U.S. patent application number 10/116635 was filed with the patent office on 2002-10-17 for guided deposition in spatial arrays.
Invention is credited to Buchko, Christopher J., Modlin, Douglas N., Zaffaroni, Alejandro C..
Application Number | 20020151085 10/116635 |
Document ID | / |
Family ID | 23276838 |
Filed Date | 2002-10-17 |
United States Patent
Application |
20020151085 |
Kind Code |
A1 |
Zaffaroni, Alejandro C. ; et
al. |
October 17, 2002 |
Guided deposition in spatial arrays
Abstract
An apparatus and method is provided for preparing and using a
very large and diverse array of compounds on a substrate having
rapidly accessible locations. The substrate contains cells in which
the compounds of the array are located. Surrounding the cells is a
non-wetable surface that prevents the solution in one cell from
moving to adjacent cells. The compounds are delivered to the
individual cells of the array by a micropipette attached to an X-Y
translation stage.
Inventors: |
Zaffaroni, Alejandro C.;
(Atherton, CA) ; Buchko, Christopher J.; (Ann
Arbor, MI) ; Modlin, Douglas N.; (Palo Alto,
CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Family ID: |
23276838 |
Appl. No.: |
10/116635 |
Filed: |
April 3, 2002 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10116635 |
Apr 3, 2002 |
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09654742 |
Sep 1, 2000 |
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09654742 |
Sep 1, 2000 |
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08327512 |
Oct 18, 1994 |
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6121048 |
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Current U.S.
Class: |
436/180 ;
422/130; 422/400 |
Current CPC
Class: |
B01J 2219/00317
20130101; B01J 2219/00605 20130101; Y10T 436/2575 20150115; B01J
2219/00536 20130101; B01J 2219/00612 20130101; G01N 2035/1039
20130101; B01J 2219/00619 20130101; B01J 2219/00635 20130101; B01J
2219/00659 20130101; B01J 2219/0072 20130101; B01J 2219/00585
20130101; C40B 60/14 20130101; B01J 2219/00621 20130101; B01J
2219/00527 20130101; B01J 2219/0061 20130101; B01J 2219/00367
20130101; Y10T 436/111666 20150115; B01L 2300/0893 20130101; Y10T
436/143333 20150115; B01J 2219/00626 20130101; B01J 19/0046
20130101; B01J 2219/00596 20130101; B01L 3/5085 20130101; B01J
2219/00378 20130101 |
Class at
Publication: |
436/180 ; 422/99;
422/102; 422/130 |
International
Class: |
B01J 019/00 |
Claims
What is claimed is:
1. A method for conducting a plurality of reactions on a substrate,
said substrate having a plurality of cells at predefined locations
thereon, the location of each cell being encoded by information
readable with electrical, magnetic or electromagnetic sensing
means, said method comprising the following steps: (a) selecting a
first set of cells; (b) decoding the locations of said first set of
cells by said sensing means and delivering a first reactant to each
of said first set cells; (c) selecting a second set of cells; (d)
decoding the locations of said second set of cells by said sensing
means and delivering a second reactant to each of said second set
of cells; and (e) simultaneously reacting said first reactant in
each of said first set of cells and said second reactant in each of
said second set of cells, wherein the first reactant is prevented
from contacting the second set of cells and the second reactant is
prevented from contacting the first set of cells.
2. The method of claim 1 where said step of delivering the first
reactant to the first set of cells includes the following steps:
(a) identifying a reference mark on the substrate; (b) moving a
dispenser of said reactants a fixed distance and direction from
said reference point such that the dispenser is positioned
approximately above a first cell of the first set of cells; (c)
delivering the first reactant to the first cell; and (d) repeating
steps b and c for each remaining cell of the first set of
cells.
3. The method of claim 1 wherein the first and second reactants are
monomers.
4. The method of claim 3 wherein the monomers are selected from the
group consisting of amino acids, nucleotides and saccharides.
5. The method of claim 1 wherein the first and second reactants are
receptors.
6. The method of claim 1 wherein said substrate is a rotatable
disk, and said steps of delivering reactants each includes the
following steps: (a) moving a dispenser of said reactants to the
radial position of a first cell from said selected set of cells;
(b) rotating of said substrate with respect to the dispenser such
tha the dispenser is positioned approximately above said first
cell; (c) delivering said reactant to said first cell; (d)
repeating steps a-c for each remaining cell of said selected set of
cells.
7. The method of claim 1 wherein the first and second reactants are
delivered to the cells from a micropipette.
8. The method of claim 1 wherein the first and second reactants are
delivered in charged droplets from capillaries having electrodes
attached thereto.
9. The method of claim 1 wherein the substrate has surface regions
surrounding the plurality of cells, the surface regions having a
hydrophobic surface.
10. A method for conducting a plurality of reactions on a
substrate, said substrate having a plurality of cells at predefined
locations thereon, the method comprising the following steps: (a)
selecting a first set of cells and a second set of cells; (b)
delivering a solution of a first reactant to the first set of cells
and a second reactant to the second set of cells; (c)
simultaneously conducting reactions of the first reactant in the
first set of cells and of the second reactant in the second set of
cells; wherein regions of the substrate surrounding the first set
of cells are covered with a layer of protecting groups that are
non-wetting with respect to the solutions of the first and second
reactants.
11. The method of claim 10 where said steps of delivering reactants
each includes the following steps: (a) identifying a reference mark
on the substrate; (b) moving a dispenser of said reactants a fixed
distance and direction from said reference point such that the
dispenser is positioned approximately above said first cell; (c)
delivering said reactant to said first cells; and (d) repeating
steps b and c for each remaining cell of the selected set of
cells.
12. The method of claim 10 wherein the protecting groups are
selected from the group consisting of alkyl silanes.
13. An array of compounds on a substrate, said array comprising:
(a) a plurality of cells on which the individual compounds of the
array are located; (b) a layer of non-wetting protecting groups
surrounding said cells.
14. The array of claim 13 wherein the protecting groups are
selected from the group consisting of alkyl silanes.
15. The array of claim 13 wherein the compounds are polymers.
16. The array of claim 13 wherein the protecting groups are
photolabile.
17. A system for conducting reactions on selected cells, said
system comprising the following elements: (a) substrate on which a
plurality of cells are disposed, the substrate having non-wetting
protecting groups on regions surrounding the cells; (b) one or more
reservoirs for storing one or more reactants; (c) means for
dispensing the one or more reactants on selected cells of said
substrate, said means for dispensing being mounted on an arm
movable with respect to the substrate; (d) an actuator for
controlling the radial position of said arm with respect to said
substrate; and (e) processing means controlling the actuator such.
that said reactants are delivered to predetermined cells in a
predetermined sequence through said means for dispensing.
18. The system of claim 17 wherein the reactants are selected from
the group consisting of receptors, amino acids, nucleotides, and
saccharides.
19. The system of claim 17 wherein the protecting groups are
photocleavably attached to the substrate.
20. The system of claim 17 wherein the protecting groups are
selected from the group consisting of alkyl silanes.
Description
BACKGROUND OF TE INVENTION
[0001] The present invention lies in the field of methods and
apparatus for preparing large arrays of polymers, receptors, and
other compounds. More particularly, it lies in the fields of
automated methods for preparing diverse arrays of polymers and of
techniques for directing specified materials to predefined
locations on a substrate.
[0002] Very diverse collections of compounds are often desired in
research and other applications. Microtiter plates conventionally
contain wells for testing as many as 96 different compounds. For
many applications, 96 represents an unacceptably small number of
samples. Further, when the compounds of interest are rare or
valuable, the test samples must be minuscule. Unfortunately,
signals from such small samples can be lost or diluted in the
relatively large volume wells of a conventional microtiter plate.
If the wells were made smaller and placed in higher densities on
microtiter plates, suitable methods would still be needed for
accurately delivering small aliquots to specified wells, and for
identifying wells containing compounds that exhibit a desired
activity.
[0003] Often the compounds of interest are polymers, such as
nucleic acids, polysaccharides, or peptides. Some attempts have
been made to synthesize a limited number of peptide sequences on,
for example, a number of "rods." See, for example, Geysen, et al.,
J. Immun. Meth. (1987) 102:259-274, incorporated herein by
reference for all purposes, which describes a procedure in which
peptide syntheses are carried out in parallel on several rods or
pins (to complement standard microtiter plates, 96 were used in the
described method). The Geysen et al. method is limited in the
number of sequences that can be synthesized in a reasonable amount
of time. For example, Geysen et al. report in the above journal
that it has taken approximately 3 years to synthesize 200,000
peptide sequences. In addition, such methods have continued to
produce fewer peptide sequences for study than are often desired.
Even if the large number of desired compounds could be produced
quickly, they would not be readily accessible for further study.
The 96 pin arrays of Geysen et al. would occupy far too much space
to rapidly screen thousands of candidate polymers.
[0004] Techniques have recently been introduced for synthesizing
large arrays of different peptides and other polymers on solid
surfaces. For example, in Pirrung et al., PCT Publication No. WO
90/15070, incorporated herein by reference for all purposes, a
technique is disclosed for generating arrays of peptides and other
materials using, for example, light-directed, spatially-addressable
synthesis techniques. See also, Fodor et al., PCT Publication No.
WO 92/10092 (incorporated herein by reference for all purposes)
which discloses, among other things, a method of gathering
fluorescence intensity data, various photosensitive protecting
groups, masking techniques, and automated techniques for performing
light-directed, spatially-addressable synthesis techniques. Arrays
containing up to 64,000 different elements have been formed using
this technology. See, U.S. patent application No. 805,727, filed
Dec. 6, 1991, which is incorporated herein by reference for all
purposes. Because of their relationship to semiconductor
fabrication techniques, these methods have come to be referred to
as "Very Large Scale Immobilized Polymer Synthesis," or
"VLSIPS.TM." technology. Such techniques have met with substantial
success in, for example, screening various ligands, such as
peptides, to determine their relative binding affinity to a
receptor such as an antibody.
[0005] In some applications, it is desirable to study pre-formed
collections of synthetic chemical compounds or natural product
extracts. For example, it would be desirable to "immortalize" a
collection of chemical samples from a rain forest threatened with
destruction. In addition, thousands of different synthetic
compounds often are cataloged in "libraries" of Universities and
corporations. Unfortunately, the compounds of these libraries are
not readily accessible for systematic study.
[0006] Methods for immobilizig collections of materials on a solid
substrate are known. For example, U.S. Pat. No. 4,562,157 issued to
Lowe et al., and incorporated herein by reference for all purposes,
discusses a technique for attaching biochemical ligands to surfaces
through a photochemically reactive arylazide. Irradiation of the
azide creates a reactive nitrene moiety which reacts irreversibly
with macromolecules in solution to form a covalent bond. The high
reactivity of the nitrene intermediate, however, results in both
low coupling efficiencies and many potentially unwanted reactions
through nonspecific reactions.
[0007] An improved method for immobilizing collections of compounds
is disclosed in Barrett et al., PCT Publication No. WO 91/07087
which is incorporated herein by reference for all purposes. This
publication discloses a technique for immobilizing arrays of
anti-ligands, such as antibodies or antigens, hormones or hormone
receptors, oligonucleotides, polysaccharides, and other materials.
