U.S. patent application number 10/778831 was filed with the patent office on 2004-08-26 for cell sorting apparatus and methods for manipulating cells using the same.
Invention is credited to Braff, Rebecca, Gray, Martha, Schmidt, Martin, Toner, Mehmet, Voldman, Joel.
Application Number | 20040166555 10/778831 |
Document ID | / |
Family ID | 31190633 |
Filed Date | 2004-08-26 |
United States Patent
Application |
20040166555 |
Kind Code |
A1 |
Braff, Rebecca ; et
al. |
August 26, 2004 |
Cell sorting apparatus and methods for manipulating cells using the
same
Abstract
A cell analysis and sorting apparatus is capable of monitoring
over time the behavior of each cell in a large population of cells.
The cell analysis and sorting apparatus contains individually
addressable cell locations. Each location is capable of capturing
and holding a single cell, and selectively releasing that cell from
that particular location. In one aspect of the invention, the cells
are captured and held in wells, and released using vapor bubbles as
a means of cell actuation. In another aspect of the invention, the
cells are captured, held and released using electric field
traps.
Inventors: |
Braff, Rebecca; (Boston,
MA) ; Voldman, Joel; (Somerville, MA) ; Gray,
Martha; (Arlington, MA) ; Schmidt, Martin;
(Reading, MA) ; Toner, Mehmet; (Wellesley,
MA) |
Correspondence
Address: |
NUTTER MCCLENNEN & FISH LLP
WORLD TRADE CENTER WEST
155 SEAPORT BOULEVARD
BOSTON
MA
02210-2604
US
|
Family ID: |
31190633 |
Appl. No.: |
10/778831 |
Filed: |
February 13, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10778831 |
Feb 13, 2004 |
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09710032 |
Nov 10, 2000 |
|
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6692952 |
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60164643 |
Nov 10, 1999 |
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Current U.S.
Class: |
435/29 ;
435/288.4; 435/34 |
Current CPC
Class: |
B01J 2219/00605
20130101; G01N 15/147 20130101; B01J 2219/00743 20130101; B01J
2219/00614 20130101; B01J 2219/00653 20130101; B01J 2219/00659
20130101; B01J 2219/00704 20130101; G01N 2015/149 20130101; G01N
2015/1415 20130101; G01N 30/0005 20130101; B01J 2219/00637
20130101; G01N 15/1484 20130101; B01J 2219/00612 20130101; G01N
15/1456 20130101; B01J 2219/00317 20130101 |
Class at
Publication: |
435/029 ;
435/034; 435/288.4 |
International
Class: |
C12Q 001/04 |
Claims
What is claimed is:
1. A method for analyzing a cell population, comprising: providing
a cell sorting apparatus having an array of sites, each site
including a capture mechanism comprising a well that is capable of
capturing a single cell, and a release mechanism comprising an
actuator coupled to the well for selectively releasing the single
cell from its site; introducing a fluid medium containing a
plurality of cells onto the apparatus; capturing at least one cell
in a well; identifying a cellular response in the at least one
captured cell; and selecting at least one captured cell based on
the cellular response.
2. The method of claim 1, wherein the step of capturing further
comprises inducing a fluid medium with a cell population to flow
across the substrate.
3. The method of claim 1, wherein the cell is captured in the well
by negative pressure.
4. The method of claim 1, wherein the cell is captured in the well
by well geometry.
5. The method of claim 1, wherein the cell is captured in the well
by gravitational forces.
6. The method of claim 1, wherein the cell is captured in the well
by electric field forces.
7. The method of claim 1, wherein the cell is captured in the well
by dielectrophoretic forces.
8. The method of claim 1, wherein the step of identifying a
cellular response further includes applying an optical probe to the
captured cell and detecting an optical signal from the captured
cell.
9. The method of claim 1, wherein a plurality of cells are
captured, and the method further comprises the step of applying a
fluorescent agent to the plurality of cells.
10. The method of claim 9, further including the step of detecting
fluorescence exhibited by captured cells within the sorting
apparatus.
11. The method of claim 10, further including the step of measuring
intensity of fluorescence at individual sites in the sorting
apparatus over time.
12. The method of claim 1, wherein the step of identifying a
cellular response further comprises coupling a photometric array to
the cell sorting apparatus to detect fluorescence at sites in the
sorting apparatus.
13. The method of claim 12, further including the step of comparing
the fluorescence intensity at particular sites.
14. The method of claim 13, wherein the step of selecting a cell
further comprises selecting a cell based on its fluorescence
intensity, and releasing the cell from its well.
15. The method of claim 13, wherein a receptor-ligand binding event
at a site in the sorting apparatus is identified by fluorescence
intensity.
16. The method of claim 13, wherein a protein to protein
interaction at a site in the sorting apparatus is identified by
fluorescence intensity.
17. The method of claim 13, wherein cellular signal transduction at
a site in the sorting apparatus is identified by fluorescence
intensity.
18. The method of claim 13, wherein intracellular calcium transport
at a site in the sorting apparatus is identified by fluorescence
intensity.
19. The method of claim 1, wherein the step of selecting at least
one captured cell further comprises releasing the captured cell
from its well by activating the release mechanism of the well.
20. The method of claim 19, wherein the step of releasing comprises
forming a microbubble to displace the captured cell from its
well.
21. The method of claim 19, wherein the step of releasing comprises
lowering an energy field surrounding the captured cell.
22. A method for sorting individual cells in a cell population,
comprising: providing a cell sorting apparatus having an array of
sites, each site including a capture mechanism comprising a well
that is capable of capturing a single cell, and a release mechanism
comprising an actuator coupled to the well for selectively
releasing the single cell from its site; introducing a fluid medium
containing a plurality of cells onto the apparatus; capturing at
least one cell in a well; selecting a captured cell; and releasing
the selected, captured cell from the apparatus.
23. The method of claim 22, wherein the step of capturing comprises
inducing a fluid medium with a cell population to flow across the
substrate.
24. The method of claim 22, wherein the cell is captured in the
well by negative pressure.
25. The method of claim 22, wherein the cell is captured in the
well by well geometry.
26. The method of claim 22, wherein the cell is captured in the
well by gravitational forces.
27. The method of claim 22, wherein the cell is captured in the
well by electric field forces.
28. The method of claim 22, wherein the cell is captured in the
well by dielectrophoretic forces.
29. The method of claim 22, wherein a plurality of cells are
captured within wells.
30. The method of claim 22, wherein the step of releasing the
captured cell further comprises activating the actuator so as to
release the captured cell from its well.
31. The method of claim 30, wherein the step of activating further
comprises forming a microbubble to displace the captured cell from
its well.
32. The method of claim 22, wherein the step of releasing the
captured cell further comprises lowering an energy field
surrounding the captured cell.
33. The method of claim 22, wherein the step of releasing the
captured cell further comprises releasing the selected, captured
cell into a fluid flow around the well.
34. The method of claim 22, further including the step of
collecting at least one released cell.
35. The method of claim 22, further including the step of coupling
a photometric array to the apparatus to detect fluorescence at
sites in the sorting apparatus.
36. The method of claim 35, further including the step of applying
a fluorescent agent to the captured cell.
37. The method of claim 36, further including the step of detecting
fluorescence at sites within the sorting array.
38. The method of claim 37, wherein a temporal response of captured
cells to stimuli is monitored.
39. The method of claim 22, wherein the step of selecting a
captured cell from the sorting apparatus further comprises
selecting the cell selected based on its response to at least one
stimulus.
40. The method of claim 39, further including the step of observing
a temporal response to stimulus.
41. The method of claim 39, wherein the captured cell is selected
based on detection of a receptor-ligand binding event at a site in
the sorting apparatus.
42. The method of claim 39, wherein the step of selecting the
captured cell is based on detection of a protein to protein
interaction at a site in the sorting apparatus.
43. The method of claim 39, wherein the captured cell is selected
based detection of cellular signal induction at a site in the
sorting apparatus.
44. The method of claim 39, wherein the captured cell is selected
based on detection of intracellular calcium transport at a site in
the sorting apparatus.
45. The method of claim 39, wherein the captured cell is selected
based on detection of a drug response at a site in the sorting
apparatus.
46. The method of claim 39, wherein the captured cell is selected
based on detection of a phenotypical response at a site in the
sorting apparatus.
47. The method of claim 46, wherein the phenotypical response of
the selected cell is different from that of other cells captured in
the sorting apparatus.
48. The method of claim 39, wherein the captured cell is selected
based on a reporter-gene based assay.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/710,032, filed on Nov. 10, 2000, now U.S.
Pat. No. 6,692,952, which claims priority to U.S. Provisional
Application No. 60/164,643, filed on Nov. 10, 1999.
FIELD OF THE INVENTION
[0002] This invention relates to cell analysis and sorting devices
and methods for manipulating cells using these devices. More
particularly, the invention relates to a cell analysis and sorting
apparatus that can capture and hold single cells at known locations
and then selectively release certain of these cells. A method of
manipulating the cells using the cell analysis and sorting
apparatus is also provided.
BACKGROUND OF THE INVENTION
[0003] Many recent technological advances have enhanced the study
of cellular biology and biomechanical engineering, most notably by
improving methods and devices for carrying out cellular analysis.
For example, in the past decade an explosion in the number of
optical probes available for cell analysis has enabled an increase
in the amount of information gleaned from microscopic and flow
cytometric assays. Microscopic assays allow the researcher to
monitor the time-response of a limited number of cells using
optical probes. Flow cytometry, on the other hand, uses optical
probes for assays on statistically significant quantities of cells
for sorting into subpopulations.
