910, to alter conditions to which the
target 800 is exposed. A fluid bath controller 610 could also be
linked with the control system 400 to regulate temperature of the
fluid bath 600.
[0048] The control system 400 can control and drive the positioning
system 300 with the motion control board 310, power amplifier
device 320, and motorized stage 330, such that the target 800 can
be positioned or moved during treatment relative to the source 230
to selectively expose the target 800 to acoustic energy.
[0049] The control system 400 can specify a process to be performed
upon a sample. In this regard, the ultrasound treatment system 200
can include an arbitrary waveform generator 210 that drives an RF
amplifier 220, such that the acoustic energy source 230 receives an
input. The output signal of the RF amplifier 220 may be conditioned
by an impedance matching network and input to the acoustic energy
source 230. The control system 400 can generate a variety of useful
alternating voltage waveforms to drive a acoustic energy source.
For instance, a high power "treatment" interval consisting of about
5 to 1,000 sine waves, for example, at 1.1 MHz, may be followed by
a low power "convection mixing" interval consisting of about 1,000
to 1,000,000 sine waves, for example, at the same frequency. "Dead
times" or quiescent intervals of about 100 microseconds to 100
milliseconds, for example, may be programmed to occur between the
treatment and convection mixing intervals. A combined waveform
consisting of concatenated treatment intervals, convection mixing
intervals, and dead time intervals may be defined by the operator
or selected from a stored set of preprogrammed waveforms. The
selected waveform may be repeated a specified number of times to
achieve the desired treatment result.
[0050] FIG. 3 depicts a control system 50 similar to control system
400 of FIG. 2 that includes a processor 54, a user interface 56,
and a memory 52. The control system 50 links to actuating systems
58 that implement processes specified by the control system 50 and
to sensors 60 that may measure processing parameters and/or detect
sample characteristics. Actuating systems 58 can include
positioning systems, ultrasound treatment systems for driving a
acoustic energy source, parameter controllers, and any other
devices capable of implementing treatment processes, such as those
described above in reference to FIG. 1. Sensors 60 may monitor the
impact and efficacy of a treatment process on a sample by detecting
visual indicators, temperature, and/or cavitation. Sensors 60 may
also detect initial characteristics of the sample like size,
solubilization level, and type of sample vessel.
[0051] The memory 52 can include preprogrammed waveforms,
protocols, and functions from which the processor 54 can select
when determining a treatment process. Protocols can include
combined or alternating waveforms and any other instructions for
any actuating systems 58. The instructions are preferably
predetermined to be advantageous for effecting a specific
objective, such as enhancing a reaction, solubilizing the sample,
or sterilization, for a specific sample type, which may be
dependent on the sample contents, size, temperature, viscosity,
level of solubility, vessel, or any other characteristics.
Functions can configure a coordinated set of instructions for the
actuating systems 58 or select a protocol based on input collected
by the processor 54. The input can be initial characteristics of
the sample and/or process parameters that can be detected by
sensors 60 or entered by an operator via the user interface 42. For
instance, a function can, given the volume and contents of a
sample, determine the necessary waveform, duty cycle, and length of
treatment to mix a sample without significant heating side effects.
Other processing variables the function can determine include
frequency, energy delivered, burst pattern, intensity, cycles per
burst, pulse shape of the waveform, maximum energy level, etc. The
processor 54 can select a process to implement based on a
combination of user input from the user interface 42 and/or
information from the sensors 60. The user interface 42 allows an
operator to design and specify a process to be performed upon a
sample. In particular, the operator can directly control
instructions to actuating systems 58, select an option from the
memory 52, indicate characteristics of the sample and an objective,
or some combination thereof. The user interface 42 can also
communicate to the operator which treatment process options are
available and data detected by the sensors 60. Information from the
sensors 60 can be used to configure a treatment process, to select
a treatment process, or as feedback to a treatment process.
[0052] In one embodiment, measurable or discernible process
attributes such as sample temperature, water bath temperature,
intensity of acoustic cavitation, or visible evidence of mixing in
the sample processing vessel, may be monitored by the control
system 50 and employed in feedback loop to modify automatically
during the treatment process any parameters controlled by actuating
systems 58, such as the treatment waveform or acoustic energy
source position. The modification of the treatment waveform may be
a proportional change to one or more of the waveform parameters or
a substitution of one preprogrammed waveform for another. For
instance, if the sample temperature deviates excessively during
treatment from a set-point temperature due to absorbed acoustic
energy, the control system 50 may proportionally shorten the
treatment interval and lengthen the convection mixing interval in
response to the discrepancy between the actual and target sample
temperatures. Or, alternatively, the control system 50 may
substitute one predetermined waveform for another. The control
system 50 may be programmed to terminate a process when one or more
of the sensors 60 signal that the desired process result has been
attained.