Cycles of irdiation on different regions of a surface and
immobilization of different anti-ligands allows formation of an
immobilized matrix of anti-ligands at defined sites on the surface.
The immobilized matrix of anti-ligands permits simultaneous
screenings of a liquid sample for ligands having high affinities
for certain anti-ligands of the matrix.
[0008] While the technique disclosed in PCT Publication No. WO
91/07087 as well as the VLSIPS.TM. technique are useful for
preparing and using large arrays of materials, other techniques
emphasizing efficient formation and use of much larger arrays of
individual compounds would be desirable. If a very large group of
compounds is used to form a diverse array, the number of individual
locations on the array can be enormous. For example, to synthesize
all dimmers from 100 monomer starting materials requires 100,000
(100.sup.2) separated locations. Forming an array of this size can
be a daunting task. Further, once the array is produced, it can
become quite difficult to locate specific members of the array for
further processing or study.
[0009] Some work has been done to automate synthesis of polymer
arrays. For instance, in Fodor et al., PCT Publication No. WO
92/10092, a method is described for using a computer-controlled
system to direct a VLSIPS.TM. procedure. Further, Southern, PCT
Application No. WO 89/10977 describes the use of a conventional pen
plotter to deposit three different monomers at twelve distinct
locations on a substrate. These monomers were subsequently reacted
to form three different polymers, each twelve monomers in length.
This reference also discusses the possibility of using an ink-jet
printer to deposit monomers on a substrate. Wong et al., European
Patent Application No. 260 965 describes a process in which a
single polymer species in solution was putatively deposited in a
single spot on a substrate by an apparatus resembling an ink-jet
printer. However, neither the method described in the Wong et al.
reference nor the method described in the Southern application
concerns very large arrays of polymers.
[0010] Further, the methods described in these two references would
be unacceptably slow in accessing specific elements of a large
array.
SUMMARY OF THE INVENTION
[0011] The present invention is directed to methods and apparatus
for preparing large arrays of chemical compounds. It is also
directed to methods for using such arrays in a variety of
applications, such as screening compounds in drug development
research. The chemical elements of the array are located on very
small predefined zones or "cells" on a substrate surface. In one
embodiment, a rotatable substrate surface is divided into tracks
and/or sectors that allow identification of each cell's location.
In another embodiment, a substrate surface is divided into cells
that are automatically accessed from a few reference points by a
dead reckoning navigation technique. Thus, reactants or other
materials (e.g. from a library of compounds) are quickly and
automatically delivered to any cell on the substrate surface by a
dispenser. In many embodiments, the sequence of steps necessary to
deliver specified reactants to certain groups of cells will be
prerecorded and controlled by a processor such as a computer. The
reactants will then be delivered to precise locations by, for
example, micropipettes, electrophorectic pumps, or mechanisms
adapted from inkjet printing technology.
[0012] In one aspect of the present invention, the reactants are
monomers and the cells of the array contain polymers having
different monomer sequences. Thus, the present invention provides a
method for synthesizing a large array of different polymers on a
substrate. A first monomer solution is delivered to a first set of
cells on a suitably derivatzed substrate. Thereafter, a second
monomer solution is delivered to a second set of cells, a third
monomer solution is delivered to a third set, and so on until a
number of cells each have one species of free monomer located
therein. These monomers are then reacted with the surface, washed,
and prepared for reaction with a new set of monomers. Dimers,
trimers, and ultimately polymers of controlled length and monomer
sequence are prepared by repeating the above steps with different
groupings of the cells. In alternative embodiments, the polymers or
other compounds of the array are delivered to the cells as complete
species and, thus, the above polymer synthesis steps become
unnecessary. Regardless of how the array is formed, the properties
of its individual components can be studied by conducting
simultaneous reactions in each cell. In this way a large number of
reactions can be studied in parallel. In some cases, samples from
one array may be accessed and moved to a second array where a
reaction is conducted.
[0013] In one embodiment, the system for conducting reactions (such
as polymer synthesis) on selected cells includes a substrate on
which a plurality of cells are located and surrounded by
non-wetting surface regions. Thus, the reactants in one cell will
not flow to adjacent cells where they could contaminate the
reaction. In preferred embodiments, the cells of the array are
defined by selective irradiation of a substrate surface containing
photolabile, hydrophobic protecting groups. In areas where the
surface is irradiated, the hydrophobic protecting groups can be
removed to define cells. When an aqueous or other polar reactant
solution is deposited in the cell, it will have a relatively large
wetting angle, thereby preventing flow to adjacent cells.
[0014] In some embodiments, the substrate includes digitally
encoded instructions governing the sequence of deposition steps
necessary to control the array of reactions. The instructions are
typically readable by optical, electrical or magnetic sensors.
Preferably, the cells and encoded instructions are located on
tracks embedded on the disk surface. In this embodiment, a "read
head" is necessary to receive the encoded information for further
processing. In addition to the sequence of deposition steps, the
encoded instructions will, in some embodiments, identify
characteristics or properties of the polymers located in individual
cells.
[0015] In preferred systems, the reactants are stored in one or
more reservoirs which are available to a dispenser. The dispenser
delivers the individual reactants from the reservoirs to the
various cells on the disk surface. Typically, the dispenser will be
mounted on an arm that can be moved across the substrate surface to
any position by an actuator. The entire process is governed by a
processor that controls the position of the dispenser with respect
to the substrate and the reactant to be delivered. In this way,
precise control over a very large array of cells and reactions is
available.
[0016] A further understanding of the nature and advantages of the
inventions herein may be realized by reference to the detailed
description of the specification and the associated drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1(A) displays a side view of a reactant solution in a
cell surrounded by a hydrophobic mask according to one embodiment
of the present invention;
[0018] FIG. 1(B) displays a top view of an array of cells having
circular perimeters;
[0019] FIGS. 2A-B display elements of an optical record/read
apparatus and bumpforming media that can be used with such
apparatus;
[0020] FIGS. 3A-D display features of a dispensing apparatus for
delivering several different reactants to a disk substrate;
[0021] FIG. 4 displays elements of a typical guided droplet
dispenser that can be used to deposit the reactant solutions of the
present invention;
[0022] FIG. 5 displays a preferred embodiment of the present
invention; and
[0023] FIGS. 6A-E show various formats for the disk arrays that can
be used in certain embodiments of the present invention.
1 DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
I. Glossary II. Overview of the Invention III. Isolation of
Reaction Areas A. Dimples or recesses B. Controlling the Wetting
Angle IV. Moving the Dispenser with Respect to the Substrate A.
Mapping the Frame of Reference B. Rotational Mechanisms V.
Delivering the Reactant Solution VI. Conducting the Reactions VII.
Example A. Apparatus B. Locating Desired Cells C. Accessing
Selected Cells D. Depositing Reactants in Selected Cells E. Peptide
Synthesis F. Imaging the Array VIII. Conclusion
[0024] I. Glossary
[0025] The following terms are intended to have the following
general meanings as they are used herein:
[0026] 1. Substrate: A material having a rigid or semi-rigid
surface. In many embodiments, at least one surface of the substrate
will be substantially flat, although in some embodiments it may be
desirable to provide dimples, wells, raised regions, etched
trenches, or the like. According to other embodiments, small beads
or pellets may be provided on the surface within dimples or on
other regions of the surface.
[0027] 2. Cell: A cell is a localI area on a substrate which is to
be used for conducting a reaction. This use of "cell" should not be
confused with the common biological use of "cell." In some
instances, the cells (reaction locales) of this invention will
contain one or more biological cells for study. When the biological
usage of "cell" is intended, it will be clearly indicated herein.
Some reactions within a cell involve binding between a ligand and a
receptor, and other reactions, for example, involve synthesis of a
selected polymer. A cell may have any convenient shape, e.g.,
circular, rectangular, elliptical, wedge-shaped, etc. In some
embodiments, cells are smaller than about 1 cm.sup.2, more
preferably less than 1 mm.sup.2, still more preferably between
about 100 .mu.m.sup.2 and 1 mm.sup.2.
[0028] 3. Substantially Pure: A polymer or other compound is
considered to be "substantially pure" when it exhibits
characteristics that distinguish it from the polymers or compounds
in other cells. For example, purity can be measured in terms of the
activity or concentration of the compound of interest. Preferably,
the compound in a cell is sufficiently pure such that it is the
predominant species in the cell. According to certain aspects of
the invention, the compound is 5% pure, more preferably more than
10% pure, and most preferably more than 20% pure. According to more
preferred aspects of the invention, the compound is greater than
80% pure, preferably more than 90% pure, and more preferably more
than 95% pure, where purity for this purpose refers to the ratio of
the number of compound molecules formed in a cell having a desired
structure to the total number of non-solvent molecules in the
cell.
[0029] 4. Monomer: In general, a monomer is a member of a set of
small molecules which are or can be joined together to form a
polymer. The particular ordering of monomers within a polymer is
referred to herein as the "sequence" of the polymer. As used
herein, monomer refers to any member of a basis set used for
synthesis of a polymer. For example, dimers of the 20 naturally
occurring L-amino acids form a basis set of 400 monomers for
synthesis of polypeptides. Different basis sets of monomers can be
used at successive steps in the synthesis of a polymer.
Furthermore, each of the sets may include protected members which
are modified after synthesis. The invention described herein can
readily be applied in the preparation of diverse types of polymers.
Such polymers include, for example, both linear and cyclic polymers
of nucleic acids, polysaccharides, phospholipids, and peptides
having either .alpha.-, .beta.-, or .omega.-amino acids,
hetero-polymers in which a known drug is covalently bound to any of
the above, polyacetates, polyamides, polyarylene sulfides,
polycarbamates, polycarbonates, polyesters, polyethyleneimines,
polyimides, polynucleotides, polyphosphonates, polysiloxanes,
polysulfones, polysulfoxides, polyureas, polyurethanes, or other
polymers which will be apparent upon review of this disclosure.
[0030] 5. Protective Group: A material which is bound to a monomer
or other compound or group and which can be selectively removed
therefrom to expose an active site such as, in the example of an
amino acid, an amine group. A protective group will typically be
used to block one reactive site of a bifunctional monomer from
reacting during an addition reaction such as in the formation of a
peptide from amino acids. A protective group can also cover certain
regions of a substrate surface to impart certain properties such as
non-wetability and to define cell perimeters or other features.
[0031] 6. Head: A head is a device that reads, records, or erases
signals stored on a suitable medium, such as magnetic or optical
storage disks. The portion of the medium along which the head moves
is called the "track." The head will typically be a transducer that
senses (reads) or generates (writes) a signal corresponding to
information provided by the medium or a processor. The transduction
will typically involve electromagnetic fields or radiation,
depending upon the type of media employed.
