U.S. patent application number 15/326892 was filed with the patent office on 2017-07-20 for ultrasound system for shearing cellular material.
This patent application is currently assigned to University of Washington. The applicant listed for this patent is University of Washington. Invention is credited to Karol Bomsztyk, Brian MacConaghy, Thomas J. Matula, Adam D. Maxwell, Justin Reed.
Application Number | 20170205318 15/326892 |
Document ID | / |
Family ID | 55079001 |
Filed Date | 2017-07-20 |
United States Patent
Application |
20170205318 |
Kind Code |
A1 |
Matula; Thomas J. ; et
al. |
July 20, 2017 |
ULTRASOUND SYSTEM FOR SHEARING CELLULAR MATERIAL
Abstract
A system for processing biological or other samples includes an
array of transducer elements that are positioned to align with
sample wells in a microplate. Each transducer element produces
ultrasound energy that is focused towards a well of the microplate
with sufficient acoustic pressure to cause inertial cavitation. In
one embodiment, the transducers are configured to direct ultrasound
energy into cylindrical wells. In other embodiments, the transducer
elements are configured to direct ultrasound energy into
non-cylindrical wells of a microplate.
Inventors: |
Matula; Thomas J.;
(Kirkland, WA) ; Bomsztyk; Karol; (Mercer Island,
WA) ; MacConaghy; Brian; (Kent, WA) ; Reed;
Justin; (Seattle, WA) ; Maxwell; Adam D.;
(Woodinville, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Washington |
Seattle |
WA |
US |
|
|
Assignee: |
University of Washington
Seattle
WA
|
Family ID: |
55079001 |
Appl. No.: |
15/326892 |
Filed: |
July 14, 2015 |
PCT Filed: |
July 14, 2015 |
PCT NO: |
PCT/US2015/040444 |
371 Date: |
January 17, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62025873 |
Jul 17, 2014 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M 47/06 20130101;
B06B 2201/70 20130101; B01L 3/5085 20130101; G10K 11/30 20130101;
B06B 1/0215 20130101; G01N 1/286 20130101; B06B 2201/55 20130101;
B06B 1/0629 20130101 |
International
Class: |
G01N 1/28 20060101
G01N001/28; B01L 3/00 20060101 B01L003/00; B06B 1/02 20060101
B06B001/02; G10K 11/30 20060101 G10K011/30; B06B 1/06 20060101
B06B001/06 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under grant
nos. 1 R21 GM 111439-01 and 1 R33 CA 191135-01 awarded by the
National Institutes of Health. The government has certain rights in
the invention.
Claims
1-13. (canceled)
14. A system for shearing biological materials in a sample well of
a microplate, comprising: a number of transducer elements, each of
which is constructed to receive a conical sample well of a
microplate at least partially through the transducer element and to
deliver ultrasound energy into the sample well at sufficient
acoustic pressures to cause inertial cavitation in a biological
sample.
15. The system of claim 14, where the transducer elements are
configured to create ultrasound energy with the ability to shear
DNA or chromatin samples down to about 50 base pairs.
16. The system of claim 14, where the transducer elements are
configured to create ultrasound energy with the ability to shear
DNA or chromatin to a range of sizes including 100-300 base
pairs.
17. The system of claim 14, wherein each transducer element is
spherical.
18. The system of claim 14, wherein each transducer element is
hemispherical.
19. The system of claim 14, wherein each transducer element is
cylindrical.
20. The system of claim 19, wherein each cylindrical transducer
element includes a lens in an interior portion of the cylindrical
transducer element.
21. The system of claim 14, wherein each transducer element is
disk-shaped with a hole through a central portion thereof and
includes a waveguide on top of the disk to direct ultrasound energy
from the transducer into a sample well of the microplate.
22-24. (canceled)
25. A microplate including a number of wells into which a samples
are to be placed, wherein each well of the microplate has a bottom
surface with a varying thickness that is configured to form a
concave lens to focus ultrasound energy from an external unfocused
ultrasound transducer towards an interior portion of the well.
26. The microplate of claim 26, wherein the bottom surface of each
well of the microplate has a thickness between 52 and 68 microns
thick.
27. A system for shearing cellular material, comprising a signal
generator configured to supply ultrasound driving pulses; an
amplifier for amplifying the driving pulses; and an number of
piezoelectric elements that receive the amplified driving pulses
and are configured to produce ultrasound energy that is directed
into a microplate having a number of wells in which cellular
material is placed, wherein each piezoelectric element is formed of
a piezoelectric substrate that is larger than a single well of the
microplate and smaller than an area of the microplate, whereby the
number of piezoelectric elements are arranged in a pattern to
underlie the wells of the microplate; wherein the amplifier is
configured to amplify the ultrasound driving pulses to a voltage
sufficient to create a negative pressure in the wells of the
microplate of greater than 5 MPa to shear cellular material by
inertial cavitation.
28. The system of claim 27, further comprising a gel layer between
the number of piezoelectric elements and the wells of the
microplate for coupling ultrasound energy into the wells of the
microplate.