[0004] However, these mechanisms alone are insufficient for
time-dependent analysis. Microscopic assays can only track a few
cells over time, and do not allow the user to track the location of
individual cells. With flow cytometry, the user can only observe
each cell once, and can only easily sort a cell population into
three subpopulations. Flow cytometry techniques fail to provide for
analysis of the same cell multiple times, or for arbitrary sorting
of subpopulations. These kinds of bulk assay techniques produce
mean statistics, but cannot provide the researcher with
distribution statistics.
[0005] Advances in microsystems technology have also influenced
many applications in the fields of cell biology and biomedical
engineering. Scaling down to the micron level allows the use of
smaller sample sizes than those used in conventional techniques.
Additionally, the smaller size and ability to make large arrays of
devices enables multiple processes to be run in parallel.
[0006] Integrated circuits have been fabricated on silicon chips
since the 1950s, and as processing techniques improve, the size of
transistors continues to shrink. The ability to produce large
numbers of complex devices on a single chip sparked interest in
fabricating mechanical structures on silicon as well. The range of
applications for micro electromechanical systems (MEMS) is
enormous. Accelerometers, pressure sensors, and actuators are just
a few of the many MEMS devices currently produced. Another
application of MEMS is in biology and medicine. Micromachined
devices have been made for use in drug-delivery, DNA analysis,
diagnostics, and detection of cell properties.
[0007] Manipulation of cells is another application of MEMS. For
example, in the early 1990's, Sato et al. described in his paper,
which is hereby incorporated by reference, Individual and Mass
Operation of Biological Cells using Micromechanical Silicon
Devices, Sensors and Actuators, 1990, A21-A23:948-953, the use of
pressure differentials to hold cells. Sato et al. microfabricated
hydraulic capture chambers that were used to capture plant cells
for use in cell fusion experiments. Pressure differentials were
applied so that single cells were sucked down to plug an array of
holes. Cells could not be individually released from the array,
however, because the pressure differential was applied over the
whole array, not to individual holes.
[0008] Bousse et al. in his paper, which is hereby incorporated by
reference, Micromachined Multichannel Systems for the Measurement
of Cellular Metabolism, Sensors and Actuators B, 1994, 20:145-150,
described arrays of wells etched into silicon to passively capture
cells by gravitational settling. Multiple cells were allowed to
settle into each of an array of wells where they were held against
flow due to the hydrodynamics resulting from the geometry of the
wells. Changes in the pH of the medium surrounding the cells were
monitored by sensors in the bottom of the wells, but the wells
lacked a cell-release mechanism, and multiple cells were trapped in
each well. Another known method of cell capture is
dielectrophoresis (DEP). DEP refers to the action of neutral
particles in non-uniform electric fields. Neutral polarizable
particles experience a force in non-uniform electric fields which
propels them toward the electric field maxima or minima, depending
on whether the particle is more or less polarizable than the medium
it is in. By arranging the electrodes properly, an electric field
may be produced to stably trap dielectric particles.
[0009] Microfabrication has been utilized to make electrode arrays
for cell manipulation since the late 1980s. Researchers have
successfully trapped many different cell types, including mammalian
cells, yeast cells, plant cells, and polymeric particles. Much work
involves manipulating cells by exploiting differences in the
dielectric properties of varying cell types to evoke separations,
such as separation of viable from non-viable yeast, and enrichment
of CD34+ stem cells from bone marrow and peripheral blood stem
cells. More relevant work on trapping cells in various two- and
three-dimensional microfabricated electrode geometries has been
shown by several groups. However, trapping arrays of cells with the
intention of releasing selected subpopulations of cells has not yet
been widely explored. Additionally, DEP can potentially induce
large temperature changes, causing not only convection effects but
also profoundly affecting cell physiology.
[0010] These studies demonstrate that it is possible to trap
individual and small numbers of cells in an array on a chip, but
without the ability to subsequently manipulate and selectively
release individual cells. This inability to select or sort based on
a biochemical measurement poses a limitation to the kinds of
scientific inquiring that may be of interest.
[0011] The currently available mechanisms for carrying out cell
analysis and sorting are thus limited in their applications. There
is thus a need for an improved method and apparatus for sorting and
releasing large quantities of cells that can easily and efficiently
be used. In addition, there is a need for an analysis and sorting
device that allows the user to look at each cell multiple times,
and to track many cells over time. Finally, there is a need for a
cell sorter that lets the user know the cell locations, and to be
able to hold and selectively release the cells so that the user can
arbitrarily sort based on any aspect of the cells' characteristic
during time-responsive assays.
SUMMARY OF THE INVENTION
[0012] The present invention provides a cell sorting apparatus that
is capable of monitoring over time the behavior of each cell in a
large population of cells. The cell analysis and sorting apparatus
contains individually addressable cell locations. Each location is
capable of capturing and holding a single cell, and selectively
releasing that cell from that particular location. In one aspect of
the invention, the cells are captured and held in wells, and
released using vapor bubbles as a means of cell election. In
another aspect of the invention, the cells are captured, held and
released using electric field traps.
[0013] According to one aspect of the present invention, the cell
analysis and sorting apparatus has an array of geometric sites for
capturing cells traveling along a fluid flow. The geometric sites
are arranged in a defined pattern across a substrate such that
individual sites are known and identifiable. Each geometric site is
configured and dimensioned to hold a single cell. Additionally,
each site contains a release mechanism to selectively release the
single cell from that site. Because each site is able to hold only
one cell, and each site has a unique address, the apparatus allows
the user to know the location of any particular cell that has been
captured. Further, each site is independently controllable so that
the user is able to arbitrarily capture cells at select locations,
and to release cells at various locations across the array.
[0014] In one embodiment of the present invention, the geometric
sites are configured as wells. As a fluid of cells is flown across
the array of specifically sized wells, cells will fall into the
wells and become trapped. Each well is sized and shaped to capture
only a single cell, and is configured such that the cell will not
escape into the laminar flow of the fluid above the well. The
single cell can be held inside the well by gravitational forces.
Each well can further be attached via a narrow channel to a chamber
located below the well. Within the chamber is a heating element
that is able to induce bubble nucleation, the mechanism for
releasing the cell from the site. The bubble creates volume
expansion inside the chamber which, when filled with fluid, will
displace a jet of fluid out of the narrow channel and eject the
cell out of the well. Fluid flow above the well will sweep the
ejected cell away to be either collected or discarded.
[0015] In another embodiment of the present invention, the
geometric sites are formed from a three-dimensional electric field
trap. Each trap comprises four electrodes arranged in a trapezoidal
configuration, where each electrode represents a corner of the
trapezoid. The electric fields of the electrodes create a potential
energy well for capturing a single cell within the center of the
trap. By removing the potential energy well of the trap, the cell
is ejected out of the site and into the fluid flow around the trap.
Ejected cells can then be washed out and collected or
discarded.
[0016] In yet another embodiment of the present invention, an
integrated system is proposed. The system can be a
microfabrication-based dynamic array cytometer (.mu.DAC) having as
one of its components the cell analysis and sorting apparatus
previously described. To analyze a population of cells, the cells
can be placed on a cell array chip containing a plurality of cell
sites. The cells are held in place within the plurality of cell
sites in a manner similar to that described above and analyzed, for
example, by photometric assay. Using an optical system to detect
fluorescence, the response of the cells can be measured, with the
intensity of the fluorescence reflecting the intensity of the
cellular response. Once the experiment is complete, the cells
exhibiting the desired response, or intensity, may be selectively
released into a cell sorter to be further studied or otherwise
selectively processed. Such an integrated system would allow
researchers to also look at the cell's time response.
[0017] Further features and advantages of the present invention as
well as the structure and operation of various embodiments of the
present invention are described in detail below with reference to
the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] This invention is pointed out with particularity in the
appended claims. The above and further advantages of this invention
may be better understood by referring to the following description
when taken in conjunction with the accompanying drawings, in
which:
[0019] FIGS. 1A, 1B, 1C, and 1D show the mechanism by which one
embodiment of the present invention uses to capture, hold and
release a single cell.
[0020] FIGS. 2A, 2B, and 2C show a process by which another
embodiment of the present invention uses to capture, hold and
release a single cell.
[0021] FIGS. 3A and 3B show a top-down view of the cell sorting
apparatus of FIG. 2.
[0022] FIG. 4 shows an exploded view of the cell sorting apparatus
of FIG. 2.
[0023] FIG. 5 shows an exploded view of yet another embodiment of
the present invention in which a cell sorting apparatus is
integrated into a fluorescence-detecting system.
[0024] FIG. 6 is the thermodynamic pressure-volume diagram for
water.
[0025] FIG. 7A shows a top view of a resistor of the present
invention.
[0026] FIG. 7B shows a cross-section of the resistor of FIG.
7A.
[0027] FIG. 8 shows thermal resistances as seen by a heater of the
present invention.
[0028] FIGS. 9A and 9B show flow lines for flow over rectangular
cavities of different aspect ratios.
[0029] FIG. 10 shows a schematic of forces on a particle in a
well.
[0030] FIG. 11A shows a top view of a heater of the present
invention.
[0031] FIG. 11B shows a cross-section of the heater of FIG.
11A.
[0032] FIG. 12A shows a side view of a cell well of the present
invention.