[0032] 7. Receptor: Receptors used with the present invention may
be naturally-occurring or manmade molecules. Also, they can be
employed in their unaltered state or as aggregates with other
species. Receptors may be attached, covalently or noncovalently, to
a binding member, either directly or via a specific binding
substance. Examples of receptors which can be employed by this
invention include, but are not restricted to, antibodies, cell
membrane receptors, monoclonal antibodies and antisera reactive
with specific antigenic determinants (such as on viruses, cells or
other materials), drugs, polynucleotides, nucleic acids, peptides,
cofactors, lectins, sugars, polysaccharides, cells, cellular
membranes, and organelles. Receptors are sometimes referred to in
the art as anti-ligands. As the term receptors is used herein, no
difference in meaning is intended. A ligand is a molecule that is
recognized by a particular receptor. Examples of ligands that can
be investigated by this invention include, but are not restricted
to, agonists and antagonists for cell membrane receptors, toxins
and venoms, viral epitopes, hormones (e.g., opiates, steroids,
etc.), hormones, peptides, enzymes, enzyme substrates, cofactors,
drugs, lectins, sugars, oligonucleotides, nucleic acids,
oligosaccharides, proteins, and antigens. A "Ligand Receptor Pair"
is formed when two macromolecules have combined through molecular
recognition to form a complex. Specific examples of receptors which
can be investigated by this invention include, but are not
restricted to the following:
[0033] a) Microorganism receptors: Determination of ligands which
bind to receptors, such as specific transport proteins or enzymes
essential to survival of microorganisms, is useful in developing
new classes of antibiotics. Of particular value would be
antibiotics against opportunistic fungi, protozoa, and those
bacteria resistant to the antibiotics in current use.
[0034] b) Enzymes: For instance, the present invention can be used
to identify ligands that bind to the binding site of enzymes such
as the enzymes responsible for cleaving neurotransmitters, and
ligands that bind to certain receptors to modulate the action of
the enzymes which cleave the different neurotransmitters, as would
be useful in the development of drugs which can be used in the
treatment of disorders of neurotransmission.
[0035] c) Antibodies: For instance, the invention may be useful in
investigating the ligand-binding site on the antibody molecule
which combines with the epitope of an antigen of interest;
determining a sequence that mimics an antigenic epitope may lead to
the development of vaccines of which the immunogen is based on one
or more of such sequences or lead to the development of related
diagnostic agents or compounds useful in therapeutic treatments
such as for autoimmune diseases (e.g., by blocking the binding of
the "self" antibodies).
[0036] d) Nucleic Acids: Sequences of nucleic acids may be
synthesized to establish DNA or RNA binding sequences.
[0037] e) Catalytic Polypeptides: These receptors are polymers,
preferably polypeptides, which are capable of promoting a chemical
reaction involving the conversion of one or more reactants to one
or more products. Such polypeptides generally include a binding
site specific for at least one reactant or reaction intermediate
and an active functionality proximate to the binding site, which
functionality is capable of chemically modifying the bound
reactant. Catalytic polypeptides and others are described in, for
example, PCT Publication No. WO 90/05746, WO 90/05749, and WO
90/05785, which are incorporated herein by reference for all
purposes.
[0038] f) Hormone receptors: Hormone receptors include, for
example, the receptors for insulin and growth hormone.
Determination of the ligands which bind with high affinity to a
receptor is useful in the development of, for example, an oral
replacement of the daily injections which diabetics must take to
relieve the symptoms of diabetes, and in the other illustrative
case, a replacement for the scarce human growth hormone which can
only be obtained from cadavers or by recombinant DNA technology.
Other examples include the vasoconstrictive hormone receptors;
determination of those ligands which bind to these receptors may
lead to the development of drugs to control blood pressure.
[0039] g) Opiate receptors: Determination of ligands which bind to
the opiate receptors in the brain is useful in the development of
less-addictive replacements for morphine and related drugs.
[0040] II. Overview
[0041] The present invention provides a method and apparatus for
conducting a number of different reactions in parallel on a single
substrate. It improves on certain prior methods (e.g. the method of
Geysen et al. discussed above) by providing a greater density of
reaction sites, a more rapid technique for conducting numerous
reactions, and a more accessible collection of reaction sites.
Because the reactions are conducted in parallel, the number of
separate washing and other reaction steps can be minimized.
Further, the reactions at different reaction sites can be
controlled independently. Thus, the reactant concentrations and
other parameters can, to some extent, be varied independently from
reaction site to reaction site, thus optimizing the procedure. The
invention can be used for a variety of purposes. For example, it
can be used as a synthesis tool (as for example in peptide
syntheses), as a screening tool (as for example in screening
compound libraries for drug activity), or as a
monitoring/diagnostic tool (as for example in medical or
environmental testing).
[0042] As a tool for studying existing materials, the present
invention can be employed to immobilize vast collections of
synthetic chemical compounds or natural product extracts. In such
methods, compounds are delivered by dispensing systems of the type
described below to specified cells where they can be assayed for
specific activities. As an example, a large complement of different
human receptors could be deposited on a disk, one in each well.
Then, a plant/animal extract could be screened for binding to
different receptors. Competitive assays or other well-known
techniques can be used to identify such activities.
[0043] In some embodiments, more than one cell of a disk will have
the same compound immobilized therein. The reaction of that
compound with various compounds such as the members of the chemical
library of the type discussed above can be tested by dispensing
small aliquots of each member of the library to a different cell. A
"caged biotin" (described in Barrett et al., U.S. patent
application Ser. No. 612,671, the PCT counterpart of which was
previously incorporated herein by reference), or other
immobilization technique can be used to immobilize the compounds to
specific cells. Such techniques permit a variety of materials to be
immobilized on the cells, including functional receptors, or
biological cells beaing receptors. When the array contains the same
or similar compounds in each cell, the immobilization procedure is
simplified, because the coupling chemistry is consistent from cell
to cell.
[0044] As a synthesis tool, the present invention can be used to
prepare arrays of diverse polymers. This is accomplished by
successively depositing monomers in groups of cells. For example,
assume a monomer "A" is to be bound to the substrate in a first
group of selected cells. If necessary, all or part of the total
array of cells is activated for binding by flowing appropriate
reagents over all or part of the substrate, or by depositing the
appropriate reagents (with a dispenser as described below) in the
selected cells for example. After the dispenser is filled with a
reagent containing monomer A, the dispenser and/or the substrate
are moved so that the dispenser can deposit a small quantity of
monomer A in each of the first group of selected cells. The
activated cells are in fluid contact with the dispensed material,
thereby binding monomer A on the substrate directly or indirectly
(via a linker) in the first selected cells.
[0045] Thereafter, the unreacted materials are removed from the
substrate and a monomer "B" is coupled to a second group of
selected cells, some or all of which may be included among the
first selected cells. The second selected cells will be available
to the dispenser through translation and rotation of the dispenser
and/or the substrate. If necessary, a step is performed for
activating at least the second group of cells. After monomer B is
appropriately deposited, it is bound or reacted at the second
selected cells. In this particular example, the resulting sequences
bound to the substrate at this stage of the synthesis process can
be, for example, A, B, and AB. The process is repeated, optionally
with additional monomers, to form a vast array of sequences of
desired length at known locations on the substrate.
[0046] Whether the present invention is used to synthesize a
collection of polymers, immobilize one type of receptor, or
immobilize a collection of compounds, the resulting array can be
used for a variety of purposes, such as drug discovery and
diagnostic assays. As described above, a collection of peptides or
other bio-active compounds can be assayed for activity against one
or more receptors. By quickly identifying materials having a strong
affinity for the receptor of interest, researchers may discover
potential new drugs. In other embodiments, the arrays produced by
the present invention can be used to diagnose, measure, and/or
monitor specific conditions in organisms. Specific binding patterns
on premade arrays can be expected to accurately identify specific
pathogens or other factors present in the sera of a patient. In
still other embodiments, the present invention can be used to
monitor the quality of phaeuticals or other materials, particularly
biomolecules produced by, for example, fermentation processes. In
such uses, very specific patterns or "fingerprints" on a very large
array will correlate with the purity of the compound of interest.
Subtle or not so subtle changes in the preparation containing the
compound of interest will show up as variations in a baseline
binding pattern of the array.
[0047] In the delivery systems of the present invention, a small,
precisely metered amount of reactant solution is dispensed into
each cell. This can be accomplished by a variety of delivery
techniques. For example, conventional micropipetting apparati can
be adapted to dispense 5 nanoliter or smaller droplets from a
capillary. Such droplets can fit within a cell having a diameter of
300 .mu.m or less when a non-wetting mask of the invention is
employed. In another embodiment, the dispenser can be a
piezoelectric pump that generates charged droplets that can be
guided to the cell by an electric field as employed in conventional
ink-jet printers.
[0048] The reactant solutions in the individual cells must often be
prevented from moving to adjacent cells. This may be ensured by
providing an appropriate barrier between the various cells of the
substrate. For example, a hydrophobic material can be used to coat
the region surrounding the individual cells. Such materials prevent
aqueous (and certain other polar) solutions from moving to adjacent
cells. Of course, when non-aqueous or nonpolar solvents are
employed, different surface coatings will be required. By choosing
appropriate materials (substrates, hydrophobic coatings, and
reactant solvents), one can control the contact angle of the
droplet with respect to the substrate surface. Large contact angles
are desired because the area surrounding the cell remains unwetted
by the solution within the cell. In one embodiment of the
invention, the perimeters of the individual cells of an array
formed on a hydrophilic substrate are defined by selectively
irradiating a surface covered with photocleavable hydrophobic
protective groups. In the irradiated areas, the protective groups
are removed from the substrate to-form lipophilic cells.
[0049] In some embodiments, the cells can be further defined by
dimples in the substrate surface. This will be especially
advantageous when a head or other sensing device must contact or
glide along a substrate surface. The dimples can also act as
identification marks directing the dispenser to the cell of
interest.
[0050] The dispenser of the present invention can be aligned with
respect to the appropriate cells by a variety of conventional
systems. Such systems, which are widely used in the microelectronic
device fabrication and testing arts, can deliver droplets to
individual cells at rates at up to 3-10 drops per second. The
translational (X-Y) accuracy of such systems is well within 1-5
.mu.m. The position of the dispenser stage of such systems can be
calibrated with respect to the position of the substrate by a
variety of methods known in the art. For example, with only one or
two reference points on the substrate surface, a "dead reckoning"
method can be provided to locate each cell of the array. The
reference marks in any such systems can be accurately identified by
using capacitive, resistive or optical sensors. Alternatively, a
"vision" system employing a camera can be employed.
[0051] In another embodiment of the present invention, the
dispenser can be aligned with respect to the cell of interest by a
system analogous to that employed in magnetic and optical storage
media fields. For example, the cell in which the monomer is to be
deposited is identified by its track and sector location on the
disk. The dispenser is then moved to the appropriate track, while
the disk substrate rotates. When the appropriate cell is positioned
below the dispenser, a droplet of monomer solution is released.
[0052] III. Isolation of Reaction Areas
[0053] A. Dimples or recesses
[0054] If the substrates used in the present invention are to
contain dimples or other recesses, the dimples must be sufficiently
small to allow close packing on the substrate. Preferably, the
dimples will be less than 1 mm in diameter, more preferably less
than 500 .mu.m in diameter, and most preferably less than 300 am in
diameter. The depth of such dimples will preferably be less than
100 .mu.m and more preferably less than 25 .mu.m below the upper
surface of the substrate.