29. The system of claim 27, wherein the ultrasound driving pulses
produced by the signal generator have a frequency of 2 MHz and a 15
microsecond duration.
30. The system of claim 29, wherein the amplifier is configured to
amplify the ultrasound driving pulses to a field of 400 volts per
mm. in the piezoelectric substrate.
31. The system of claim 27, further comprising a plate positioned
between the number of piezoelectric elements and the microplate,
wherein the plate includes a number of lenses formed therein that
focus the ultrasound energy produced by the piezoelectric elements
into the wells of the microplate.
Description
CROSS-REFERENCE TO RELATED APPLICATION(S)
[0001] The present application is related to, and claims the
benefit of, U.S. Provisional Patent Application No. 62/025,873
filed Jul. 17, 2014, which is herein incorporated by reference in
its entirety.
TECHNICAL FIELD
[0003] The disclosed technology relates to systems for preparing
materials for analysis and in particular to systems for shearing
cellular material by cavitation.
BACKGROUND
[0004] Sample preparation is one of the preliminary steps that is
performed before biological samples are analyzed. Sample
preparation often involves the breakdown of the material into
cellular or subcellular fragments. One particular application is
the breaking up (or shearing) of DNA or Chromatin into smaller
fragments. Ultrasound is one known method of breaking down
material. In some prior art devices, biological samples are placed
into a test tube that is put into a liquid bath and subjected to
high intensity ultrasound waves--similar to a jewelry cleaner, but
with much high power. To avoid an uneven exposure of the sample,
the test tube is moved around within the ultrasound field as it is
processed. While this approach does work, it is limited to
processing a single test tube sample at a time.
[0005] To increase the throughput of cellular processing, some
systems have proposed analyzing cellular samples in microplates. As
will be appreciated by those skilled in the art, a microplate is a
tray that contains an array of wells in which samples can be placed
for analysis. Advantages of using microplates include the fact that
such trays are easily processed with automated equipment and that
multiple samples can be processed at the same time without moving
the samples from one vessel to another. One system for shearing
cellular samples in a microplate uses ultrasonically vibrating pins
that extend into the wells. However, this can lead to cross
contamination between the various wells and requires extensive
cleaning of the pins. It is also not very useful for tissue
samples. Furthermore, the quality of the results depends greatly on
the exact position of the tips in the sample. Another approach uses
a large ultrasound transducer that is positioned below a single
well and focuses the energy within the well. The focused ultrasound
energy creates cavitation in the sample material that is in the
well but only one well is processed at a time. For a 96 element
microplate, the processing time to shear all the samples can exceed
several hours during which some samples may degrade.
[0006] Another suggested approach to processing cellular material
in a microplate is to place a single ultrasound transducer below
each well. See for example U.S. Pat. No. 6,699,711 to Hahn et al.
("Hahn"). However, when trying to experiment with the system
described in the Hahn patent for use in analyzing biological
materials including DNA and chromatin, it was found that the system
was ineffective in shearing chromatin without causing the
transducers to break.
[0007] Given these problems, there is a need for a system that can
both process cellular samples in parallel using high (negative)
acoustic pressures to induce or facilitate shearing, and can be
operated in a manner that doesn't destroy the transducers.
SUMMARY
[0008] The disclosed technology relates to systems for applying
ultrasound to a number of samples that simultaneously induces
and/or enhances cavitation in the samples. As will be described in
further detail below, the disclosed technology uses transducer
elements that are configured such that the stresses generated while
the transducer is producing ultrasonic energy are not concentrated
at a normal vibrational mode of the transducer element itself. In
one embodiment, two or more transducers are formed on a sheet of
piezoelectric material to form an array. In another embodiment, an
array of transducer elements is created by securing individual
transducers to a common support that absorbs the stresses created
by the individual transducer elements.
[0009] In one embodiment, an array of two or more transducers is
formed from a single sheet of piezoelectric substrate material. In
one embodiment, a lens is positioned in front of each transducer
and focuses the ultrasound produced by the transducers towards a
well of a microplate. The transducers are driven to a level that
induces inertial cavitation in a biological sample that is in the
well. Multiple transducers are driven in parallel to simultaneously
process the material in the wells of the microplate. In another
embodiment, the transducers are curved to focus the acoustic energy
so that a separate lens is not needed.
[0010] In one embodiment, an array of transducer elements is formed
from a sheet of piezoelectric material having one side with a
conductive material disposed on the majority of the surface and a
second side with a conductive material that is patterned into two
or more transducer elements that are not electrically connected and
have a shape that corresponds substantially to the shape of the
wells in the microplate. Electrical connections are made to supply
a varying voltage across the transducer elements. A microplate
having flat well bottoms is placed over the transducers. A lens is
positioned between each transducer element and a well of a
microplate to focus ultrasound generated by each transducer towards
the corresponding well.
[0011] In another embodiment of the disclosed technology, the wells
of the microplate are conical in shape. For this style of
microplate, the transducer elements are shaped to surround a
portion of the wells. The acoustic energy passes into the well from
the sides, not from the bottom. The transducer elements may be
generally spherical, hemispherical, cylindrical or annular with a
center region that receives a portion of a conical well of a
microplate.