[0033] FIG. 12B shows a top-down view of the cell well of FIG.
12A.
[0034] FIGS. 13A, 13B, and 13C shows a top-down view of a silicon
processing mask set for use in the present invention.
[0035] FIG. 14 shows a top-down view of a glass processing
mask.
[0036] FIG. 15 shows a diagram of a flow system for testing devices
of the present invention.
[0037] FIG. 16A shows a top-down view of a flow chamber of the
present invention.
[0038] FIG. 16B shows a side view of the flow chamber of FIG.
16A.
[0039] FIG. 17 is a graph of pressure drop vs. flow rate for the
flow chamber of FIGS. 16A and 16B.
[0040] FIG. 18A shows a top-down view of the chamber base of flow
chamber of FIG. 16A and 16B.
[0041] FIG. 18B shows a side view of the chamber base of FIG.
18A.
[0042] FIG. 18C shows a top-down view of the chamber lid of flow
chamber of FIG. 16A and 16B.
[0043] FIG. 18D shows a side view of the chamber lid of FIG.
18C.
[0044] FIGS. 19A-19C show a process of fabricating a glass slide of
the present invention.
[0045] FIGS. 20A-20H show a process of fabricating a silicon wafer
of the present invention.
[0046] FIGS. 21A-21D show a process of assembling the silicon wafer
of FIGS. 20A-20H onto the glass slide of FIGS. 19A-19C.
[0047] FIG. 22 is a graph of temperature v. resistance for platinum
resistors of the present invention.
[0048] FIG. 23 shows a configuration for a resistor testing
apparatus used in the present invention.
[0049] FIG. 24 is a graph of current v. voltage for the onset of
boiling in platinum line resistors of the present invention.
[0050] FIG. 25 is a graph of current v. temperature for the
platinum line resistors of FIG. 24.
[0051] FIG. 26 is a graph of temperature v. resistance for a set of
annealed platinum line resistors of the present invention.
[0052] FIG. 27 is a graph of temperature v. resistance for a set of
annealed platinum line resistors which were heated on a hot
plate.
[0053] FIG. 28 is a graph of current v. voltage for a set of
annealed platinum line resistors of the present invention.
[0054] FIG. 29 is a graph of current v. temperature for the
resistors of FIG. 28.
[0055] FIG. 30 is a graph of current v. voltage for the resistors
of FIG. 28 under repeated boiling tests.
DETAILED DESCRIPTION OF THE INVENTION
[0056] FIGS. 1A-1D illustrate an exemplary system of the present
invention. A cell site 10, shown in cross-section, contains a well
12 sized and shaped to hold a single cell 18. Connected to the
bottom of the well 12 is a narrow channel 14 that opens into a
chamber 16 situated below the well. In this particular example, the
well 12 and narrow channel 14 are etched out of a silicon wafer.
The silicon wafer is attached to a glass slide on which there is a
platinum heater 20, and the alignment is such that the heater 20 is
sealed inside the chamber 16, which is filled with a fluid such as
water.
[0057] The well 12 functions as a capture and hold mechanism. In
operation, fluid containing cells is flown over the top of the
apparatus, and then the flow is stopped. As shown in FIG. 1A, the
cells then settle and gravitational forces will allow one cell 18
to fall into and become trapped within the well 12. At this point
the flow is started again, and the cell in the well is trapped
while the cells not in wells are flushed away by convection. FIG.
1B shows how the well 12 is dimensioned and configured to hold only
one cell 18 within the well 12 at a time. In addition, the well 12
is configured such that the cell 18 will not be swept out of the
well due to laminar or fluid flow above.
[0058] Experiments may be performed on the trapped cells, such as
by adding a reagant. When the experiments are concluded, the cells
exhibiting the desired characteristics may be selectively released
from the wells. In this example, when it is desired to release cell
18 from the well 12, the operator can apply a voltage to the
heating element 20 within the chamber 16. The heating element 20 is
then heated to a temperature above the superlimit of the fluid
contained within the chamber 16 to initiate vapor bubble nucleation
at the surface of the heating element 20, as seen in FIG. 1C. In
FIG. 1D, a microbubble 22 is formed inside the chamber, creating a
volume displacement. By adjusting the voltage of the heating
element 20, the operator can control the size of the microbubble
22. When the microbubble 22 is of sufficient size, the volume
expansion in the chamber will displace a jet of fluid within the
chamber 16 out of the narrow channel 14, ejecting the cell 18 out
of the well 12. The released cell 18 can be swept into the fluid
flow outside the well 12, to be later collected or discarded.
[0059] In another exemplary system of the present invention, the
cell site 30 includes electric field traps. FIGS. 2A-2C show, in
cross-section, two cell sites on a substrate such as a
microfabricated chip 36. Each site includes a plurality of
electrodes 32. Preferably, each cell site 30 contains four
electrodes, positioned in a trapezoidal configuration, as seen in
FIGS. 3A and 3B. The cell site 30 is configured and positioned such
that only one cell can be held within the site. The electrodes 32
create a non-uniform electric field trap within which a single cell
34 can be held and subsequently released. FIG. 4 illustrates how
the location and polarity of the electrodes 32 can create an
electric field trap for capturing the cell 34.
[0060] In use, cells in fluid medium flow over the cell sites 30,
as shown in FIG. 2A. By adjusting the electric field of each
electrode 32, a potential energy well can be created within each
cell site 30. The potential energy well is of sufficient strength
to capture a single cell 34 traveling along the fluid flow and to
hold the cell 34 within the center of the trap, as seen in FIG. 2B.
When the operator selects to release a cell 34, he can adjust the
electric fields of the electrodes 32 forming the trap. FIG. 2C
shows how this in turn removes the potential energy well, releasing
the cell 34 back into the fluid flow. The cell 34 can then be
collected or discarded.
[0061] The electrodes forming the electric field trap are
preferably thin-film poles formed of gold. This creates a
three-dimensional electric field trap that is effective in holding
a cell against the laminar flow of the fluid surrounding the
electrodes. Further, while only one or two cell sites are
illustrated, it is understood that the drawings are merely
exemplary of the kind of site that can be included in the cell
sorting apparatus of the present invention. The cell sorting
apparatus can contain anywhere from a single cell site to an
infinite number of cell sites, for sorting mass quantities of
cells. Moreover, while the embodiments herein are described as
holding cells, it is understood that what is meant by cells
includes biological cells, cellular fragments, particles,
biological molecules, ions, and other biological entities.
[0062] Because the cell sorting apparatus of the present invention
allows the operator to know the location of each cell in the array
of cell sites, the operator is able to manipulate the cells and
arbitrarily sort the cells based on their characteristic under
time-responsive assays. One such method contemplates using scanning
techniques to observe dynamic responses from cells. As shown in
FIG. 5, an integrated cellular analysis system 100 is proposed in
which cells are tested using light-emitting assays to determine the
cell's response to stimuli over time. The integrated system can be
a microfabrication-based dynamic array cytometer (.mu.DAC). The
tested cells are placed on a cell array chip 110 similar to the
cell sorting apparatus above, to be held in place within the
plurality of cell sites, such as those described above. Using an
optical system 120 to detect fluorescence, the response of the
cells can be measured, with the intensity of the fluorescence
reflecting the intensity of the cellular response. Once the
experiment is complete, the cells exhibiting the desired response,
or intensity, may be selectively released, to be collected or later
discarded. Such an integrated system would allow researchers to
look at the cell's time response.
[0063] Any light-emitting assay in which the cell's response may
vary in time is suited for study using this proposed system. It is
ideally suited for finding phenotype inhomogeneities in a nominally
homogeneous cell population. Such a system could be used to
investigate time-based cellular responses for which practical
assays do not currently exist. Instead of looking at the
presence/absence or intensity of a cell's response to stimulus, the
researcher can look at its time response. Furthermore, the
researcher can gain information about a statistically significant
number of cells without the potential of masking important
differences as might occur in a bulk experiment. Specific
applications may include the study of molecular interactions such
as receptor-ligand binding or protein-protein interactions. Signal
transduction pathways, such as those involving intracellular
calcium, can also be investigated.
[0064] An advantage of the proposed integrated system is that the
full time-response of all the cells can be accumulated and then
sorting can be performed. This is contrasted with flow cytometry,
where each cell is only analyzed at one time-point and sorting must
happen concurrently with acquisition. Geneticists can look at gene
expression, such as with immediate-early genes, either in response
to environmental stimuli or for cell-cycle analysis. Another large
application area is drug discovery using reporter-gene based
assays. The integrated system can also be used to investigate
fundamental biological issues dealing with the kinetics of drug
interactions with cells, sorting and analyzing cells that display
interesting pharmacodynamic responses. Another application is
looking at heterogeneity in gene expression to investigate
stochastic processes in cell regulation. Finally, once temporal
responses to certain stimuli are determined, the integrated system
can be used in a clinical setting to diagnose disease and monitor
treatment by looking for abnormal time responses in patients'
cells.
[0065] One objective of the present invention is to provide a cell
analysis and sorting apparatus which uses hydraulic forces to
capture individual cells into addressable locations, and can
utilize microbubble actuation to release these individual cells
from their locations. In developing this apparatus, it was
necessary to model and understand many physical phenomena, not the
least important of which includes the theory behind bubble
nucleation on micro-heaters. Further, it was necessary to design a
device with the proper dimensions so that single particles, or
cells, could be held in wells against a flow. Biological cells were
not used in these experiments, as polystyrene microspheres of the
same dimensions were thought to be more robust for testing
purposes. The fabrication process had to be designed in order to
build chips with the desired attributes, and various problems which
arose needed to be resolved. Finally, it was necessary to
understand the heating of the resistors so that sufficiently high
temperatures could be reached.
