U.S. patent application number 12/018439 was filed with the patent office on 2008-07-24 for sorting of microdevices.
This patent application is currently assigned to Arrayomics, Inc.. Invention is credited to David Rothwarf.
Application Number | 20080176765 12/018439 |
Document ID | / |
Family ID | 39641866 |
Filed Date | 2008-07-24 |
United States Patent
Application |
20080176765 |
Kind Code |
A1 |
Rothwarf; David |
July 24, 2008 |
Sorting Of Microdevices
Abstract
Particles or other microdevices are disposed in an array having
discreet regions (e.g. magnetic bars), oriented within a magnetic
field, and then sorted through application of a removing force
under conditions that remove a proper subset of the microdevices
from the array as a function of differing orientations of the
microdevices. Methods are also contemplated for using magnetic
patterns to sort collections of microdevices by magnetic
complementarity. Preferred methods use a capture and release
process to sort microdevices (microdevices), and unlike
conventional sorters, do not require high particle flow rates. Also
contemplated are microdevice libraries in which microdevices have
mutually distinct magnetic codes, and a region with a mutually
distinct polymeric or other chemical moiety.
Inventors: |
Rothwarf; David; (La Jolla,
CA) |
Correspondence
Address: |
FISH & ASSOCIATES, PC;ROBERT D. FISH
2603 Main Street, Suite 1050
Irvine
CA
92614-6232
US
|
Assignee: |
Arrayomics, Inc.
San Diego
CA
|
Family ID: |
39641866 |
Appl. No.: |
12/018439 |
Filed: |
January 23, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60886370 |
Jan 24, 2007 |
|
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60886373 |
Jan 24, 2007 |
|
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Current U.S.
Class: |
506/16 ; 209/636;
506/13; 506/18; 506/20; 506/31 |
Current CPC
Class: |
B01J 19/0046 20130101;
B01J 2219/00549 20130101; C40B 50/00 20130101; G06K 19/06187
20130101; G01N 33/54366 20130101; G01N 33/54326 20130101; B01J
2219/00563 20130101; C40B 40/00 20130101; B01J 2219/00497 20130101;
B01J 2219/00709 20130101; B01J 2219/00585 20130101; B01J 2219/00655
20130101 |
Class at
Publication: |
506/16 ; 506/31;
506/13; 506/20; 506/18; 209/636 |
International
Class: |
C40B 40/06 20060101
C40B040/06; C40B 50/16 20060101 C40B050/16; C40B 40/00 20060101
C40B040/00; B07C 5/344 20060101 B07C005/344; C40B 40/14 20060101
C40B040/14; C40B 40/10 20060101 C40B040/10 |
Claims
1. A method of sorting microdevices, comprising: providing an array
having discrete regions that exert magnetic forces; using the
magnetic forces to orient the microdevices with respect to the
regions; and applying a removing force to the oriented microdevices
under conditions that remove a proper subset of the microdevices
from the array as a function of differing orientations of the
microdevices.
2. The method of claim 1, wherein the microdevices have a longest
linear dimension of 0.1 to 500 .mu.m, inclusive.
3. The method of claim 1, further comprising repeating the steps of
using the magnetic force and applying a removing step.
4. The method of claim 1, further comprising utilizing a set of the
microdevices that utilize a magnetic coding space that supports at
least 10 choices.
5. The method of claim 1, further comprising utilizing a set of the
microdevices that utilize a magnetic coding space that supports at
least 10.sup.3 choices.
6. The method of claim 1, further comprising utilizing a set of the
microdevices that utilize a magnetic coding space that supports at
least 10.sup.6 choices.
7. The method of claim 1, further comprising utilizing a set of the
microdevices that have a predetermined preferential axis of
magnetization, and an aspect ratio of at least 1.2.
8. The method of claim 1, wherein the step of using the magnetic
forces to orient the microdevices comprises causing members of the
subset of microdevices to stand up relative to ones of the
microdevices outside the subset.
9. A method of sorting, comprising: using microdevices that have a
predetermined preferential axis of magnetization, and that utilize
a magnetic coding space that can support at least 10 choices; and
using a magnetic force to orient the microdevices.
10. The method of claim 1, wherein the microdevices have a longest
linear dimension of 0.1 to 500 .mu.m, inclusive, and at least some
of the microdevices utilize a magnetic coding space that supports
at least 10.sup.3 choices.
11. The method of claim 9, wherein at least some of the
microdevices utilize a magnetic coding space that supports at least
10.sup.6 choices.
12. A method of sorting: positioning a set of microdevices on an
array; applying a magnetic field to the microdevices in a manner
that alters magnetic interactions of a subset of the microdevices
with the array; and selectively removing the subset of microdevices
from the array.
13. The method of claim 1, wherein each of the microdevices in the
set have a longest linear dimension of 0.1 to 500 .mu.m, inclusive,
and a chemically active site.
14. The method of claim 13, further comprising repeating the steps
of applying the magnetic force and selectively removing at least
five times.
15. The method of claim 13, further comprising utilizing a set of
the microdevices that utilize a plurality of magnetic coding
regions that utilize a coding space that supports at least 10
choices.
16. A method of performing combinatorial chemistry, comprising:
providing a plurality of magnetically orientable microdevices, each
of which includes a chemically reactive site, and each of which
includes a magnetic code; using magnetic orientation of the
microdevices to divide the microdevices into at least first and
second sets; performing different reactions at the reactive sites
of the microdevices in the first and second sets, and then
recombining at least portions of the first and second sets of
microdevices; using magnetic orientation of the microdevices to
divide dividing the microdevices from the recombined first and
second sets into at least third and fourth sets; and performing
different reactions at the reactive sites of the microdevices in
the third and fourth sets, and then recombining at least portions
of the third and fourth sets of microdevices.
17. The method of claim 16, further comprising at least partially
sorting at least some of the microdevices as a function of the
orientation of such microdevices on an array.
18. The method of claim 16, further comprising using the step of
sorting to facilitate dividing the microdevices from the recombined
first and second sets into the at least third and fourth sets.
19. The method of claim 17, wherein at least some of the codes
support at least 10.sup.3 distinct choices.
20. The method of claim 16, wherein at least some of the codes
support at least 10.sup.6 distinct choices.
21. A method of displaying, comprising: proving a set of
microdevices, different ones of which include different magnetic
codes; providing an array having a first and second arraying sites
that complement different ones of the magnetic codes, adding the
microdevices to the array; and applying an external magnetic field
to the array such that distinct subsets of the microdevices select
to the first and second sites, respectively.
22. The method of claim 21 further comprising at least an
additional 8 arraying sites that complement different magnetic
codes from the first and second arraying sites.
23. A library comprising first, second and third microdevices, each
of which has a mutually distinct magnetic code, and a region with a
mutually distinct chemical moiety.
24. The library of claim 23, wherein each of the mutually distinct
chemical moieties are polymers.
25. The library of claim 23, wherein each of the mutually distinct
chemical moieties are peptides or nucleic acids.
26. A chemical entity researched through use of the library of
claim 23.
Description
[0001] This application claims priority to U.S. provisional
application Ser. No. 60/886370 filed Jan. 24, 2007, and to U.S.
provisional application Ser. No. 60/886373 filed Jan. 24, 2007.
FIELD OF THE INVENTION
[0002] This invention relates to the use of physical forces to sort
collections of microdevices into subsets.
BACKGROUND
[0003] Commonly used particle sorting technology is based on the
movement of particles through a device or channel. Sorting rates
are dependent on the flow rate of the particles and high flow rates
can damage particles. Since sorting particles in a channel narrow
enough to direct the flow of the particle is slow and subject to
channel blockage, channels typically used are substantially larger
in diameter than the particles to be sorted. Consequently sorting
processes often involve using electrostatic charge to direct
particles to a particular stream--a method not well suited for
dense particles. Most systems rely on reading an optical signal
(generally fluorescence) to identify particles during the sorting
process. While cell and particle sorters are able to achieve speeds
on the order of 10.sup.6 particle/min this rate refers to the
detection of the particles, separation of particles into high
purity fractions is generally .about.two orders or more of
magnitude slower.
[0004] This application references various patents, patent
applications, and publications. The contents of all of these items
are hereby incorporated by reference in their entirety. Where a
definition or use of a term in a reference, which is incorporated
by reference herein is inconsistent or contrary to the definition
of that term provided herein, the definition of that term provided
herein applies and the definition of that term in the reference
does not apply.
SUMMARY OF THE INVENTION
[0005] The present invention provides systems and methods in which
particles or other microdevices are disposed in an array having
discreet regions (e.g. magnetic bars), oriented within a magnetic
field, and then sorted through application of a removing force
under conditions that remove a proper subset of the microdevices
from the array as a function of differing orientations of the
microdevices.
[0006] In one aspect of preferred embodiments at least some of the
discreet regions can be sufficiently aligned to appear as one or
more bands, with another one or more of the regions oriented
parallel to, but offset from the band. Adjacent regions can
advantageously be separated by an inter region distance of 0.1 to
500 .mu.m. Unless otherwise apparent from the context, all ranges
described herein are inclusive of the endpoints.
[0007] Regions preferably contain substantially parallel bars of a
magnetized material, which can be permanently or only transiently
magnetized. Bars can have any combination of low or high
coercivity, and low or high remanence. A given region can contain
1, 2, 3 or an even higher number of such bars.
[0008] A preferred method of sorting comprises the steps of:
positioning a set of microdevices on an array; applying a magnetic
field to the microdevices in a manner that alters magnetic
interactions of a subset of the microdevices with the array; and
selectively removing the subset of microdevices from the array. The
steps of applying the magnetic force and selectively removing can
be advantageously performed at least five times.