[0055] Dimples having these characteristics can be produced by a
variety of techniques including laser, pressing, or etching
techniques. For example, a suitable dimpled substrate surface can
be provided by pressing the substrate with an imprinted "master"
such as those commonly used to prepare compact optical disks. In
addition, an anisotropic etching technique employing
photolithography can be employed. In such techniques, a mask is
used to define the cell regions of the substrate. After the
substrate is irradiated through the mask, selected regions of the
photoresist are removed to define the arrangement of cells on the
substrate. The dimples may be cut into the substrate with standard
plasma or wet etching techniques. If the substrate is a glass or
silicon material, suitable wet etch materials can include hydrogen
fluoride or other common wet etchants used in the field of
semiconductor device fabrication. Suitable plasma etchants commonly
used in the semiconductor device fabrication field can also be
employed. Such plasma etchants include, for example, mixtures of
halogen containing gases and inert gases. Typically, a plasma etch
will produce dimples having a depth of less than 10 .mu.m, although
depths of up to 50 .mu.m can be obtained under some conditions.
[0056] Another method for preparing a suitably dimpled surface
employs photochemically etchable glass or polymer sheets. For
example, a photochemically etchable glass known as "FOTOFORM" is
available from Corning Glass Company (New York). Upon exposure to
radiation through a mask, the glass become soluble in aqueous
solutions. Thereafter, the exposed glass is simply washed with the
appropriate solution to form the dimpled surface. With this
material, well-defined dimples can be made having aspect ratios of
10 to 1 (depth to diameter) or greater and depths of up to 0.1
inches. Dimple diameters can be made as small as 25 .mu.m in a 250
.mu.m thick glass layer.
[0057] B. Controlling the Wetting Angle
[0058] Even when a dimpled surface is employed, it is often
important to ensure that the substrate material is not wetted
beyond the cell parameters. To ensure that the solutions in the
individual cells do not wet the surrounding surface and contaminate
other cells, various techniques can be applied to control the
physical interactions that affect wetting. Whether or not a liquid
droplet will wet a solid surface is governed by three tensions: the
surface tension at the liquid-air interface, the interfacial
tension at the solid-liquid interface and the surface tension at
the solid-air interface. If the sum of the liquid-air and
liquid-solid tensions is greater than the solid-air tension, the
liquid drop will form a bead (a phenomenon known as "lensing"). If,
on the other hand, the sum of the liquid-air and liquid-solid
tensions is less than the solid-air tension, the drop will not be
confined to a given location, but will instead spread over the
surface. Even if the surface tensions are such that the drop will
not spread over the surface, the contact or wetting angle (i.e.,
the angle between the edge of the drop and the solid substrate) may
be sufficiently small that the drop will cover a relatively large
area (possibly extending beyond the confines of a cell). Further,
small wetting angles can lead to formation of a thin (approximately
10 to 20 .ANG.) "precursor film" which spreads away from the liquid
bead. Larger wetting angles provide "taller" beads that take up
less surface area on the substrate and do not form precursor films.
Specifically, if the wetting angle is greater than about
90.degree., a precursor film will not form.
[0059] The contact angle is determined by the same three parameters
that determine whether a liquid will spread. Specifically, the
contact angle is given by the following expression, known as
Young's equation:
cos .theta.=(.theta..sub.sa-.theta..sub.sl)/.theta..sub.la
[0060] where .theta. is the wetting angle, .theta..sub.sa is the
solid-air tension, .theta..sub.sl is the solid-liquid tension, and
.theta..sub.la is the liquid-air surface tension.
[0061] The surface tensions that determine the wetting properties
of a liquid-solid interface are governed by thermodynamic
considerations including the chemical constituents of the liquid
and the solid substrate. The liquid-air surface tension for various
chemicals is easily measured by a variety of techniques such as
those described in Adamson, Physical Chemistry of Surfaces (John
Wiley and Sons, 5th Ed. (1990)), which is incorporated herein by
reference for all purposes. The solid-air tension is not determined
so easily. Nevertheless, the difference of the solid-liquid and
solid-air tensions can, for a given system, be determined
empirically from a Zisman plot. In this approach, the contact
angles are measured for a homologous series of liquids on a given
solid surface. For some liquid in the series, a "critical contact
angle" is observed, beyond which lower surface tension liquids wet
the surface. The liquid-air surface tension of the liquid at this
critical contact angle is assumed to be the surface tension of the
solid. This approach has been found to provide quite reasonable
results for low energy solids such as Teflon, polyethylene,
hydrocarbons, etc. The information gained from such studies can be
used to optimize substrate compositions to increase wetting angles
for a given reactant solution in the array.
[0062] The surface chemistry can be varied from position to
position on the substrate to control the surface free energy and,
hence, the contact angle of the reactant solution drops. In this
way, an array of cells can be defined on the substrate surface. For
example, if an aqueous reactant solution is used, the region inside
the cells can be hydrophilic, while the region surrounding the
cells can be hydrophobic. Methods for controlling the local surface
free energy of a substrate include a variety of techniques that
will be apparent to those skilled in the art.
[0063] In one method, various protecting groups are used to control
the chemical composition of the surface. For example, a mono-layer
of hydrophobic photoprotecting groups can be coupled to, for
example, linker molecules attached to the substrate surface. The
surface is then selectively irradiated (or otherwise activated)
through a mask to expose those regions where the cells are to be
located. This cleaves the protecting groups from the substrate
surface, causing the cell regions to be less hydrophobic than the
surrounding area. Because hydrophobic materials have lower surface
free energies (surface tensions) than water, the solution droplet
in the cell beads rather than spreads. Suitable hydrophobic
protecting groups for use with the present invention include, but
are not limited to, the alkyl silanes (e.g., octadecyl silane).
[0064] For some systems, other protecting groups will be
preferable. For instance, if a non-aqueous solution chemistry is
employed, the protecting groups can be tailored to increase the
wetting angle for the particular solution being used. Further, if
different solvents are to be contacted with the same cell during
polymer synthesis, the protecting groups surrounding that cell can
be changed during the course of the process by a VLSIPS.TM.
technique. This will ensure that no matter what type of solution is
added to the cell, a large wetting angle is obtained.
[0065] FIG. 1A shows a droplet 700 after it was deposited by a
micropipette on a non-wetting mask 702 above a substrate 704
according to the present invention. The aspect ratio shown (dome
height equals one-half the width) was empirically determined to be
stable using a non-wetting mask having a two millimeter by two
millimeter square area (the cell diameter was 0.2835 millimeter).
FIG. 1B illustrates a top view of a high-density droplet array 710.
The four cells of the array are shown at 712, 714, 716 and 718.
Because the cell array is defined with photolithographic techniques
and the hydrophobic mask is on the order of one monolayer thick,
the droplets can be packed very closely together.
[0066] IV. Moving The Dispenser With Respect To The Substrate
[0067] A. Mapping the Frame of Reference
[0068] To consistently deposit reactant droplets at precisely
specified locations, a common frame of reference between the
delivery instrument and the substrate is required. In other words,
the reference coordinates of the instrument must be accurately
mapped onto the reference coordinates of the substrate. Ideally,
only two reference points on the substrate are required to
completely map the array of cells. The dispenser instrument can
locate these reference points and then adjust its internal
reference coordinates to provide the necessary mapping. Of course,
in this situation, the dispenser instrument must provide precisely
repeatable movements. Further, the individual cells must not move
with respect to the reference marks on the substrate after the
reference marks have been formed. Unfortunately, pressing or other
mechanical operations commonly encountered during fabrication and
use of a substrate can warp the substrate such that the
correspondence between the reference marks and the cells is
altered.
[0069] To allow for this possibility, a substrate containing both
"global" and "local" reference marks can be employed. Only two
global reference marks are needed define the initial frame of
reference. From these marks, the dispenser instrument can determine
where each cell of the originally imprinted substrate is located.
In an initial, "course" adjustment, the dispenser is positioned in
the local area of the cell of interest. Once in the local region,
the dispensing instrument looks for local reference marks to define
a local frame of reference. From these, the dispenser is accurately
positioned over the cell of interest. In this manner, the effects
of warpage or other deformation can be minimized. The number of
local reference marks is determined by the amount of deformation
expected in the substrate. If the substrate is sufficiently rigid
that little or no deformation will occur, very few local reference
marks will be required. If substantial deformation is expected,
however, more local reference marks will be required.
[0070] Starting at a single reference point, the micropipette or
other dispenser can be translated to other cells of the substrate
by a correct distance in the correct direction (as noted above,
this is the "dead reckoning" navigational technique). Thus, the
dispenser can move from cell to cell, dispensing correctly metered
amounts of reactant. In order to initially locate the reference
point and align the dispenser directly over it, a vision or blind
system can be employed. In a vision system, a camera is rigidly
mounted to the dispenser nozzle. When the camera locates the
reference point(s), the dispenser is known to be a fixed distance
and direction away from the point, and a frame of reference is
established. Blind systems locate the reference point(s) by
capacitive, resistive, or optical techniques, for example. In one
example of an optical technique, a laser beam is transmitted
through or reflected from the substrate. When the beam encounters a
reference mark, a change in light intensity is detected by a
sensor. Capacitive and resistive techniques can be applied
similarly. A sensor registers a change in capacitance or
resistivity when a reference point is encountered.
[0071] For purposes of this invention, the spacing between the
individual cells preferably will be on the order of 10 .mu.m or
less. Further, the angular relation between the cells is preferably
consistent, to within 0.1 degrees. Of course, the photolithographic
or other process used to define the arrangement of cells will
accurately define the angle and spacing. However, in subsequent
processes (e.g., pressing processes), the angle can be distorted.
Thus, in some embodiments, it may be necessary to employ "local"
reference points throughout the array.
[0072] Translational mechanisms capable of moving with the desired
precision are preferably equipped with position feedback mechanisms
(encoders) of the type used in devices for semiconductor device
manufacturing and testing. Such mechanisms will preferably be
closed loop systems with insignificant backlash and hysteresis. In
preferred embodiments, the translation mechanism will have a high
resolution, i.e. greater than five motor ticks per encoder count.
Further, the electromechanical mechanism will preferably have a
high repeatability relative to the cell diameter travel distance
(preferably .+-.1-5 .mu.m).
[0073] To accurately deposit a drop of reactant solution on the
substrate, the dispenser nozzle must be placed a correct distance
above the surface. For a drop having a volume of approximately five
nanoliters, the dispenser tip preferably will be located about 5-50
.mu.m above the substrate surface when the drop is released. More
preferably, the drop will be about 10 .mu.m above the substrate
surface when the drop is released. The degree of control necessary
to achieve such accuracy can be attained with a repeatable
high-resolution translation mechanism of the type described above.
In one embodiment, the height above the substrate is determined by
moving the dispenser toward the substrate in small increments,
until the dispenser tip touches the substrate. At this point, the
dispenser is moved away from the surface a fixed number of
increments which corresponds to a specific distance. From there,
the drop is released to the cell below. Preferably, the increments
in which the dispenser moves will be less than about 5 .mu.m and
more preferably less than about 2 .mu.m.