[0066] Under the theory of bubble nucleation, pool boiling takes
place when a heater surface is submerged in a pool of liquid. As
the heater surface temperature increases and exceeds the saturation
temperature of the liquid by an adequate amount, vapor bubbles
nucleate on the heater. The layer of fluid directly next to the
heater is superheated, and bubbles grow rapidly in this region
until they become sufficiently large and depart upwards by a
buoyancy force. While rising the bubbles either collapse or
continue growing depending on the temperature of the bulk
fluid.
[0067] There are two modes of bubble nucleation: homogeneous and
heterogeneous. Homogeneous nucleation occurs in a pure liquid,
whereas heterogeneous nucleation occurs on a heated surface.
[0068] In a pure liquid containing no foreign objects, bubbles are
nucleated by high-energy molecular groups. According to kinetic
theory, pure liquids have local fluctuations in density, or vapor
clusters. These are groups of highly energized molecules which have
energies significantly higher than the average energy of molecules
in the liquid. These molecules are called activated molecules and
their excess energy is called the energy of activation. The
nucleation process occurs by a stepwise collision process that is
reversible, whereby molecules may increase or decrease their
energy. When a cluster of activated molecules reaches a critical
size, then bubble nucleation can occur.
[0069] In order to determine at what temperature water will begin
to boil in the homogeneous nucleation regime, it was useful to know
the thermodynamic superheat limit of water. FIG. 6 is the
thermodynamic pressure-volume diagram for water, which shows a
region of stable liquid to the far left, stable vapor to the far
right, metastable regions, and an unstable region in the center of
the dashed curve. The dashed line is called the spinodal, and to
the left of the critical point represents the upper limit to the
existence of a superheated liquid. Along this line, Equation (1-1)
holds true, and within the spinodal, Equation (1-2) applies. 1 ( P
v ) T = 0 ( 1 - 1 ) ( P v ) T > 0 ( 1 - 2 )
[0070] The van der Waals and Berthelot equations of state were used
to calculate the superheat limit of water. 2 ( P + a T n v 2 ) ( v
- b ) = RT ( 1 - 3 )
[0071] Where .nu. is the specific volume, R is the gas constant,
and a and b are constants. n=0 for the van der Waals equation, n=1
for the Berthelot equation, and n=0.5 for the modified Berthelot
equation. a and b were computed using Equation (1-3), given the
fact that at the critical point, Equations (1-4) and (1-5) are
true. 3 ( P v ) T cr = 0 ( 1 - 4 ) ( 2 P v 2 ) T cr = 0 ( 1 - 5
)
[0072] Using the above equations, the thermodynamic superheat limit
of water was computed. The results are shown below in Table 1.
1 Equation of State T/T.sub.cr (T.sub.cr = 647.degree. K.)
Superheat Limit (.degree. C.) Van der Waals 0.844 273 Modified
Berthelot 0.893 305 Berthelot 0.919 322
[0073] These values represent the temperature above which
homogeneous nucleation must begin.
[0074] A kinetic limit of superheat may also be computed using the
kinetic theory of the activated molecular clusters. The kinetic
limit of superheat for water is about 300.degree. C.
[0075] When liquid is heated in the presence of a solid surface,
heterogeneous nucleation usually occurs. In this regime, bubbles
typically nucleate in cavities (surface defects) on the heated
surface. The degree of superheat necessary to nucleate a bubble in
a cavity is inversely dependent on the cavity radius, as shown in
Equation (1-6). 4 T w - T sat = 2 T sat h lv v r c ( 1 - 6 )
[0076] Where T.sub.w is the surface temperature, T.sub.sat is the
saturation temperature (100.degree. C. for water), .sigma. is the
surface tension, h.sub.fg is the latent heat of vaporization,
p.sub.v is the vapor density, and r.sub.c is the cavity radius. For
example, the surface temperature necessary to nucleate bubbles in
water with a surface that has a 1 .mu.m cavity radius is about
133.degree. C. For a 0.1 .mu.m cavity radius the temperature to
nucleate a bubble is about 432.degree. C., well above the highest
thermodynamic water superheat limit of 322.degree. C.
[0077] Accordingly, for surfaces with cavity sizes well below 1
.mu.m, it is likely that homogeneous nucleation will occur since
the liquid will reach the superheat limit before a bubble nucleates
in a cavity. Micromachined surfaces tend to have very smooth
surfaces. For instance, the platinum resistors are only 3-6 .mu.m
wide, and 0.1 .mu.m thick, so it is unlikely that cavities will
exist on the surface which are large enough for heterogeneous
nucleation to occur. The largest likely nucleation cavity would be
the thickness of the resistor, which is 0.1 .mu.m, and results in a
boiling temperature for heterogeneous nucleation above the
thermodynamic superheat limit as shown above. Thus, it was assumed
that homogeneous nucleation was the most likely method of bubble
nucleation to occur for the resistors of this invention.
[0078] However, when platinum films are annealed, thermal grooving
and agglomeration can take place at the grain boundaries. A groove
will develop on the surface of a hot polycrystalline material where
a grain boundary meets the surface. As the surface gets hotter, the
grooves deepen, initiating holes, and the platinum begins the
process of balling up in order to reduce surface area. This process
is called agglomeration. The agglomeration rate is insignificant at
anneal temperatures below 700.degree. C. However, for a 600.degree.
C. anneal of platinum for 1 hour, the onset of agglomeration can
cause small voids in the platinum with radii of up to about 0.5
.mu.m. In this case, heterogeneous nucleation would be possible at
a temperature of about 166.degree. C.
[0079] Next, it was desirable to predict the electrical current
necessary to achieve a certain temperature of the resistor. The
schematic and boundary conditions for this resistor model are shown
in FIG. 7A and 7B. For the cross-sectional slice through the
resistor (7B), the water above the heater was 450 .mu.m thick,
corresponding to the height of the silicon chamber containing the
water. It was assumed that the ambient temperature was maintained
at the top of the water in the well since above this there was
silicon with water at the ambient temperature flowing over the top
of it. The bottom of the glass slide was also assumed to be at the
ambient temperature since it was contacting a surface at the
ambient temperature. The resistor was about 10,000 times thinner
than the glass slide and had ohmic heating, or power generation
equal to I.sup.2R for the entire volume of the resistor.
[0080] First, the characteristic time for the heat to conduct
through the two bounding surfaces was calculated using Equation
(1-7). 5 L 2 ( 1 - 7 )
[0081] Where L is the characteristic length for conduction and
.alpha. is the thermal diffusivity of the material.
[0082] Using this relation, it was found that the characteristic
time for conduction through 1 mm of glass was about 2.3 seconds.
Similarly, the characteristic time for conduction through 450 .mu.m
of water was 1.38 seconds. Accordingly, for this system the time to
reach steady state would be about four times greater than the
highest characteristic time, about 9 seconds. As established above,
homogeneous bubble nucleation was likely to occur, which is a
molecular process and thus may be assumed to be approximately
instantaneous. The time for a bubble to nucleate was therefore far
shorter than the 9 seconds necessary for the system to reach steady
state, so steady state conditions are unlikely to be achieved
before the bubble nucleates.
[0083] It was then necessary to determine the dominant modes of
heat transfer from the resistor to its surroundings. The purpose of
this model was to predict the temperature of the heater for a given
current, before the onset of boiling. For this model, heat transfer
due to radiation was neglected.
[0084] A lumped model approach was taken for this analysis. This
approximation was checked by computing the Biot number for the
resistor. 6 Bi = ht k Pt = 7 .times. 10 - 9 << 1 ( 1 - 8
)
[0085] Where t is the platinum resistor thickness (0.1 .mu.m) and
k.sub.Pt is the thermal conductivity of platinum (71.5 W/mK). It
was assumed in this model a heat transfer coefficient of h=5
W/m.sup.2K as a high bound for natural convection. The Biot number
measures the ratio of internal conduction resistance to external
convection resistance. Since the Biot number was much less than
unity, the lumped body approximation was used and an assumption was
made that the entire resistor was at a uniform temperature.
[0086] FIG. 8 shows the thermal resistances between the resistor
and the ambient temperature. For the purpose of this order of
magnitude estimate of the heat transfer mechanisms, steady state
conditions were used in determining thermal resistances. First, the
thermal resistance due to convection through the water was
computed. For this case it was assumed there was natural convection
since the water above the heater was stagnant, and boiling was not
occurring. The thermal resistance due to convection was calculated
below. 7 R convection = 1 hA = 1 hwL = 6.67 .times. 10 7 K W ( 1 -
9 )
[0087] Where w is the resistor width (3 .mu.m) and L is the
resistor length (1000 .mu.m).