[0009] In practice, the arrays can be used to perform combinatorial
chemistry. For example, one can provide a plurality of magnetically
orientable microdevices, each of which includes an active site
having a relatively high chemical reactivity (a "chemically active
site"), and each of which includes an individual and optionally
unique code; divide the microdevices into at least first and second
sets; perform different reactions at the reactive sites of the
microdevices in the first and second sets, and then recombining at
least portions of the first and second sets of microdevices. The
process can be repeated for third and fourth sets, and so forth. In
line with the descriptions above, it is especially contemplated
that one could at least partially sort at least some of the
microdevices as a function of the orientation on an array, using a
code that can support 10, 10.sup.3, 10.sup.6 or even a greater
number of choices. It is also contemplated that one could use the
step of sorting to facilitate dividing the microdevices from the
recombined first and second sets into the at least third and fourth
sets.
[0010] Viewed from another perspective, the inventive subject
matter can be seen to include methods of using of magnetic patterns
to sort collections of microdevices by magnetic complementarity.
Preferred methods use a capture and release process to sort
microdevices (microdevices), and unlike conventional sorters, do
not require high particle flow rates. The density and size of a
particle does not interfere with sorting by magnetic
complementarity. Because the sorting can be carried out in a batch
process, very high effective rates of particle sorting and
separation are achievable--on the order of 10.sup.9
microdevices/min. This is 4-6 orders of magnitude greater than the
rates of conventional FACS (Fluorescence Activated Cell Sorting) or
flow cytometry-based instruments. Moreover, although some specific
applications can benefit from use of an optical reading device,
many embodiments of the inventive subject matter do not require use
of such a device.
[0011] Contemplated physical embodiments can include the following
components:
[0012] 1) A set of magnetically encoded microdevices. Said
microdevices comprise a nonmagnetizable substrate and magnetizable
material that contain a magnetically distinguishable code.
Individual microdevices can range in size from 500 micron to less
than 1 micron.
[0013] 2) A sorting chip that, by means of magnetic
complementarity, is able to divide a set of microdevices into
subsets, comprising bound and non-bound microdevices. Said sorting
chip comprises a substrate and a magnetically distinguishable
coding region. In a preferred embodiment the sorting chips each
contain a plurality of coding regions. A coding region can be
substantially identical to its neighboring coding regions or it can
be distinct from its neighboring coding regions.
[0014] 3) A magnetic field generator. Said field generator can be
electromagnetic or it can include permanent magnets or a
combination of the two. Preferred embodiments include
electromagnetic generators capable of generating uniform fields
over the surface of the sorting chip.
[0015] 4) A force generator for removing non-bound microdevices
from the sorting chip. Preferred embodiments include those using
fluidic force either alone or in combination with a magnetic force
generator.
[0016] The inventive subject matter also includes methods of
displaying, comprising: proving a set of microdevices, different
ones of which include different magnetic codes; providing an array
having a first and second arraying sites that complement different
ones of the magnetic codes, adding the microdevices to the array;
and applying an external magnetic field to the array such that
distinct subsets of the microdevices select to the first and second
sites, respectively. Such methods can advantageously include at
least an additional eight arraying sites that complement different
magnetic codes from the first and second arraying sites.
[0017] Also contemplated are microdevice libraries that include at
least first, second and third microdevices, each of which has a
mutually distinct magnetic code, and a region with a mutually
distinct chemical moiety. In such libraries the mutually distinct
chemical moieties can be polymers (e.g., peptides or nucleic acids)
or non-polymers. Still further, the inventive subject matter
includes chemical entities that are invented, developed or
otherwise researched through use of such libraries.
[0018] For purposes of summarizing the claimed inventions and their
advantages achieved over the prior art, certain objects and
advantages of the inventive subject matter have been described
herein. Of course, it is to be understood that not necessarily all
such objects or advantages can be achieved in accordance with any
particular embodiment. Thus, for example, those skilled in the art
will recognize that the inventive concepts can be embodied or
carried out in a manner that achieves or optimizes one advantage or
group of advantages as taught herein without necessarily achieving
other objects or advantages as can be taught or suggested
herein.
[0019] All of the embodiments described herein are intended to be
within the scope of the inventive subject matter. These and other
embodiments will become readily apparent to those skilled in the
art from the following detailed description of the preferred
embodiments having reference to the attached figures, the subject
matter not being limited to any particular preferred embodiment(s)
disclosed.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1. Left panel: Face up and face down orientations of
the same type of microdevice containing 2-bar (each bar having
three fingered ends) magnetic code and non-magnetic optical code
(OCR characters 437). Magnetic code is located symmetrically within
microdevice with respect to the x- and y-axes (the long axes of a
microdevice), but asymmetrically located with respect to the z-axis
being 1 micron from the bottom surface of the microdevice and 1.8
micron from the top surface of the microdevice. Magnetic elements
on arraying chip located 0.46 micron below top surface. Center
panel: Arrayed mixture of face-up and face down microdevices in the
presence of an arraying field parallel to the long axis of the
magnetic bars. Right panel: Same view during application of a
lifting field (z-axis) that lifts only the face-down
microdevices.
[0021] FIGS. 2A-2C. A six position coding scheme (n=6): A.
Enumerated representations for one element (k=1) and two element
(k=2) codes; B. Enumerated representations for three element (k=3)
code; C. Enumerated representations for four element (k=4), five
element (k=5), and six element (k=6) codes.
[0022] FIG. 3. Ten pair of three element orthogonal arraying
patterns suitable for sorting the coding schemes that are shown in
FIG. 2.
[0023] FIG. 4. Eighteen position code corresponding to three
six-position codes. By using three elements (k=3) for each
six-position code twenty representations for each six-position code
can be obtained (as shown in FIG. 2B). Such a code can be used to
encode a tripeptide library consisting of all combinations of the
naturally occurring amino acids.
[0024] FIG. 5. A four position coding scheme (n=4): Enumerated
representations for one element (k=1), two element (k=2), three
element (k=3), and four element (k=4) codes.
[0025] FIG. 6. Examples of asymmetric microdevices. Microdevices
comprise either an asymmetrical shape or an asymmetrical
arrangement of magnetic elements or both. Magnetic elements may
consist of a magnetic code and magnetic alignment elements; in the
examples magnetic alignment elements are shown as thicker bars.
[0026] FIG. 7. Enumerated representations for microdevices
containing an eight position coding scheme (n=8) containing five
element (k=5) codes and asymmetric arraying bars.
[0027] FIG. 8. Thirty-five pair of four element orthogonal arraying
patterns that are suitable for sorting the microdevices shown in
FIG. 7.
[0028] FIG. 9. Schematic of a multi-split sorting process to
produce four sets of microdevices. In the multi-split sorting
processes a pool of microdevices is divided into groups and the
groups are divided further. In this example the pool is first
divided into two subgroups and then each subgroup is then further
divided into two groups.
[0029] FIG. 10. Schematic of a sequential sorting process to
produce four groups of microdevices. In the sequential sorting
processes a pool of microdevices is divided into groups in a
stepwise manner. In this example the pool is divided into four
groups.
[0030] FIG. 11. Schematic of a sequential sorting process to
produce four groups of microdevices using the minimum number of
sorting chips. Microdevices that are unbound after the third
sorting step are all members of the same group. Consequently, the
final sorting chip in FIG. 10 captures all members of the group and
is not strictly required.
[0031] FIG. 12. Schematic of a multi-split sorting process to
produce four groups of microdevices using the minimum number of
sorting chips. Microdevices that are unbound after each sorting
step are all members of the same group or sub-group. Consequently,
three sorting chips in FIG. 9 capture all members of the group or
sub-group and are not strictly required. Sorting chips are numbered
as in FIG. 9.
[0032] FIG. 13. Schematic of a multi-split sorting process to
produce four groups of microdevices wherein each group is captured
and eluted from a sorting chip. Sorting chips are numbered as in
FIGS. 9 and 12.
[0033] FIG. 14. Fifteen sets of four element orthogonal arraying
patterns suitable for sorting the n=6 k=5 coding schemes shown in
FIG. 2C into three groups.
[0034] FIG. 15. Sixteen position coding space shown as one
8-position code with two arrangements per position (upper
representation), two 4-position codes with two arrangements per
position (lower representation).
[0035] FIG. 16. Enumerated representations for a four position
coding scheme (n=4, k=3, m=2).
[0036] FIG. 17. Upper panel: 8-position code with two arrangements
per position (n=8 m=2). Lower panel: 8-position code with three
arrangements per position (n=8 m=3). Left side shows general
representation with all possible positions filled and right side
show specific example of a k=7 representation. Magnetic elements
are the same size in the microdevices shown in the upper and lower
panels.
[0037] FIG. 18. Schematic representation of arraying of low
coercivity microdevices on a low coercivity arraying chip. Left
side shows a pattern of arraying bars on an arraying chip. Right
side shows that same pattern with a microdevice arrayed. Arrow
indicates the direction of the external magnetic field.
[0038] FIG. 19. Schematic representation of arraying of low
coercivity microdevices containing a four position single-element
code (n=4 k=1) on a low coercivity sorting chip. Left side shows a
pattern of sorting bars on a sorting chip--all arraying positions
are equivalent. Right side shows the same sorting chip with
microdevices containing all four codes in arrayed form. Arrow
indicates the direction of the external magnetic field.
[0039] FIG. 20. Schematic representation of sorting of low
coercivity microdevices containing a four position single-element
code (n=4 k=1) on a low coercivity sorting chip. Left side shows
microdevices containing all four codes of a four position code
arrayed on sorting chip. Right side shows the same arrayed
microdevices after application of a magnetic lifting force and
removal of the lifted microdevices. Only those microdevices with a
code complementary to the sorting chip are retained--selection
criterion was one coding element being aligned. Arrow indicates the
direction of the external magnetic field.