[0074] In an alternative embodiment, the dispenser nozzle is
encircled by a sheath that rigidly extends a fixed distance beyond
the dispenser tip. Preferably, this distance corresponds to the
distance at which the solution drop will be most easily delivered
to the cell. Thus, when the sheath contacts the substrate surface,
the movement of the dispenser is halted and the drop is released.
It is not necessary in this embodiment to move the dispenser back,
away from the substrate, after contact is made. In this embodiment,
as well as the previous embodiment, the point of contact with the
surface can be determined by a variety of techniques such as by
monitoring the capacitance or resistance between the tip of the
dispenser (or sheath) and the substrate below. A rapid change in
either of these properties is observed upon contact with the
surface.
[0075] B. Rotational Mechanisms
[0076] In some embodiments, the substrate rotates on an axis and
the dispenser moves radially on a line from the substrate axis to
the substrate perimeter. These embodiments employ mechanisms
analogous to those encountered in the computing and recording arts.
In magnetic media, a rigid or floppy disk contains magnetized and
unmagnetized regions that correspond to bits of data. In some
instances, the direction of magnetization corresponds to the bits
of data. The magnetization of these bits can be changed by moving a
"recording head" close to the bit. The recording head generates
magnetic flux that will either magnetize, demagnetize, or leave
unchanged a selected region on the disk. Thus, by carefully
controlling both the magnetic flux on a write head and the relative
position of the magnetic disk with respect to the write head, data
can be recorded at preselected locations on the disk surface.
Further, by using standard formats for subdividing and labelling
the disk, recorded data can be rapidly located and retrieved by a
"read head."
[0077] Two widely used types of magnetic disk are the floppy disk
typically made from a mylar film and the hard disk typically made
from a rigid material such as an aluminum disk. Both disks require
very close correspondence between the read/write head and the
recording media (i.e. the disk). In a floppy disk, the read and
write head actually touches a magnetic disk, and must be lubricated
to avoid excessive wear. In contrast, a hard disk read/write head
never touches the surface, but rather "flys" at submicron distances
above the hard disk surface on a hydrodynamic bearing surface. The
hydrodynamic bearing is created by the rapid rotation of the disk
(typically, about 3600 rpm) and overcomes the force of gravity,
thus preventing the head from "crashing" onto the disk. In this
way, disk wear is minimized and the head can be rapidly moved
across the disk surface.
[0078] Large disk memories employ a stack of hard disks coated on
both sides with the magnetic material and rotating together on a
common spindle. A set of read/write heads is used: two are required
for each plate. Typically, large disk memory will have an access
time of about 20 milliseconds.
[0079] In optical media, a beam of laser light is used to either
read from or write to a disk. When the read laser beam is passed
over the surface of the disk, it normally encounters the flat
unrecorded regions of the surface and is reflected back in the
direction from which it originated. A sensor of a form well-known
in the art monitors the reflected light to detect recorded
information on the disk surface. Most prerecorded optical disks
(commonly known as "compact disks") have a series of very small
pits cut into the disk surface. Typically, the pit size can be as
small as about 1 .mu.m. When the laser beam passes over a pit, the
path length of the reflected beam increases. At the edge of the
pit, the beam will be divided into portions having different path
lengths which can destructively interfere with one another. This
change in intensity is interpreted as recorded information that can
be interpreted by appropriate processing circuitry. Preferably, the
pit depth will be one quarter of the wavelength of the read laser
light. Thus, the total change in path length of a laser beam
passing over a pit will be one half wavelength, a distance
corresponding with the maximum possible destructive
interference.
[0080] Information can be recorded on an optical disk by using a
laser beam to change the optical properties of the disk surface.
For example, the laser beam can ablate small regions of the surface
to create pits. Other means of recording information include,
heating regions of the substrate above the Curie point and,
thereafter, changing the magnetization of the region, and heating
regions of the substrate to create readable bumps such as described
in Marchant, Optical Recording, Addison-Wesley (1990), incorporated
herein by reference in its entirety for all purposes. Other methods
of optical recording suitable for use with the present invention
will be known to those of skill in the art.
[0081] FIG. 2A illustrates one example of an electro-optical system
for recording data on a data storage medium in accordance with
various embodiments of the present invention. The recording system
includes a digital data processing circuit 30 whose output on line
31 controls the pulsed variable-intensity laser 32. The laser beam
33 emerging from the laser 32 is collimated by a lens 34 and then
reflected by a mirror 35. The reflected beam from the mirror is
propagated through a beam splitter 36.
[0082] The laser beam emerging from the beam splitter 36 is passed
through a filter 37, which can be a quarter-wavelength plate, and
then propagated through an objective lens 38 which focuses the
laser beam on the moving optical data storage medium 39. Light
reflected back from the medium 39 is collected by the lens 38 and
propagated through the filter 39 to the beam splitter 36, which
propagates the reflected light to a light sensor 40.
[0083] The laser 32 is preferably a high-power laser (2-15 mW at
the media surface) and is either continuous or pulsed. The
wavelength of the laser beam 33 is the "write" or "record"
wavelength, and is either continuous, shaped, or pulsed. The write
beam typically enters the medium at the substrate side, as shown in
FIG. 2B, and passes through a transparent substrate 4 into an
expansion layer 6. The expansion layer, which is absorptive of
light at the laser wavelength, rises in temperature due to the
absorption, but is kept from localized expansion by the rigid
substrate 4 and a retention layer 8 (which is in its glassy state).
Expansion pressure thus builds up and the retention layer begins to
deform in a broad manner. Meanwhile, the temperature of the
retention layer rises by conduction from the expansion layer, and
possibly by light absorption as well. As the temperature of the
retention layer increases, it approaches the glass transition
temperature and a small weak area is formed around the axis of the
incident beam. The expansion layer then flows into this weak area
allowing expansion to be localized, thereby creating a well-defined
bulge or bump 18. The retention layer 8 deforms accordingly to
follow the contour of the bulge, and protrude into a soft
reflective layer 10. When the laser is turned off, the various
layers cool. The reflective layer 10 acts as a heat sink rapidly
drawing heat away from the retention layer 8, and the retention
layer 8 cools down below its glass transition temperature,
increasing its shear modulus to lock in the deformation while the
expansion layer 6 is still in its expanded state.
[0084] Erasure is achieved by using a laser beam of a different
wavelength, one which is absorbed primarily by the retention layer
8. The expansion layer 6 may also be absorptive at this wavelength,
to some degree, provided that the resulting temperature increase in
the expansion layer is not great enough to record a mark.
Absorption of the light from this beam by the retention layer will
raise it to its rubbery state, at which point elastic forces in the
expansion layer as well as the viscoelastic properties in the
retention layer will draw the retention layer back to a bump-free
configuration. Reflective layer 10 will naturally flow back into
the void left by the retention layer.
[0085] In both magnetic and optical storage media, information must
be quickly accessed. As mentioned, a diskette typically spins at
about 300 RPM and a hard disk typically spins at about 3600 RPM.
The actuator that moves the recording/reading head across the disk
radius is also fast: for a diskette, it takes an average of about
one-sixth of a second to move to any radial position on the disk,
and for a hard disk, it takes only about {fraction (1/25)}th of a
second.
[0086] In computer operating systems such as DOS, particular data
such as program applications (e.g. spreadsheets, databases, etc.)
and data files are stored as binary magnetic information at various
locations on the surface of the disk. This information is organized
on a series of concentric circles or "tracks." Hard disks have
hundreds of such tracks, each of which is identified by a number,
starting with track zero at the outer edge of the disk. Each track
is divided into various "sectors" containing a predefined number of
bytes. Like the tracks, the sectors are also numbered, starting
with one (sector zero is reserved for identification rather than
data storage). Thus, any particular piece of data can be located by
defining the sector (or at least the first sector if the data file
is large) on which it is recorded. By analogy to the above
discussion on frames of reference, the tracks and sectors can be
viewed as local reference marks.
[0087] On DOS formatted storage disks, the locations of the
recorded data files are stored on a small "system area" of the
disk. Specifically, the list of sectors on which a given data file
resides is recorded in the system area of a DOS formatted storage
disk. Thus, by inputting a file name, the system area of the disk
can identify the location of the file on the magnetic disk. Steps
can then be taken to move the read/write head to that location to
access the file.
[0088] Like conventional data storage disks, the disks used to hold
the compound arrays of the present invention can provide "system"
information (i.e., tracking/location information) prerecorded on a
blank disk. However, unlike conventional storage disks, the disks
of the present invention also have "cells" in which the individual
compounds of the matrix are synthesized or stored. These cells can
be interspersed among the information tracks (including both system
and data regions) in a variety of formats, as will be explained in
the example below. The cells can take the form of dimples on the
disk, especially if information is stored on magnetic media.
However, other forms of cells can be used if the individual
solution drops can be adequately isolated from each other. Using
these and similar disk designs, the user can record and quickly
access the location and cells by techniques well-known in the
art.
[0089] Alternatively, the monomer (or other compound) solutions can
be distributed from a closely packed array of capillaries, or from
a slit-shaped container covered with a conductive screen having
holes small enough to preclude leakage due to gravity alone. In
these embodiments, the individual dispensers will have a limited
range of movement over the disk radius and may even be fixed in
space. In any of the above apparati, electrodes can be provided in
the individual nozzles to provide electrical contact to the monomer
solutions.
[0090] A preferred precision placement dispenser is shown in FIGS.
3A-3D. FIG. 3C is a side view of cartridge 100 which contains
several capillaries 102 filled with various monomers. Electrodes
(not shown) run down the center of each capillary 102. FIG. 3D is a
top view of cartridge 100, showing an arrangement of leads 115 to
the electrodes. The cartridges are loaded into a delivery plate 104
such that terminals 106 on the cartridge ends mate with terminals
108 on a spindle 110 as shown in top view FIG. 3B. Thus, a central
power supply can control all the electrodes. The delivery plate 104
rotates above the substrate 112 as shown in FIG. 3A. Substrate 112
will, in many embodiments, be made from a conductive material held
at ground, or a non-insulating material covering a plate held at
ground. A servo motor 114 rotates the delivery plate 104 and the
power supply selectively charges the electrodes so that drops of
desired monomer will cross from the dispenser to the desired cells
on substrate 112. If the basis set of monomers is sufficiently
small, the capillaries may be held in fixed positions, relying on
rotation of the substrate and electric fields to guide the charged
droplets to the cells.
[0091] V. Delivering the Reactant Solution
[0092] As explained above, commercially available micropipettes are
a preferred delivery mechanism for the present invention.
Commercially available micropipettes, such as the A203XVY Nanoliter
injector, are able to reproducibly dispense 4.6 nanolter droplets.
In some embodiments, the micropipette is accurately and precisely
positioned above the cell, as described above, before the reactant
solution is deposited.