[0088] Next the thermal resistance due to conduction through the
platinum resistor, glass slide, and water were computed. The
resistance due to conduction was given by: 8 R conduction = L kA (
1 - 10 )
[0089] Where L is the length through which heat conducts, and A is
the cross-sectional area. For the platinum, the length through
which heat conducts was very long (12 mm) and the cross-sectional
area was very small, resulting in a high thermal resistance: 9 R
platinum = L Pt k Pt tw = 5.4 .times. 10 8 K W ( 1 - 11 )
[0090] Where t is the platinum film thickness (0.1 .mu.m), L.sub.Pt
is the length through which heat conducts (12 mm), w is the width
of the resistor (3 .mu.m), and k.sub.Pt is the conductivity of
platinum (71.5 W/mK). Similarly, the thermal resistances of the
glass and water were computed. 10 R glass = L g k g Lw = 4.1
.times. 10 5 K W ( 1 - 12 ) R water = L w k w Lw = 2.2 .times. 10 5
K W ( 1 - 13 )
[0091] Where L.sub.g is the length of glass through which heat
conducts (1 mm), k.sub.g is the conductivity of glass (0.81 W/mK),
L is the length of the resistor (1000 .mu.m), w is the width of the
resistor (3 .mu.m), L.sub.w is the length of water through which
heat conducts (450 .mu.m), and k.sub.w is the conductivity of water
(0.67 W/mK).
[0092] From this it was shown that R.sub.glass and R.sub.water were
the dominant thermal resistances for the system. Thus, heat
transfer due to convection in the water and conduction through the
platinum were negligible.
[0093] An estimate the temperature of the resistor as a function of
time for a given current using semi-infinite body theory was then
made. For small times (t<1 ms) it was assumed that both the
water and glass are semi-infinite bodies with initial temperature
T.sub.a. At t=0, a constant heat flux (due to the resistor) is
applied at the water-glass interface (x=0). The one-dimensional
temperature profile was computed using the infinite composite solid
solution. The region x>0 is water, x=0 is the resistor, and
x<0 is the glass. A one-dimensional model was used for short
times since the length of the resistor (L=1000 .mu.m) was much less
than the width of the resistor (L=6 .mu.m). The temperature was
assumed to be constant along the resistor, and lateral conduction
was neglected for small times. This model will break down when the
lateral conduction becomes significant, and when the assumption of
semi-infinite bodies becomes invalid. The boundary conditions for
this problem are given below. 11 q 1 1 1 2 K 1 = q 2 2 1 2 K 2 , x
= 0 , t > 0 ( 1 - 15 )
[0094] Where K is the thermal conductivity (0.61 W/mK for water and
0.88 W/mK for glass), q is the heat flux, and the subscript `1`
denotes water, and `2` denotes glass.
[0095] The solution for the temperature profiles in water and air
for a constant heat flux q (W/m.sup.2) applied at x=0 is given by
Equations (1-17) and (1-18). 12 T 1 - T o = 2 q 1 2 t K 1 2 + K 2 1
erfc x 2 1 t ( 1 - 17 ) T 2 - T o = 2 q 1 2 t K 1 2 + K 2 1 erfc x
2 2 t ( 1 - 18 )
[0096] Where .alpha. is the thermal diffusivity
(1.47.times.10.sup.-7 m/s.sup.2 for water and 4.4.times.10.sup.-7
m/s.sup.2 for glass) and T.sub.o is the initial temperature of the
body.
[0097] The solution was also used to check the semi-infinite body
assumption. For times equal to or less than 1 ms, and a reasonable
heat flux such as 2.5.times.10.sup.7 W/m.sup.2, the heat
penetration depths into the glass and water were less than 100
.mu.m. The total thickness of the water was 450 .mu.m and of the
glass was 1 mm, so the semi-infinite body assumption held true. The
one-dimensional model was sufficient for determining the
temperature of the resistor at small times.
[0098] Using the theory described above, it was possible to predict
the power necessary to form a bubble. Since homogeneous bubble
nucleation was assumed, the bubbles would form at approximately the
superheat limit of water. The value of 305.degree. C. given by the
modified Berthelot equation (Table 1) was used. Next, the infinite
composite solid solution was used to calculate the temperature of
the heater for a given time, say 1 ms. Rearranging equation (1-17)
to solve for the heat flux, or power per unit area at position x=0,
it was derived: 13 P Lw = ( K 1 2 + K 2 1 ) ( T - T o ) 2 1 2 t ( 1
- 19 )
[0099] For an initial temperature of 20.degree. C., and the other
properties given above, the heat flux necessary to heat the
resistor to 305.degree. C. in 1 ms was computed from (1-19) to be
1.32.times.10.sup.7 W/m.sup.2. For typical resistor dimensions of
w=6 .mu.m and L=1500 .mu.m, the necessary power was about 120
mW.
[0100] The micromachined wells must be of the proper dimensions to
ensure that particles which settle into them remain held in the
wells once a flow above them is initiated. The theory of slow
viscous flow over cavities has been well characterized and the
streamlines for various geometries have been calculated and
experimentally verified.
[0101] FIG. 9 shows the flow pattern for laminar flow over a
rectangular cavity for two different width to height aspect ratios.
From these flow patterns it was seen that there was a separating
flow line which penetrates slightly into the cavity. Below this
line there were one or two vortices, depending on the aspect ratio
of the cavities. A particle below the separating flow line would
not be swept out of the cavity by a slow flow in the laminar range,
though the vortex may agitate the particle.
[0102] An order of magnitude calculation was performed in order to
compare the relative sizes of the gravity force pulling a particle
down, compared to the viscous shear force pulling a particle out of
the well. A diagram of a particle in a well with flow over the top
is shown in FIG. 10.
[0103] The force of gravity acting on the particle was dependent on
the difference in density between the particle and the water,
.DELTA..rho.. The density of water is approximately 1000
kg/m.sup.3, and the density of the polystyrene beads used in the
experiments was given by the manufacturer as 1060 kg/m.sup.3. The
density of cells ranges from 1050-1100 kg/m.sup.3. Accordingly, the
force of gravity, F.sub.g was computed as shown: 14 F g = 4 3 a 3 g
( 1 - 20 )
[0104] Where a is the particle radius (5.times.10.sup.-6 m), and g
is the gravitational constant.
[0105] The viscous shear force acting on the particle was computed
by assuming the top of the particle was at the top of the well, and
that the flow profile was parabolic. The shear stress at the wall
was: 15 w = u y y = 0 ( 1 - 21 )
[0106] Where .mu. is the viscosity of water (1.times.10.sup.-3
kg/ms) and u(y) is the velocity profile as a function of y, the
distance from the wall.
[0107] Assuming a parabolic velocity profile in the flow chamber,
the flow profile was calculated for a known chamber height and
volume flow rate. 16 u ( y ) = 6 V _ h 2 y ( h - y ) ( 1 - 22 ) V _
= Q wh ( 1 - 23 ) u ( y ) = 6 Q wh 3 y ( h - y ) ( 1 - 24 ) u y y =
0 = 6 Q wh 2 ( 1 - 25 )
[0108] Where {overscore (V)} is the average flow velocity, w is the
chamber width, and h is the chamber height.
[0109] The viscous shear force on the cell was estimated as the
wall shear stress multiplied by the area being effected,
approximately .pi.a.sup.2. 17 F v = w a 2 = 6 Q wh 2 a 2 ( 1 - 26
)
[0110] Where a is the cell radius. Finally the ratio of gravity to
viscous force was computed. 18 F g F v = 4 3 a 3 g 6 Q wh 2 a 2 = 2
agwh 2 9 Q ( 1 - 27 )
[0111] Using the flow chamber dimensions in FIG. 16, and a range of
reasonable flow rates, this ratio was computed. 19 Q = 1 L min ( V
_ = 2.1 m s ) -> F g F v = 292 ( 1 - 28 ) Q = 10 L min ( V _ =
21 m s ) -> F g F v = 29 ( 1 - 29 ) Q = 100 L min ( V _ = 210 m
s ) -> F g F v = 3 ( 1 - 30 )
[0112] It was necessary that the ratio of forces be greater than
one so that the gravity force was stronger than the viscous force.
These numbers were used to aid in determining a range of acceptable
operating flow rates.
[0113] Another relevant piece of information was the time it took
for the particles to settle. At low Reynolds number, an isolated
rigid spherical particle will settle with its Stokes velocity. 20 U
o = 2 a 2 ( s - ) g 9 ( 1 - 31 )
[0114] Where a is the sphere radius (5 .mu.m for a polystyrene
bead), .rho..sub.s is the density of the bead (about 1060
kg/m.sup.3), .rho. is the density of water (1000 kg/m.sup.3), and
.mu. is the viscosity of water. Using these values a Stokes
velocity was calculated as: 21 U o = 5 .times. 10 - 6 m s = 5 m s (
1 - 32 )
[0115] Using this velocity to check the associated Reynolds number
it was found that: 22 Re = U o a = 3 .times. 10 - 5 << 1 ( 1
- 33 )
[0116] Thus, the assumption of low Reynolds number was valid. The
Reynolds number is the ratio of inertial effects to viscous forces.
For this case, only the highly viscous regime applied and inertial
effects were negligible.
[0117] Another value which was checked was the Peclet number. This
is the ratio of sedimentation to diffusion. For the particles to
settle, the Peclet number must be sufficiently high, otherwise the
particles will diffuse throughout the liquid. 23 Pe = aU o D o ( 1
- 34 ) D o = kT 6 a = 4 .times. 10 - 14 m 2 s ( 1 - 35 )
[0118] Where D.sup.o is the Brownian diffusivity, and k is the
Boltzmann's constant (1.38.times.10.sup.-16 erg/cm). Thus the
Peclet number was sufficiently high for settling to dominate over
diffusion.