[0040] FIG. 21. Schematic representation of a low coercivity
sorting chip where bars in the arrayed microdevice will
simultaneously partially and fully overlap bars on the arraying
chip.
[0041] FIG. 22. Actual representation of a portion of a low
coercivity sorting chip where bars in the arrayed microdevice will
simultaneously partially and fully overlap bars on the arraying
chip. On the right are a pair of microdevices that can be sorted on
the sorting chip; the upper microdevice exactly matches the pattern
of five bars on the sorting chip, while the lower microdevice only
matches two of the bars on the sorting chip.
[0042] FIG. 23. Actual representation of a sorting process. Left
panel: portion of sorting chip of the type shown in FIGS. 21 and 22
containing an arrayed mixture of two different microdevices of the
type shown in FIG. 22; Center panel: same view during application
of a lifting field that lifts only one of the microdevices; Right
panel: same view after application of fluidic force to remove the
lifted (non-bound) microdevice.
[0043] FIG. 24. Schematic representation of arraying of low
coercivity microdevices on a high coercivity arraying chip. Left
side shows a microdevice arrayed when the external field is aligned
in parallel with the direction of magnetization of the magnetic
elements on the arraying chip. Right side shows a microdevice
arrayed when the external field is aligned in antiparallel with the
direction of magnetization of the magnetic elements on the arraying
chip. Arrow indicates the direction of the external magnetic
field.
[0044] FIG. 25. Schematic representation of arraying of low
coercivity microdevices containing a 32 position 15-element code
(n=32 k=15; >565 million codes) on a high coercivity sorting
chip. Left side shows a pattern of sorting bars on a sorting
chip--all arraying positions are equivalent. Right side shows the
same sorting chip with microdevices containing a 32-position code
arrayed. Arrow indicates the direction of the external magnetic
field. External magnetic field is aligned in antiparallel with the
direction of magnetization of the magnetic elements on the sorting
chip.
[0045] FIG. 26. Schematic representation of sorting of low
coercivity microdevices containing a 32 position 15-element code
(n=32 k=15) on a high coercivity sorting chip. Left side shows
microdevices containing a 32-position code arrayed on sorting chip.
Right side shows the same arrayed microdevices after application of
a magnetic lifting force and removal of the lifted microdevices.
Only those microdevices with a code complementary to the sorting
chip are retained--selection criterion was greater or equal to
eight coding elements being aligned. Arrow indicates the direction
of the external magnetic field.
[0046] FIG. 27. Schematic representation of a portion of a high
coercivity sorting chip where each arraying position is unique.
[0047] FIG. 28. Schematic representation of a portion of a low
coercivity sorting chip that contains two different arraying sites.
On the right are a pair of microdevices that can be arrayed on the
sorting chip.
[0048] FIG. 29. Actual representation of a portion of a low
coercivity sorting chip that contains two different arraying sites.
On the right are a pair of microdevices that can be arrayed on the
sorting chip.
[0049] FIG. 30. Actual representation of the non-random arraying
process showing portion of sorting chip of the type shown in FIGS.
28 and 29 containing an arrayed mixture of two different
microdevices of the type shown in FIG. 29.
DETAILED DESCRIPTION
[0050] Definitions
[0051] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of ordinary skill in the art to which this invention belongs. All
patents, applications, published applications and other
publications referred to herein are incorporated by reference in
their entirety. If a definition set forth in this application is
contrary to or otherwise inconsistent with a definition set forth
in the patents, applications, published applications and other
publications that are herein incorporated by reference, the
definition set forth in this application prevails over the
definition that is incorporated herein by reference. In instances
where a definition is not set forth in this application and
conflicting definitions arise amongst definitions incorporated
herein by reference, those definitions given in a co-pending U.S.
patent application Ser. No. 12/018319, entitled "Microdevice Arrays
Formed by Magnetic Assembly," filed on even date herewith shall
prevail.
[0052] As used herein, "coercivity" of a material refers to the
intensity of the applied magnetic field required to reduce the
magnetization of that material to zero after the magnetization of
that material has been driven to saturation. Coercivity is usually
measured in oersted units. A magnetic field greater than the
coercivity of a material must be applied to that material in order
to coerce it to change the direction of its magnetization. A "high
coercivity" material is often referred to as a permanent
magnet.
[0053] As used herein, a "predetermined preferential axis of
magnetization" means a preferential axis of magnetization that can
be predetermined through knowledge of the manufacturing process and
design of the microdevice. The "predetermined preferential axis of
magnetization" of a microdevice is a fundamental aspect of the
design of that microdevice, for example, bar-shaped elements of
CoTaZr as used in many of the examples presented in this
application have a predetermined preferential axis of magnetization
that is parallel to the long axis of the magnetic bar. A
"predetermined preferential axis of magnetization" is a property of
a microdevice that depends on the geometry, composition, and
structural configuration of the magnetic elements of the
microdevice. Bar-shaped elements of CoTaZr as used in many of the
examples presented in this application have a predetermined
preferential axis of magnetization that is parallel to the long
axis of the bar; in contrast conventional magnetic beads which have
a random distribution of magnetic material do not have a
predetermined preferential axis of magnetization. The induced
magnetization along the predetermined preferential axis of
magnetization (in its absolute magnitude) is larger than or at
least equal to induced magnetization along any other axis of the
microdevice. In general, for a microdevice of the present invention
to rotate or orient itself under the interaction of the applied
magnetic field and the induced magnetization, the induced
magnetization (in its absolute magnitude) along the predetermined
preferential axis of magnetization of the microdevice should be at
least 20% more than the induced magnetization of the microdevice
along at least one other axis. Preferably, the induced
magnetization (in its absolute magnitude) along the predetermined
preferential axis of magnetization of the microdevices of the
present invention should be at least 50%, 70%, or 90% more than the
induced magnetization of the microdevice along at least one other
axis. Even more preferably, the induced magnetization (in its
absolute magnitude) along the predetermined preferential axis of
the magnetization of the microdevices of the present invention
should be at least two, five times, ten times, twenty times, fifty
times or even hundred times more than the induced magnetization of
the microdevice along at least one other axis. As used herein, an
"orthogonal" set of sorting chips divides a space into groups such
that through appropriate choice of a magnetic selection criterion
all members of a group can be captured (remain bound) by one member
of the orthogonal sorting set.
[0054] As used herein, a "bound" microdevice is one that is in an
arrayed position during a sorting step on a sorting chip. A
"non-bound" microdevice is one that is not in an arrayed position
during a sorting step on a sorting chip. During a sorting step an
"arrayed" microdevice is one that is held in a position that is
substantially parallel to the surface of the sorting chip by
magnetic association with the sorting chip. An "eluted" microdevice
is one that was a "bound" microdevice at an earlier step in the
sorting process but that has become "non-bound". Bound, non-bound,
and eluted refer to processes that occur on a single sorting chip.
For example, a collection of magnetically encoded microdevices are
placed on a sorting chip and arrayed. A magnetic selection
criterion corresponding to a magnetic field is applied resulting in
a subset of the microdevices becoming non-bound by being lifted
from the surface of the sorting chip and orienting substantially
perpendicular to the surface of the sorting chip. These non-bound
microdevices are removed using some type of force generator. The
remaining bound microdevices are then eluted through application of
a magnetic field and/or some type of force generator. As used
herein, non-bound refers to the first subset of microdevices
removed from a sorting chip during a sorting process while all
subsequent subsets of microdevices removed from a sorting chip are
referred to as eluted. In some instances the only non-bound subset
corresponds to broken and defective microdevices and may have no
members.
[0055] Microdevice, Detailed Description.
[0056] The microdevice contains magnetically distinguishable code
that enables the microdevice to be sorted. Said microdevice
comprises a magnetizable substance and can have a preferential axis
of magnetization. Additional features can be incorporated into the
microdevice, including, but not limited to, photorecognizable
coding patterns. The properties of such microdevices containing
photorecognizable coding patterns are enumerated in U.S. Pat. No.
7,015,047. U.S. Pat. No. 7,015,047 discusses a subset of
microdevices compatible with the magnetic assembly process.
[0057] The microdevices can have any shape. They can have planar
surfaces, but they need not have planar surfaces; they can resemble
beads. Flat disks are a preferred implementation. Microdevices
shaped as circles, squares, ovals, rectangles, hexagons, triangles,
and irregular shapes are all amenable to the magnetic assembly
arraying process. The microdevices can be of any suitable
dimension(s). For example, the thickness of the microdevice can be
from about 0.1 micron to about 500 microns. Preferably, the
thickness of the microdevice can be from about 1 micron to about
200 microns. More preferably, the thickness of the microdevice can
be from about 1 micron to about 50 microns. In a specific
embodiment, the microdevice is the form of a rectangle having a
surface area from about 10 squared-microns to about 1,000,000
squared-microns (e.g., 1000 micron by 1000 micron). In another
specific embodiment, the microdevice is an irregular shape having a
single-dimension from about 1 micron to about 500 microns.
[0058] The microdevices can contain one or many magnetizable
elements. The microdevices can have a predetermined preferential
axis of magnetization.
[0059] The individual magnetic elements within the microdevice can
be of any width, length, thickness and shape. The individual
magnetic elements within a microdevice can be composed of different
materials having similar or different magnetic properties.
[0060] Any suitable magnetizable material can be used in the
present microdevices. In one example, the magnetizable substance
used is a paramagnetic substance, a feltimagnetic substance, a
ferromagnetic substance, or a superparamagnetic substance.