[0053] In another embodiment, initial characteristics of the sample
may be used by the control system 50 to assess whether treatment is
needed and/or to select a protocol or function optimized for those
characteristics. Initial sample characteristics can include sample
content, vessel, size, viscosity, temperature, pressure, and
position relative to any of the actuating systems 58. In addition,
one of the sensors 60 may be adapted to recognize an identifying
mark either affixed to the sample or scanned in separately by the
operator. The control system 50 may associate the identifying mark
with a corresponding waveform, parameter, protocol, or function,
e.g., such that the corresponding aspect of the treatment is set
automatically by the controller upon detection of the mark and/or
executed automatically upon activation of the ultrasonicator 10. If
the computer has sufficient control over the effects of the
actuating systems 58 to yield the desired objective, the use of a
preprogrammed protocol or function may eliminate the need for
monitoring sensors during the treatment process. In particular, the
control system 50 upon recognition of sufficient sample
characteristics can implement a protocol optimized to render the
desired effect on that sample while maintaining the sample within
certain constraints such as temperature or pressure ranges, without
relying on feedback from sensors 60 while the process is in
progress. An embodiment without process monitoring capabilities may
be advantageous in cases where a simplified acoustic treatment
apparatus is desirable, such as a benchtop apparatus for processing
a single sample.
[0054] Acoustic treatment may be applied to many types of samples
for a variety of purposes. Chemical and biological samples, as well
as other types of samples, may be sterilized, mixed, or heated by
acoustic treatment. Other applications are described in U.S. Pat.
No. 6,719,449 entitled "Apparatus and Method for Controlling Sonic
Treatment," which is hereby incorporated by reference herein. One
application in particular is the acoustic treatment of blood or
blood-based samples. Treatments can be configured to sterilize a
blood sample, to ensure homogeneity of a blood sample, to mix a
blood sample with an agent such as an anti-coagulant or a test
compound that tests for antibodies, and any other suitable
applications that may arise. The ultrasonicator 10, described above
in reference to FIG. 1, can be adapted for blood treatment as well
as for other applications, particularly applications involving
small numbers or quantities of samples. In addition to clinical
uses, the ultrasonicator 10 can be used in hospitals and doctor's
offices to prepare or test blood or other samples.
[0055] Other applications outside of laboratory settings can
utilize the agitating effects of acoustic treatment. Acoustic
treatment may be used to break up and/or mix components during food
or beverage preparation, to prepare cosmetics, and to homogenize
mixtures/suspensions/solutions that separate or otherwise become
heterogeneous during storage, such as paint. For instance, acoustic
treatment can break up food at the cellular level and/or form
emulsions or suspensions. Possible uses include making milkshakes,
mayonnaise, purees, foams, sauces, juices (e.g., from fresh
produce), ice cream, and butter. Acoustic treatment may be used to
prepare cosmetics, such as lipsticks, moisturizers, creams,
emollients, liquid soaps, perfumes, astringents, and other suitable
colloidal or liquid products, or agitate paint to uniformly mix
colors and components. The ultrasonicator 10 can be adapted for
these non-clinical applications and be useful in settings in which
customization and/or portability of the device is desired. For food
preparation applications, the ultrasonicator 10 can be a countertop
appliance in consumer or commercial settings, allowing a user to
freshly prepare a customized food product. For cosmetic
applications, customers in a retail setting can specify and mix
colors, scents, and other ingredients for cosmetic products. For
paint applications, the ultrasonicator 10 can allow a user to
prepare paint for use. Due to the size and relative portability of
the ultrasonicator 10, paint may be mixed at any desired location,
for instance, by contractors or painters at a job site. The
ultrasonicator 10 may also be used to create small samples of
customized paint as an alternative to color swatches.
[0056] FIGS. 4 and 5 depict illustrative processes for treating a
pressurized sample with acoustic energy. In particular, FIGS. 4A
and SA depict sample vessels for containing the sample and
isolating it from other components of an acoustic energy apparatus.
FIGS. 4B and 5B depict the sample vessels of FIGS. 4A and 5A,
respectively, after samples have been deposited within the
respective vessels. FIGS. 4C and SC depict the samples and sample
vessels of FIGS. 4B and 5B, respectively, after they have been
sealed closed and pressurized to pressurize the samples. Methods
and apparatus for pressurizing samples within sample vessels are
described in more detail below. FIGS. 4D and 5D depict acoustic
energy sources applying acoustic energy to the pressurized samples
and sealed sample vessels of FIGS. 4C and 5C, respectively.