[0119] The value calculated above for the Stokes velocity is that
for an isolated particle; however, in the case at hand there were
many beads settling at once. This was taken into account in the
calculation of the hindered velocity. A function of the particle
volume fraction is multiplied by the Stokes velocity to result in
the hindered velocity of particles in the suspension. 24 U = 4.75
.times. 10 - 6 m s = 4.75 m s ( 1 - 39 )
[0120] Where .phi. is the particle volume fraction (about 0.01 for
this case). Accordingly, the time necessary for all the particles
to settle to the bottom of the flow chamber was calculated using
the hindered velocity and the chamber height, the maximum distance
to be traveled. 25 t s = h U = 166 s = 2.76 min ( 1 - 40 )
[0121] Where h is the chamber height (790 .mu.m). This settling
time was used as a guideline in experiments.
[0122] A more reasonable assumption for calculating the settling
time was that the distance the particles fell is an average of half
the chamber height. For this case a settling time of about 83
seconds was obtained.
[0123] For the given pressure increase associated with the bubble
formation in the large sealed well, the flow rate out of the
channel in the top of the well was calculated. Since the Reynolds
number was in the creeping flow regime (Re<1), inertial effects
neglected, and the initial, instantaneous flow out of the channel
was computed using the steady state equation for flow through a
circular aperture at low Reynolds number. 26 Q = Pc 3 3 ( 1 - 41
)
[0124] Where Q is the volume flow rate, .DELTA.P is the pressure
drop, c is the aperture radius (.about.2.5 or 4 .mu.m), and .mu. is
the water viscosity.
[0125] Since the pressure change due to the bubble formation was
not easily calculable, the volume flow rate out of the chamber was
estimated in a different way. Because water is incompressible, it
was assumed in the model that the bubble formation as a volume
injection into the chamber resulted in the same volume being
ejected from the chamber over the characteristic bubble formation
time. For instance, if it took 1 ms to form a 10 .mu.m diameter
bubble, then the resulting volume flow rate out of the chamber was
calculated as follows. 27 V = 4 3 r 3 = 5.24 .times. 10 - 16 m 3 (
1 - 42 ) Q = V t = 5.24 .times. 10 - 13 m 3 s ( 1 - 43 )
[0126] Using the volume flow rate the average velocity of fluid out
of the channel was calculated, and it is seen that the Reynolds
number of the flow was indeed low. 28 V _ = Q c 2 = 27 mm s ( 1 -
44 ) Re = V _ c = 0.067 < 1 ( 1 - 45 )
[0127] Where c is the channel radius (2.5 .mu.m). The force of the
fluid jet on the particle was calculated using the Stokes drag
force:
F.sub.D=6.pi..mu.a{overscore (V)}=2.5.times.10.sup.-9 N (1-46)
[0128] Where a is the radius of the spherical particle (5 .mu.m for
polystyrene beads). Comparing this to the gravitational force
(1-20) pulling the particle down, it was found that the force of
the jet on the particle was much greater than the force of gravity.
29 F D F g V _ a 2 ( 1 - 48 )
[0129] Where .DELTA..rho. is the difference in densities between
the water and the polystyrene beads (60 kg/m.sup.3). It was seen
that as the particle radius increased, the effect of gravity
increased. For typical cells, the radius ranges from 5 .mu.m (red
blood cells) to 20 .mu.m (most other cells) to 100 .mu.m (embryos
and eggs). This device will most likely be used for cells on the
order of 5-10 .mu.m in radius so the above calculation was
representative of the expected applications.
Design of the Components
[0130] A. Resistive Heaters
[0131] In order to heat the water to a sufficiently high
temperature for microbubble formation, resistive heaters were used.
The heaters were made of thin-film platinum on standard glass
slides. In designing the heaters it was necessary first to
determine a range of resistances and currents to attain the desired
power output. The design constraint for this step was the need to
keep the current density below the electromigration limit of
platinum, while retaining an adequate degree of ohmic heating. The
electromigration limit is the maximum current density which
platinum can endure before the atoms begin to migrate leaving the
resistor inoperable.
[0132] The electromigration limit of platinum was reported to be
J=9.times.10.sup.6 A/cm.sup.2. It was necessary to design the
resistors to operate at a current density below this limit.
[0133] The resistance of a line heater is calculated as follows. 30
R = L tw ( 1 - 49 )
[0134] Where R is the resistance (.OMEGA.), L is the length of the
resistor (m), t is the film thickness (m), w is the width of the
resistor (m), and .rho. is resistivity of platinum (.OMEGA.m).
[0135] The power output of a resistor is a function of the current
and resistance, as shown below. 31 J = I wt < 9 .times. 10 6 A
cm 2 ( 1 - 51 )
[0136] Where I is the current (A) and J is the current density.
[0137] Accordingly, as the currents were limited by the
electromigration limit, the resistances needed to be sufficiently
high to achieve the desired power output. The power output
necessary to form a bubble was estimated by using the numbers from
Lin et al.'s paper, `Microbubble Powered Actuator`, herein
incorporated by reference, where microbubbles were formed on a
polysilicon line heater. Their resistor was on top of a thin
dielectric layer, which was on a silicon wafer. It was reasonable
to assume that the heat dissipation of this configuration might
well be greater than the heat dissipation of the platinum line
resistor fabricated on a glass slide. Also, a liquid with a higher
boiling necessary to nucleate bubbles under these conditions was
approximately 65 mW.
2TABLE 2 Resistor dimensions, resistances, and electromigration
limits. Resis- Max Slide Re- Length Width tance Electromigration
Power Name sistor (um) (um) (Ohms) Limit (mA) (mW) Slide 1 1 3000 3
1000 22 467 2 2500 3 833 22 389 3 500 3 167 22 78 4 1000 3 333 22
156 5 1000 4 250 29 207 6 2000 3 667 22 311 7 1500 3 500 22 233 8
1000 5 200 36 259 Slide 2 1 3000 3,6 483 22 226 2 2500 3,6 400 22
187 3 500 3 167 22 78 4 1000 3,6 150 22 70 5 1000 6 167 43 311 6
2000 3,6 317 22 148 7 1500 3,6 233 22 109 8 1000 3,5 180 22 84
Slide 3 1 3000 6 625 43 1166 2 2500 6 521 43 972 3 500 3 208 22 97
4 1000 3 417 22 194 5 1000 6 208 43 389 6 2000 6 417 43 778 7 1500
6 313 43 583 8 1000 6 208 43 389
[0138] Using this as a guideline, the resistances were chosen to
range from 167 .OMEGA.-1000 .OMEGA., yielding maximum powers before
electromigration of 70-1166 mW. These powers were chosen to be up
to an order of magnitude greater than necessary to avoid reaching
the electromigration limit in the operation of the resistors.
[0139] The resistivity of platinum actually varies with temperature
and film deposition conditions, but for these calculations it was
taken to be 1.times.10.sup.-7 .OMEGA.m. This is the value for bulk
platinum, however the resistivity of thin film platinum can vary
widely. Heater widths range from 3-6 .mu.m and lengths range from
500-3000 .mu.m. Some heaters were designed to have a narrow region,
100 .mu.m long in the center, which would be hotter than the rest
of the resistor. FIG. 11A is a top view of a heater configuration,
while FIG. 11B shows a cross-sectional view of the heater and its
dimensions. A table of resistor dimensions, maximum currents, and
maximum power outputs is also shown in Table 2.
[0140] The lines connecting the contact pads to the heaters were
designed to have a far lower resistance than the heaters. This was
done to ensure that the lines did not heat up, and that they
remained approximately at the ambient temperature. The connector
line widths were chosen to be 1500 .mu.m with lengths of 12 mm. The
total resistance of each line was about 7.7 .OMEGA..
[0141] B. Wells
[0142] Square wells were micromachined into silicon in order to
hold cells. It was necessary to choose a range of dimensions for
these wells to allow for tests with different particle sizes and
flow rates. The final goal was to have the ability to trap one
particle in each of an array of wells.
3TABLE 3 Well Dimensions Chip Number Well Dimension (um) Hole
Dimension (um) 1 16 5 b 16 8 2 10 5 2b 10 8 3 20 5 3b 20 8 4 30 5
4b 30 8 5 40 5 5b 40 8 6 50 5 6b 50 8
[0143] Side lengths of the wells were chosen to range from 10
.mu.m, corresponding to the smallest test bead size, up to 50
.mu.m. Well sizes ranging from 10-50 .mu.m were chosen. Narrow
channel widths of 5 .mu.m and 8 .mu.m were chosen since both these
sizes are smaller than the minimum test particle size of 10 .mu.m
and it is necessary that particles not be able to settle down into
the narrow channel. The table of well dimensions is shown in Table
3. A diagram of the well geometry is shown in FIG. 12A, which shows
a side view, while FIG. 12B shows a top-down view.
[0144] Photomasks for use in the device fabrication were created
using standard mask layout software. The mask set for the silicon
processing are shown in FIGS. 13A-13C and the glass mask set is
shown in FIG. 14A.
[0145] Three masks were designed for the silicon portion of the
device processing. One mask was created for the cell wells (FIG.
13A), one for the narrow channels within the wells (FIG. 13B), and
one for the large wells (FIG. 13C) etched from the backside of the
wafer to enclose the heaters. Two masks were made for the
fabrication of the platinum heaters on the glass slides. One mask
(FIG. 14A) was designed to pattern the metal.