Preferably, the magnetizable substance is a transition metal
composition or an alloy thereof such as iron, nickel, copper,
cobalt, manganese, tantalum, and zirconium. In a preferred example,
the magnetizable substance is a metal oxide. Further preferred
materials include nickel-iron (NiFe) and cobalt. Additional
preferred materials include alloys of cobalt such as CoTaZr,
cobalt-iron (CoFe), cobalt-nickel-iron (CoNiFe),
cobalt-niobium-zirconium (CoNbZr), cobalt niobium hafnium (CoNbHf),
and cobalt tantalum hafnium (CoTaHf). Preferably such features are
bar shapes that have a preferential axis of magnetization. The term
"bar", in addition to rectangular shapes, includes rod-like shapes
as well as slightly irregular shapes that still exhibit a
preferential axis of magnetization, e.g., elongated pyramidal
shapes. A bar need not be solid and can contain cutouts or holes as
described below. The magnetizable substance can be situated
completely inside (encapsulated) the non-magnetizable substrate
comprising the microdevice, completely outside yet attached to the
non-magnetizable substrate comprising the microdevice, or anywhere
in between. Preferably the magnetizable substance is patterned, for
example using micromachining or lithographic techniques, so that
its three-dimensional shape is a known feature of the design of the
microdevice.
[0061] Because the microdevices are used to carry out assays in a
liquid array format, it is advantageous that they can be
conveniently aliquoted or dispensed using conventional liquid and
bead handling devices (e.g. pipettors). Consequently, it is
desirable that they do not self-associate in the absence of a
magnetic field. Therefore, low remanence (i.e., magnetization left
behind in a medium after an external magnetic field is removed) is
a desirable quality. Cobalt alloys such as CoTaZr and iron oxides
(Fe.sub.3O.sub.4) are preferred examples of magnetic materials that
meet this criterion.
[0062] In a preferred embodiment, microdevices include a
non-magnetic substrate composed of multiple layers, as described in
U.S. Pat. No. 7,015,047. This non-magnetic substrate can contain
other features including optical encoding patterns and wells.
Additional features can be included and any of the wide range of
features compatible with planar microfabricated devices such as
those used in Micro-Electro-Mechanical Systems (MEMS) can be
incorporated into the non-magnetizable substrate of the
microdevice. In a preferred embodiment the microdevice contains
electrical contact pads and circuitry that allow MEMS type sensors
within the microdevice to be utilized. This circuitry is composed
of electrically conductive material that is preferably encapsulated
within the substrate of the microdevice such that only contact pads
and sensor elements are exposed on the surface of the microdevice.
Contact pads on the surface of the microdevice can be used to
connect the microdevice to a power source(s) and/or sensing
device(s) by means of complementary contact pads on the arraying
chip. In a preferred embodiment, electrical circuitry is placed
within each microdevice in a unique configuration, thus the
connection between the microdevice contact pads and the
complementary pads on the arraying chip may be used to determine
the identity of the microdevice.
[0063] In one embodiment the microdevices comprise a chemically
reactive surface that is suitable for attachment of a chemical or
biological moiety. In another embodiment this surface is present in
a well or indentation. In one embodiment this surface is produced
by means of a silane (e.g. aminopropyltrimethoxysilane,
gycidoxypropyltrimethoxy silane). In another embodiment a reactive
surface is produced by means of a thiol containing reagent (e.g.
11-mercaptoundecanoic acid). In another embodiment the reactive
surface is a self-assembled monolayer (for example as reviewed in
"Formation and structure of self-assembled monolayers" by Ulman
Chem. Rev. 96:1533-1554 (1996) and "Self-assembled monolayers of
thiolates on metals as a form of nanotechnology" by Love et al.
Chem. Rev. 105:1103-1169 (2005)). The reactive surface can be
generated on the microdevice using batch techniques (e.g. a set of
microdevices placed in an aqueous solution of the appropriate
reagent, such as silane to generate a reactive surface on exposed
silicon dioxide surface of the microdevice). Alternatively, the
reactive surface can be generated on the microdevices prior to
their release from the wafer (during or after the fabrication
process). The reactive surface can be applied to all the
microdevices on the wafer (e.g. by gas or liquid phase
silanization) or at particular positions on the wafer using
position specific deposition (e.g. inkjet) or masking (e.g.
photolithography) such that the reactive surface is applied only to
a subset of microdevices on the wafer or even to specific locations
on individual microdevices. In a further embodiment such position
specific processes can be used to produce unique chemical compounds
on individual microdevices. Such techniques are widely used to
produce DNA microarrays and are well-established art (e.g.
"Spatially addressable combinatorial libraries" by Pirrung Chem.
Rev. 97, 473-488 (1997) and "In situ synthesis of oligonucleotide
microarrays" by Gao et al. Biopolymers, 73:579-596 (2004)). In a
further embodiment the locations of reactive surface on individual
microdevices can be patterned. Such patterning can be generated by
masking in which a material is used to protect a surface from being
modified, for example a layer of photoresist can be used to
surround a silicon dioxide well and then following the silanization
of the well surface the photoresist can be dissolved away to reveal
a unsilanized surface. Patterning can also be achieved through the
use of different materials, for example a gold surface can be
created on a silicon dioxide surface, reaction with a carboxylated
alkyl thiol will yield a carboxylated surface only over the gold.
Individual microdevice can contain one or many patterned reactive
surfaces. Such methods are well established in the fabrication and
chemical literature particularly as applied to the manufacture of
DNA and protein microarrays. In additional embodiments the
chemically reactive surface corresponds to a linker molecule used
in solid phase synthesis. Many such linker molecules are known to
those practiced in the art of combinatorial chemistry (e.g. as
referenced in Jung, G., Combinatorial Chemistry,
Weinheim,Wiley-VCH, 1999; "Comprehensive survey of chemical
libraries for drug discovery and chemical biology; 2006" by Dolle
et al. Journal of Combinatorial Chemistry, 9:855-902 (2007)).
[0064] When a microdevice that contains magnetic elements is placed
in an external magnetic field, a magnetic dipole(s) is induced in
the microdevice. Because the microdevice has a preferential axis of
magnetization it will, unless impeded, rotate so as to align its
preferential axis of magnetization with the force lines of the
external magnetic field. When placed in a rotating external
magnetic field the microdevices, unlike conventional magnetic
beads, will rotate and, in effect, serve as mini stir-bars.
Consequently it is desirable, apart from any considerations with
respect to arraying, that the microdevices respond strongly to
external magnetic fields. Magnetic elements composed of materials
with high saturation magnetizations such as CoTaZr alloys are a
preferred embodiment.
[0065] Sorting Chip, Detailed Description.
[0066] The sorting chip is comprised of both magnetic and
non-magnetic material. A sorting chip performs it function by first
arraying microdevices and as such is a specialized arraying
chip--the properties and features of general arraying chips are
described in a co-pending U.S. patent application Ser. No.
12/018319 entitled "Microdevice Arrays Formed by Magnetic
Assembly," filed on even date herewith and can be applied to the
sorting chips disclosed here. Any suitable magnetizable material
can be used in the sorting chip. In one example, the magnetizable
substance used is a paramagnetic substance, a ferromagnetic
substance, a ferrimagnetic substance, or a superparamagnetic
substance. Preferably, the magnetizable substance is a transition
metal composition or an alloy thereof such as iron, nickel, copper,
cobalt, manganese, tantalum, and zirconium. In a preferred example,
the magnetic substance is a metal oxide. Further preferred
materials include NiFe and cobalt. Additional preferred materials
include alloys of cobalt such as CoTaZr, CoFe, CoNiFe, CoNbZr,
CoNbHf, and CoTaHf. Preferably such features are bar shapes that
have a preferential axis of magnetization. In many applications
residual magnetization in the sorting chip is a desirable quality.
Similar to the microdevice, the magnetizable substance in the
sorting chip can be situated completely inside (encapsulated) the
non-magnetizable substrate comprising the sorting chip, completely
outside yet attached to the non-magnetizable substrate comprising
the sorting chip, or anywhere in between. A preferred embodiment
places the magnetic elements on top of a glass substrate and
encapsulates them with silicon dioxide such that the silicon
dioxide forms a planar or substantially planar surface. A further
preferred embodiment places the magnetic elements on top of a
silcon substrate and encapsulates them with silicon dioxide such
that the silicon dioxide forms a planar or substantially planar
surface.
[0067] Although the examples presented in this application use a
sorting chip containing CoTaZr bars that have low remanence and low
coercivity, these properties are not necessary for the assembly of
magnetic arrays or the sorting process. Since high remanence will
cause microdevices to magnetically assemble into chains or clumps
in the absence of an external magnetic field, in general, it is not
desirable for the microdevices to contain such; although, it can be
desirable that the magnetic elements contained within the sorting
chip have said qualities in order to allow assembled arrays to
remain intact once the arraying field is removed.
[0068] The individual magnet elements within the sorting chip can
be composed of different designs. The magnetic elements can be of
any shape and size. Individual magnetic elements can be distinct
from all other elements or comprise a subset of such elements. The
individual magnetic elements can be composed of different materials
having similar or different magnetic properties. Preferably the
magnetic elements are bar shapes that have a preferential axis of
magnetization. More preferably the magnetic elements have a
predetermined preferential axis of magnetization. The term "bar",
in addition to rectangular shapes, includes rod-like shapes as well
as slightly irregular shapes that still exhibit a preferential axis
of magnetization, e.g., elongated pyramidal shapes. A bar need not
be solid and can contain cutouts or holes.
[0069] A preferred embodiment is magnetic elements that are bars
composed of a high permeability ferromagnetic material. These bars
can be rectangular or substantially rectangular. Bars containing
"fingers" such as those shown in FIG. 1 and described in U.S. Pat.
