U.S. patent application number 10/959639 was filed with the patent office on 2005-05-26 for array fabrication.
Invention is credited to Webb, Peter G..
Application Number | 20050112029 10/959639 |
Document ID | / |
Family ID | 24519009 |
Filed Date | 2005-05-26 |
United States Patent
Application |
20050112029 |
Kind Code |
A1 |
Webb, Peter G. |
May 26, 2005 |
Array fabrication
Abstract
Methods, apparatus, and computer program, for use in fabricating
a chemical array. The method may use an apparatus which includes a
head system, a transport system, and a processor. The head system
has multiple groups of drop dispensers which move in unison, each
group having multiple dispensers. The transport system can move the
head system with respect to a substrate with different dispensers
of the groups following respective paths. The processor can
dispense droplets from dispensers during operation of the transport
system, in a pattern along a selected path for each group so as to
form the array. The method includes identifying an error in one or
more dispensers. When a dispenser of a first group is in error,
then a second dispenser of each group is moved along the selected
path for its group while dispensing droplets from at least the
second dispenser of the first group in at least part of the pattern
for the selected path of the first group.
Inventors: |
Webb, Peter G.; (Menlo Park,
CA) |
Correspondence
Address: |
AGILENT TECHNOLOGIES, INC.
Legal Department, DL429
Intellectual Proerty Administration
P. O. Box 7599
Loveland
CO
80537-0599
US
|
Family ID: |
24519009 |
Appl. No.: |
10/959639 |
Filed: |
October 5, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10959639 |
Oct 5, 2004 |
|
|
|
09628470 |
Jul 31, 2000 |
|
|
|
Current U.S.
Class: |
422/400 |
Current CPC
Class: |
B01J 2219/00596
20130101; B01J 2219/00659 20130101; Y10T 436/2575 20150115; B01J
2219/00725 20130101; C40B 40/10 20130101; B01J 2219/00475 20130101;
B01J 2219/00722 20130101; B01J 2219/00378 20130101; B01J 2219/00605
20130101; B01J 2219/00585 20130101; B01J 2219/00693 20130101; B01J
2219/00626 20130101; C40B 40/06 20130101; B01J 2219/00637 20130101;
B01J 2219/00695 20130101; B01J 2219/0061 20130101; B01J 2219/00527
20130101; C40B 60/14 20130101; B01J 2219/0059 20130101; B01J
2219/00689 20130101; B01J 19/0046 20130101 |
Class at
Publication: |
422/100 |
International
Class: |
B01L 003/00 |
Claims
1-13. (canceled)
14. An apparatus for fabricating a chemical array, comprising: (a)
a head system with multiple groups of drop dispensers which move in
unison, each group having multiple drop dispensers; (b) a transport
system to move the head system with respect to a substrate with
different dispensers of the groups following respective paths; (c)
a processor which: dispenses droplets from dispensers during
operation of the transport system, in a pattern along a selected
path for each group so as to form the array; and when an error
indication is identified by the processor indicating an error in a
first dispenser of a first group, then moves a second dispenser of
each group along the selected path for its group while dispensing
droplets from at least the second dispenser of the first group in
at least part of the pattern for the selected path of the first
group.
15. An apparatus according to claim 14 wherein the dispensers are
pulse jets.
16. An apparatus according to claim 14 wherein the processor, when
the error indication is identified, dispenses droplets from second
dispensers of multiple groups in at least part of the pattern for
the selected path of the same group.
17. An apparatus according to claim 16 wherein: a series of
dispensers within a group communicate with a corresponding common
reservoir for that series; and when the error indication is
identified, the processor dispenses droplets from a non-error
dispenser of the same series as the error dispenser.
18. An apparatus according to claim 16 wherein, when the error
indication is identified, the processor dispenses droplets from at
least one second dispenser of a group in the complete pattern for
the selected path of the same group.
19. An apparatus according to claim 16 wherein, when the error
indication is identified, the processor dispenses droplets from at
least the second dispenser of the first group in the complete
pattern for the selected path of the first group.
20. A method according to claim 16 wherein when another error
indication is identified by the processor additionally indicating
an error in a dispenser of a second dispenser of a second group,
the processor alternately moves the first and second dispensers of
each group along the selected path for that group while dispensing
droplets from non-error dispensers of the first and second groups
in different parts of the pattern for the selected path for the
first group.
21. An apparatus for fabricating a chemical array, comprising: (a)
a head system with multiple groups of drop dispensers which move in
unison, each group having multiple rows and columns of dispensers;
(b) a transport system to move the head system with respect to a
substrate with different rows following respective paths; (c) a
processor which: dispenses droplets from dispensers during
operation of the transport system, in a pattern along a selected
path for each group so as to form the array; when an error
indication is identified by the processor indicating an error in
dispensers of different columns within first and second rows of a
first group, then: (i) positions the head with a first row of each
group aligned with the selected path for that group; (ii) moves the
head with respect to the substrate while dispensing droplets from
non-error dispensers in the first row of the first group in
accordance with a part of the pattern for the selected path for
that group; (iii) re-positions the head such that a second row of
each group is aligned with the selected paths; and (iv) moves the
head with respect to the substrate while dispensing droplets from
non-error dispensers in the second row of the first group in
accordance with a part of the pattern for that group.
22. An apparatus according to claim 21 wherein the dispensers are
pulse jets.
23. An apparatus according to claim 21 wherein in step (iv)
droplets are dispensed from non-error dispensers in the second row
of each of multiple groups in accordance with at least part of the
pattern for that group.
24. An apparatus according to claim 21 wherein: a series of
dispensers in each of multiple columns of each of multiple groups,
communicates with a corresponding common reservoir for that series;
and in step (iv) droplets are dispensed from a non-error dispenser
of the same series as the error dispenser.
25. An apparatus according to claim 21 wherein in step (iv)
droplets are dispensed from at least one second row of a group in
the complete pattern for the selected path of the same group.
26. An apparatus according to claim 21 additionally comprising a
sensor to monitor dispensers for an error and provide corresponding
data to the processor.
27. A computer program product for use with an apparatus for
fabricating a chemical array having: a head system with multiple
groups of drop dispensers which move in unison, each group having
multiple dispensers; a transport system to move the head system
with respect to a substrate with different dispensers of the groups
following respective paths; and a processor; the computer program
product comprising a computer readable storage medium having a
computer program stored thereon which, when loaded into the
processor, performs the steps of: a) identifying for an error in
one or more dispensers; b) when a dispenser of a first group is in
error, then moving a second dispenser of each group along a
selected path for its group while dispensing droplets from at least
the second dispenser of the first group in at least part of the
pattern for the selected path of the first group.