[0057] Without wishing to be bound by theory, by increasing the
pressure of the fluid to be processed, the acoustic energy dose
required to cavitate the solution may be greater. This may increase
the shear forces consequent to cavitation bubble collapse. This may
also result in greater retention time of the sample in the focal
zone of the applied acoustic field and/or reduced rate of sample
escaping the focal zone. This in turn may effectively increase the
collision frequency of the sample with the acoustic bubbles
generated by the applied energy and/or increase their resultant
shear forces upon bubble collapse. Without wishing to be bound by
theory, it is possible that the pressurization of the sample during
the ultrasonic treatment may effect a transient increase in the
effective viscosity of the sample, and that the acoustic energy has
a greater effect in this altered state. This increase in effective
strength may result in the observation of finer particle formation,
faster tissue homogenization, accelerated lysis of microbial
organisms, or otherwise provide for increased precision or speed of
processing using the acoustic energy treatment process.
[0058] The sample may include a liquid or solution comprising a
sample (e.g., tissue, cell, crystal, buffer, solvent, gel, gum,
slurry, blend, single-walled carbon nanotubes, etc., or combination
thereof). In certain embodiments, acoustic energy is applied to a
solid sample, e.g., in a liquid or gaseous environment, to form
particles of the solid material. For example, the application of
focused acoustic energy to a solid can cause it to break apart into
increasingly smaller fragments than unfocused acoustic energy.
Similarly, acoustic energy can agitate sample pieces or particles,
inducing collisions that promote further fracturing and/or
fragmenting of the solids. In other embodiments, acoustic energy is
applied to a liquid sample, thereby inducing the formation of
particles. For example, acoustic energy can be applied to, a
supersaturated solution, causing a solute to precipitate out of
solution. Alternatively, acoustic energy can be applied to a
biphasic liquid sample, inducing mixing or the phases and causing
the precipitation of a solid. Similarly, acoustic energy can be
applied to a hot solution in conjunction with cooling, so that
solids that precipitate during cooling are formed into particles of
a desired size. The subject systems and methods can be applied to
any procedure that results in the formation of a solid material in
order to control the size and size distribution of the solid
material that forms. Other procedures and desired results are
described below.
[0059] An additional benefit for a system is that the entire
acoustic circuit, which in some embodiments includes a series of
acoustic interfaces such as the transducer-couplant,
couplant-vessel, vessel wall, vessel-inner sample, and
sample-air/vapor headspace, may be pressurized, which may improve
the efficiency of the treatment process. For example, just as a
more dense acoustic couplant may transmit acoustic energy more
efficiently, a pressurized fluid may transmit acoustic energy more
efficiently than a non-pressurized fluid.
[0060] FIG. 6 depicts an illustrative process for treating a
pressurized sample with acoustic energy. In addition, FIG. 6 allows
higher pressures to be obtained readily without requiring a special
vessel, materials, or a custom design. In particular, FIG. 6A
depicts an acoustic energy treatment system which includes an
acoustic energy source 6002 coupled via a coupling medium 6004 to a
sample vessel 6006 containing a sample 6008, similar to the sample
vessel 4010 depicted in FIG. 4B. The medium 6004 may be a fluid,
such as water or buffer, or other compressible medium. FIG. 6B
depicts the system of FIG. GA after it has been placed within an
air-tight chamber 6010 and pressurized. More particularly, both the
sample 6008 and the medium 6004 coupling the acoustic energy source
6002 to the sample vessel 6006 are pressurized due to the elevated
atmospheric pressure within the chamber 6010. Methods and apparatus
for pressurizing acoustic energy treatment systems are described in
more detail below. FIG. 6C depicts the acoustic energy source
applying acoustic energy 6012 via the pressurized medium 6004 to
the pressurized sample 6008.
[0061] The pressurizing atmosphere may be compressed air, nitrogen,
argon, helium, or any other suitable gases or combination thereof.
Certain gases may be preferred in certain applications, e.g., for
their intrinsic physical properties such as inhibiting biological
events, such as nitrogen, or because they may beneficially alter
the cavitation threshold energy, such that an altered headspace
over a fluidic or partially fluidic/solid sample more readily
enables bubble formation and collapse.
[0062] In various embodiments, certain vessel designs may be used
to apply pressure to a sample. These vessel designs may allow lab
technician to apply pressure to a sample efficiently so that
multiple samples can be processed with minimal time and effort. For
example, a sample vessel may have a sealing mechanism, for sealing
the interior of the sample vessel from the external atmosphere,
with which the lab technician, or any other user, may pressurize
the sample.