No. 7,015,047 are another preferred embodiment. These fingers can
be short (e.g., 1-2% of the total length of the bar) or long (e.g.,
comprising almost the entire length of the bar) or anywhere in
between.
[0070] The non-magnetizable substrate can be comprised of any
suitable material including silicon, silicon dioxide, silicon
nitride, plastic, glass, ceramic, polymer, metal (e.g., gold,
aluminum, titanium, etc.) or other similar materials or
combinations of such materials. In a preferred example the material
is silicon dioxide. In another preferred example the material is
glass. The substrate can comprise a single layer or it can comprise
multiple layers. The sorting chip substrate can, but need not be,
planar or substantially planar. There can exist indentations in the
sorting chip that allow for "seating" of the microdevices to assure
exact alignment of said microdevices, which can be desirable for
some applications. These indentations, for example, can have planar
faces for seating of microdevices that are flat-ish, or they can be
spherical for seating of beads or bead-like microdevices. In one
preferred embodiment the indentations are designed to match the
shape of individual planar microdevices, e.g. rectangular wells to
hold rectangular microdevices.
[0071] The number of arraying sites per unit area is dependent on
the size and spacing of the magnetic elements on the sorting chip.
For example, sorting chips that are arraying microdevices of the
size shown in FIG. 1 that are 60.times.75 micron in size can array
approximately 100 microdevices per square millimeter. In other
embodiments the density will be much higher. For example,
microdevices that are 20.times.25 micron in size can be arrayed and
sorted at a density of approximately 1,000 microdevices per square
millimeter.
[0072] The sorting chip can contain additional features that are
not necessarily required to facilitate the sorting process. Any of
the wide range of features compatible with planar microfabricated
devices can be incorporated into the non-magnetizable substrate of
the sorting chip, such as those used in MEMS (for example as
reviewed in Liu, C., Foundations of MEMS, Pearson Prentice Hall,
Upper Saddle River, N.J., 2006; Gad-el-Hak, M., MEMS (Mechanical
Engineering), CRC Press, Boca Raton, 2006). A preferred example is
microchannels. Such channels can be used to deliver and/or remove
reagents and other materials such as microdevices from the sorting
chip surface. Additional preferred examples include electronic and
optical microsensors including those used in MEMS (for example as
reviewed in Gardner, J. W. et al., Microsensors, MEMS, and Smart
Devices, John Wiley & Sons, West Sussex, 2001).
[0073] The magnetic elements of the sorting chip should be
complementary to those of the microdevice, but need not exactly
match those of the microdevice in dimension or shape.
[0074] Fabrication
[0075] Microdevices and sorting chips may be fabricated using any
of a variety of processes. In preferred embodiments they are
produced using variations of conventional micromachining and
semiconductor fabrication methods. Such methods are described and
referenced in U.S. Pat. No. 7,015,047 and US Patent Application
2002/0081714 as well as in reviews and textbooks that discuss
photolithographic or MEMS fabrication techniques (for example in
Banks, D., Microengineering, MEMS, and Interfacing: A Practical
Guide, CRC Press, 2006).
[0076] Distinguishable Magnetic Codes
[0077] Magnetically encoded microdevices contain magnetically
distinguishable codes that enable the microdevices to be sorted. A
magnetically distinguishable code is a code that can be
distinguished from another by magnetic means. Magnetically
distinguishable codes differ in the strength and or distribution of
their magnetic materials. Preferred embodiments include the
distribution of magnetic elements within a substrate. Such
distributions can occur along any axis (x, y, or z, where the
x-axis is the long axis of the microdevice (length), the y-axis is
the second longest axis of the microdevice (width), and the z-axis
is the short axis of the microdevice (height)). FIG. 1 shows an
example of how microdevices that contain magnetic elements that
differ only in the placement of those magnetic elements along the
z-axis of the microdevice can be distinguished. In that example,
arrayed microdevices containing magnetic bars are subjected to a
magnetic arraying field that is parallel to the long axis of the
magnetic bars in the arraying chip. A second magnetic field
perpendicular to the plane of the arraying chip is then provided.
Face-up microdevices that have their magnetic elements 1.46 micron
from the magnetic elements of the arraying chip; by contrast
face-down microdevices have their magnetic elements 2.26 micron
from the magnetic elements of the arraying chip. This difference in
distance results in a difference in magnetic force between the
arraying chip magnetic elements and the face-up and face-down
microdevices that is sufficiently large such that face-down
microdevices can be selectively lifted from arraying chip surface
by magnetic means.
[0078] Magnetic codes can comprise only a single element and still
be distinguishable (e.g. a single bar magnet encapsulated by 1
micron of silicon dioxide can be distinguishable from a single bar
magnet encapsulated by 1.1 micron of silicon dioxide when arrayed
on an arraying chip of the type shown in FIG. 1). Similarly if the
microdevice docking location on the sorting chip restricts the
motion of a properly docked microdevice (e.g. with a well or posts)
then a single element code in the x,y plane can also be
distinguishable. Moreover, single element codes that differ in the
size, shape, and material comprising the magnetic element can be
distinguishable.
[0079] Magnetic elements that form the codes do not need to be
uniform in size so long as there is a discrete difference between
the magnetic force of a properly arrayed microdevice containing a
code to be retained and a properly arrayed microdevice containing a
code to be removed in the unbound fraction, such that an
appropriate threshold exists for lifting the unbound microdevice
while retaining the bound microdevice.
[0080] A preferred embodiment is that the differences in
distribution of magnetic material for distinguishable codes reside
within the x,y plane of the microdevice. Exemplarily
representations of such codes include barcodes. Two primary
components of the barcode design processes are the number of codes
and the manner in which they can be divided.
[0081] Coding Space
[0082] The total number of codes encoded by a space n containing k
elements is defined in eqn 1.
Coding Space = n ! k ! ( n - k ) ! ( 1 ) ##EQU00001##
[0083] For example, consider a bar code with 6 available positions
in the code as shown in FIG. 2. This corresponds to n=6 in eqn 1.
There are 6 possible values of k. For k=1 there are 6 possible
codes; for k=2 there are 15 possible codes; for k=3 there are 20
possible codes; for k=4 there are 15 possible codes; for k=5 there
are 6 possible codes; for k=6 there is only one possible code. In
order to divide all the codes into orthogonal groups some subset of
bars must be used for capture. For k=1 the coding space can be
divided in 2,3,4,5, or 6 groups, since any combination of
orthogonal capture bars can be used as shown in FIG. 2. For
example, consider two orthogonal capture groups each member
containing three bars as shown in FIG. 3. Each coded microdevice
has either one or zero bar overlap with each capture chip--if it
has one bar overlap with one capture chip it will have zero overlap
with the other members.
[0084] For k=2 the situation is more complicated. Using capture
bars divided into two orthogonal groups of the type shown in FIG.
3, each coded microdevice has two, one, or zero bar overlap with
the capture chip. For any given pair there will be only 6
microdevice codes that will have two bar overlap and 9 codes that
will have only a one bar overlap and thus fail to be
distinguishable. One can always sort using a trivial solution, i.e.
arraying patterns that precisely match only one code and such an
approach can be of great value as discussed below.
[0085] For k=3, again using the capture bar sets shown in FIG. 3,
each coded microdevice has three, two, one, or zero bar overlap
with the capture chip. However, unlike the k=2 example, there is no
way to evenly divide three bars, consequently for any capture pair
every code will have at least 2 bars overlap with only one member
of an orthogonal capture pair. Therefore, a capture (selection)
criterion of 2 or more bar overlap can be used to evenly divide the
coding space.
[0086] For k=4 each coded microdevice has three, two, or one bar
overlap with the capture chip the same problem exists as for k=2,
there are 9 codes that will be indistinguishable using any capture
criterion (two bars overlap with each member of a capture
pair).
[0087] For k=5 each coded microdevice has three or two bar overlap.
There is no way to evenly divide five bars consequently for any
capture pair every code will have 3 bars overlap with only one
member of an orthogonal capture pair. Therefore, a capture
criterion of 3 bars overlapping can be used to evenly divide the
coding space. For k=6 there is only one code.
[0088] There are other ways of dividing a coding space other than
into two equal groups and coding spaces can be selected so as to
tailor the sorting space to meet the specific application. One
simple example is peptide synthesis. If one desires to produce all
combinations of naturally occurring tri-peptides this requires 8000
codes and the ability to split the microdevices into 20 groups (one
for each of the naturally occurring amino acids). FIG. 4 shows one
method of encoding such a process by using three separate codes for
20 (n-6 k=3). The selection criterion becomes the individual
representations for each code. This is one of the powerful uses of
the "trivial solution" mentioned above. By combining easily
divisible codes a large easily assigned coding space can be
generated.
[0089] For example consider coding spaces targeted towards DNA
synthesis where splitting into four groups is desired. It can be
carried out using the same approach as the previous example by
using multiple copies of a four member code (e.g. n=4, k=1 or n=4,
k=3) as shown in FIG. 5.