[0146] In order to test the finished devices, a fluidic system as
illustrated in FIG. 15 was designed and assembled. A syringe pump
150 was used as the flow source for the bulk fluid, and flow rates
ranging from 1 to 100 .mu.L/min were specified. Beads, cells, or
cell stimuli were injected through the sample injection valve 152.
A pressure sensor 154 was located before the flow chamber 156 so
that the pressure drop across the chamber could be monitored. All
fluid was outlet into a waste beaker 158 which could be reused if
desired.
[0147] A schematic of the flow chamber 156 is shown in FIGS. 16A
and 16B. The flow chamber was machined from plexiglass so that it
was clear and a microscope was used to observe cell behavior from
above the chamber. HPLC (high-performance liquid chromatography)
fittings were used with tube dimensions of {fraction (1/16)} inch
outer diameter and 0.020 inch inner diameter. The gasket between
the slide and the top cover were made from PDMS (poly dimethyl
siloxane), a flexible polymer. A seal was formed by screwing the
top plate down onto the bottom plate. Aluminum molds were machined
in order to create PDMS gaskets of the proper dimensions. Gaskets
were compressed until a hard stop was reached. The stop was
provided by the spacers, made of metal shim stock, in order to
accurately specify the channel height. The aspect ratio of the
channel's width to height was greater than 10, allowing the
assumption of a parabolic velocity profile-plane Poiseuille
flow.
[0148] The height of the flow chamber was 790 .mu.m (determined by
thickness of metal spacer). Flow rates ranged from 1 to 100
.mu.L/min and corresponded to Reynolds numbers of 0.001-0.1. In
this creeping flow regime, the entrance length for fully developed
flow was calculated to be negligible. These calculations are shown
below. 32 V _ min = Q min A c = 1.77 m s ( 1 - 52 ) V _ max = Q max
A c = 177 m s ( 1 - 53 ) Re min = hV min v = 0.0011 ( 1 - 54 ) Re
max = hV max v = 0.11 ( 1 - 55 ) X e h Re max 30 = 2.6 m ( 1 - 56
)
[0149] Where {overscore (V)}.sub.min is the minimum average
velocity, Q.sub.min is the minimum volume flow rate (1 .mu.L/min),
A.sub.c is the cross-sectional area of the channel (h=790 .mu.m,
w=12 mm), V.sub.min is the maximum average velocity, Q.sub.max is
the maximum volume flow rate (100 .mu.L/min), Re is the Reynolds
number, v is the kinematic viscosity of water (1.times.10.sup.-6
m.sup.2/s), and X.sub.e is the entrance length for fully-developed
flow.
[0150] Electrical connections to the contact pads were made using a
probe station. Contact pads were positioned outside of the PDMS
gasket and were thus kept outside of the fluid flow.
[0151] In order to ensure the proper flow characteristics of the
flow chamber, dye was injected into the flow and the resulting
profile was observed. The results were used to discover problems
such as blockages in the flow chamber and correct them. When a
uniform flow was established, 1 .mu.m diameter beads were injected
into the flow and observed under a microscope.
[0152] The pressure drop across the flow chamber was monitored
using a pressure transducer. The majority of the pressure drop was
caused by the connector tubing, but by comparing the pressure
reading to the theoretical value, the presence of bubbles and other
blockages to the flow may be detected.
[0153] The pressure versus flow rate plot for the flow chamber is
shown in FIG. 17. The theoretical value is plotted with the
experimental measurements. When these two values do not match, a
blockage in the chamber or tubing is probable.
[0154] The pressure drop through the tubing was calculated using
the following equation. 33 P = - 8 Q r 4 x ( 1 - 57 )
[0155] Where .mu. is the viscosity of water (1.times.10.sup.-3
kg/ms), r is the tube radius (0.254 mm), and .DELTA.x is the tube
length (m). The pressure drop through the chamber was calculated to
be negligible in comparison. The flow chamber schematic with
dimensions is shown in FIGS. 18A-18D.
Fabrication of the Components
[0156] The platinum heaters were fabricated on standard 1.times.3
in glass slides using a lift-off process. The process flow is shown
in FIGS. 19A-19C. In the first step illustrated as FIG. 19A,
photoresist was spun onto the glass slide, exposed using mask 4,
and developed. Next, 100 .ANG. of titanium and 1000 .ANG. of
platinum were evaporated onto the slide, as seen in FIG. 19B. The
titanium served as an adhesion layer between the glass and the
platinum. In the following step, the slide was submerged in acetone
to dissolve the photoresist and lift away the metal which was
deposited on top of the photoresist, as depicted in FIG. 19C. Only
the platinum resistors were left on the glass slide. Some slides
were then annealed in a tube furnace at 600.degree. C. for 1 hour.
While not used in this example, it is contemplated that photoresist
may be applied manually to the slide to attach the silicon chip to
the slide.
[0157] The silicon chip process flow is shown in FIGS. 20A-20H.
Double Side Polished (DSP) four inch diameter silicon wafers were
used. In the first step shown as FIG. 20A, 1 .mu.m of thermal oxide
was grown on the wafer. Next the oxide was patterned using mask 1,
FIG. 20B. Resist was spun on top of the oxide and patterned using
mask 2. The resulting configuration was called a nested mask, shown
as FIG. 20C.
[0158] First the photoresist mask was used to etch the narrow 5
.mu.m trenches, then the oxide mask was used to etch the cell
wells, as shown in FIGS. 20D and 20E. Next the wafer was turned
over and photoresist was deposited and patterned on the back side
using mask 3 (FIG. 20F). A deep silicon etch was then performed to
etch through the wafer and intersect the narrow trenches etched
previously (FIG. 20G) to obtain a finished wafer (FIG. 20H).
[0159] A complete device consisted of a silicon chip attached to a
glass slide by photoresist, as shown in FIGS. 21C and D. The resist
provided a water-tight seal so that volume expansion in the bubble
wells resulted in a burst of fluid being pushed through the narrow
channel and ejecting a cell.
[0160] To facilitate the assembly process, alignment marks were
fabricated on the glass slide and matching holes were etched in the
silicon chip. The alignment tolerances were sufficiently large
(about 2 mm) that the chip could be aligned to the slide by hand
using just the naked eye, while still positioning the bubble wells
over the platinum heaters.
[0161] Photoresist was painted onto the silicon chip around the
bubble wells using a toothpick. Drops of water were deposited into
each well using a pipette, then the glass slide was visually
aligned from above and stuck down onto the chip. The drops of water
served to fill the bubble wells and get pushed through the narrow
channel to fill it with water. The device was now ready to be
tested in the flow chamber.
[0162] Next, the resistance of the platinum resistors were studied.
The film thickness was first measured using a profilometer. The
platinum thickness measurements ranged from about 800-900 .ANG., so
the average value of 850 .ANG. was used in the subsequent
calculations. The resistance along metal lines wide enough not to
be strongly affected by variation of a few microns was measured
using a multimeter. The lines used for this measurement were
measured in an optical microscope to be about 1510 .mu.m wide. The
length of the lines was about 8 mm. Knowing the width, thickness,
and length of these lines, as well as the measured resistance, the
resistivity of the thin film platinum at room temperature was
determined. The measured resistance was 15 .OMEGA., and the
computed resistivity was calculated below. 34 = twR L = 2.41
.times. 10 - 7 m ( 1 - 58 )
[0163] Where t is the film thickness (850 .ANG.), w is the line
width (1513 .mu.m), R is the measured resistance (15 .OMEGA.), and
L is the length of the line (8 mm). This resistivity was more than
twice the value for bulk platinum (1.times.10.sup.-7 .OMEGA.m), but
was a reasonable value for thin film platinum. This is because bulk
platinum is a crystalline material, whereas thin film platinum is
polycrystalline and the grain boundaries significantly increase
resistance.
[0164] Next, the resistance of the resistors was measured with a
multimeter. Using the value of resistivity from above, the line
width of each resistor was determined. The line widths were also
measured using an optical microscope to an accuracy of about .+-.1
.mu.m. The results of for two different resistor slides are shown
in Table 4.
4TABLE 4 Resistance measurements and calculated, measured, and
designed line widths. Computed Re- R Line Measured Design sistor #
L (um) (Ohms) Width (um) (um) (um) Slide 1 1 3000 1020 8.34 8 3 2
2500 845 8.39 8 3 3 500 185 7.66 8 3 4 1000 347 8.17 8 3 5 1000 272
10.42 10 4 6 2000 672 8.44 9 3 7 1500 504 8.44 8 3 8 1000 260 10.90
10 5 Slide 3 1 3000 850 10.01 10 6 2 2500 728 9.74 10 6 3 500 247
5.74 6 3 4 1000 479 5.92 6 3 5 1000 316 8.97 9 6 6 2000 620 9.15 9
6 7 1500 450 9.45 10 6 8 1000 270 10.50 10 6
[0165] From this it was determined that the measured and calculated
line widths were within the range of error for the measurements,
confirming the resistivity calculation. The resulting plot of
normalized resistance versus temperature is shown in FIG. 22. The
resistance was normalized using the resistance at room temperature.
This curve was used later to predict the temperature of a resistor,
knowing the resistance at room temperature and measuring the
resistance during operation.
[0166] Using the cross-sectional area of the resistors, the maximum
current before electromigration was calculated. It was known that
maximum current density before electromigration is 9.times.10.sup.6
A/cm.sup.2. Using this the maximum current for each resistor was
calcualted. The results of this are shown in Table 5.