[0012] In one embodiment, a plate containing a separate lens for
each transducer element is positioned between the transducer
element and a microplate well. In another embodiment, a lens is
built into each microplate well itself, which operates to focus
ultrasound towards an interior portion of the well.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a diagram of an ultrasound shearing system in
accordance with one embodiment of the disclosed technology;
[0014] FIG. 2 illustrates a simulated focal zone created in a well
of a microplate in accordance with an embodiment of the disclosed
technology;
[0015] FIG. 3 illustrates a cross section of a transducer assembly
showing a relationship between a number of microplate wells and a
corresponding number of ultrasound transducers in accordance with
an embodiment of the disclosed technology;
[0016] FIG. 4 shows an embodiment of the transducer assembly with a
cover in an unlocked position;
[0017] FIG. 5 shows the transducer assembly of FIG. 4 with the
cover in a locked position;
[0018] FIGS. 6A and 6B show one embodiment of an array of
transducers for use in a transducer assembly in accordance with an
embodiment of the disclosed technology;
[0019] FIG. 7 shows an exploded view of a number of ultrasound
transducers and a lens plate in a transducer assembly;
[0020] FIGS. 8A-8C show one method of making electrical connections
to the transducers in a transducer assembly in accordance with an
embodiment of the disclosed technology;
[0021] FIG. 9 illustrates how a circuit board can be used to make
connections to the transducer elements in accordance with an
embodiment of the disclosed technology;
[0022] FIG. 10 shows one example of a lens plate in accordance with
an embodiment of the disclosed technology;
[0023] FIG. 11 shows a transducer assembly with spacers that allows
for the adjustment in height between different microplates and the
transducers/lenses in accordance with an embodiment of the
disclosed technology;
[0024] FIGS. 12A and 12B illustrate measured pressures in a water
tank, and a model of the focal zone caused by a transducer element,
superimposed with a well in accordance with an embodiment of the
disclosed technology;
[0025] FIG. 13A shows a number of spherical transducers that accept
conical wells of a microplate in accordance with an embodiment of
the disclosed technology;
[0026] FIG. 13B shows a wraparound electrode that can be used with
the transducers shown in FIG. 13A;
[0027] FIG. 14 shows a number of cylindrical transducers that
accept conical wells of a microplate in accordance with an
embodiment of the disclosed technology;
[0028] FIG. 15 shows a number of cylindrical transducers with built
in lenses that accept conical wells of a microplate in accordance
with an embodiment of the disclosed technology;
[0029] FIG. 16 shows a number of annular transducers that form a
phased array and accept conical wells of a microplate in accordance
with an embodiment of the disclosed technology;
[0030] FIG. 17 shows a number of semi-spherical transducers that
accept conical wells of a microplate in accordance with an
embodiment of the disclosed technology;
[0031] FIG. 18 shows a number of semi-spherical transducers that
accept conical wells of a microplate in accordance with one
embodiment of the disclosed technology;
[0032] FIG. 19 shows a number of annular transducers with
waveguides on one surface that accept conical wells of a microplate
in accordance with an embodiment of the disclosed technology;
[0033] FIG. 20 shows a number of spherical transducers that accept
conical wells of a microplate in accordance with one embodiment of
the disclosed technology;
[0034] FIG. 21 is a graph of the expected transmission efficiency
for ultrasound into a flat bottomed well of a microplate as a
function of the base thickness of the bottom of the well; and
[0035] FIG. 22 illustrates a microplate well having an integrated
lens to focus ultrasound energy from a transducer into an interior
portion of the well in accordance with an embodiment of the
disclosed technology.
DETAILED DESCRIPTION
[0036] As will be discussed in further detail below, the disclosed
technology relates to a system for applying a sufficient amount of
ultrasound energy to a number of samples in order to cause some
shearing of the molecular bonds in the samples. In one embodiment,
the system simultaneously subjects a number of samples that are in
the wells of a microplate to a sufficient level of ultrasound
energy that causes inertial cavitation to occur in the samples.
[0037] As shown in FIG. 1, a system 100 includes a transducer
assembly 110 that receives a microplate 120 having a number of
sample wells (not individually shown) that contains samples of
biological or other materials. A signal generator 130 provides a
driving signal that is applied to an amplifier 140 that in turn
increases the power of the signals and supplies the amplified
signals to the transducer assembly 110. Individual transducers in
the transducer assembly convert the amplified driving signals into
acoustic energy that is sufficient to shear the materials into
components. In one embodiment, the acoustic energy is
simultaneously applied to each well of the microplate, and the
processing time for the samples is reduced. Furthermore, the
samples may remain in the individual wells of the microplate and
therefore may not need to be moved to another container for further
processing. In the embodiment shown, a spill cover 142 is placed
over the individual wells of the microplate 120 to prevent cross
contamination of the wells of the microplate. The spill cover may
or may not have fingers (not shown) that protrude into the samples
when the cover is in place and can be temperature controlled by,
for example, running a cooling liquid through passageways (not
shown) in a top cover.