[0063] In one embodiment, an acoustic energy apparatus processes a
single sample, e.g., which is inserted into the device.
Alternatively, a collection of samples may be inserted into the
device, e.g., in a suitable rack, container, or other array for
holding the collection of samples. In either scenario, a sealing
cap is applied to an individual sample to simultaneously close and
pressurize the sample. For example, the sealing cap, while engaging
the vessel containing the sample, acts as a piston to pressurize
the sample. FIG. 4C depicts an exemplary sealing cap 4002 for
pressurizing a sample 4004. In particular, the sealing cap 4002 has
a bayonet portion 4006 encircled by a rim 4008 of approximately the
same length as the bayonet portion 4006. The size and shape of the
rim 4008 is selected such that when the sealing cap 4002 initially
engages the sample vessel 4010, the rim 4008 seals off exposure of
the sample 4004, and the rest of the vessel interior 4012, to the
environment external to the vessel 4010. As the sealing cap 4002 is
further engaged with the vessel 4010, such that the rim 4008
overlaps more of the walls of the sample vessel 4010, the bayonet
portion 4006 protrudes into the vessel interior 4012 to increase
the pressure within the vessel 4010, thereby pressurizing the
sample 4004. Generally, a sealing mechanism may include a
displacement portion, such as the bayonet portion 4006, that can
protrude into the interior of the sample vessel to decrease the
volume of the interior, thereby pressurizing the sample contained
within the interior.
[0064] Alternatively, a sample may be sealed and then pressurized
after sealing. For example, FIG. 5 depicts an embodiment of a
vessel 5000 used to apply pressure to sample or samples 5010. The
vessel chamber 5008 may be accessed through input 5004. Input 5004
may be covered by a protective seal 5006 that is bonded or engaged
with vessel chamber 5008. Protective seal 5006 may be reversibly
sealable so that sample or samples 5010 can be introduced into
vessel chamber 5008. Protective seal 5006 may be made from any
combination of metal, glass, plastic, rubber, plastic film, or any
other material suitable to form a bond with vessel chamber 5008 in
order to provide a seal, e.g., to prevent sample from exiting the
chamber during treatment. In certain embodiments, the seal may be
air-tight, water-proof, and/or hermetic. Vessel chamber 5008 may be
made from any combination of metal, glass, plastic, rubber, plastic
film or any other suitable nonporous material that enables vessel
chamber 5008 to provide a barrier between the sample or samples
5010 and an external environment. In certain embodiments,
protective seal 5006 may comprise port 5002. Port 5002 may be a
reversibly sealable port, such as a one-way valve or a rubber
septum, for accessing the vessel chamber 5008, e.g., for supplying
pressure to vessel chamber 5008, and thus sample or samples 5010.
In one embodiment, port 5002 may be shaped and sized to interface
with a needle, such as a hypodermic needle, air injector, cannula,
or similar feature. The needle may be used to add or remove
materials, such as a sample, a liquid, or a gas, to or from the
vessel chamber. In certain embodiments, the vessel chamber may be
pressurized (e.g, with air, argon, nitrogen, or another suitable
gas) prior to the introduction of the sample or sample medium via a
needle. In other embodiments, a needle may be used to inject a
liquid or gas into vessel chamber 5008 after introduction of the
sample in order to increase the pressure within vessel chamber
5008. For example, the liquid or gas so introduced may be an inert
gas, compressed air, a solvent for the sample, or a suitable
treatment medium for the sample. In certain embodiments, the needle
may be coupled to a mechanical or manual air pump to increase the
pressure of the environment within vessel chamber 5008. For
example, in certain embodiments, the needle may be attached to a
bulb. The bulb may be made out of a malleable and durable material
such as rubber, plastic, or any other suitable material that is
able to deform nondestructively and preferably reversibly when
mechanically squeezed. The bulb may be shaped and sized so that the
volume of the bulb is greater than the volume of the vessel chamber
5008. In certain embodiments, the bulb may be squeezed or otherwise
deformed to inject a liquid or gas into vessel chamber 5008 in
order to increase the pressure within vessel chamber 5008. Thus,
vessel 5000 permits sample or samples 5010 to be exposed to higher
pressures in order to facilitate processing of the sample or
samples.