[0093] In a different preferred embodiment, the reactant solutions
will be delivered from a reservoir to the substrate by an
electrophoretic pump. In such a device, a thin capillary connects a
reservoir of the reactant with the nozzle of the dispenser. At both
ends of the capillary, electrodes are present to provide a
potential difference. As is known in the art, the speed at which a
chemical species travels in a potential gradient of an
electrophoretic medium is governed by a variety of physical
properties, including the charge density, size, and shape of the
species being transported, as well as the physical and chemical
properties of the transport medium itself. Under the proper
conditions of potential gradient, capillary dimensions, and
transport medium rheology, a hydrodynamic flow will be set up
within the capillary. Thus, bulk fluid containing the reactant of
interest can be pumped from a reservoir to the substrate. By
adjusting the appropnate position of the substrate with respect to
the electrophoretic pump nozzle, the reactant solution can be
precisely delivered to predefined cells.
[0094] In one particularly useful embodiment, the electrophoretic
pump can be used to produce an array containing various fractions
of an unknown reactant solution. For example, an extract from a
biological material, such as a leaf or cell culture, might contain
various unknown materials, including receptors, ligands, alkaloids,
nucleic acids, and even biological cells, some of which may have a
desired activity. If a reservoir of such extract is pumped
electrophoretically, the various species contained therein will
move through the capillary at different rates. Of course, the
various components being pumped should have some charge so that
they can be separated. If the substrate is moved with respect to
the dispenser while the extract components are being separated
electrophoretically, an array containing various independent
species will be produced. This array can then be tested for
activity in a binding assay or other appropriate test. Those
elements of the array that show promising activity will be
correlated with a fraction of the extract which can be isolated
from another source for further study. In some embodiments, the
components in the extract solution can be tagged with, for example,
a fluorescent label. Then, during the process of delivering the
solution with the electrophoredc pump, a fluorescence detector can
determine when labeled species are being deposited on the
substrate. In some embodiments, the tag will selectively bind to
certain types of compounds within the extract, and impart a charge
to those compounds.
[0095] In other embodiments, the present invention employs a
solution depositing apparatus that resembles devices commonly
employed in the ink-jet printing field. In fact, some ink-jet
printers can be used with minor modification by simply substituting
a monomer containing solution for ink. As mentioned, Wong, et al.,
European Patent Application 260 965, incorporated herein by
reference for all purposes, describes the use of a commercial
printer to apply an antibody to a solid matrix. In the process, a
solution containing the antibody is forced through a small bore
nozzle that is vibrating in a manner that fragments the solution
into discrete droplets. The droplets are subsequently charged by
passing through an electric field and then deflected onto the
matrix material.
[0096] A conventional ink drop printer includes a reservoir in
which ink is held under pressure. The ink reservoir feeds a pipe
which is connected to a nozzle. An electromechanical transducer is
employed to vibrate the nozzle at some suitable high frequency. The
actual structure of the nozzle may have a number of different
constructions, including a drawn glass tube which is vibrated by an
external transducer, or a metal tube vibrated by an external
transducer (e.g. a piezoelectric crystal) or a magnetostrictive
metal tube which is magnetostrictively vibrated. Accordingly, the
ink is ejected from the nozzle in a stream which shortly thereafter
breaks into individual drops. An electrode may be present near the
nozzle to impart a charge to the droplets.
[0097] Because these drops are to be charged and thereafter
deflected by electrical signals, it is desirable to make these
drops be as uniform in size as possible. It is also desirable to
form these drops with a close spacing, because closer spacings
result in better resolution. Also, it is desirable to form the
drops into a small size so that the amplitude of the signals
required to deflect these drops should not be excessive.
[0098] A schematic drawing of an ink drop dispenser (such as is
described in U.S. Pat. Nos. 3,281,860 and 4,121,222, which are
incorporated by reference herein for all purposes) which can be
employed in the present invention is shown in FIG. 4. This
apparatus comprises a reservoir 210 which contains a solution under
pressure. Tubing 212 is connected to the reservoir 210 and
terminates in a metal nozzle 242. Nozzle 242 is disposed within a
hole provided in piezoelectric crystal 240. The end of the metal
tube and of the piezoelectric crystal are made to coincide. The
tubing and the piezoelectric crystal are soldered together to form
a permanent waterproof attachment. The coincident ends of the
crystal and the tubing are covered with a washer 244 which is
termed an orifice washer. This washer has an opening 246 drilled
therethrough through which the solution is emitted under pressure.
A source of oscillations 218 is connected between the outside of
the metal tubing 242 and the outside of the piezoelectric crystal
240. The construction is such that hermetic sealing can be employed
which protects against electrochemical and atmospheric attack of
the components.
[0099] The piezoelectric crystal 240 is vibrated substantially at
the frequency of the source of oscillations causing the tubing and
nozzle to vibrate whereby the solution stream breaks down into
droplets 246. A signal source 224 which is synchronized by the
source of oscillations is connected between the nozzle and the
charging cylinder 226. As a result, each of the drops, which should
be substantially the same mass, receives a charge, the amplitude of
which is determined by the amplitude of the signal applied from the
source 224 and the charging cylinder 226.
[0100] The charged drops, after passing through the charging
cylinder, pass into an electric field which is established between
two plates respectively 230 and 232 which are connected to a field
potential source 234. As a result of the action between the field
and the charge of each drop, the drops are deflected from their
center line path between the plates in accordance with the charge
which they carry. Thus, when they fall on an optionally moving
writing medium 236, a deposition pattern occurs on the writing
medium representative of the information in the signals.
[0101] Other suitable delivery means include osmotic pumps and cell
(biological) sorters. An osmotic pump will deliver a steady flow of
solution for a relatively long period. The construction of such
pumps is well-known in the art, generally incorporating a solution
of the extract of interest within a solvent permeable bag. Osmotic
pressure is applied to the extract solution by solvent molecules
diffusing across the bag to equalize a concentration difference.
The extract is thus forced out of a nozzle in the bag at a constant
rate. Cell sorters are also well-known in the art, and can be used
in applications wherein it is desirable to apply single biological
cells to distinct locations on the substrate.
[0102] Although the above embodiments have been directed to.
systems employing liquid droplets, minuscule aliquots of each test
substance can also be delivered to the cell as miniature pellets.
Such pellets can be formed from the compound of interest (e.g.
ligands for use in an affinity assay) and one or more kinds of
inert binding material. The composition of such binders and methods
for the preparation of the "pellets" will be apparent to those of
skill in the art. Such "mini-pellets" will be compatible with a
wide variety of test substances, stable for long periods of time,
suitable for easy withdrawal from the storage vessel and dispensing
(i.e., non-tacky, preferably suspendable in a liquid such as
physiological buffer), and inert with respect to the binding
activity of receptors.
[0103] VI. Conducting the Reactions
[0104] Methods for synthesizing desired polymer sequences such as
peptide sequences are well known to those of skill in the art. For
example, the so-called "Merrifield" solid-phase peptide synthesis
has been in common use for several years and is described in
Merrifield, J. Am. Chem Soc. (1963) 85:2149-2154 and Atherton,
Solid Phase Peptide Synthesis, IRL Press, Oxford (1989),
incorporated herein by reference for all purposes. Methods of
synthesizing oligonucleotides are found in, for example,
Oligonucleotide Synthesis: A Practical Approach, Gait, ed., IRL
Press, Oxford (1984), incorporated herein by reference in its
entirety for all purposes.
[0105] The preferred processes involve steps of first binding a
"protected" monomer to a solid substrate such as glass, polymer or
other non-swellable, insoluble, and otherwise reaction-compatible
materials. By immobilizing the growing polymer, one simplifies
subsequent purification procedures to washing steps, rather than
the complicated recrystalliation steps traditionally used. The
monomers are protected by attaching chemical groups on a reactive
terminus of the monomer so that the immobilized monomer--which has
another reactive terminus coupled to the substrate--cannot further
react with other dissolved monomers to form dimers. Merrifield
originally used carbobenzoxy groups to protect the amine terminus
of amino acid monomers from further reaction. After the first
monomer was immobilized and washed free of dissolved monomer
species, the carbobenzoxy groups were cleaved from the monomer by a
mixture of hydrogen bromide in glacial acetic acid. Of course,
other protective groups and cleavage mixtures can be used for
particular monomers as is well-known in the art. For example,
tert-butyloxycarbonyl-protected amino groups, cleavable with
trifluoroacetic acid treatment can also be used in peptide
synthesis. Additional .alpha.-amino protecting groups include acyl
type protecting groups (e.g., formyl, trifluoroacetyl, acetyl),
aromatic urethane type protecting groups (e.g., benzyloxycarboyl
(Cbz) and substituted Cbz), aliphatic urethane protecting groups
(e.g., isopropyloxycarbonyl, cyclohexyloxycarbonyl) and alkyl type
protecting groups (e.g., benzyl, triphenylmethyl).
[0106] Alternatively, the protecting groups on individual monomers
can be photocleavable. Various suitable photoprotecting groups are
disclosed in, for example, Fodor, et al., published PCT Application
No. W092/10092. In this embodiment, the entire substrate or
selected cells can be exposed to radiation for activation. This
approach is especially advantageous when the disk information is
provided in an optically readable format. The read/write head in
such a system can be used to direct light radiation to selectively
activate various cells by deprotecting the monomers therein.
[0107] After the first monomer in a polymer sequence has been
deprotected, a solution of the second monomer (with one reactive
terminus protected) may be reacted with the immobilized first
monomer. One well-known coupling reaction employs
N,N'-dicyclohexylcarbodiimide in dimethylformrnamide solvent. Other
coupling methods can be employed depending upon monomers used, -as
is well-known in the art. Subsequent washing, deprotection, and
reaction steps will generate a polymer of the desired sequence. The
polymer, in an array of many different polymers, can then be used
in, for example, screening assays. Alternatively, individual
polymers can be cleaved from the substrate and used elsewhere.
[0108] It may, of course, be necessary to "wash" certain cells of
the array to remove reactant solutions between synthesis cycles.
Such steps can be accomplished by removing the disk from the
synthesizer and immersing it in a cleaning or other solution.
Alternatively, the disk can be left on the platter in the
synthesizer and a stream of fluid passed over it to clean off
unreacted species. In other embodiments, a high pressure injector
is used to "randomly access" cells and to wash off residues or left
over solutions. In still other embodiments, a charged plate is
lowered over the substrate surface to draw off the solutions. This
is particularly advantageous when the reactant droplets were
deposited by a process in which they were charged, as by a corona
discharge or an electrophoretic pump. Because some residual charge
will remain on the droplets when they are present in the cells, a
charged plate of polarity opposite to that of the drops will draw
them off of the substrate. This approach allows the substrate to be
cleaned without removing it from the compartment in which the
reactions take place.
[0109] Evaporation should be minimized, especially if the reagents
used have low vapor pressures and the substrate or dispenser is
spinning at sufficiently high rates of speed to cause convection at
the surface of liquid drops. If the drops are sufficiently small,
evaporation can cause the reactant concentration to increase and
ultimately cause precipitation. The effects of evaporation can be
minimized by seaing selected regions of the disk when they need not
be accessible. Alternatively, the partial pressure of volatile
reagents can be controlled to equalize the liquid and vapor phase
fugacities so that there is a reduced thermodynamic driving force
for evaporation. The partial pressure of the reagents may be
increased by providing a relatively large reservoir of volatile
reagents in a sealed chamber. Alternatively, solvents having a low
vapor pressure under the conditions of interest can be used. In
some cases, evaporation can be further controlled by application of
a low vapor-pressure film or coverplate having a reverse cell
pattern Other methods of preventing evaporation are well-known in
the physical chemical arts and may be used in the present
invention.