5TABLE 5 Computed electromigration limits for resistors. Computed
Line Maximum Resistor # Width (um) Current (mA) Slide 1 1 8.3 71.3
2 8.4 71.7 3 7.7 65.5 4 8.2 69.9 5 10.4 89.1 6 8.4 72.1 7 8.4 72.1
8 10.9 93.2 Slide 3 1 10.0 85.6 2 9.7 83.2 3 5.7 49.1 4 5.9 50.6 5
9.0 76.7 6 9.1 78.2 7 9.5 80.8 8 10.5 89.8
[0167] These results were used as guidelines during testing of
microbubble devices to avoid burning out the resistors.
[0168] The main objective for the resistors was that they be able
to reach high enough temperatures to boil water. The resistors were
tested on a probe station using an HP4145b to vary the voltage and
measure the resulting current through the resistor. A PDMS gasket
was place on top of the slide and filled with water. The gasket
contained the water and kept it from touching the electrical
contacts and probes. FIG. 23 is a schematic of this
configuration.
[0169] Upon ramping the voltage across resistors from zero to about
20-30 V, there was violent bubbling originating not from the hot
part of the resistor, but from the edges of the wide connector
lines. It was evident that the bubbles were gas bubbles and not
water vapor bubbles because the bubbles did not condense when the
heater was turned off. Further experimentation revealed that
electrolysis of the water was occurring and the water was being
broken down into hydrogen and oxygen. After flushing the slides,
gaskets, and glassware for several minutes with deionized water,
and testing again, the problem of electrolysis was eliminated.
[0170] When the problem of electrolysis was eliminated, the
resistors were once again tested in water. When the resistor
reached a sufficient temperature, boiling occurred along the length
of the heater. After the power was turned off, small air bubbles
remained on the resistor due to the dissolved gas coming out of
solution, as described previously. In subsequent tests, the air
bubbles served as nucleation sites for boiling, the inception of
boiling occurred at a much lower temperature. When boiling begins
and bubbles form on the resistor, the heat dissipation into the
water increases drastically. This is a favorable phenomenon for the
operation of the device because the onset of boiling is represented
as a sharp increase in current on the I-V curve. This is because
when the heat dissipation increases, the temperature decreases,
resulting in a lower resistance and thus a higher current through
the resistor. An I-V curve for the onset of boiling on a line
resistor is shown in FIG. 24.
[0171] In this I-V curve it is shown that for the first run when no
bubbles were present on the line, there is a sharp jump in current
at the onset of boiling. For the second run, residual bubbles were
left on the heater and served as nucleation sites for boiling
resulting in a smooth I-V curve with boiling beginning at a lower
temperature. The two curves are very close after the boiling begins
for run 1.
[0172] In later tests, when no dissolved gas came out of solution,
the jump in the I-V curve occurred during each heating cycle for
the resistors, since there were no residual air bubbles left when
the power was turned off.
[0173] Using the calibration given in FIG. 22 for the
temperature-resistance relationship of the resistor, the
temperature of the resistor for each current was plotted to find
the boiling temperature. The current vs. temperature plot
corresponding to the I-V curve shown above is in FIG. 25. On this
plot water is shown to boil at approximately 308.degree. C., at
which point the temperature drops rapidly due to the increased
convective heat transfer associated with boiling.
[0174] The boiling points for the 5 resistors tested ranged from
250.degree. C. to 308.degree. C. The lowest calculated value for
the superheat limit of water was found to be 273.degree. C., so
these measured boiling points suggest that the bubble nucleation
occurs either in the homogeneous regime, or by a weak heterogeneous
mechanism.
[0175] After a considerable amount of testing of the resistors
characterized above, a drift in the boiling temperature became
apparent. In order to determine the reason for this, the resistors
were recalibrated as described in the previous section. The
temperature versus normalized resistance curve is shown in FIG. 26.
The dramatic change in temperature-resistance characterization led
to the testing of a second generation of resistors. It is thought
that these change characteristics are caused over time by the
heating of the resistors. The operation of the resistors
effectively caused them to anneal themselves. Annealing changed the
geography of the platinum grain boundaries and thus changed the
resistivity of the resistors.
[0176] In order to avoid this effect in future testing, new
resistor slides were annealed at 600.degree. C. for 1 hour as the
last step in their process. This temperature is higher than
operating temperatures are likely to reach, but not so high that
major agglomeration will result. Once the anneal was complete, the
new resistors were characterized as described above for the first
generation resistors.
[0177] First, the resistivity of the platinum at room temperature
was found to be 2.056.times.10.sup.-7 .OMEGA.m, less than the
unannealed resistors that were 2.41.times.10.sup.-7 .OMEGA.m. Next
the resistances were measured using a multimeter, and the line
widths were computed as before, as shown in Table 6.
6TABLE 6 Measured resistances and computed line widths of second
generation resistors. Computed Design Resistor # L (um) R (Ohms)
Line Width (um) (um) Slide 3 1 3000 553 13.12 6 2 2500 481 12.57 6
3 500 146 8.28 3 4 1000 281 8.61 3 5 1000 205 11.80 6 6 2000 409
11.83 6 7 1500 310 11.70 6 8 1000 186 13.00 6
[0178] The temperature-resistance characteristic or the resistors
was then measured on a hotplate as described above, and is shown in
FIG. 27.
[0179] At this point, the bubble formation characteristics of the
resistors were tested as described previously with boiled,
deionized water. Voltages were ramped up by 0.5V steps with delay
times of 1 ms using the HP4145b, as before. None of these tests
resulted in residual gas bubbles since the delay time was short,
and the maximum voltage used was just above the bubble nucleation
voltage, determined by testing. All resulting vapor bubbles
condensed back into the liquid phase within on minute of stop of
current flow.
[0180] A resulting I-V curve is shown in FIG. 28, and the
corresponding temperature curve is shown in FIG. 29. From the curve
we can see that the onset of boiling occurred at about 200.degree.
C. a much lower temperature than for the first generation
resistors, and well below the superheat limit of water. For the 8
second generation resistors tested, boiling points ranged from
128.degree. C.-200.degree. C. with the majority of the temperatures
above 180.degree. C. This suggests that the boiling is in the
heterogeneous nucleation regime as discussed earlier. The cavity
radii corresponding to these boiling inception temperatures are
calculated from Equation (1-59). 35 r c = 2 T sat h fg v ( T w - T
sat ) ( 1 - 59 )
[0181] The result of this calculation are shown in Table7.
7TABLE 7 Bubble nucleation cavity radii corresponding to measured
boiling temperatures. Resistor # Boiling Temperature (C) Cavity
Radius (um) 1 200.7 0.33 2 198.3 0.34 3 170.4 0.47 4 183.2 0.40 5
128 1.19 6 188 0.38 7 189 0.37 8 169 0.48
[0182] From this we can see that bubbles were nucleated in radii
ranging from 0.3-1.2 .mu.m. As discussed previously, these cavities
were most likely formed during the 600.degree. C. anneal, during
which the grooves at the grain boundaries widened creating
cavities.
[0183] The second generation resistors were also tested for the
repeatability of their boiling temperatures. I-V curves were
measured as in the previous section, and then remeasured for the
same conditions several times. Between measurements, time was given
for the vapor bubbles to dissipate so that the characteristic jump
in the I-V curve at boiling could be observed with each
measurement. The boiling point was found to be very repeatable, and
an example of the results is shown in FIG. 30. This result
demonstrated the potential of a control system based on a jump in
the I-V curve at the onset of boiling, since the boiling point
remained fixed.
[0184] Another interesting result from this testing is that for a
particular resistor, the bubbles tended to nucleate in the same
locations on the resistor each time. This strengthens the
hypothesis that the bubbles are nucleating in the heterogeneous
regime, in cavities created by thermal grooving caused by the
annealing.
Results
[0185] The cell chip was attached to the glass resistor slide as
described earlier, and then tested in two ways. First tests were
done with stagnant fluid on the device. Then the device was put
into the flow chamber for testing. The results of these tests are
described below.
[0186] For these tests, several drops of bulk solution were placed
on top of the cell chip, and contained by the PDMS gasket. A drop
of the polystyrene bead solution was then added to the bulk fluid
and allowed to settle. The bulk solution was a 0.05% solution of
Triton x-100 surfactant in deionized water. The bead solution was
about 1% beads diluted in the same bulk solution. Some of the beads
settled into wells, as shown in FIG. 42. When voltage across the
resistor was ramped up by the HP4145b, an I-V curve with a jump
similar to that in FIG. 24 was produced, demonstrating that boiling
had occurred. Consequently, the bubble formation under the well
caused a volume expansion which rapidly ejected the beads from the
well. First the beads are in the well, and then they are rapidly
expelled. This sequence was also captured on videotape, and the
process was repeated multiple times with the same success.
[0187] Preliminary dynamic testing was performed in the flow
chamber. Beads were ejected in a similar way to the static test,
and carried away in the flow. The preliminary tests suggested that
the beads are held in the wells against a reasonable flow rate, and
are ejected into the flow when a microbubble forms.
[0188] While the invention has been particularly shown and
described above with reference to several preferred embodiments and
variations thereon, it is to be understood that additional
variations could be made in the invention by those skilled in the
art while still remaining within the spirit and scope of the
invention, and that the invention is intended to include any such
variations, being limited only by the scope of the appended
claims.
* * * * *