[0065] FIG. 7 depicts an embodiment of an automatic pressurizing
device 7002 to increase the pressure of the environment within a
sealed vessel chamber, such as the sealed vessels of FIG. 5. The
device includes a needle 7006 adapted to reversibly penetrate a
seal, such as a rubber septum, a gas source 7008, (e.g., a canister
of compressed air, nitrogen, argon, or other suitable gas or
mixture thereof), and optionally a pressure sensor 7004. Pressure
sensor 7004 measures the pressure of the environment at the end of
the needle 7006, and may be mechanical or digital. Digital pressure
sensors may include piezoresistive semiconductors,
microelectromechanical system chips, variable capacitors, or other
hardware to suitably detect the pressure of an environment. In
embodiments that include pressure sensor 7004, the automatic
pressurizing device 7002 may be configured to automatically
pressurize the chamber in an automated fashion when the needle 7006
is inserted into a sample until a desired level of pressure is
reached within the sample chamber. In such embodiments, the
automatic pressurizing device 7002 may be triggered by the force
exerted on the needle when it is pushed through a septum, or it may
be triggered by force applied to a trigger external to the needle
7006. The trigger may comprise, for example, a guard 7010 in the
vicinity of the needle that contacts the septum as the needle
penetrates the septum, or a manual trigger such as a button or
lever operated by the user (not shown). Upon activation, the
automatic pressurizing device 7002 may add gas to the chamber until
a predetermined pressure is reached, e.g., the pressure of the gas
source 7008, or a pressure set by the user or the manufacturer of
the device, e.g., via an interface (not shown). In embodiments
employing a pressure sensor 7004, the pressure sensor 7004 may
communicate with a controller that can operate to open or close a
pathway between the gas source 7008 and the needle 7006 so that the
correct amount of pressure is reached in a vessel chamber. This
communication may occur through electrical means, such as a wire,
RFID, or wireless communication, or any other suitable means.
[0066] In an exemplary embodiment, a sample vessel may be sealed
with a pierceable septum. For example, the sample may be contained
within a 13 mm.times.65 mm round bottom borosilicate glass culture
tube (Chromocol, UK) with a screw cap end that is sealed with a
Bakelite cap having a rubber septum center. A needle, such as a
22-gauge, 1.5-inch hypodermic needle, can be inserted through the
septum to apply elevated pressure through the needle. The pressure
equilibrates through the needle bore, pressurizing the interior of
the tube and the sample. High-intensity focused ultrasound ("HIFU")
acoustic energy (as used with the Covaris, Woburn, Mass., USA
S-series instruments) can then be applied to process the sample in
the tube. The pressure may be allowed to equilibrate to atmospheric
pressure, and then the sample may be removed from the tube, e.g.,
by inserting the needle further into the tube to collect the
fluidic sample in a manual manner. Alternatively, the pressure in
the tube may be used to eject the sample, e.g., through a cannula
inserted through the septum into the sample. All or part of the
above-described process, including sample preparation, sealing,
pressurization, treatment, depressurization, and sample removal,
may be readily automated. In some such embodiments, the needle,
though inserted into the sample chamber, is not in the focused
acoustic energy field. In other embodiments, the needle may be
located in the acoustic focal zone, e.g., as a nucleation site for
cavitation events.
[0067] In another embodiment, a single sample or a batch of single
samples are inserted into an apparatus and pressurized prior to the
application of acoustic energy. For example, a microtitre plate
with 96 350-microliter sample wells may be loaded with 100
microliters per well. The open plate may be inserted into an
apparatus which allows the atmosphere at the fluid/air interface of
the samples to be elevated prior to an acoustic dose.
Alternatively, the plate may be sealed prior to treatment with a
lid that either pressurizes each well (e.g., via a piston-type
approach, as discussed above) or allows the sealed compartment to
be pressurized (e.g., through a septum or one-way valve, as
discussed above).
[0068] In another embodiment, a sample vessel may have one or more
flexible or elastic walls to allow the volume of the interior of
the sample vessel to decrease, thereby increasing the pressure. One
of the walls, or portion of a wall, of the sample vessel may be
sufficiently pliable to allow increased pressure external to the
pliable wall to be transmitted to the contained sample. For
example, a plastic bag, a balloon, a test tube having a slidably
engaged piston plunger, or other sample vessel having a deformable
structure may shrink in volume when placed in an elevated pressure
atmosphere, thereby pressurizing the sample, even though the sample
remains sealed from the external environment. Alternatively or in
addition, an external force may be applied to the sample vessel to
shrink the volume of the vessel interior. The application of the
external force may be automated or may comprise a user of an
acoustic treatment apparatus physically manipulating the sample
vessel to deform its structure.