[0110] VII. Example
[0111] A. Appartus
[0112] This example and the following discussion are directed to
the polymer synthesis embodiments of the present invention. This is
not meant to suggest that the invention is to be used solely for
synthesizing arrays of polymers. As described above, the methods
and apparatus of the present invention can be applied with equal
utility to other chemical and biological systems. For instance, in
preparing and screening arrays of existing compounds, one can use
methods and instruments similar to those discussed below for making
and screening new compounds. Moreover, the following example is
directed to a rotating disk substrate in which reference
information is encoded on the disk. As explained above, other
methods, such as those employing rectangular substrates with global
and local reference marks, can be employed. Rectangular or
spherical navigational coordinates can also be used.
[0113] FIG. 5 illustrates an example of the invention in which
various monomers are to be bound at selected cells of the
substrate. The substrate may be biological, nonbiological, organic,
inorganic, or a combination of any of these, existing as gels,
sheets, tubing, containers, capillaries, pads, slices, films,
plates, slides, etc. It is preferably flat, but may take on a
variety of alternative surface configurations. For example, the
substrate may contain raised or depressed regions on which the
synthesis takes place.
[0114] In this embodiment, the substrate contains a magnetic or
optical storage media coating on its surface at defined regions
where digital information is stored. Of course, the entire
substrate can be made from such storage media, so long as the
material is compatible with the desired polymer synthesis. If a
magnetic storage media is employed, the read/write head typically
rides directly on or near the surface of the substrate. Thus, the
cells are preferably located such that the polymers and oligomers
are not rubbed away by the contact between the substrate and the
recording head. One way to accomplish this is by providing dimples
on the substrate surface to serve as cells. Thus, as the magnetic
head rides over the surface, it does not shear off the polymers in
the cells. If information is encoded on an optical storage media,
the read/write head does not contact the surface, and dimpled cells
are therefore optional.
[0115] Referring now to the synthesizer shown in FIG. 5, a disk
substrate 301 includes a number of cell tracks 302 and encoded
information tracks 304. The actual polymers of the array are
synthesized in the individual cells on the cell tracks. The disk is
mounted on a spindle 307 and a platter 306 which is rotated by a
motor 308 at variable speds. The individual monomer solutions are
provided to the cells as droplets from dispenser 310. As explained
above in connection with ink drop dispensers, the droplets can be
charged by an electrode or conductive device inside the dispenser
or dispenser nozzle. An attachment to the dispenser head can
provide variable direction electric fields to guide the charged
droplets to the appropriate cells. The dispenser itself can be a
piezoelectric pump or other device capable of producing fine
droplets of controllable size and spacing, as discussed above in
connection with the ink drop dispenser. Alternatively, the droplets
can be provided as neutral drops from a standard micropipette.
[0116] The dispenser is located at the end of an arm 312 capable of
having its radial position controlled by an actuator 314 such as a
voice coil actuator or other translation control device commonly
used in the optical or magnetic disk storage technologies. In
addition, the dispenser can have a plurality of capillary nozzles
316 for delivering individual monomer solutions. These nozzles can
be movable along a track (not shown) on arm 312 or can be fixed on
the arm, relying on translation by actuator 314 to reach the proper
radial position.
[0117] The various monomer solutions are supplied from reservoirs
318 to the dispenser and nozzles through lines 322. The reservoirs
can be pressurized to ensure adequate delivery rates of the monomer
solutions to the dispenser. Typically, a control valve is used to
ensure that the monomers are dispensed only when required. Other
feed systems can also be used. For example, if reservoirs 318 are
disposed above the dispenser, a gravity feed system can be used to
ensure adequate delivery rates. In some embodiments, electric
fields between the disk 301 and the dispenser 310 are used to
"pull" the droplets of monomer solution out of capillary nozzles
316.
[0118] The pertinent commands directing the radial translation of
arm 312, the rotation of disk 301, and the dispensing of
appropriate monomer solutions are controlled by processor 320.
Processor 320 can be a computer or workstation, such as an IBM PC
or compatible system, an Apple MacIntosh, a Sun Microsystems SPARC
station, or any other conventional system. In addition, it can be a
dedicated microprocessor or hardware logic designed specifically to
control the operation of the synthesizer. Processor 320 can also
obtain data from and/or store data in internal or external storage
devices such as RAM or cache memories, hard disk drives, CD ROMs,
or other well known memory devices. Alternatively, data can be
obtained from or written to disk 301 through read/write head 324.
In one embodiment, head 324 senses information stored on
information tracks 304, converts that information to electrical
signals which are then interpreted by processor 320. Read/write
head 324 utilizes standard principles used in magnetic and optical
recording heads, as outlined above. The position of head 324 is
controlled by an actuator 327 which controls the radial position of
arm 325.
[0119] Although dispenser 310 and read/write head 324 are shown
opposite one another in FIG. 5, it is desirable in some embodiments
to mount them on the same arm or on arms proximate to one another.
This is especially advantageous when the information governing the
deposition in certain cell tracks 302 is recorded on information
tracks 304 adjacent to the cell tracks where deposition is to take
place. The information in tracks 304 can be slightly ahead of the
"phase" of the cells in cell tracks 302, as will be described
below.
[0120] In defining a protocol for rapid delivery to a variety of
sectors, it is advantageous to deposit one monomer solution on
certain cell tracks in succession. For example, when the first set
of monomers is being deposited, monomer A is deposited in every
cell of track 3, sector 7, or at least some fraction of the cells
in sector 7. Of course, to obtain a maximally diverse array, this
approach cannot be used at every stage of the deposition process,
but it does help build some local redundancy into the system. The
local redundancy in the polymer sequences has other advantages as
explained below.
[0121] B. Locating Desired Cells
[0122] Two preferred types of information processing can be used in
the practice of the present invention. The first relates to
procedures for depositing the specific monomers at specific
locations on the disk. This includes instructions directing the
movement of the dispensers and the disk during deposition so that
the desired array of polymers is synthesized. Thus, for instance,
when the first monomers of the polymer are being deposited,
instructions are provided to deposit alanine at track x, sectors 3,
4, and 6, track y, sectors 1, 2, and 3, track z, sectors 2, 4, 7,
and 8, etc. In many instances, it will also be necessary to specify
specific cells within the sector.
[0123] The second type of information processing relates to the
monomer sequence listing, or identity, of the polymer associated
with each cell. Related information such as the physical properties
(polarity, degree of branching, etc.) and the binding constants for
certain ligands can also be recorded for each cell, as well as
other pertinent information useful and apparent to those of skill
in the art. Preferably, the information is digitally encoded and
presented in a format that can be read and written by conventional
magnetic or optical heads. Desired characteristics of such heads
have been described above.
[0124] The information can be recorded and stored in various
locations. For example, completely prerecorded disks can be used.
These will have both major types of information (the deposition
protocol and the monomer sequence listing associated with each
cell) recorded on a "blank" disk (i.e., a disk on which no monomers
have yet been deposited). The synthesizer reads the deposition
protocol from the disk and directs the monomer dispensers to the
correct cells at correct time, ultimately synthesizing the desired
array of polymers. The blank disk also has a listing of the monomer
sequence for each cell so that cells of interest can be quickly
identified. Alternatively, the blank disks can have only one type
of information recorded. For instance, the deposition protocol can
be stored elsewhere in the synthesizer, such as in a mass storage
device, RAM, or a cache memory. Thus, the blank disk does not have
to be read to determine the deposition protocol, allowing
additional flexibility in specifying what polymers are to be
synthesized. Contemporaneously with the deposition and polymer
synthesis, the sequence listing in each cell can be written on the
disk. In this way, the finished disk has a complete listing of the
deposited polymer sequences and their locations.
[0125] Of course, it is also possible that the disk contains no
monomer sequence listing or deposition protocol information. This
information can be stored elsewhere in the synthesizer or archived
on removable storage media. However, it will typically be
preferable to keep at least the sequence listing information on the
completed disk to reduce the risk of losing the information.
[0126] If information is to be recorded on the disk, it can be
stored at any of several possible locations. On optical disks, the
bit size is on the order of 1 .mu.m, whereas the preferred cell
size is about 100 .mu.m. Thus, the pertinent information can be
stored on thin tracks readable by the read/write head, while the
cells can be located on larger tracks ("cell tracks"), parallel
with the information tracks. The two types of tracks can form
concentric circles (as in optical and magnetic disks), or they can
form continuous spirals (as is possible in optical disks).
[0127] Referring to FIG. 6, various arrangements of information on
the disk are provided. Information tracks are denoted as dotted
lines, while the cell tracks are denoted by a series of circles.
For example, when the information and cell tracks are concentric
circles, all the information tracks can be located together near
the inner perimeter of the disk as shown in FIG. 6A or near the
outer perimeter of the disk as shown in FIG. 6B. Also, the
information and cell tracks can alternate across the radius of the
disk as shown in FIG. 6C. If no information is provided on the
disk, the disk can appear as shown in FIG. 4D.
[0128] The information provided in these arrangements can be used
in a variety of manners. For instance, if the alternating track
format of FIG. 6C is employed, the read/write head can move to a
first information track, read the deposition protocol for the first
cell track, thereafter move to the first cell track, and deposit
appropriate monomers in the cells as prescribed by the first
information track. Thus, a first layer of monomers can be deposited
by moving the dispensers and read write head radially across the
surface of the disk. These first monomers can then be reacted, and
the process repeated for the second layer of monomers. After a
sufficient number of these track-by-track read and deposition
cycles are completed, the polymer array is completed. During this
process, the sequence listing information for each cell can also be
recorded, either on a blank track provided for that purpose or over
the deposition protocol track containing information that has
already been read (and is therefore no longer necessary). If the
deposition protocol information is overwritten by the monomer
sequence information, more space will be available on the disk for
cells.
[0129] The above discussion has provided examples in which the
information and cells were provided on different tracks, but they
can be provided on the same track as shown in FIG. 6E. In this
approach, less information needs to be read, stored, and
interpreted between the successive deposition steps.
[0130] The arrangements shown in FIGS. 6A-6E can easily be adapted
for use in a spiral rather than concentric format. If the
read/write head and the dispenser travel together along the spiral,
the deposition information can be read by the read/write head and
almost immediately translated into deposition instructions for the
dispenser. Of course, a short "phase lag" between the information
track and the cell track is necessary to account for the
information processing time. The lag can be provided by simply
locating the cells slightly upstream from their corresponding
deposition information. With this format, it is possible to quickly
guide the dispenser for a single monomer along a track, stopping or
slowing, if necessary, to deposit the monomer in the desired cells.
This process can be repeated for each monomer dispenser, until all
the cells are filled. After the polymerization reactions are
conducted, the deposition process for the next layer of monomers is
then started. Alternatively, each of several monomer dispensers can
be used to deposit all of the necessary monomers during one
traverse of the spiral track.