28. A computer program product according to claim 27 wherein in
step (b) droplets are dispensed from each second dispenser of
multiple groups in at least part of the pattern for the selected
path of the same group.
29. A computer program product according to claim 27 wherein when a
dispenser of a second set of the first group is additionally
identified as being in error, the program causes the first and
second dispensers of each group to be alternately moved along the
selected path for that group while droplets are dispensed from
non-error dispensers of the first and second groups in different
parts of the pattern for the selected path for the first group.
Description
[0001] This application claims priority to copending application
Ser. No. 09/628,470, filed Jul. 31, 2000, under 35 U.S.C. 120, the
entirety of which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to arrays, particularly
polynucleotide arrays such as DNA arrays, which are useful in
diagnostic, screening, gene expression analysis, and other
applications.
BACKGROUND OF THE INVENTION
[0003] Polynucleotide arrays (such as DNA or RNA arrays), are known
and are used, for example, as diagnostic or screening tools. Such
arrays include regions of usually different sequence
polynucleotides arranged in a predetermined configuration on a
substrate. These regions (sometimes referenced as "features") are
positioned at respective locations ("addresses") on the substrate.
The arrays, when exposed to a sample, will exhibit an observed
binding pattern. This binding pattern can be detected upon
interrogating the array. For example all polynucleotide targets
(for example, DNA) in the sample can be labeled with a suitable
label (such as a fluorescent compound), and the fluorescence
pattern on the array accurately observed following exposure to the
sample. Assuming that the different sequence polynucleotides were
correctly deposited in accordance with the predetermined
configuration, then the observed binding pattern will be indicative
of the presence and/or concentration of one or more polynucleotide
components of the sample.
[0004] Biopolymer arrays can be fabricated by depositing previously
obtained biopolymers (such as from synthesis or natural sources)
onto a substrate, or by in situ synthesis methods. Methods of
depositing obtained biopolymers include dispensing droplets to a
substrate from dispensers such as pin or capillaries (such as
described in U.S. Pat. No. 5,807,522) or such as pulse jets (such
as a piezoelectric inkjet head, as described in PCT publications WO
95/25116 and WO 98/41531, and elsewhere). For in situ fabrication
methods, multiple different reagent droplets are deposited from
drop dispensers at a given target location in order to form the
final feature (hence a probe of the feature is synthesized on the
array stubstrate). The in situ fabrication methods include those
described in U.S. Pat. No. 5,449,754 for synthesizing peptide
arrays, and described in WO 98/41531 and the references cited
therein for polynucleotides. The in situ method for fabricating a
polynucleotide array typically follows, at each of the multiple
different addresses at which features are to be formed, the same
conventional iterative sequence used in forming polynucleotides
from nucleoside reagents on a support by means of known chemistry.
This iterative sequence is as follows: (a) coupling a selected
nucleoside through a phosphite linkage to a functionalized support
in the first iteration, or a nucleoside bound to the substrate
(i.e. the nucleoside-modified substrate) in subsequent iterations;
(b) optionally, but preferably, blocking unreacted hydroxyl groups
on the substrate bound nucleoside; (c) oxidizing the phosphite
linkage of step (a) to form a phosphate linkage; and (d) removing
the protecting group ("deprotection") from the now substrate bound
nucleoside coupled in step (a), to generate a reactive site for the
next cycle of these steps. The functionalized support (in the first
cycle) or deprotected coupled nucleoside (in subsequent cycles)
provides a substrate bound moiety with a linking group for forming
the phosphite linkage with a next nucleoside to be coupled in step
(a). Final deprotection of nucleoside bases can be accomplished
using alkaline conditions such as ammonium hydroxide, in a known
manner.
[0005] The foregoing chemistry of the synthesis of polynucleotides
is described in detail, for example, in Caruthers, Science 230:
281-285, 1985; Itakura et al., Ann. Rev. Biochem. 53: 323-356;
Hunkapillar et al., Nature 310: 105-110, 1984; and in "Synthesis of
Oligonucleotide Derivatives in Design and Targeted Reaction of
Oligonucleotide Derivatives", CRC Press, Boca Raton, Fla., pages
100 et seq., U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S.
Pat. No. 5,153,319, U.S. Pat. No. 5,869,643, EP 0294196, and
elsewhere
[0006] In array fabrication, the quantities of polynucleotide
available, whether by deposition of previously obtained
polynucleotides or by in situ synthesis, are usually very small and
expensive. Additionally, sample quantities available for testing
are usually also very small and it is therefore desirable to
simultaneously test the same sample against a large number of
different probes on an array. These conditions require use of
arrays with large numbers of very small, closely spaced features.
It is important in such arrays that features actually be present,
that they are put down accurately in the desired target pattern,
are of the correct size, and that the DNA is uniformly coated
within the feature. Failure to meet such quality requirements can
have serious consequences to diagnostic, screening, gene expression
analysis or other purposes for which the array is being used.
However, for mass production of arrays with many features, a
dispensing system is required which typically has many drop
dispensers.
[0007] The present invention realizes that one or more of such drop
dispensers may suffer from errors, such as failure to dispense a
drop at all or failure to dispense in the correct location. To
reduce errors in arrays formed with such an apparatus non-error
dispensers can be used to dispense droplets which would otherwise
have been dispensed by an error dispenser. However, this can
involve multiple additional movements of the dispensers relative to
the substrate which requires further time (and hence can decrease
array quantity produced over a given time) and can lead to further
errors.
[0008] It would be desirable then, to provide a means by which
drops to be dispensed by dispensers in error, can instead be
dispensed by non-error dispensers while maintaining a relatively
simple pattern of movement of the drop dispensers relative to a
substrate.
SUMMARY OF THE INVENTION
[0009] The present invention provides a method for fabricating a
chemical array using an apparatus having a head system with
multiple groups of drop dispensers which move in unison. The groups
may, for example, include respective columns and rows of
dispensers. The apparatus also has a transport system to move the
deposition system with respect to a substrate with different
dispensers of the groups following respective paths. A processor
controls the dispensers so as to cause them to dispense droplets
during operation of the transport system, in a pattern along a
selected path for each group so as to form the array. The method
may include identifying an error in one or more dispensers. Such
identification may, for example, be based on data specifically
identifying an error dispenser (such as operator input data) or
upon data received from a sensor which monitors dispensers for an
error and provides corresponding data to the processor (in which
case the processor can identify an error from the received data).