[0038] FIG. 2 is a simulation of the ultrasound energy that is
created by a transducer in a single well of a microplate. In this
example, a single cylindrical well 150 is part of a larger
microplate (not shown) and contains a biological sample 160 in the
well. A piezoelectric transducer 170 is positioned below the well
150 and produces ultrasonic energy. The energy is focused by a
concave lens 172 that is positioned between the transducer 170 and
the bottom of the well 150. Typically there would also be some
coupling fluid such as water or a gel (not shown) that is located
between the lens 172 and the bottom of the well 150 in order to
provide a good acoustic coupling and to reduce reflections. The
lens focuses the acoustic energy into a focal zone 176 that is
towards the well 150 at pressure levels that are sufficient to
cause inertial cavitation in the sample 160. Inertial cavitation
causes bubbles to be created in the sample that collapse with an
energy that is sufficient to shear the biological material into
smaller components. In one embodiment, the focused acoustic energy
is sufficient to cause chromatin shearing to yield 100-300 base
pair fragments in each well of the microplate (e.g. an acoustic
pressure amplitude of >1 Megapascal). This figure is
representative only and does not consider the complications of
having a mixture of bubbles in the sample, nor having a finite
volume of liquid in the well. It will be appreciated that the focus
of the transducer element need not necessarily be in the well
itself. Cavitation can still occur in a sample that is located in a
pre-focal or post-focal area. Therefore, the lens need only focus
the ultrasound towards the well and not necessarily into the well
itself.
[0039] FIG. 3 illustrates a cross-sectional view of one possible
embodiment of the ultrasound transducer assembly 110 and the
microplate 120. The microplate 120 includes a number of individual
generally cylindrical wells 122a, 122b, 122c . . . 122h. In one
embodiment, the microplate 120 has 96 individual wells arranged in
an 8.times.12 grid. However, it will be appreciated that other
sizes of microplates could be used, and the transducer array could
be adjusted to fit the appropriate microplate well configuration
(number of elements and size of each element).
[0040] Positioned below each individual well of the microplate is
one or more corresponding ultrasound transducers. For example, an
ultrasound transducer 170a is positioned below well 122a. An
ultrasound transducer 170b is positioned below well 122b etc. A
coupling material 180 such as degassed water or a gel is positioned
between the ultrasound transducers and the individual wells to
provide a good acoustic coupling for the acoustic energy produced
by the ultrasound transducers into the material contained in each
of the wells.
[0041] In some embodiments, the transducer assembly 110 can include
a locking top cover that is placed over the wells of the microplate
hold the microplate in relation to the transducers.
[0042] FIG. 4 shows a top cover 200 having an inner surface 202
that is sized to fit over the outer perimeter of the microplate 120
and over the spill cover 142. The top surface of the top cover 200
includes first and second cylindrical detents 204, 206 on opposite
sides thereof. The detents 204 and 206 are shaped to receive
corresponding cylindrical rods 208 and 210 that are located on arms
212 and 214 that are hinged to the transducer assembly 110. When
the hinged arms are positioned in the unlocked position, the rods
208 and 210 swing away from the detents 204, 206 on the top of the
cover and the cover can be lifted off the microplate. When the arms
are rotated to a locked position, the rod 208 engages the detent
204 and the rod 210 engages the detent 206 as shown in FIG. 5. With
the arms in the locked position, the top cover 200 is secured over
the microplate and to the ultrasound transducer assembly 110 as
shown in FIG. 5. This is one possible embodiment for a cover, and
it is recognized that many other ways to hold the microplate in
place can be used. The top cover can also be kept cool to help
reduce sample heating by running coolant through slots in the top
cover, or by directly cooling the cover itself. In one embodiment,
the coupling material 180 that is positioned between the wells of
the microplate and the individual transducer elements remains
static. In another embodiment, the coupling material can be kept
moving through ports (not shown) on the transducer assembly with a
pump mechanism or the like in order to remove heat that is created
during the application of ultrasound energy to the individual
wells.
[0043] One of the problems encountered in applying ultrasound
energy to the wells with an acoustic pressure that is sufficient to
create shearing in a sample is that the transducers can crack or be
damaged. To overcome this problem, one embodiment of the disclosed
technology groups the ultrasound transducer elements into a
multi-element array in order to spread the stresses created by any
single transducer over a larger area, or offsetting the stresses
from the normal vibrational modes of the transducer element. FIGS.
6A and 6B illustrate one embodiment of a transducer array for
applying acoustic energy to corresponding wells in a microplate. In
the embodiment shown, an array comprises a sheet of piezoelectric
material 250 having a first side 252 and a second side 254. The
first side 252 is coated with a conductive material such as silver
over its entire surface. The second side 254 includes two or more
transducer patterns 256 and 258. The transducer patterns can be
made via a number of techniques such as etching. In one embodiment,
the transducer patterns 256, 258 are created with a
photolithographic process by coating the side 254 with a conductive
material followed by a resist material. The resist material is
exposed with a mask pattern and then chemically etched to remove
the conductive material where it is not desired. After etching the
conductive material, the patterns of the transducers 256 and 258
are left on the surface of the piezoelectric sheet. In the
embodiment shown, the transducers 256 and 258 are circular in shape
to correspond to the shape of the bottom surface of the wells of
the microplate. However, it would be appreciated that other shapes
could be used if desired, or even no patterning at all.