[0131] Disk operating systems have been designed to use the
concentric format of magnetic disks. Therefore, a concentric
optical disk format is preferable if the system is to be compatible
with existing disk memories. The concentric format is also
desirable when frequent multiple passes over the same track region
are necessary. However, a spiral format requires less track
jumping, thus speeding up the read/write process.
[0132] The disk tracks employed in the present invention can be
divided into cell and information "sectors" analogous to the
sectors in conventional magnetic and optical storage media. This
allows the recorded material to be provided in standard size chunks
that can be understood by conventional information processors.
Further, in standard formats, sectors contain "header" information
that allows the synthesizer to rapidly gain its "bearings" as it
jumps from track to track seeking arbitrary cells or pieces of
recorded information. The function and design of such headers is
well-known in the optical and magnetic storage media arts, and
discussed in, for example, Marchant, Optical Recording, Chapter 10,
Addison-Wesley (1990), which was previously incorporated herein by
reference for all purposes.
[0133] The amount of recording space needed to describe the monomer
sequence of the polymer can be provided with a few bytes of data.
The precise amount of data required is a function of the numbers of
monomers in the longest polymer and the number of monomers in the
basis set used to construct the polymer. If the basis set includes
fewer than 256 monomers, a single byte can be used to identify each
monomer in the polymer. If the basis set is larger, two bytes will
serve to distinguish 65,536 different monomers. Of course, the
entire sequence for each cell can be saved on the storage media,
but this can be inefficient. In most instances, all sequences of a
given sector or other unit are synthesized with some degree of
redundancy, so that less data must be recorded for each cell.
-Thus, the header for a given sector might indicate that all cells
within the sector contain specific isomers or derivatives of
glucose, mannose, and ribose at the 2, 3, and 7 polymer positions,
respectively. As described above, it is often efficient to use a
deposition protocol in which a single monomer is deposited in a
group of neighboring cells. Thus, it is expected that the polymer
sets in many sectors will have the redundancy necessary to take
advantage of the above recording strategy.
[0134] C. Accessing the Selected Cells
[0135] The synthesizer in this embodiment preferably employs one or
more computerized servo motor systems to orient a given cell under
the dispenser for a specified monomer, and to keep track of which
monomers are applied to each cell. The read/write and dispenser
head described above provides one structure for this purpose. An
aliquot of the specified first monomer can be applied to cells 1,
101, 201, . . . to 9901, a second can be applied to cells 2, 102,
202 . . . to 9902, and so on for all the cells and monomers.
[0136] With only slight modifications, a conventional disk drive
seeking method can be used to access a specific cell or group of
cells from anywhere on the disk. Thus, if the track and sector of a
cell are known, the read/write head and dispenser, if necessary,
can be moved radially, counting the tracks crossed, until the
desired track is located. After settling and confirming that the
proper track has been reached, the synthesizer can be synchronized
with the disk by reading the appropriate control information on the
track. The appropriate sector and cell is then identified by
reading the header information.
[0137] In some embodiments, the disk is moving continuously during
the deposition step. By carefully controlling the disk speed and
dispenser position, the monomer drops are expelled in short pulses,
timed to correspond with the passage of particular cells under the
dispenser head. The electrostatic control mechanisms described
above helps guide the monomer-containing drops to their desired
locations. To realize the full potential for polymer diversity in
the present invention, some deposition steps must take place over
different tracks and noncontiguous sectors. Thus, the monomer is
deposited at various unrelated points over the disk surface. The
disk rotates and stops at various angular positions, while the
monomer dispenser jumps radially (or tangentially) to the location
of the cell where the monomer is dispensed.
[0138] For many deposition steps, however, the system takes
advantage of the disk's rotation to rapidly access the desired
cells. Very rapid processing can be realized when some deposition
steps deliver one monomer to all or most of the cells on a track of
the disk. This greatly speeds the deposition process, as the
dispenser does not have to move from a single radial position
during deposition. Deposition in accordance with this approach is
accomplished by first moving the dispenser radially an appropriate
distance until it is positioned over the appropriate track, and
then expelling metered amounts of the monomer solution for a
sufficient period to cover the desired angular displacement on the
disk.
[0139] The position of the dispenser can be moved across the
rotatable disk by a variety of actuators. Typically, the dispenser
is mounted on an arm that can be driven by voice-coil motors in
conjunction with servo systems which sense position information
from disks. Such actuators systems are commonly employed in
magnetic hard disk drives. These are described in, for example,
Magnetic Recording Vol. II, Chptr. 2 by C. D. Mee and E. D. Daniel,
McGraw-Hill (1988), which is incorporated by reference herein for
all purposes. If individual jets or capillaries for each reagent
are involved, they can be individually positioned over the desired
zones by sliding precisely along a track mounted on the arm.
[0140] D. Depositing Reactants in the Selected Cells
[0141] A variety of methods can be used to direct the monomer
solutions from the reservoirs to the desired cells on the disk. As
described above, micropipette, ink-jet, and pen plotter
technologies are sufficiently developed and flexible to be useful
in the present invention. In addition, electrophoretic and osmotic
pumps, also described above, can be employed. These technologies
are capable of delivering very small amounts of material to a
selected location, thus permitting very small cells to be used in
the present invention. The droplets produced by the dispensers of
the present invention preferably range in diameter from about 200
.mu.m to about 500 .mu.m depending upon the application, although
smaller sizes (to, for example, 50 .mu.m) may be possible. As noted
above, standard micropipettes can provide 4.6 nanoliter droplets
having a diameter of approximately 0.21 mm. Droplet size can be
controlled by a variety of methods well-known in the art. For
example, the solution feed rate affects the drop size. In addition,
pulsed electrical fields or vibrations at the nozzle (created by a
piezoelectric pump, for example) affect the drop size.
[0142] In some embodiments, it will be desirable to charge the
drops so that a controlled electric field can direct the droplets
to the desired cells. Typically, the liquid will be stored in
capillary tubes in which a charged electrode is present. Hence, the
liquid surface will develop a charge. The liquid will therefore be
drawn to a grounded substrate. When the capillary is charged to a
few hundred volts, the liquid leaves in a fine stream of
electrically charged droplets. If the capillary is charged to a few
thousand volts, a jet of electrically charged liquid flies from the
nozzle.
[0143] In some embodiments, there will be a charge on the nozzle of
the dispenser to produce the electric field useful in guiding the
droplets (or jet) to the substrate. Of course, other charged
surfaces (such as plates or cylinders) can be placed near the path
of the liquid to alter the electric field and hence the trajectory
of the droplets. By carefully controlling the electric field in
this manner, the deposition location of the monomer solution
droplets can be tailored for the particular characteristics of the
disk system.
[0144] Other means of charging the droplets can also be employed,
such as, for example, standard "corona charging" methods. With
these techniques, a stream of particles or drops passes through a
corona discharge (a region where an intense electric field created
by a sharply pointed and highly-charged electrode which ionizes the
surrounding air molecules). When particles or droplets are passed
through the corona discharge, they are bombarded by the ionized air
molecules and they, themselves, receive a charge, thus permitting
them to be guided by an electric field to the disk for deposition.
Corona charging techniques do not require the use of an electrode,
thereby avoiding a possible source of contamination in corrosive
solutions. Related techniques are widely used to precipitate
particulate emissions from power-station chimneys and other
industrial plants, and to filter dust from air in offices and other
public places.
[0145] After the monomer solution has been directed to the desired
cell, it should remain there during subsequent processing steps
until it is washed off the substrate. As mentioned above in the
discussion with respect to the substrate, the cells may take the
form of wells or dimples surrounded by a non-wetting coating. This
helps to localize the monomers at the positions of the elements of
the array, thus avoiding cross-reactions with the reactants in
neighboring cells.
[0146] In some embodiments, especially optical-type read/record
systems, a dimpled surface is unnecessary. As alluded to above,
high surface tension liquids (and hydrophobic substrate coatings)
can be employed to increase the surface forces that will tend to
keep the reactant solutions in place. Of course, such properties
are also desirable in dimpled systems, especially when the disk
substrate is rotated at high speeds. As is well-known in the art,
various inert materials can be added to the monomer solutions to
control the surface tension, viscosity, and other physical
properties of the monomer solutions.
[0147] E. Peptide Synthesis
[0148] The surface to which the reagents are to be applied is
suitably derivatized, with a spacer molecule if necessary, in order
to form covalent bonds with the applied monomers. Heat, catalyst
(e.g., gaseous acid or base), etc. are subsequently supplied to the
entire surface or to individual cells, as required by the coupling
chemistry, so that the first monomers attach to the substrate. The
surface is typically washed to remove unreacted reagent after the
first round of monomers is bound. A second round of reagents is
then squirted or otherwise deposited into predetermined cells and
the disk surface is once again incubated at reaction conditions
selected to minimize cleavage of the preceding covalent link and
preserve the sequence of all the products. Solution phase
polymerization of the monomers should be discouraged by, for
example, preventing one end of each monomer from reacting. As
described above, this can be accomplished by attaching a cleavable
protecting group on a reactive end of the monomer.
[0149] The basic reaction strategy of Merrifield, described above,
can be used in each cell of the disk. The particular chemical
reagents used to effect the reactions can be any of those commonly
used in the art, as discussed above.
[0150] Because many of the reactions can take place under similar
conditions, it may be desirable to adjust the concentrations or
ingredients (e.g., monomer or buffers) of the different reagent
formulations to compensate for differences in reactivity or
tendency to undergo side reactions. It is highly desirable to push
every reaction to near quantitative conversion to minimize the
development of deletion compounds. Capping the unreacted chains
before deprotection can be employed to minimize the interference
which such deletion compounds might introduce.
[0151] F. Imaging the Array
[0152] According to preferred embodiments, the array of polymer
sequences is utilized in one or more of a variety of screening
processes. For example, according to one embodiment, the entire
disk substrate is exposed to a receptor of interest such as an
enzyme or antibody. According to preferred embodiments, the
receptor is labelled with fluorescein, or other detectable
material, so as to provide for easy detection of the location(s) at
which the receptor binds. According to still other embodiments, the
binding signal is provided by exposing the substrate to the
receptor of interest, and then exposing the substrate to a labelled
material which is complementary to the receptor of interest and
which preferably binds at multiple locations of the receptor of
interest. For example, in one specific embodiment, if a mouse
antibody is to be studied, a labelled second antibody can be
exposed to the substrate which is, for example, goat-antimouse
antibody. Such techniques are described in PCT Application
WO/10092, previously incorporated herein by reference.
[0153] In addition to fluorescence, absorbance, ellipsometry,
reflectance, and various types of spectroscopy may be used to study
the various elements of the array. The entire array can be imaged
by techniques well known in the art, including spot reading, spot
scanning, and area imaging.
[0154] Conclusion
[0155] The above description is illustrative and not restrictive.
Many variations of the invention will become apparent to those of
skill in the art upon review of this disclosure. For example,
although the substrate has been described as having cells on only
two dimensions, it can have cells on three dimensions. Further, the
bottom of a flat substrate, as well as the top, can be utilized in
some arrays. Such a substrate would be analogous to the double
sided magnetic storage disks in wide use today. These and other
embodiments show that the scope of the invention should be
determined not with reference to the above description, but instead
with reference to the appended claims along with their full scope
of equivalents.
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