[0069] FIG. 8 depicts another embodiment of a vessel 8000 used to
apply pressure to sample or samples 8010. The vessel chamber 8008
may be accessed through input 8002. Vessel chamber 8008 may be made
from any combination of metal, glass, plastic, rubber, plastic film
or any other suitable nonporous material that enables vessel
chamber 8008 to provide a barrier between the sample or samples
8010 and an external environment. Input 8002 may be shaped and
sized to interface with plunger 8004. Plunger 8004 may comprise a
stopper 8006 coupled to a stem 8007. The length of stem 8007 of
plunger 8004 may be equal to or greater in length than the length
of vessel chamber 8008, but is typically sufficiently long to
facilitate movement of the stopper 8006 through a range of
positions within the vessel chamber. Stopper 8006 of plunger 8004
may be shaped and sized to form a seal with input 8002. Stopper
8006 of plunger 8004 may be made out of rubber, plastic, or any
other suitable material to form a seal with input 8002. The seal is
preferably airtight. Stem 8007 of plunger 8004 may be made out of
glass, plastic, or any other suitable material that allows a user
or an automated device to employ stem 8007 of plunger 8004 and
deliver a downward force to reduce the volume of the vessel chamber
8008, thereby increasing the pressure in the chamber. Stem 8007 of
plunger 8004 may also be shaped and sized so that it may be
employed to apply an upward force to remove stopper 8006 from the
vessel chamber 8008 so that the pressure within vessel chamber 8008
may be reduced and/or the vessel chamber can be accessed. Thus,
vessel 8000 may be employed to expose sample or samples 8010 to
higher pressures in order to facilitate processing of the sample or
samples.
[0070] FIG. 9 depicts another embodiment of a vessel 9000 used to
apply pressure to sample or samples 9010. Sample or samples 9010
may be placed inside vessel chamber 9008 through input port 9002.
Vessel chamber 9008 may be made out of a substantially malleable
yet durable material such that vessel chamber 9008 deforms
nondestructively when physically manipulated and provides a barrier
between the sample or samples 9010 and an external environment.
Suitable materials include rubber, metal foil (e.g., aluminum),
plastic, or any combination thereof. Vessel 9000 also includes
bottom 9006. Bottom 9006 may be shaped and sized to fit into a
sample holding device such as a 96-well plate, or any suitable
sample holding device. In certain embodiments, input port 9002 may
be shaped and sized to be clamped by clip 9012. Clip 9012 may be
made out of plastic, rubber, or any material suitable to clamp
input port 9002. Clip 9012 may be shaped and sized to slide up and
down the body of the vessel chamber 9008 and hold a fixed position
after movement. Clip 9012 may be removable (e.g., to access vessel
chamber 9008 through input port 9002), and preferably seals vessel
chamber 9008, e.g., for treatment, such as with an airtight seal.
In a variant embodiment, input port 9002 may be located on the side
of the vessel 9000, such that the clip 9012 slides up and down the
body and over the port 9002, thus reversibly sealing the vessel
chamber 9008. By manipulating clip 9012, parts of the vessel body
9008 may deform nondestructively. In certain embodiments,
manipulating clip 9012 may reduce the volume of vessel chamber
9008. By reducing the volume of vessel chamber 9008 in this manner,
the vessel chamber 9008 may be pressurized. Thus, vessel 9000 may
be employed to expose sample or samples 9010 to higher pressures in
order to facilitate processing of the sample or samples.
[0071] In another embodiment, focused acoustic energy is applied to
a flowing fluid stream exposed to elevated pressure prior to and/or
during acoustic energy treatment. This embodiment may form part of
a flow-through or intermittent flow system that may be employed in
the production of fine chemicals, food products, pharmaceuticals,
cosmetics, and in other manufacturing settings. For example, this
system may include a cell with a quartz window for acoustic energy
transmission. In another example, the flowing fluid stream passes
through a constriction which elevates the pressure on the fluid in
the region preceding the constriction. The entire transducer may be
contained within the flowing stream of a sample to be processed
that is intermittently pressurized. In certain embodiments, a
focused acoustic energy apparatus may synchronize the flow with the
acoustic dose to achieve the desired result (e.g., crystal
dissolution, sonocrystallization, and the like). This continuous
flow process may also be automated.
[0072] Various embodiments of the present invention will be further
understood by reference to the following non-limiting examples.
Example One
[0073] Long nucleic acid strands, such as genomic DNA, are too
large to use in certain applications without first shearing the
strands to fragments of smaller size, e.g., for library
construction or for certain methods of DNA sequencing. Cleaving the
DNA strands to fragments with lengths of 500 base pairs ("bp"), 200
bp, or less can thus be an important step in the preparation of DNA
samples.