When a first dispenser of a first group is in error, then a second
dispenser of each group is moved along the selected path for its
group while dispensing droplets from at least the second dispenser
of the first group in at least part of the pattern for the selected
path of the first group. This step may involve re-positioning the
head system from a position in the which the first dispensers will
follow the selected path for each group upon operation of the
transport system, to a position in which second dispenser (and
optionally further dispensers) follow the selected path for each
group.
[0010] Droplets may be dispensed from each second dispenser of
multiple groups in at least part of the pattern for the selected
path of the same group. Alternatively, droplets may be dispensed
from at least one second dispenser of a group (such as the first
group, or for each of the groups) in the complete pattern for the
selected path of the same group. In a preferred arrangement, a
series of dispensers within each of different groups, may be loaded
with a same fluid for dispensing. For example, members of a given
series may communicate with a corresponding common reservoir for
that series. In this case, droplets may be dispensed from a
non-error dispenser of the same series as the error dispenser. For
example, in the case where the dispensers of the group are arranged
in rows and columns, there may be multiple series in each group
with each series comprising a column of dispensers within the
corresponding group.
[0011] In a particularly preferred operation of a method of the
present invention, when a second dispenser of a second group is
additionally in error, the first and second dispensers of each
group are alternately moved along the selected path for that group
while droplets are dispensed from non-error dispensers of the first
and second groups in at least part of the pattern for the selected
path for the respective groups. This same procedure can be extended
to the general case where multiple identified dispensers of
different order (including first or second) in respective groups
are in error. That is, the corresponding non-error dispensers of
each group are alternately moved along the selected path for that
group while droplets are dispensed from the non-error dispensers
each at least in part of the pattern for the selected path for the
corresponding group. In any event, typically the pattern for the
selected path of each group would be completed.
[0012] The present invention further provides an apparatus for
fabricating a chemical array. The apparatus includes a deposition
system and transport system, of any of the constructions as
described above. The apparatus may further include a processor
which co-ordinates dispensing of droplets and movement of the
deposition system, in accordance with one or more methods of the
present invention.
[0013] The present invention further provides a computer program
product for use with an apparatus as described above, and which
provides the instructions to the processor such that it can cause
the deposition system and transport system to execute one or more
methods of the present invention. The program product includes a
computer readable storage medium having a computer program stored
thereon which, when loaded into a computer (which is a
"processor"), causes it to perform the steps required of it in such
that the apparatus can perform a method of the present invention.
Optionally, the present invention may further provide for exposing
the array to a sample, and interrogating the array following the
exposure and optionally processing results of the interrogation.
Such an interrogation or processing result may be forwarded to a
remote location.
[0014] The various aspects of the present invention can provide any
one or more of the following and/or other useful benefits. For
example, drops can be dispensed by non-error dispensers while
maintaining a relatively simple pattern of movement of the drop
dispenser system relative to a substrate. Further, re-loading of
the head system with fluids to be dispensed, is not required by
methods of the present invention in order to have a non-error
dispenser dispense drops in place of an error dispenser.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Embodiments of the invention will now be described with
reference to the drawings, in which:
[0016] FIG. 1 illustrates a substrate carrying multiple arrays,
such as may be fabricated by methods of the present invention;
[0017] FIG. 2 is an enlarged view of a portion of FIG. 1 showing
ideal spots or features;
[0018] FIG. 3 is an enlarged illustration of a portion of the
substrate in FIG. 2;
[0019] FIG. 4 schematically illustrates one embodiment of a method
of the present invention;
[0020] FIG. 5 schematically illustrates another embodiment of a
method of the present invention; and
[0021] FIG. 6 is a schematic diagram of an apparatus at a user site
which can execute a method of the present invention.
[0022] To facilitate understanding, identical reference numerals
have been used, where practical, to designate identical elements
that are common to the figures.
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
[0023] In the present application, unless a contrary intention
appears, the following terms refer to the indicated
characteristics. A "biopolymer" is a polymer of one or more types
of repeating units. Biopolymers are typically found in biological
systems (although they may be made synthetically) and particularly
include peptides or polynucleotides, as well as such compounds
composed of or containing amino acid analogs or non-amino acid
groups, or nucleotide analogs or non-nucleotide groups. This
includes polynucleotides in which the conventional backbone has
been replaced with a non-naturally occurring or synthetic backbone,
and nucleic acids (or synthetic or naturally occurring analogs) in
which one or more of the conventional bases has been replaced with
a group (natural or synthetic) capable of participating in
Watson-Crick type hydrogen bonding interactions. Polynucleotides
include single or multiple stranded configurations, where one or
more of the strands may or may not be completely aligned with
another. A "nucleotide" refers to a sub-unit of a nucleic acid and
has a phosphate group, a 5 carbon sugar and a nitrogen containing
base, as well as functional analogs (whether synthetic or naturally
occurring) of such sub-units which in the polymer form (as a
polynucleotide) can hybridize with naturally occurring
polynucleotides in a sequence specific manner analogous to that of
two naturally occurring polynucleotides. For example, a
"biopolymer" includes DNA (including cDNA), RNA, oligonucleotides,
and PNA and other polynucleotides as described in U.S. Pat. No.
5,948,902 and references cited therein (all of which are
incorporated herein by reference), regardless of the source. An
"oligonucleotide" generally refers to a nucleotide multimer of
about 10 to 100 nucleotides in length, while a "polynucleotide"
includes a nucleotide multimer having any number of nucleotides. A
"biomonomer" references a single unit, which can be linked with the
same or other biomonomers to form a biopolymer (for example, a
single amino acid or nucleotide with two linking groups one or both
of which may have removable protecting groups). A "peptide" is used
to refer to an amino acid multimer of any length (for example, more
than 10, 10 to 100, or more amino acid units). A biomonomer fluid
or biopolymer fluid reference a liquid containing either a
biomonomer or biopolymer, respectively (typically in solution).