[0044] In one embodiment, the conductive coating on the first side
252 of the substrate is connected via one or more electrical leads
to one electrical potential such as ground, while the transducers
256, 258 are connected via individual leads to a positive
potential. Upon the application of sufficient voltage signals to
the transducers 256, 258, the transducers will produce ultrasonic
sound waves that can be coupled into the individual wells of the
microplate. The electrodes can be wired such that each transducer
element is driven in parallel with other transducer elements or in
a manner such that each individual transducer element can be driven
separately from other elements.
[0045] FIG. 7 is an exploded view of a transducer assembly 110
without the top cover that is constructed in accordance with one
possible embodiment of the disclosed technology. The transducer
assembly 110 includes a base 260, an electrode support plate 270, a
plate 280 having transducers bonded to a bottom surface thereof and
a top cap 290. The base 260 is a generally rectangular enclosure a
closed bottom surface, and an opening 262 in a sidewall through
which conductors to the individual transducer elements can be
routed. The support plate 270 is constructed to support a number of
spring loaded contacts or "pogo pins" in an array that corresponds
to the arrangement of the transducer elements. In one embodiment,
the contacts are wired in parallel so that individual wires do not
need to be routed from each individual transducer element to a
position outside the assembly. However, it is possible to wire each
individual transducer element separately if desired, which may or
may not include pogo pins.
[0046] Above the support plate 270 is the plate 280 with the one or
more arrays of transducer elements secured to a bottom surface
thereof with an acoustically matched epoxy or other adhesive. As
will be explained in further detail below, in one embodiment the
plate 280 includes a number of lenses positioned over a
corresponding transducer element in order to focus ultrasound
energy created by the transducer element towards a well of a
microplate. In one embodiment, the plate 280 is made of a metal
such as aluminum having the lenses formed directly into the plate
280. However, other materials such as ceramics could be used if
desired. In yet another embodiment, separate lens elements may be
secured to the plate 280. The top cap 290 fits over the surface of
the plate 280 and is secured to the base plate 260 with screws or
the like in order to secure the plate 280 and transducers against
the number of spring loaded pins that are held in the support plate
270. A rim 296 extending around an inner perimeter of the top cap
290 supports a microplate (not shown) at a fixed distance from the
top surface of the plate 280 so that ultrasound is focused at the
correct location towards the wells of the microplate. In one
embodiment, liquid, gel or other material is placed into an opening
of the cap 290 prior to the placement of a microplate in order to
effectively couple the acoustic energy produced by the transducers
into the wells of the microplate.
[0047] FIG. 8A shows one embodiment of the support plate 270 that
supports a number of electrical contacts that connect to the
various transducer elements of the transducer assembly. The support
plate 270 has an outer rim 272 that surrounds an arrangement of
cylindrical bores 274 in which individual contact pins are fitted.
In one embodiment, the cylindrical bores 274 are supported by a
honeycomb arrangement of fins 276 that extend outwardly from each
of the bores. Spaces between the fins 276 serve to decrease the
weight of the support plate 270. In the embodiment shown, the top
surface of each cylindrical bore has a flat top section that is
joined at its corners to an adjacent flat top section of another
cylindrical bore. The pattern of cylindrical bores 274 is designed
to match the corresponding pattern of transducer elements and also
to the pattern of wells in the microplate. The support plate 270
can be molded, created by a 3-D printer or constructed using other
techniques.
[0048] FIG. 8B illustrates how the conductive pins 278 are secured
within the support plate 270 and engaged against the surface of the
corresponding transducer elements that are on the bottom surface of
the plate 280. The conductive pins can be press fit into a
cylindrical bore or can be secured by an adhesive. Alignment pins
at the corners of the support plate 270 align the plate when it is
placed in the base section and also provide a vertical space for
the pins. As shown in FIG. 8C, the support plate 270 is secured to
the cap 290 of the ultrasound assembly with fasteners such as
screws or the like. The top cap 290 is secured to the base 260 to
complete the transducer unit.
[0049] As an alternative to using spring-loaded conductors, other
mechanisms can be used to supply the required current and voltages
to the transducer elements. FIG. 9 illustrates one embodiment that
uses a printed circuit board to supply driving signals to the
transducer elements. In this embodiment, a lens plate 350 has a
number of transducer elements 352 secured to a rear surface
thereof. In the embodiment shown, each of the transducer elements
is formed as an array 354 of two transducer elements on a section
of piezoelectric substrate. Groups of these transducer element
pairs are arranged in a pattern corresponding to the pattern of
wells in a corresponding microplate. In this embodiment, a printed
circuit board 360 has a number of openings 362 that correspond to
the position of the transducer elements. The dimensions of the
openings 362 are slightly smaller than the dimensions of the
transducer elements so that a portion of the printed circuit board
360 overlaps a portion of the outer perimeter of the transducer
elements. Therefore, electrical contacts placed on the surface of
the printed circuit board that engages the outer portion of the
transducer elements can be used to deliver signals to the
transducer element. Traces can be routed through the printed
circuit board in order to wire the transducer elements in parallel,
in groups of transducer elements or individually. It is recognized
that the pins or printed circuit board are only to deliver
electrical signals to the transducer elements. Direct soldering and
other methods exist to deliver electrical signals.