[0090] The codes that have been shown thus far have been numbered
for easy identification. However, the patterns shown are
symmetrical and as such some codes can not be distinguishable from
other codes after rotation, thereby reducing the number of
distinguishable codes available for a given coding space. Should it
be desired to use the coding space in a more efficient manner,
there are a number of ways to overcome symmetry. Several examples
are shown in FIG. 6. These examples illustrate the use of different
patterns of alignment bars that are asymmetrical. While the
alignment bars shown in FIG. 6 are the bars of greater width this
need not be the case and alignment bars that are similar in width
to magnetic code elements can be used. Alternatively, if an
arraying process is carried out in a well that is complementary in
shape to the microdevice then asymmetrical shaped microdevices can
be used to eliminate symmetry in the code. It is the overall
symmetry of the microdevice that is at issue. Consequently, a
symmetrical code and a symmetrical shape can yield an asymmetrical
microdevice if none of the symmetry planes and axes of the code and
the shape are coincident. FIG. 6 shows several asymmetric
microdevices. These examples are illustrative and are in no way
exhaustive. In a preferred embodiment the microdevices will contain
an alignment bar. Alignment bar placement can occur at any position
within the microdevice. In another preferred embodiment the
microdevices contain asymmetric alignment bars. In a further
preferred embodiment the microdevices will contain both an
alignment bar and an asymmetric shape. While FIG. 6 shows
asymmetrical arrangements of codes in the x,y plane, asymmetrical
arrangements can also be generated through the asymmetric
arrangement of magnetic bars along the z-axis (height of the
microdevice) as in the microdevice shown in FIG. 1.
[0091] For simplicity in further schematic examples when displaying
magnetic codes within microdevices an asymmetric pair of alignment
bars will be used.
[0092] The examples given thus far have been straightforward to
encode and sort but they do not most effectively utilize the space.
In general, for sorting applications where the entire space is to
be divided into equal size groups it is usually simplest to have
the total number of positions (coding space) in the magnetic code
be even (divisible by two) and the number of occupied positions
(elements) in the code be odd as this guarantees that the total
number of codes can be evenly divided using two orthogonal
(non-overlapping) arraying-based sorting steps as demonstrated
above for the exemplary coding spaces n=6, k=3 and n=6, k=5.
[0093] Consider n=8, there are a large number of ways to encode and
sort an 8 position matrix code--the maximum number of codes is
8!/4!4! or 70 by choosing any 4 bars out of the 8 rather than
restricting the position to one bar in every four positions.
Sorting such a representation into two equally sized orthogonal
groups using a unique code is more complex, since as discussed
above k is even. The n=8 coding space can be divided into two n=4
spaces or four n=2 spaces each of which would contain 16 unique
codes. However, consider another example choosing 5 bars out of 8
this yields 56 codes and since the value of k is odd it is easily
divided into two equal sized groups. FIG. 7 shows the exhaustive
enumeration of this representation, which for convenience of
referral have been denoted numerically 1-56. To array these coded
microdevices into two equal groups, magnetic arraying bars
consisting of 4 bars can be used. Microdevices that have at least
three bars magnetically aligned with the sorting chip will be bound
while the remaining microdevices will be eluted. For the exemplary
coding space shown in FIG. 7 there are 35 unique pairs of 4 bars
that can be used. FIG. 8 shows the possible orthogonal arraying bar
patterns for dividing the entire coding space into two groups.
Table 1 shows the complete representation of a splitting process
for each pair of orthogonal four arraying bar sets.
[0094] Each group can then be subdivided by repeating a sorting
process with a different orthogonal set. Consequently, to divide
any given space into four groups using 3 pairs of orthogonal sets
would require a total of 6 sorting steps as shown schematically in
FIG. 9. However, when dividing a space into groups it is not
strictly necessary to carry out the final step since those
microdevices that remain after the next-to-last step are all
members of the same group. Consequently the final splitting step is
not really a splitting step as it captures all members of the
target group. For example when dividing a group into four, as shown
in FIG. 10, only the first three steps are strictly necessary as
shown in FIG. 11. Similarly, when using sets of sorting chips that
divide the space into two groups, only three steps are strictly
required, as shown in FIG. 12. Therefore to divide a group of
microdevices into four groups, the multi-split sorting method and
the sequential sorting method each only require three sorting
steps. There can be, however, an advantage in retaining the final
step since it removes any microdevices that should have been
captured in the previous step as well as removing any damaged or
defective microdevices. A multi-split sorting process can still
maintain this quality control process with only five steps. Such a
preferred embodiment is shown in FIG. 13.
[0095] Comparing a simple sequential sorting approach to a
multi-split sorting approach to produce a set of all possible
10-mer oligonucleotides (using A,C,T,G)--this requires 1,048,576
codes (4.sup.10). Using a four code approach could use a code space
of 40 and 40 sorting steps. Using a multi-split sorting approach
could use a coding space of at least 23 (n=23 k=11 encodes
1,352,078 patterns) and 50 sorting steps. The appropriate choice of
coding depends on the specific application.
[0096] There are a variety of other coding options that could be
used that permit a space to subdivided in a manner that allows
sorting. This can involve choosing a subset of the larger
space--the example shown above in FIG. 4 of three sets of n=6 k=3
represents a subset of the n=18 k=9 space. It can also involve
picking a particular selection criterion--there are a wide range of
orthogonal sets of arraying chips that can be used. For example, in
FIG. 2C the n=6 k=5 group can be divided into two equal groups by
using the orthogonal pairs shown in FIG. 2C where the selection
criterion is using three bars to select three bars. However, the
same coding space can be divided into three equal groups by using
four bars to select four bars as shown in FIG. 14. This example
also demonstrates that while the sorting process divides the space
into three orthogonal groups the sorting chips within each set each
have two elements in common with any other member of the set.
[0097] A coding space can also be divided into more complex coding
schemes. One such example is more readily described by rearranging
the spacing such that each position is depicted as a column where
each column contains more than one possible arrangement of
elements; the number of element arrangements per column is denoted
by the letter m. The examples shown in FIG. 15 illustrate a 16 bar
code containing either n=8 m=2 or two sets of n=4 m=2. The total
number of codes is defined in the following equation:
Coding Space = n ! k ! ( n - k ) ! m k ( 2 ) ##EQU00002##
[0098] For a given value of n and m there are a range of values of
k that can be used. For simplicity consider the lower panel in FIG.
15 showing two sets of n=4 m=2. In order to easily split the
microdevices into four groups, k=1 can be used leading to 64 codes.
However, k=3 is equally effective at subdividing the space into
four groups and leads to a 16-fold increase in the coding space,
i.e. 1024 codes. FIG. 16 shows the 32 coding patterns for an n=4
m=2 k=3 coding representation.
[0099] One of the advantages of coding representations such as
those shown in FIGS. 15 and 16 is that they can more effectively
utilize space. Since only one bar in a column will be used there is
no need for there to be a space between the bars and in fact the
positions can have considerable overlap. Consequently, in the space
of a fully independent 16 element code as shown in FIG. 15 could
contain 24 elements of the same size as shown in FIG. 17. The
corresponding n=8, k=7, m=3 representation would result in 17496
codes. This is 36% more codes than the n=16 k=8 representation and
over 50% more than the sorting friendly n=16 k=7 representation.
Formally, the n=8, k=7, m=3 representation is a subset of the n=24
k=7 representation.
[0100] In a preferred embodiment the patterns include a common set
of alignment bars on all microdevices, such that all microdevices
whatever their code will array on the sorting chip. While a sorting
process can be performed using more than one pattern per sorting
chip a preferred embodiment is to use only a single code per
sorting chip such that all arraying positions on the sorting chip
are equivalent. This results in arraying processes reducing to the
robust arraying processes described in a co-pending U.S. patent
application Ser. No. 12/018319, entitled "Microdevice AlTays Formed
by Magnetic Assembly," filed on even date herewith.
[0101] The arrangement of magnetic elements on the sorting chip is
dependent on the magnetic properties of the magnetic elements on
the sorting chip and the magnetic properties of the magnetic
elements of the microdevices. A preferred embodiment for
microdevices is that their magnetic elements have low coercivity
and low remanence so that they will not strongly self-associate in
the absence of an external magnetic field. For microdevices of this
type, sorting chips containing a wide range of magnetic materials
can be used. One preferred embodiment is that the magnetic elements
in the sorting chips have low coercivity. To array and sort
microdevices on sorting chips with these type of elements, magnetic
overlap can be used, where the North seeking poles overlap South
seeking poles. FIG. 18 shows a schematic example of a magnetic bar
arrayed using such bars. FIGS. 19 and 20 show schematic examples of
a sorting process using low coercivity elements for a n=4 k=1
encoded microdevice with asymmetrical alignment bars.
[0102] Apart from any consideration of overall magnetic strength,
the length of the bars on the arraying chip relative to the length
of the bars in the microdevices can be important. Since the
"magnetic charge" is concentrated near the ends of the magnetic
regions, for low coercivity sorting chips the interaction between
fully overlapping bars is repulsive. However in the case of the
overlap between a long and a short bar the interaction can be
attractive especially when the short bar overlaps the central
region of the long bar. The ability of long bars to have favorable
magnetic interactions when overlapping with much smaller bars can
be used to create arraying chip patterns that increase the overall
strength of desirable arraying interactions and improve the
efficiency of arraying and sorting. In this procedure a magnetic
bar of an arrayed microdevice fully overlaps a smaller bar while
still engaging in favorable interactions by partially overlapping
two other bars. In a preferred embodiment the fully overlapped bar
on the array is less than 50% of the length of the overlapping bar
on the arrayed microdevice. In a preferred embodiment the sorting
chips contain alternating large and small bars. In a further
preferred embodiment the small bars are less than 60% of the gap
between the larger bars. FIG. 21 shows a schematic example of a
sorting chip containing magnetic bars that are smaller than the
magnetic bar on the microdevice to be arrayed. FIG. 22 shows an
actual example of a sorting chip and microdevices that can be
sorted using this type of bar pattern. In this example the sorting
chip corresponds exactly to one of the microdevices while the other
microdevice has only two out of its five magnetic elements in
common with the sorting chip. Consequently, a selection criterion
of 3, 4, or 5 aligned magnetic elements can be used to distinguish
these microdevices. The microdevices are 60.times.75.times.3 micron
in size. FIG. 23 shows an example of these microdevices being
sorted. The left most panel of FIG. 23 shows a portion of the
sorting chip containing each of the type of microdevices in FIG. 22
in arrayed form. After application of a lifting field the
noncomplementary microdevice is raised from the surface as shown in
the central panel. Application of fluidic force (supplied by means
of a laboratory micropipettor) removes the raised microdevice
(removes the non-bound microdevice as represented in FIGS. 9-13).