[0024] A "set" or "sub-set" of any item (such as a set of nozzles)
may contain only one of the item, or only two, or three, or any
number of multiple items. An "array", unless a contrary intention
appears, includes any one, two or three dimensional arrangement of
addressable regions bearing a particular chemical moiety to
moieties (for example, biopolymers such as polynucleotide
sequences) associated with that region. An array is "addressable"
in that it has multiple regions of different moieties (for example,
different polynucleotide sequences) such that a region (a "feature"
or "spot" of the array) at a particular predetermined location (an
"address") on the array will detect a particular target or class of
targets (although a feature may incidentally detect non-targets of
that feature). Array features are typically, but need not be,
separated by intervening spaces. In the case of an array, the
"target" will be referenced as a moiety in a mobile phase
(typically fluid), to be detected by probes ("target probes") which
are bound to the substrate at the various regions. However, either
of the "target" or "target probes" may be the one which is to be
evaluated by the other (thus, either one could be an unknown
mixture of polynucleotides to be evaluated by binding with the
other). An "array layout" refers collectively to one or more
characteristics of the features, such as feature positioning, one
or more feature dimensions, and some indication of a moiety at a
given location. "Hybridizing" and "binding", with respect to
polynucleotides, are used interchangeably.
[0025] When one item is indicated as being "remote" from another,
this is referenced that the two items are at least in different
buildings, and may be at least one mile, ten miles, or at least one
hundred miles apart. "Communicating" information references
transmitting the data representing that information as electrical
signals over a suitable communication channel (for example, a
private or public network). "Forwarding" an item refers to any
means of getting that item from one location to the next, whether
by physically transporting that item or otherwise (where that is
possible) and includes, at least in the case of data, physically
transporting a medium carrying the data or communicating the
data.
[0026] It will also be appreciated that throughout the present
application, that words such as "top", "upper", and "lower" are
used in a relative sense only. "Fluid" is used herein to reference
a liquid. Reference to a singular item, includes the possibility
that there are plural of the same items present. Furthermore, when
one thing is "moved", "moving", "re-positioned ", "scanned", or the
like, with respect to another, this implies relative motion only
such that either thing or both might actually be moved in relation
to the other. For example, when dispensers are "moved" relative to
a substrate, either one of the dispensers or substrate may actually
be put into motion by the transport system while the other is held
still, or both may be put into motion. All patents and other cited
references are incorporated into this application by reference.
[0027] Referring first to FIGS. 1-3, typically methods and
apparatus of the present invention generate or use a contiguous
planar substrate 10 carrying one or more arrays 12 disposed across
a front surface 11a of substrate 10 and separated by inter-array
areas 13. A back side 11b of substrate 10 does not carry any arrays
12. The arrays on substrate 10 can be designed for testing against
any type of sample, whether a trial sample, reference sample, a
combination of them, or a known mixture of polynucleotides (in
which latter case the arrays may be composed of features carrying
unknown sequences to be evaluated). While ten arrays 12 are shown
in FIG. 5 and the different embodiments described below may use
substrates with particular numbers of arrays, it will be understood
that substrate 10 and the embodiments to be used with it, may use
any number of desired arrays 12. Similarly, substrate 10 may be of
any shape, and any apparatus used with it adapted accordingly.
Depending upon intended use, any or all of arrays 12 may be the
same or different from one another and each will contain multiple
spots or features 16 of biopolymers in the form of polynucleotides.
A typical array may contain from more than ten, more than one
hundred, more than one thousand or ten thousand features, or even
more than from one hundred thousand features. All of the features
16 may be different, or some or all could be the same. In the case
where arrays 12 are formed by the conventional in situ or
deposition of previously obtained moieties, as described above, by
depositing for each feature a droplet of reagent in each cycle such
as by using a pulse jet such as an inkjet type head, interfeature
areas 17 will typically be present which do not carry any
polynucleotide. It will be appreciated though, that the
interfeature areas 17 could be of various sizes and configurations.
It will also be appreciated that there need not be any space
separating arrays 12 from one another. Each feature carries a
predetermined polynucleotide (which includes the possibility of
mixtures of polynucleotides). As per usual, A, C, G, T represent
the usual nucleotides. It will be understood that there may be a
linker molecule (not shown) of any known types between the front
surface 11a and the first nucleotide.
[0028] For the purposes of the discussions below, it will be
assumed (unless the contrary is indicated) that the array being
formed in any case is a polynucleotide array formed by the
deposition of previously obtained polynucleotides using pulse jet
deposition units. However, the applicability of the method to
arrays of other polymers or chemical moieties generally, whether
formed by multiple cycle in situ methods or deposition of
previously obtained moieties, or using other types of dispensers,
will be understood from these discussions.
[0029] Referring to FIG. 4, operation of a method of the present
invention is illustrated. FIG. 4 is a view from above looking down
(using the orientation of FIG. 6) toward a head system 210 and
substrate 10 (not shown in FIG. 4 for clarity) onto which an array
is to be fabricated. Head system 210 has two heads 210a and 210b.
In FIG. 4 each head 210a and 210b is illustrated with fifteen
parallel rows and two columns (all parallel) of dispensers.
However, as described below in connection with FIG. 6, each head
may in practice have many more rows and columns although the number
of rows and columns have been kept low in FIGS. 4 and 5 for the
purposes of clarity. Each dispenser is illustrated by its drop
dispensing outlet (the drop outlet orifice, for example, in a
corresponding pulse jet) represented by a hollow circle in FIG. 4.
Deposited droplets are represented by solid black circles. Since,
as described below in connection with FIG. 6, heads 210a and 210b
are both mounted to the same head retainer 208, all drop dispensers
will be moved in unison by the transport system (see FIG. 6). These
drop dispensers are identified as groups A, B, C, D, and E in FIG.
4, each group having respective three rows of dispensers x, y, and
z, in four columns 1, 2, 3, and 4. It will be understood though,
that it is possible that each group may have only one column of
dispensers (that is, each group may have only three dispensers).
The center to center spacing of rows of dispensers within a group,
is equal for all groups. In the discussion of FIGS. 4 and 5, any
particular drop dispenser will be referenced by its group number,
followed by row and column number. For example, drop dispenser
A.sub.y1 refers to the dispenser in group A, row y, column 1, and
drop dispenser B.sub.y2 refers to the drop dispenser in group B,
row y, column 2. Each group A, B, C, D, E has four series of
dispensers, each series being the three dispensers in a column.