[0050] FIG. 10 illustrates further details of the plate 280 in
accordance with the embodiment of the disclosed technology. As
indicated above, the plate can be made of a metal such as aluminum,
ceramic or graphite or other materials having good acoustic
transmission characteristics that couple the acoustic energy
produced by the transducers into the wells of a corresponding
microplate. In the embodiment shown, the top surface of the plate
280 includes a number of concave lenses 282 formed therein. In one
embodiment, each lens is constructed to focus ultrasound energy
from a transducer element at a distance corresponding to the
diameter of the lens (i.e. an F1 lens). However, it will be
appreciated that other lens designs could be used. The lens plate
can be cast or machined.
[0051] FIG. 11 illustrates one embodiment of a system for securing
a microplate at a desired level above the lens plate. In one
embodiment, the transducer assembly includes a number of adjustable
spacers 366 having a height selected to position the bottom of the
microplate wells at a desired height above the surface of the
lenses. Different spacers can be selected depending upon the type
of microplate being used in order to ensure that the focal zone of
the transducers is positioned in a desired portion of the wells. In
one embodiment, the spacers 366 are rods having a diameter selected
in accordance with the type of microplate being used. The rods are
positioned at opposite ends of the lens plate. The microplate is
spaced from the lens plate by a distance corresponding to the
diameter of the rods set to the height above the lenses.
[0052] FIGS. 12A and 12B illustrate the acoustic pressures
generated by a single transducer element. FIG. 12A shows the
measured acoustic pressure in a water tank at the focus of the
transducer element. A 15 microsecond driving pulse at a frequency
of 2 MHz and a driving field of 400 Volts/mm creates a positive
pressure in excess of 30 MPa and a negative pressure of over 15 MPa
in approximately 10 microseconds. The pressures rise and fall as
the driving signal is applied to the transducer element and is then
turned off. FIG. 12B shows a mathematical simulation of the
pressures in a microplate well. As can be seen, the area of
greatest absolute pressure is created at a distance 4-7 mm above
the bottom surface of the well and in the center of the well.
Pressures in this range have been determined to have sufficient
power to create inertial cavitation in a biological sample. The
lower limit of pressures required to induce or facilitate inertial
cavitation has not been determined and may be lower than the
pressures described. As an example, it has been determined that
negative pressures should be greater than 5 MPa. In practice, the
voltages applied to the transducer elements are increased until
cavitation can be detected. The focus can be adjusted to
accommodate more or less sample in the well by changing one or more
of the lens geometry, the frequency of the ultrasound signals
applied or the spacing between the transducer elements and
microplate. As previously stated, one does not have to have the
focus inside the well. Cavitation can also be generated in the
pre-focal or post-focal region.
[0053] In some instances, the wells of the microplate may not be
cylindrical. Therefore, embodiments of the disclosed technology are
constructed and arranged to receive non-cylindrical wells in order
to focus the ultrasound energy into the biological samples held by
the wells. In an embodiment shown in FIG. 13A, a number of
transducer elements 400 are generally spherical having a hole at
their top and bottom that receive a conical well 420 of a
microplate. The transducer elements 400 have an inner surface 402
and an outer surface 404. The inner surface 402 includes a first
electrode thereon and the outer surface 404 includes a second
electrode thereon. In one embodiment, a wire can be bonded to the
inner surface 402 and routed through a hole in the transducer
element to a signal source. In another embodiment as shown in FIG.
13B, a wraparound electrode 408 is electrically connected to the
electrode on the inner surface and terminates on the exterior
surface of the spherical transducer. An outer electrode 410 on the
outer surface of the transducer element surrounds but does not
touch the electrode 408. In one embodiment, the electrodes 408 and
410 are connected by wires to a signal source to cause the
spherical transducer element 400 to vibrate and produce ultrasound
energy.
[0054] In one embodiment, a coupling material such as liquid or a
gel is disposed between the interior surface of the transducer
element 400 and a conical well 420 of a microplate. The spherical
shape of the transducer elements 400 cause the acoustic energy
created by the application of a positive and negative voltage of
the interior and exterior electrodes of the spherical transducer
elements to be focused within the conical well of the microplate
element. In one embodiment, the spherical transducer elements are
cast as hemispheres and are sintered together once the electrodes
are patterned on the inside and outside surfaces of the electrode
elements.