The bound microdevice can then be "eluted" as represented in FIGS.
9-13 by increasing the lifting field and the application of fluidic
force to complete the sorting step.
[0103] Another preferred embodiment is that the magnetic elements
in the sorting chips have high coercivity. To array and sort
microdevices on sorting chips with these type of elements, magnetic
overlap can be used. Unlike the magnetic overlap that occurs
between low coercivity magnetic elements, magnetic overlap between
a low coercivity magnetic element and a high coercivity magnetic
element is dependent on the specific direction on the external
magnetic field. FIG. 24 shows a schematic example of a magnetic bar
arrayed using such bars with the external field running parallel
and anti-parallel to the direction of the magnetization of the high
coercivity elements.
[0104] For high coercivity elements there is no need for gaps to be
present in order to array and sort. A preferred embodiment is an
arrangement of magnetic elements arranged so as to provide no
well-defined gaps between elements. FIGS. 25 and 26 demonstrate a
microdevice being sorted where the microdevices and sorting chips
meet this criterion. In this example the coding space (n=32, k=15)
can contain over 565 million different codes, but a single sorting
chip can be used to effectively divide the microdevices into two
groups of known composition.
[0105] The examples above are in no way intended to be limiting.
Sorting does not need to be into equal sized groups. In addition,
the same criteria do not need to be used in successive sorting
cycles. For example, a first selection could include a set of three
bars being used to select all microdevice patterns containing those
bars, while a second selection step could correspond to 4 bars
requiring perfect matching or even 5 bars selecting for all
combinations containing 3 or more bars. A coding space can be
sub-divided in a wide variety of ways as will be readily apparent
to one skilled in the art. Moreover, a single sorting chip can be
used to divide a space by sequentially eluting microdevices. Such a
sequential elution process involves eluting in succession more
weakly held microdevices. For example, in a code containing 5
magnetic elements such as that shown in FIG. 7 the microdevices
could be eluted into four separate groups from a single 4-element
sorting based on the number of bars that overlap (1, 2, 3, or 4).
For example such sequential elution procedures could be used to
isolate a specific code using a small number of sorting chips.
[0106] It is also possible to target a single microdevice for
elution by generating a localized lifting field such that only one
microdevice or a small number of microdevices in the region of the
lifting field will be eluted. This can be done using a small
electromagnet or a small permanent magnet. Under high-density
arraying conditions it is possible that more than one microdevice
will be lifted, but the eluted microdevices can be re-arrayed at
lower density and the process repeated to isolate the microdevice
of interest. Additionally, sequential elution and localized lifting
fields can be used in combination to rapidly isolate an individual
microdevice.
[0107] Non-Random Arraying
[0108] A sorting process can be performed using more than one
pattern per sorting chip. In that situation microdevices will be
directed to array through magnetic complementarity to specific
locations on an arraying chip. In one embodiment the array contains
a subset of the magnetic codes and selection criteria can be used
to select the subset of microdevices that will be retained at a
particular array location. In another preferred embodiment the
arraying chip contains a unique pattern of magnetic elements
corresponding to each magnetic coded microdevice. Such particle
arrays are no longer random as the location of specific coded
microdevice on the arraying chip is determined by the location of
its complementary magnetic pattern. FIG. 27 shows a schematic
diagram of such an array on a high coercivity arraying chip with
each arraying position being unique. FIG. 28 shows a schematic
example of a much simpler low coercivity arraying chip in which two
different arraying patterns are being used as well as two
microdevices that can be arrayed on such as an array. FIG. 29 shows
an actual example that corresponds to the schematic example in FIG.
28. The microdevices are 60.times.75.times.3 micron and contain 5
magnetic elements that are 50.times.3.times.0.4 micron. FIG. 30
shows an actual non-random array formed using the microdevices and
arraying chip shown in FIG. 29. Microdevices are only arrayed in
the locations on the array that fully match their magnetic
codes.
[0109] Magnetic Field Generator
[0110] A magnetic field generator can be electromagnetic, or it
could include permanent magnets, or a combination of the two.
Preferred embodiments include electromagnetic generators capable of
generating uniform fields over the surface of the sorting chip
[0111] The external magnetic field generators can also be
electromagnetic, or it can include permanent magnets, or a
combination of the two. The initial step in a sorting process is
the arraying of the microdevices. This process is described in a
co-pending U.S. patent application Ser. No. 12/018319, entitled
"Microdevice Arrays Formed by Magnetic Assembly," filed on even
date herewith. The suitability of any particular external field
generator is dependent upon the specific application, particularly
the coding space and the selection criterion. In a preferred
embodiment the magnetic field generator consists of a set of nested
electromagnetic coils (e.g. Helmholtz coils) that direct magnetic
fields along multiple axes (e.g. x,y,z).
[0112] In a preferred embodiment, the magnetic field generators
include individual nested sets of electromagnetic coils, similar to
Helmholtz coils but wherein the individual coils that would
comprise a Helmholtz coil can be independently regulated. In a
further preferred embodiment the coils contain magnetic cores such
as iron or ferrite. In another preferred embodiment the magnetic
field generating system contains a DC power supply capable of
producing outputs of either positive or negative polarity. In
another preferred embodiment the magnetic field generating system
contains an AC power supply or a frequency generator coupled with
an amplifier capable of driving the electromagnetic coil. In a
further preferred embodiment the magnetic field generating system
contains an AC power supply suitable for generating a demagnetizing
pulse.
[0113] In a preferred embodiment the magnetic field generator is
controllable such that sequences of magnetic field changes can be
executed in a programmed manner (for example by means of a set of
electromagnetic coils powered by digitally controllable power
supplies).
[0114] Force Generator for Removing Non-bound Microdevices
[0115] As shown in FIG. 1, magnetic discrimination can be used to
separate microdevices into a bound and a non-bound state. Non-bound
microdevices are microdevices that do not remain arrayed upon
application of a lifting force.
[0116] Because of the distance dependence of magnetic interactions
the force required to lift the microdevices is much greater than
the force required to maintain them in a lifted state.
Consequently, once non-bound microdevices have been lifted from the
surface the magnetic fields (e.g. z-axis field) holding them up can
be decreased weakening the strength of the magnetic force holding
the non-bound microdevices to the sorting chip surface. Once the
z-axis field has been decreased a larger magnetic field bias can be
introduced along any axis to draw the non-bound microdevices into a
collection area. Fluidic force is advantageous and can be used
alone or in combination with a magnetic field gradient--at low
z-axis fields (e.g. after that field has been reduced) microdevices
that are in their upright form are easily dislodged--even the
addition of a drop of alcohol to an aqueous solution on the sorting
chip surface generates sufficient turbulence to dislodge non-bound
microdevices. Laboratory micropipettors are particularly effective
at removing non-bound microdevices as demonstrated in the results
shown in FIG. 23.
[0117] Preferred embodiments include those using fluidic force
either alone or in combination with a magnetic force generator.
Additional preferred embodiments include the use of vibratory
forces, fluidic force, acoustic force, diaelectrophoretic force,
etc. as described in US Patent Application 20020137059. These
forces can be used alone or in combinations and these combinations
can include a magnetic force generator.
[0118] Synthesis
[0119] Magnetic sorting and arraying offer significant advantages
in the area of library synthesis and screening. Particle-based
libraries can be produced by synthesizing compounds directly onto
microdevices. Solid phase synthesis methods are widely used and
microdevice surface chemistry can be constructed so as to be
compatible with existing solid phase synthesis protocols.
Embodiments described herein can be used to track microdevices in a
random split and mix approach. In a preferred embodiment one can
assign specific synthesis steps to a microdevice, such that a
compound to be synthesized can be associated with a particular
magnetic code or portion of a magnetic code. In a preferred
embodiment the microdevices comprise an optical code. This code can
be the magnetic code or it can be an independent nonmagnetic layer.
A sorting process results in all microdevices being displayed in an
arrayed format. This feature in concert with an optically
detectable code can be utilized to monitor the microdevices during
a sorting process. In a preferred embodiment an optical quality
check can be performed to verify arraying accuracy. In another
preferred embodiment a synthesis process can be carried out using
optically detectable protecting groups (e.g. fluorescently labeled)
or an optically detectable test of coupling efficiency can be
carried out such that at any step in that synthesis process
coupling efficiencies can be estimated (e.g. as discussed in "The
one-bead-one-compound combinatorial method" by Lam et al. Chem.
Rev. 97:411-448 (1997)). In another preferred embodiment each
microdevice has a unique optical code, such that coupling
efficiency for compounds on each microdevice can be determined at a
step in the synthesis process. In another preferred embodiment
microdevices contain an independent magnetic group code allowing a
predetermined subset of codes to be collected.