Dispensers of each series communicate with a common reservoir for
that series and thus in operation dispensers of a same series are
loaded with the same fluid, as described below.
[0030] In the following discussion row y in each group will be
regarded as a first row, with row x being a second row in each
group (and row z being a third row). However, designation of rows
as "first", "second", "third", and the like is merely an arbitrary
naming for identification purposes only and does not imply that the
rows are in the physical sequence of first, second and third one
after the other. Nor does such naming imply that during operation
of the method the "first" row should dispense drops first, followed
by the "second" row. Instead, the order of dispensing from the rows
may be in any convenient order with, for example, the "second" row
dispensing drops before the "first" row. However the identification
of rows implies that when a given named row (or dispenser within a
row) of one group is aligned for movement along a selected path
A.sub.s through E.sub.s for that group, the same named row (or
dispenser) of the other groups are simultaneously aligned for
movement along the respective selected paths for their groups. For
example, when the second row of group A is moved along the selected
path A.sub.s for group A, the second row of groups B, C, D, and E
will be simultaneously moved along the selected paths of their
respective groups. Such similarly named rows (or dispensers) are
therefore corresponding rows (or dispensers) of different groups.
Dispensing of all droplets in the required pattern along all
selected paths will result in at least a portion of the target
array. Similarly, reference to a group as the "first", "second", or
the like, is an arbitrary designation only, and does not imply that
the groups are in any sequence spatially with respect to one
another.
[0031] In FIG. 4 when the transport system moves head system 210 in
a straight line in the direction of any of parallel paths A.sub.s
through E.sub.s, the different rows of dispensers will follow
respective parallel paths across the substrate which extend in the
direction. However, during operation of the transport system
processor 140 (FIG. 6) only dispenses droplets from dispensers (by
controlling dispenser operation) in a pattern along the selected
path A.sub.s through E.sub.s for each group. The selected path is
the paths which will form the array when drops are dispensed
therealong in the required pattern. In FIG. 4A head system 210 has
initially been positioned so that the first rows A.sub.y, B.sub.y,
C.sub.y, D.sub.y, E.sub.y, are aligned with will respective
selected paths A.sub.s through E.sub.s (that is, they will be moved
along paths A.sub.s through E.sub.s upon activation of the
transport system). However, processor 140 has identified an error
in a first row y of the first group A while no errors have been
identified in any of the dispensers of the secondrows. Specifically
dispenser A.sub.y1 does not dispense a droplet when required to do
so. With the position of head system 210 shown in FIG. 4A, if two
repetitions of dispensing from head system 210 were to be performed
during operation of the transport system, the result would be an
array shown in FIG. 4A with two sets of deposited drops (each
having columns 1-4) and in which no drops were deposited at the two
positions A1.
[0032] To avoid the foregoing problem, prior to dispensing drops,
processor 140 positions head system 210 as shown in FIG. 4B, with t
the second rows y aligned with respective selected paths A.sub.s
through E.sub.s. Processor 140 then activates the transport system
such that the second row x of each group is moved along the
selected path for its group, while dispensing droplets from each
second row x of the groups A, B, C, D, and E in all of the pattern
for the selected path of the first group. As in FIG. 4A two
repetitions of dispensing from head system 210 may be performed to
obtain the complete array as illustrate in FIG. 4B. In FIG. 4B
then, droplets are dispensed from each of the second rows x of each
of the groups, in the complete pattern for the selected path of the
corresponding group. By processor 140 moving the second rows x
along the selected paths A.sub.s through E.sub.s, after identifying
the error in dispenser A.sub.y1, rather than using the first rows
y, errors in the array are avoided which might require further
passes of head 210 over the same region of the substrate to
correct. Further, since dispensers of a same series in each group
are loaded with the same fluid, re-loading of the head system is
not required to compensate for dispenser errors.
[0033] The method illustrated in FIG. 4 represents the simple case
where there are no error dispensers in all of the second rows
A.sub.x, B.sub.x, C.sub.x, D.sub.x, and E.sub.x. However, in
practice when it is desired to use a head system with a much larger
number of rows, it is less likely that all dispensers in
corresponding row will not have any errors. Operation of a method
of the present invention in such a situation is illustrated in FIG.
5 using the same head system in FIG. 4. For simplicity, no
deposition repetitions are illustrated in FIG. 5. In FIG. 5 the
following dispensers in the first rows y have been identified as
being in error: A.sub.y1, B.sub.y3, C.sub.y2, D.sub.y1, E.sub.y1,
and E.sub.y3. Additionally, in FIG. 5 at least one or more
dispensers in at least one or more of the second rows y has been
found to additionally be in error so that it is not possible to use
the method described in connection with FIG. 4. In this situation,
as illustrated in FIG. 5, the first row y and second row x of each
group A, B, C, D, E are alternately moved along the selected path
for that group while droplets are dispensed from non-error
dispensers of the first and second rows of each group in different
parts of the pattern for the selected path for the respective
groups. In particular, in FIG. 5A the first rows A.sub.y, B.sub.y,
C.sub.y, D.sub.y, E.sub.y are aligned with the selected paths
A.sub.s through E.sub.s and moved along those paths with respect to
the substrate while dispensing drops from non-error dispensers in
the first rows of the groups in accordance with a part of the
pattern for those groups, as illustrated in FIG. 5A. Head system
210 can then be re-positioned such that the second rows A.sub.x,
B.sub.x, C.sub.x, D.sub.x, E.sub.x are aligned with selected paths
A.sub.s through E.sub.s. The second rows A.sub.x, B.sub.x, C.sub.x,
D.sub.x, E.sub.x are then moved along selected paths A.sub.s
through E.sub.s with respect to the substrate while dispensing
drops from non-error dispensers in the second rows of the groups in
accordance with a part of the pattern for those groups, as
illustrated in FIG. 5B.
[0034] While in FIG. 5 only first and second rows of the groups are
used to complete the pattern for those groups, it will be
appreciated that this concept can be extended to use other
additional rows where error dispensers are identified such that the
first and second rows cannot complete the pattern for all
groups.
[0035] Note that in the methods of FIGS. 4 and 5, the columns of
deposited droplets 1-4 are spaced closer together than the columns
1-4 of respective dispensers (the dispensed drop columns are
"compressed" relative to the respective dispensers). This decrease
in deposited drop spacing in a direction of travel of the head
system, is readily obtained with pulse jet dispensers by processor
140 correctly timing dispenser actuation as head system 210 moves
over the substrate. Such compression allows for arrays with
deposited drop spacing as measured in the direction of head travel,
to be independent of the spacing of the respective dispensers which
deposited them.