[0055] FIG. 14 illustrates yet another alternative embodiment of a
number of transducer elements that are designed to transmit
ultrasound energy into a non-cylindrical well of a microplate. In
this embodiment, a number of transducers 450 are generally
cylindrical with an inner diameter that is large enough to accept a
portion of a conical well of a microplate. The transducer elements
include an inner surface 452 and an outer surface 454. Electrodes
placed on the inner and outer surfaces allow the application of a
voltage and current signal to the transducer element to cause it to
vibrate and produce ultrasound energy which is focused in a zone
within the well of the microplate. In one embodiment, a coupling
fluid is placed between the inner surface of the transducer 450 and
the exterior surface of the microplate wells to couple the acoustic
energy into the microplate well. In one embodiment, wires can be
used to connect the electrodes on the interior surface 452 and the
outer surface 454 to a signal source. In an alternative embodiment,
wrap around electrodes can be used to route an electrode that is
electrically coupled to the electrode on the interior surface 452
to a position that is on the exterior of the cylindrical transducer
450. Wires or other conductors can then be used to connect the
electrodes to a current and voltage source.
[0056] FIG. 15 illustrates a number of transducer elements in
accordance with another embodiment of the disclosed technology.
Each of these transducer elements is designed to focus ultrasound
energy into a non-cylindrical well of a microplate. In this
example, each transducer 460 has a generally cylindrical shape with
an inner diameter that is sized to receive a portion of the conical
well of a microplate. An electrode 462 on an interior surface of
the transducer element and an electrode 464 on the exterior surface
of the transducer element are used to supply a current and voltage
to the transducer element. In this embodiment, a lens 470 is
positioned between the interior surface of the transducer element
and the well of the microplate. In the embodiment shown, the lens
470 has a radial thickness that varies parabolically whereby it is
thinner at a top end 472 of the transducer element and radially
thicker at a bottom surface 474 of the transducer element. In one
embodiment, the lens 470 is made of an acoustically transparent
material such as aluminum or a ceramic. The design of the lens is
selected to focus ultrasound energy created by the cylindrical
electrode into the middle of the well.
[0057] Yet another alternative embodiment of a transducer element
in accordance with the disclosed technology is shown in FIG. 16. In
this embodiment, a transducer 500 includes a number of annular
transducer elements 502a, 502b . . . 502i. Each of the transducer
elements 502a-502i is annular in shape with an outer electrode 506
disposed on the outer perimeter of the transducer element and an
inner electrode 504 on an inner perimeter of the transducer. Again,
as described above, connections to the electrodes can be made with
wires or a wraparound electrode can be used to electrically connect
to the inner electrode. Application of a current and voltage signal
to the inner and outer electrodes cause the transducer elements
502a-502i to produce ultrasound signals. Because the elements
502a-502i are stacked, they can be driven as a phased array to
control focusing of the ultrasound waves into a desired portion of
the wells of a microplate.
[0058] FIG. 17 shows yet another embodiment of a transducer
designed to direct ultrasound energy into the non-cylindrical wells
of a microplate. In this embodiment, each transducer 520 comprises
a hemispherical shell having an inner surface 522 and an outer
surface 524. The shell is arranged as a cup whereby the open
portion of the hemisphere faces toward the top of the well. The
transducer elements have electrodes on the inner and outer surfaces
of the hemisphere such that application of a current and voltage
signal to the inner and outer electrodes causes the transducer
element 520 to produce ultrasound energy and direct it into a
desired zone within the conical portion of the microplate well.
Because the transducer elements are partially spherical, ultrasound
energy is generally directed towards the geometric center of the
hemisphere. The transducer element includes a hole through its
lower surface through which a tip of the microplate well extends.
In one embodiment, separate wires can be attached to the inner and
outer electrodes of the transducer element 520. Alternatively,
wrap-around electrodes can be used to allow the wires that connect
to both electrodes to be located on the exterior surface of the
electrode.
[0059] FIG. 18 illustrates a number of transducer elements that are
designed to direct ultrasound energy into a non-cylindrical well of
a microplate in accordance with an embodiment of the disclosed
technology. In this embodiment, the transducer elements 540 are
hemispherically shaped with an interior surface 542 and an exterior
surface 544. Electrodes are placed on the interior and outer
surfaces such that application of a current and voltage signal to
the electrodes causes the transducer element 540 to produce
ultrasonic energy which is focused at a desired location in the
well of the microplate. In this embodiment, the transducer elements
are arranged as umbrellas whereby the larger diameter of the
hemisphere is pointed towards the bottom of the microplate well.
Again, because the transducer element 540 is hemispherically
shaped, the ultrasound energy produced will be focused at roughly
the geometric center of the transducer. A hole positioned at the
top surface of the electrode receives the tip of the microplate
well such that the microplate well extends through the center of
the transducer element 540. Connections to the electrodes on an
interior of the transducer elements can be made with individual
wires. Alternatively, a wraparound electrode can be used to allow
connections to be made on the outside of the transducer
element.