[0074] In this example, 20 .mu.g of lambda DNA in 400 .mu.l of 1 mM
EDTA was placed in 13.times.65 mm round bottom glass tubes. Each
tube was then treated in an S2 turbo at 10% duty cycle, 10
intensity, and 200 cycles/burst for 6 minutes in the power track
mode with a bath temperature of 7-8.degree. C. The treatments were
conducted at or near the following pressures: 14.7 pounds per
square inch ("psi"), 29 psi, 44 psi, and 57 psi. The sizes of the
DNA fragments were then determined by running 22 Al aliquots from
each tube on a 0.7% agarose gel along with size markers. The DNA
was visualized with ethidium bromide staining. The size of the
starting lambda DNA was 48,502 bp. After treatment at 14.7 psi, the
majority of the DNA fragments were in the 100 to 650 bp size range.
After treatment at 29 psi, the majority of the DNA fragments were
in the 100 to 500 bp size range; and at pressures of 44 and 57 psi,
the majority of the DNA fragments were in the 100 to 375 bp range,
as summarized in Table 1 below.
TABLE-US-00002 TABLE I Ranges of sizes for the majority of DNA
fragments after treatment at various pressures Pressure DNA
Fragment Sizes 14.7 psi 100-650 bp 29 psi 100-500 bp 44 psi 100-375
bp 57 psi 100-375 bp
Example Two
[0075] The DNA shearing described in example one may also be
controlled by the variation of the pressure level, e.g., 1, 2, and
3 atmospheres ("atms") and beyond. A dose response was observed by
variations in the pressure of the sample while keeping the
treatment duration, temperature, and acoustic dose constant
[0076] In this example, 20 .mu.g of lambda DNA in 400 .mu.l of 1 mM
EDTA was placed into 13.times.65 mm round bottom glass tubes. Each
tube was then treated as in Example One, except that only pressures
of 14.7 psi and 44 psi were used. Aliquots of 22 Al were removed
after 1, 3, and 6 minutes of treatment. The size of DNA fragments
was determined by agarose gel electrophoresis as in Example One. At
14.7 psi, the size ranges for the majority of the DNA fragments at
various treatment durations are summarized by Table 2 below.
TABLE-US-00003 TABLE 2 Range of sizes for the majority of DNA
fragments after treatment at various durations and pressures DNA
Fragment DNA Fragment Sizes with Sizes with Treatment Duration
Treatment at 14.7 psi Treatment at 44 psi 60 seconds 200-2,000 bp
150-1,000 bp 180 seconds 100-1,000 bp 100-500 bp 360 seconds
100-700 bp 100-375 bp
Example Three
[0077] Particle generation of hydroxyapatite (HAP) may be faster
and result in smaller particles. Treating crystals while at
elevated pressures may produce more and smaller fragments at a
faster rate.
[0078] A suspension of ceramic hydroxyapatite particles was
prepared by placing 3.1 mg of ceramic hydroxyapatite particles (20
Am from Bio-Rad, Hercules, Calif.) in a 13.times.65 mm glass round
bottom screw cap tube and adding 2.0 ml of 50 mM trisodium citrate.
The tube was capped and immediately processed in a Covaris S2
instrument with the following treatment parameters: power track
mode, water bath temperature 8 degree C., 10% duty cycle, 10
intensity, 200 cycles per burst, and 30 seconds of treatment. The
contents of the glass tube were then transferred to a cuvette and
left undisturbed for 300 seconds to allow any large particles
present to settle out. The cuvette was then placed in a
spectrophotometer and the absorbance at 600 nm measured. The
absorbance is due to light scatterings by the small particles that
remain in suspension and thereby provides a measure of the amount
of small particles generated by the acoustic treatment.
[0079] For the elevated pressure experiments, the cap of the
13.times.65 mm tube was fitted with a tubing connection and a
section of Tygon tubing that connected the glass tube to a pressure
regulator that was, in turn, connected to a compressed air supply.
Control experiments run at atmospheric pressure (approximately 15
psi) had an absorbance reading of 0.699 after 30 second of the
above treatment. Experiments at elevated pressure were run at
approximately 45 psi with compressed air and had an absorbance
reading of 1.442 after 30 seconds of acoustic treatment. Thus the
rate of fragmentation during the first 30 seconds of acoustic
treatment was doubled when the pressure was increased to 45
psi.
Example Four
[0080] Yeast spores are more readily disrupted with the elevated
pre-pressurization prior to application of focused acoustic energy.
This pressurization may render the spores more susceptible to the
effects of HIFU. Treating yeast while at elevated pressures may
increase the lysis of yeast cells.
[0081] Yeast cells from a frozen stock were suspended in 1.5 ml of
33 mM potassium phosphate pH 7.5 buffer in a 13.times.65 mm glass
round bottom screw cap tube. 25 .mu.l of a lyticase (Sigma, St.