[0036] Referring to FIG. 6 an apparatus of the present invention
includes a substrate station 20 on which can be mounted a substrate
10. Pins or similar means (not shown) can be provided on substrate
station 20 by which to approximately align substrate 10 to a
nominal position thereon. Substrate station 20 can include a vacuum
chuck connected to a suitable vacuum source (not shown) to retain a
substrate 10 without exerting too much pressure thereon, since
substrate 10 is often made of glass.
[0037] A dispensing head system 210 is retained by a head retainer
208. Head system 210 can be positioned at any position facing
substrate 10 by means of a transport system. The transport system
includes a carriage 62 connected to a first transporter 60
controlled by processor 140 through line 66, and a second
transporter 100 controlled by processor 140 through line 106.
Transporter 60 and carriage 62 are used execute one axis
positioning of station 20 (and hence mounted substrate 10) facing
the dispensing head system 210, by moving it in the direction of
nominal axis 63, while transporter 100 is used to provide
adjustment of the position of head retainer 208 in a direction of
nominal axis 204 (and hence move the rows of dispensers as
described in connection with FIGS. 4 and 5). In this manner, head
system 210 can be scanned line by line, by scanning along a line
over substrate 10 in the direction of axis 204 using transporter
100, while line by line movement of substrate 10 in a direction of
axis 63 is provided by transporter 60. Head system 210 may also
optionally be moved in a vertical direction 202, by another
suitable transporter (not shown). However, it will be appreciated
that other scanning configurations could be used. However, it will
be appreciated that both transporters 60 and 100, or either one of
them, with suitable construction, could be used to perform the
foregoing scanning of head system 210 with respect to substrate 10.
Thus, when the present application refers to "positioning" one
element (such as head system 210) in relation to another element
(such as one of the stations 20 or substrate 10) it will be
understood that any required moving can be accomplished by moving
either element or a combination of both of them. An encoder 30
communicates with processor 140 to provide data on the exact
location of substrate station 20 (and hence substrate 10 if
positioned correctly on substrate station 20), while encoder 34
provides data on the exact location of holder 208 (and hence head
system 210 if positioned correctly on holder 208). Any suitable
encoder, such as an optical encoder, may be used which provides
data on linear position. Angular positioning of substrate station
20 is provided by a transporter 120, which can rotate substrate
station 20 about axis 202 under control of processor 140.
Typically, substrate station 20 (and hence a mounted substrate) is
rotated by transporter 120 under control of processor 140 in
response to an observed angular position of substrate 10 as
determined by processor 140 through viewing one or more fiducial
marks on substrate 10 (particularly fiducial marks 18) with a
camera (not shown). This rotation will continue until substrate 10
has reached a predetermined angular relationship with respect to
dispensing head system 210. In the case of a square or rectangular
substrate, the mounted substrate 10 will typically be rotated to
align one edge (length or width) with the scan direction of head
system 210 along axis 204.
[0038] Head system 210 may contain one or more (for example, two)
heads mounted on the same head retainer 208. Each such head may be
of a type commonly used in an ink jet type of printer and may, for
example, have one hundred fifty drop dispensing orifices in each of
two parallel rows, six chambers for holding polynucleotide solution
communicating with the three hundred orifices, and three hundred
ejectors which are positioned in the chambers opposite a
corresponding orifice. Each ejector is in the form of an electrical
resistor operating as a heating element under control of processor
140 (although piezoelectric elements could be used instead). Each
orifice with its associated ejector and portion of the chamber,
defines a corresponding pulse jet with the orifice acting as a
nozzle. Thus, there are three hundred pulse jets in this
configuration, although it will be appreciated that head system 210
could, for example, have more or less pulse jets as desired (for
example, at least ten or at least one hundred pulse jets). In this
manner, application of a single electric pulse to an ejector causes
a droplet to be dispensed from a corresponding orifice. In the
foregoing configuration, typically about twenty orifices in each
group of six reservoirs (many of the orifices are unused and are
plugged with glue), will be dispensing the same fluid. Thus, each
"series" in such a configuration has twenty dispensers. Certain
elements of each head can be adapted from parts of a commercially
available thermal inkjet print head device available from
Hewlett-Packard Co. as part no. HP51645A. The foregoing head system
210 and other suitable dispensing head designs are described in
more detail in U.S. patent application entitled "A MULTIPLE
RESERVOIR INK JET DEVICE FOR THE FABRICATION OF BIOMOLECULAR
ARRAYS" Ser. No. 09/150,507 filed Sep. 9, 1998. However, other head
system configurations can be used.
[0039] As is well known in the ink jet print art, the amount of
fluid that is expelled in a single activation event of a pulse jet,
can be controlled by changing one or more of a number of
parameters, including the orifice diameter, the orifice length
(thickness of the orifice member at the orifice), the size of the
deposition chamber, and the size of the heating element, among
others. The amount of fluid that is expelled during a single
activation event is generally in the range about 0.1 to 1000 pL,
usually about 0.5 to 500 pL and more usually about 1.0 to 250 pL. A
typical velocity at which the fluid is expelled from the chamber is
more than about 1 m/s, usually more than about 10 m/s, and may be
as great as about 20 m/s or greater. As will be appreciated, if the
orifice is in motion with respect to the receiving surface at the
time an ejector is activated, the actual site of deposition of the
material will not be the location that is at the moment of
activation in a line-of-sight relation to the orifice, but will be
a location that is predictable for the given distances and
velocities.
[0040] The sizes of the features can have widths (that is,
diameter, for a round spot) in the range from a minimum of about 10
.mu.m to a maximum of about 1.0 cm. In embodiments where very small
spot sizes or feature sizes are desired, material can be deposited
according to the invention in small spots whose width is in the
range about 1.0 .mu.m to 1.0 mm, usually about 5.0 .mu.m to 500
.mu.m, and more usually about 10 .mu.m to 200 .mu.m. Spot sizes can
be adjusted as desired, by using one or a desired number of pulses
from a pulse jet to provide the desired final spot size.