[0060] FIG. 19 illustrates yet another alternative embodiment of a
transducer. In this embodiment, a transducer 560 is formed as a
generally flat disk with a circular perimeter and a hole in the
center. A first electrode is formed on the bottom surface 562 of
the disk and a second electrode is formed on the top surface 564 of
the disk. The tip of the conical well of the microplate extends
through a hole at the center of the disk. Above the top surface of
the transducer element 560 is an acoustic reflector 570. The
acoustic reflector has a lower surface that engages the top surface
564. The diameter of the acoustic reflector tapers from a diameter
equal to the diameter of the disk and gets smaller as it extends
upwards away from the top surface 564 of the transducer 560 until
it has a lesser diameter, thereby giving the acoustic reflector 570
a generally bell shape. The acoustic reflector 570 operates as an
acoustic waveguide to focus the ultrasound energy produced by the
transducer element 560 at a desired location in the well of the
microplate. Suitable materials for the acoustic reflector 570
include metals, such as aluminum, or ceramics.
[0061] In yet another alternative embodiment of the disclosed
technology, the transducer elements can be capacitively coupled to
the well of the microplate. FIG. 20 illustrates an example where a
pair of transducer elements 602a and 602b are generally spherical
in shape with a top hole 606 on its top surface and a smaller hole
608 on its bottom surface. Dimensions of the holes 606 and 608 are
designed to accept a correspondingly conical shaped well 620 of a
microplate. In this embodiment, the outer portions of the
transducers 602a, 602b are seated in a conductive material such as
a conductive epoxy that forms an electrode on the exterior surface
of the transducer elements. The second electrode is formed on the
exterior surface of the microplate wells. As shown, an electrode
624 is disposed on the outer conical portion of a microplate well.
The holes through the transducers are tapered such that insertion
of the conical well into the holes of the transducer elements
causes the electrode 624 to be positioned against an interior
surface of the transducer element. Application of a suitable
current and voltage signal between the conductive material 618 and
the corresponding electrodes 624 on the outer surface of the well
causes the transducer elements 602a, 602b to produce ultrasound
energy which is focused at a location in the interior of the
microplate wells. In another embodiment, there may be a gap between
the electrode on the microplate well and the interior of the
transducer element (e.g. a spherical transducer element).
[0062] In some embodiments, it has been determined that the
thickness of the bottom portion of the well of a microplate affects
the transmission of acoustic energy into the well. The efficiency
has been determined to increase to a maximum efficiency at some
point between a minimum and maximum thickness of the well bottom.
In one embodiment shown in FIG. 21, it has been calculated that the
efficiency rises as the thickness is increased from 52 microns to a
maximum efficiency occurring at approximately 59 microns and then
decreases as the thickness increases from 59 microns to 68 microns.
The exact values depends on the material properties of the
microplate. Therefore, it can be seen that the thickness of the
base of the microplate well can be selected in order to achieve
maximum transmission efficiency. In one embodiment, the base
thickness is selected to be approximately 59 microns in order to
achieve maximum transmission of acoustic energy into the well.
[0063] In yet an alternative embodiment, the bottom of the
microplate well can be molded as a lens to focus ultrasound energy
into the well, and as such may not require a separate focusing lens
between the transducer and the bottom of the microplate. FIG. 22
shows a cutaway view of a microplate well 656 having a lens 660
integrally formed therein. In the example shown, a sheet of
piezoelectric material 650 has a transducer element 652 formed onto
a bottom surface. The transducer element 652 has a generally
circular shape that matches the diameter of the cylindrical
microplate well 656. A coupling material such as water or gel 654
is positioned between the bottom of the well 656 and the attachment
plate for the transducer, 650. The attachment plate 650 may be
metal and used to remove heat from the water or gel pad. In this
embodiment, the bottom and/or sidewalls of the well of the
microplate well are not of a uniform thickness but have a thickness
that varies to focus acoustic energy from the transducer element
652 towards an interior portion of the well 656. In one embodiment,
the bottom of the well has a concave shape to act as a lens that
focuses ultrasound energy into an interior portion of the well. The
well 656 can be injection-molded to form the lens 660 in its
desired shape and focus the ultrasound energy into the desired
portion of the well. Biological materials within the well 656 are
sheared due to the inertial cavitation occurring in the focal zone
670. After application of the ultrasound energy to the wells of the
microplate, the samples are ready for further processing.
[0064] From the foregoing, it will be appreciated that specific
embodiments of the invention have been described herein for
purposes of illustration, but that various modifications may be
made without deviating from the scope of the invention. For
example, although the disclosed embodiments show the use of a
single transducer element positioned to direct ultrasound into a
single sample well, it will be appreciated that two or more
transducer elements could be positioned to direct ultrasound into a
single well. Furthermore, although the samples are described as
being held in the wells of a microplate, it will be appreciated
that the size of the system can be adjusted to direct ultrasound
into other sample holders (e.g. an array of petri dishes etc.) In
yet another embodiment, the transducer elements are formed from a
sheet of piezoelectric material with a conductor one side and a
flex circuit joined to the other side, where the flex circuit
includes conductors that form the transducers when secured to a
piezoelectric material. Alternatively, the array of transducers can
be made by grouping individual transducer elements and securing
them via an adhesive or the like to a common support structure
(e.g. a piece of aluminum) such that the support structure absorbs
a portion of the stresses created from each of the transducers
secured thereto. Accordingly, the invention is not limited except
as by the appended claims.
* * * * *