Louis, Mo.) stock (500 units/ml in cold distilled H20 made fresh
daily) was added to the tube. The tube was capped and processed
with a Covaris S2 instrument. The acoustic treatments were run in
the power tracking mode with a water bath temperature of 26+/-1
degree C. The control sample was run at atmospheric pressure with
brief low power acoustic mixing to keep the yeast cells in
suspension. The low power treatment parameters were as follows: 1%
duty cycle, 3 intensity, 200 cycles per burst, for 10 seconds; then
1% duty cycle, 0.1 intensity, 50 cycles per burst, for 50 seconds.
These steps were repeated to generate the selected total treatment
time. The parameters for high power treatment were: 20% duty cycle,
10 intensity, 200 cycles per burst, 30 seconds; then 1% duty cycle,
0.1 intensity, 50 cycles per burst 60 seconds; and again 20% duty
cycle, 10 intensity, 200 cycles per burst, 30 seconds. For the
elevated pressure experiment the pressure in the tube was increased
to 45 psi as described in Example 3 above. 60 .mu.l aliquots were
removed from the glass tubes at selected time intervals and
transferred to a 0.65 ml microcentrifuge tube. The tube was
centrifuged at 10,000 rpm for 1.5 minutes to pellet the cells; 50
.mu.l of the supernatant was assayed for soluble protein using the
Bradford dye binding assay (the absorbance at 595 nm is
proportional to the protein concentration).
[0082] FIG. 10 depicts a graph showing absorbance of yeast samples
after acoustic treatment at various durations, pressures, and
acoustic intensities. The line labeled "Aco. Mix" represents
results from using an acoustic mix that was low power and
sub-cavitation energy to gently resuspend yeast during incubation.
The line labeled "15 psi" represents results from using a high
power focused acoustic field at atmospheric pressure. The line
labeled "45 psi" represents results from using a high power focused
acoustic field at elevated pressure.
[0083] After two minutes of treatment, as shown in the graph
depicted in FIG. 10, the control had a protein assay absorbance of
0.049; the high power acoustic treatment at atmospheric pressure
(15 psi) had an absorbance of 0.174; and the high power acoustic
treatment at 45 psi had an absorbance of 0.415. High power acoustic
treatment at atmospheric pressure resulted in a 3.5-fold increase
in the protein assay absorbance over the control. High power
acoustic treatment at elevated pressure, namely 45 psi, resulted in
an 8-fold increase in the protein assay absorbance over the control
and a 2-fold increase over the high power treatment at atmospheric
pressure, and appeared to reach end-point in 12 minutes.
[0084] Those skilled in the art will know, or be able to ascertain
using no more than routine experimentation, many equivalents to the
embodiments and practices described herein. Accordingly, it will be
understood that the invention is not to be limited to the
illustrative embodiments disclosed herein. Other illustrative
devices, systems, methods, applications, and features of the
invention are described in the following documents which are hereby
incorporated by reference herein: [0085] 1. U.S. Pat. No. 6,719,449
entitled "Apparatus and Method for Controlling Sonic Treatment"
[0086] 2. U.S. application Ser. No. 11/001,988, filed Dec. 2, 2004,
and entitled "Apparatus and Methods for Sample Preparation" [0087]
3. U.S. Pat. No. 6,948,843 entitled "Method and apparatus for
acoustically controlling liquid solutions in microfluidic
devices"
[0088] The first-named reference above discloses apparatuses and
methods for exposing a sample to acoustic energy and for
selectively controlling acoustic energy and/or the location of the
sample relative to acoustic energy that may be used in conjunction
with the invention disclosed herein. In particular, the first
reference discloses various acoustic energy sources, electronics
and waveforms, positioning systems, sensors, control systems,
sample vessels, materials for treatments, and applications of
acoustic treatment.
[0089] The second-named reference above discloses systems, methods,
and devices relating to processing a sample that may be used in
conjunction with the invention disclosed herein. In particular, the
second reference discloses various sample vessels and systems and
methods for collecting, stabilizing, fragmenting and/or analyzing
samples.
[0090] The third-named reference above discloses systems, methods,
and devices relating to coupling acoustic energy to a sample vessel
to lower acoustic energy requirements to obtain desired process
results, such as mixing.
[0091] The subject matter discussed above can readily be adapted
for use in the systems and methods disclosed in the above
references. It should be noted that Applicants consider all
operable combinations of the disclosed illustrative embodiments to
be patentable subject matter including combinations of the subject
matter disclosed in the above references.
* * * * *