[0041] The apparatus further includes a sensor in the form of a
camera 304, to monitor dispensers for errors (such as failure to
dispense droplets) by monitoring for drops dispensed onto substrate
10 when required of a dispenser. Camera 304 communicates with
processor 140, and should have a resolution that provides a pixel
size of about 1 to 100 micrometers and more typically about 4 to 20
micrometers or even 1 to 5 micrometers. Any suitable analog or
digital image capture device (including a line by line scanner) can
be used for such camera, although if an analog camera is used
processor 140 should include a suitable analog/digital converter. A
detailed arrangement and use of such a camera to monitor for
dispenser errors, is described in U.S. patent application Ser. No.
09/419447 entitled "Biopolymer Array Inspection" by William D.
Fisher. Particular observations techniques are described, for
example, in co-pending U.S. patent application Ser. No. 09/302,898
filed Apr. 30, 1999 by Caren et al., assigned to the same assignee
as the present application, incorporated herein by reference.
Alternatively, the sensor can be a drop detector which detects an
electrical charge on a dispensed drop, in accordance with the
apparatus and methods described in U.S. Ser. No. 09/558,532
entitled "Array Fabrication with Drop Detection" filed by
Christopher A. Schantz et al. Monitoring can occur during formation
of an array and the information used during fabrication of the
remainder of that array or another array, or test-print patterns
can be run before array fabrication. A display 310, speaker 314,
and operator input device 312, are further provided. Operator input
device 312 may, for example, be a keyboard, mouse, or the like.
Processor 140 has access to a memory 141, and controls print head
system 210 (specifically, the activation of the ejectors therein),
operation of the transport system, operation of each jet in print
head system 210, capture and evaluation of images from the camera
304, and operation display 310 and speaker 314. Memory 141 may be
any suitable device in which processor 140 can store and retrieve
data, such as magnetic, optical, or solid state storage devices
(including magnetic or optical disks or tape or RAM, or any other
suitable device, either fixed or portable). Processor 140 may
include a general purpose digital microprocessor suitably
programmed from a computer readable medium carrying necessary
program code, to execute all of the functions required of it as
described below. It will be appreciated though, that when a
"processor" such as processor 140 is referenced throughout this
application, that such includes any hardware and/or software
combination which will perform the required functions. Suitable
programming can be provided remotely to processor 140, or
previously saved in a computer program product such as memory 141
or some other portable or fixed computer readable storage medium
using any of those devices mentioned below in connection with
memory 141. For example, a magnetic or optical disk 324 may carry
the programming, and can be read by disk reader 326.
[0042] Operation of the apparatus of FIG. 6 in accordance with a
method of the present invention, will now be described. First, it
will be assumed that memory 141 holds a target drive pattern. This
target drive pattern is the instructions for driving the apparatus
components as required to form the target array (which includes
target locations and dimension for each spot) on substrate 10 and
includes, for example, movement commands to transporters 60 and 100
as well as firing commands for each of the pulse jets in head
system 210 coordinated with the movement of head system 210 and
substrate 10, as well as instructions for which polynucleotide
solution (or precursor) is to be loaded in each pulse jet (that is,
the "loading pattern"). This target drive pattern is based upon the
target array pattern and can have either been input from an
appropriate source (such as input device 312, a portable magnetic
or optical medium, or from a remote server, any of which
communicate with processor 140), or may have been determined by
processor 140 based upon an input target array pattern (using any
of the appropriate sources previously mentioned) and the previously
known nominal operating parameters of the apparatus. Further, it
will be assumed that drops of different biomonomer or biopolymer
containing fluids (or other fluids) have been placed at respective
regions of a loading station (not shown). Operation of the
following sequences are controlled by processor 140, following
initial operator activation, unless a contrary indication
appears.
[0043] For any given substrate 10, the operation is basically
follows: (i) determine a target drive pattern (if not already
provided) to obtain target array pattern, based on nominal
operating parameters and target polynucleotide array pattern; (ii)
evaluate data from the sensor for errors in the operation of the
dispensers (for example, dispenser does not dispense a drop upon
command) (iii) if there is no error in one or more operating
parameters then the apparatus is operated according to the target
drive pattern; (iv) if there is an error in one or more dispensers
then processor 140 derives, based on the error, a corrected drive
pattern from the target pattern such that firing by error
dispensers is replaced by firing from non-error dispensers in
accordance with the methods already described above. Note that a
corrected drive pattern can either be determined initially prior to
dispensing droplets to fabricate the array, or may be determined
(and continually corrected) during formation of an array as
different dispenser errors are detected. The target drive pattern
may be saved in memory or just derived during the actual array
fabrication and sent as instructions directly to the apparatus
components.
[0044] It will be appreciated that any discrepancy between a
nominal dispenser parameter and an actual parameter, may optionally
only be classified as an "error" if it meets or exceeds a
predetermined threshold value. Particular examples of dispenser
errors which may occur in the apparatus of FIG. 6 include any one
or more of the following:
[0045] 1. A dispenser does not fire upon command or dispenses a
drop which is of unsatisfactory volume.
[0046] 2. A dispenser dispenses a drop at a misplaced position (for
example, due to the dispenser firing at an angle from the expected
trajectory).
[0047] The apparatus is then operated as follows: (a) load head
system 210 with a first set of polynucleotide containing solutions
or their precursors (for example, a given head may be able to hold
n different members); (b) dispense droplets from head system 210
onto substrate 10 or a set of substrates in accordance with the
target or corrected drive patterns to provide the target array
pattern for the first set on each of multiple arrays 12; and (c)
repeat the foregoing sequence starting at step (i) with a second
set and subsequent sets of polynucleotide containing solutions or
their precursors, until all required solutions have been dispensed
onto substrate 10 (for example, if each array has m.multidot.n
members, and presynthesized polynucleotides are being dispensed,
then the sequence will be repeated m times). It will be appreciated
though, that techniques of this application or the two applications
(incorporated herein by reference) entitled "Array Fabrication" by
Peter Webb filed the same date as the present application and both
assigned to Agilent Technologies, Inc. (Attorney Docket Nos.
10004004-1 and 10003513-1, may be combined as appropriate.