[0120] Embodiments of the present invention provide a substantial
improvement over existing methods in the manufacture and screening
of libraries. The ability to rapidly sort and display microdevices
allows large compound specific particle libraries to be produced
and researched. A compound specific particle library is one in
which the compounds in the library are assigned to individual
particles prior to a synthetic step. Most particle-based libraries
are random involving split-and-mix type procedures (reviewed in Lam
et al. 1997). However, the specific compounds contained in
split-and-mix libraries can not be determined unless the libraries
generated are "fully combinatorial", meaning that the library
contains all possible combinations of building blocks (e.g. amino
acids, nucleotides, etc). Since such combinatorial libraries are
generally extremely large, in practice the actual composition of
the random library is not known. By arraying before and/or after
each split and mix step and keeping track of the identities of the
microdevices through identification of their coding patterns the
precise composition of the random library can be determined. In
addition such information allows the identity of the compound on
each encoded microdevice to be known facilitating the screening
process. By contrast a compound specific library allows the library
to contain any desired subset of compounds. Such compound specific
libraries are produced by a directed sorting process in which at
each step in the synthesis process particles of known identity are
directed to a particular reaction chamber. Libraries produced using
NEXUS Biosystems IRORI solid phase combinatorial chemistry
synthesis systems are a widely used commercially available example
of compound specific libraries produced by a directed sorting
process. Such commercial libraries generally contain less than
10,000 compounds.
[0121] As an example, consider a library comprising 10-residue
peptides composed of the 20 naturally occurring amino acids. There
are over 10.sup.13 different possible peptides (20.sup.10) in the
library. A random particle-based library would need to contain many
more particles than possible compounds in order to have a library
that contained all possible peptides. Alternatively, the random
library could contain only a random subset of the 10-residue
peptides. By contrast the compound specific particle library can
contain any desired predetermined subset of 10-residue peptides
since each magnetically coded particle can be assigned to a
specific peptide. For example, a 10-residue peptide library that
contains all the possible peptides in the human genome could be
used to screen for a physiologically relevant process, e.g.,
enzymatic specificity, binding of a soluble receptor, antibody
binding, kinase activity, etc. There are .about.10.sup.7 such
peptides encoded by the human genome, but a random library would
need to contain 10.sup.13 compounds to include them. Ignoring the
large volume needed to screen such a random library there would
still be considerable interference during screening from the
10.sup.13 non-physiologically relevant peptides. By contrast the
compound specific library can be constructed so as to only contain
the 10.sup.7 desired peptides.
[0122] An additional advantage of arraying the microdevices at each
step in the synthetic process is that in addition to the identity
of the microdevice a measure of the coupling efficiency of that
synthetic step on each individual microdevice can be determined
through the use of nondestructive assays (e.g. colorometric or
fluorogenic). For example in the case of peptide synthesis, there
are established assays that can be used to determine the completion
of coupling at the level of individual beads (Lam et al. 1997).
However, in a random bead library since it is not feasible using
current bead encoding technologies to routinely decode the entire
library this information is of limited utility; determining that
the efficiency of a coupling step was greater than 95% on 95% of
the beads does not determine the level or purity or the composition
of the major side products on any individual bead. Such particle
specific information is of great importance when interpreting
results obtained from researching (e.g. screening for function or
activity) the library. For example, a group of microdevices
containing very different main products could contain significant
amounts of similar side products due to incomplete reactions
occurring at various steps in the synthesis. By tracking this
information at every step in the synthesis the distribution of side
products can be recorded. The ability of the microdevice to contain
sensors or other types of MEMS devices offers additional advantages
in researching the library by allowing the microdevices to serve as
both the substrate for synthesis as well as the analysis
device.
[0123] The following example serves to illustrate the utility of
these embodiments. Synthesis of a library of 100,000 compounds is
carried out using 10 million microdevices. The microdevices contain
a chemically reactive site (e.g. a well comprising a chemically
reactive surface displaying an appropriate linker). The magnetic
coding space used for sorting the library has greater than one
million representations. The magnetic coding space is divided such
that there at least 100,000 magnetic sorting codes and 10 group
codes. A group code is simply another way of dividing a magnetic
coding space such that specific sets of microdevices can be sorted
as a group. In this particular example the group codes are assigned
uniformly to each sorting code such that for the 100 copies of each
sorting code in the library, exactly 10 contain any particular
group code. Therefore, each microdevice has a unique optical code,
a magnetic sorting code shared by 100 other microdevices, and a
group code shared by one million other microdevices. Prior to the
start of the synthesis process each compound to be synthesized is
assigned to a magnetic sorting code. This assignment will determine
the reaction chambers each microdevice will be placed into at each
step in the synthesis process. Microdevices are placed on a sorting
chip and sorted into eluted groups (e.g. see FIG. 13) and the
groups placed into the appropriate reaction vessels for the first
synthesis step. After an appropriate reaction time the microdevices
are washed and arrayed on a sorting chip and placed in an optical
reader (e.g. microscope or fluorescence scanner) and their optical
codes are determined and recorded along with the results of the
fluorogenic or colorometric assay used to monitor the reaction.
Another sorting process is carried out and the next synthesis step
is started. This process is repeated until the synthesis of the
library is complete (e.g. a library of 10-residue peptides would
require 10 such cycles). At the end of the synthesis process the
percent yield and the distribution of impurities for each optical
code has been determined. Following synthesis the microdevices are
sorted based on their group code to generate 10 groups. Each group
contains 10 copies of each type of magnetic code. This process
generates 10 copies of the library with each copy of the library
containing 10 copies of each compound wherein the purity of the
compound synthesized on each microdevice has been
characterized.
[0124] In libraries where each microdevice contains a unique
optical code, each compound can be represented by multiple optical
codes (e.g. 10). This allows errors to be identified during a
synthesis process and ignored or accounted for during the analysis
of the library. For example the optical codes on the microdevices
can be monitored at the end of every arraying step in the sorting
process, consequently if during step 8 in a 15 step process the
microdevice containing optical code 2341 was misarrayed, the
composition of the compound on that microdevice can be known and
therefore any analysis results for that microdevice will not
adversely affect the analysis of other microdevices that share the
same magnetic sorting code and were supposed to contain the same
compound.
[0125] The use of group codes provides a significant advantage over
other methods of making multiple copies of a library since it
overcomes the poisson distribution problem that occurs when
randomly dividing large collections of objects into groups.
[0126] In addition to enabling compound specific library synthesis,
embodiments of the current invention also provide a substantial
improvement in the manufacture and screening of random libraries.
All of the advantages that apply to compound-specific libraries
that arise from the ability to display particles in an oriented
form also apply to random libraries.
[0127] The ability to synthesize a library of known predetermined
composition and large size allows for sequential library synthesis
to be carried out to rapidly identify a compound of interest. For
example, after screening a first library a new second library can
be designed based on the results obtained by studying the first
library. The process can be continued until the desired screening
result is obtained and a synthetic compound with the desired
properties is identified. Such synthetic compounds include
inhibitory molecules, drugs, binding molecules, catalysts, etc. Any
compound that can be synthesized on a solid support falls within
the scope of this invention.
[0128] Target Isolation
[0129] Embodiments described herein can be used to isolate any item
of interest that can be attached to a microdevice and distinguished
by some optical procedure (either directly or indirectly). A
preferred example is rare cell isolation. A mixture of cells can be
attached to microdevices such that a plurality of microdevices
contains a single cell. After arraying microdevices containing the
cells of interest can be identified by optical methods (e.g.
fluorescence) and those microdevices isolated. Cells can be
targeted with an optical marker at any step of this process prior
to the optical detection step. Isolated cells can then be used for
further analysis (e.g. gene expression, SNP analysis, proteomic
profiling, etc).
[0130] It should be apparent to those skilled in the art that many
more modifications besides those already described are possible
without departing from the inventive concepts herein. Moreover, in
interpreting the disclosure, all terms should be interpreted in the
broadest possible manner consistent with the context. In
particular, the terms "comprises" and "comprising" should be
interpreted as referring to elements, components, or steps in a
non-exclusive manner, indicating that the referenced elements,
components, or steps could be present, or utilized, or combined
with other elements, components, or steps that are not expressly
referenced. Where the specification claims refers to at least one
of something selected from the group consisting of A, B, C . . .
and N, the text should be interpreted as requiring only one element
from the group, not A plus N, or B plus N, etc.
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51, 54, 56 ##STR00058## 1, 4-7, 9, 10, 13,18, 19, 23, 28, 29,36-38,
40-45, 47,48, 50, 52, 53, 55 30 ##STR00059## 2, 4, 9, 11,
13-16,19-21, 23-26, 28,30-35, 40, 47, 49,52, 54, 56 ##STR00060## 1,
3, 5-8, 10, 12,17, 18, 22, 27, 29,36-39, 41-46, 48,50, 51, 53, 55
31 ##STR00061## 3, 4, 10, 12-16, 18,20, 22-26, 29-35, 41,48, 49,
53, 54, 56 ##STR00062## 1, 2, 5-9, 11, 17, 19,21, 27, 28,
36-40,42-47, 50-52, 55 32 ##STR00063## 5, 6, 8, 11, 12, 14,17-22,
24, 27-35,42, 46, 50, 51, 55,56 ##STR00064## 1-4, 7, 9, 10, 13,15,
16, 23, 25, 26,36-41, 43-45, 47-49,52-54 33 ##STR00065## 5, 7, 9,
11, 13, 15,17-21, 23, 25, 27-35,43, 47, 50, 52,55, 56 ##STR00066##
1-4, 6, 8, 10, 12,14, 16, 22, 24, 26,36-42, 44-46, 48,49, 51, 53,
54 34 ##STR00067## 6, 7, 10, 12, 13,16-20, 22, 23, 26-35,44, 48,
50, 53, 55, 56 ##STR00068## 1-5, 8, 9, 11, 14, 15,21, 24, 25,
36-43,45-47, 49, 51, 52, 54 35 ##STR00069## 8-10, 14-20, 24-35,45,
49, 50, 54-56 ##STR00070## 1-7, 11-13, 21-23,36-44, 46-48,
51-53
* * * * *