[0048] A loading sequence for head system 210 is more completely
described in co-pending patent applications "FABRICATING BIOPOLYMER
ARRAYS", by Caren et al., Ser. No. 09/302,922, and "PREPARATION OF
BIOPOLYMER ARRAYS" by A. Schleifer et al., Ser. No. 09/302,899,
both filed Apr. 30, 1999 and both assigned to the same assignee as
the present application, and the references cited therein,
including the possibility of using a flexible microtitre plate as
described in U.S. patent application "Method and Apparatus for
Liquid Transfer", Ser. No. 09/183,604. Those references and all
other references cited in the present application, are incorporated
into this application by reference. Note that since the dispensers
of each series communicates with the same corresponding reservoir
for that series, the dispensers of a given series are effectively
loaded with the same polynucleotide fluid. Processor 140 can
control pressure within head system 210 to load each polynucleotide
solution into the chambers in the head by drawing it through the
orifices as described in one or more of the foregoing
applications.
[0049] Substrate 10 is loaded onto substrate station 20 either
manually by an operator, or optionally by a suitable automated
driver (not shown) controlled, for example, by processor 140.
[0050] The deposition sequence is then initiated to deposit the
desired arrays of polynucleotide containing fluid droplets on the
substrate to provide drops on the substrate according to the target
pattern each with respective feature locations and dimensions. As
already mentioned, in this sequence processor 140 will operate the
apparatus according to the target or corrected drive pattern, by
causing the transport system to position head system 210 facing
substrate station 20, and particularly the mounted substrate 10,
and with head system 210 at an appropriate distance from substrate
10. Processor 140 then causes the transport system to scan head
system 210 across substrate 10 line by line (or in some other
desired pattern), while coordinating activation of the ejectors in
head system 210 so as to dispense droplets as described above. If
necessary or desired, processor 140 can repeat the load and
dispensing sequences one or more times until head system 210 has
dispensed droplets in to obtain the target arrays 12 to be formed
on substrate 10. The number of spots in any one array 12 can, for
example, be at least ten, at least one hundred, at least one
thousand, or even at least one hundred thousand.
[0051] At this point the droplet dispensing sequence is
complete.
[0052] When a user receives an array made by an apparatus or method
of the present invention, it will typically be exposed to a sample
and the array interrogated following exposure. Interrogation is
usually accomplished by a suitable scanner which can read the
location and intensity of fluorescence at each feature of an array
following exposure to a fluorescently labeled sample (such as a
polynucleotide containing sample). For example, such a scanner may
be similar to the GENEARRAY scanner available from Hewlett-Packard,
Palo Alto, Calif. Results from the interrogation can be processed
such as by rejecting a reading for a feature which is below a
predetermined threshold and/or forming conclusions based on the
pattern read from the array (such as whether or not a particular
target sequence may have been present in the sample). The results
of the interrogation or processing can be forwarded (such as by
communication) to a remote location if desired, for further
use.
[0053] The present methods and apparatus may be used to deposit
biopolymers or other chemical moieties on surfaces of any of a
variety of different substrates, including both flexible and rigid
substrates. Preferred materials provide physical support for the
deposited material and endure the conditions of the deposition
process and of any subsequent treatment or handling or processing
that may be encountered in the use of the particular array. The
array substrate may take any of a variety of configurations ranging
from simple to complex. Thus, the substrate could have generally
planar form, as for example a slide or plate configuration, such as
a rectangular or square or disc. In many embodiments, the substrate
will be shaped generally as a rectangular solid, having a length in
the range about 4 mm to 1 m, usually about 4 mm to 600 mm, more
usually about 4 mm to 400 mm; a width in the range about 4 mm to 1
m, usually about 4 mm to 500 mm and more usually about 4 mm to 400
mm; and a thickness in the range about 0.01 mm to 5.0 mm, usually
from about 0.1 mm to 2 mm and more usually from about 0.2 to 1 mm.
However, larger substrates can be used, particularly when such are
cut after fabrication into smaller size substrates carrying a
smaller total number of arrays 12.
[0054] In the present invention, any of a variety of geometries of
arrays on a substrate 10 may be fabricated other than the
rectilinear rows and columns of arrays 12 of FIG. 1. For example,
arrays 12 can be arranged in a sequence of curvilinear rows across
the substrate surface (for example, a sequence of concentric
circles or semi-circles of spots), and the like. Similarly, the
pattern of features 16 may be varied from the rectilinear rows and
columns of spots in FIG. 2 to include, for example, a sequence of
curvilinear rows across the substrate surface (for example, a
sequence of concentric circles or semi-circles of spots), and the
like. In such cases, the arrangement of dispensers in head system
210 may be altered accordingly. The configuration of the arrays and
their features may be selected according to manufacturing,
handling, and use considerations.
[0055] The substrates may be fabricated from any of a variety of
materials. In certain embodiments, such as for example where
production of binding pair arrays for use in research and related
applications is desired, the materials from which the substrate may
be fabricated should ideally exhibit a low level of non-specific
binding during hybridization events. In many situations, it will
also be preferable to employ a material that is transparent to
visible and/or UV light. For flexible substrates, materials of
interest include: nylon, both modified and unmodified,
nitrocellulose, polypropylene, and the like, where a nylon
membrane, as well as derivatives thereof, may be particularly
useful in this embodiment. For rigid substrates, specific materials
of interest include: glass; plastics (for example,
polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate,
and blends thereof, and the like); metals (for example, gold,
platinum, and the like).
[0056] The substrate surface onto which the polynucleotide
compositions or other moieties is deposited may be smooth or
substantially planar, or have irregularities, such as depressions
or elevations. The surface may be modified with one or more
different layers of compounds that serve to modify the properties
of the surface in a desirable manner. Such modification layers,
when present, will generally range in thickness from a
monomolecular thickness to about 1 mm, usually from a monomolecular
thickness to about 0.1 mm and more usually from a monomolecular
thickness to about 0.001 mm. Modification layers of interest
include: inorganic and organic layers such as metals, metal oxides,
polymers, small organic molecules and the like. Polymeric layers of
interest include layers of: peptides, proteins, polynucleic acids
or mimetics thereof (for example, peptide nucleic acids and the
like); polysaccharides, phospholipids, polyurethanes, polyesters,
polycarbonates, polyureas, polyamides, polyethyleneamines,
polyarylene sulfides, polysiloxanes, polyimides, polyacetates, and
the like, where the polymers may be hetero- or homopolymeric, and
may or may not have separate functional moieties attached thereto
(for example, conjugated).
[0057] Various further modifications to the particular embodiments
described above are, of course, possible. Accordingly, the present
invention is not limited to the particular embodiments described in
detail above.
* * * * *