U.S. patent application number 10/338158 was filed with the patent office on 2003-07-24 for high density molecular arrays on porous surfaces.
Invention is credited to Ellson, Richard N., Foote, James K., Mutz, Mitchell W..
Application Number | 20030138852 10/338158 |
Document ID | / |
Family ID | 46204264 |
Filed Date | 2003-07-24 |
United States Patent
Application |
20030138852 |
Kind Code |
A1 |
Ellson, Richard N. ; et
al. |
July 24, 2003 |
High density molecular arrays on porous surfaces
Abstract
The present invention provides a unique and highly accurate
method for generating molecular arrays of very high density on
porous surfaces. The method involves the application of focused
acoustic energy to each of a plurality of fluid-containing
reservoirs to eject a small fluid droplet--on the order of 1
picoliter or less--from each reservoir to a site on a porous
substrate surface. High density molecular arrays are provided as
well, in which greater than about 62,500 molecular moieties,
serving as array elements, are present on a porous surface.
Biomolecular arrays that can be generated using focused acoustic
ejection include oligonucleotide arrays and peptidic arrays.
Inventors: |
Ellson, Richard N.; (Palo
Alto, CA) ; Mutz, Mitchell W.; (Palo Alto, CA)
; Foote, James K.; (Cupertino, CA) |
Correspondence
Address: |
REED & EBERLE LLP
800 MENLO AVENUE, SUITE 210
MENLO PARK
CA
94025
US
|
Family ID: |
46204264 |
Appl. No.: |
10/338158 |
Filed: |
January 7, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10338158 |
Jan 7, 2003 |
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09964215 |
Sep 25, 2001 |
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09964215 |
Sep 25, 2001 |
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09727392 |
Nov 29, 2000 |
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09727392 |
Nov 29, 2000 |
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09669996 |
Sep 25, 2000 |
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Current U.S.
Class: |
506/32 ;
427/2.11; 435/6.11; 435/7.1; 436/518 |
Current CPC
Class: |
B01J 2219/00659
20130101; B01J 2219/0059 20130101; B01J 2219/00608 20130101; B01J
2219/00641 20130101; B01J 2219/00617 20130101; B05B 17/0607
20130101; B41J 2/14008 20130101; C40B 80/00 20130101; B05B 17/0615
20130101; Y10T 436/2575 20150115; B01J 19/0046 20130101; B01J
2219/00637 20130101; B01J 2219/00351 20130101; C40B 60/14 20130101;
B01J 2219/00722 20130101; B01J 2219/00612 20130101; B01L 2400/0433
20130101; B01J 2219/0063 20130101; B41J 2/04 20130101; B01J
2219/00527 20130101; C40B 40/10 20130101; C07B 2200/11 20130101;
B01J 2219/00605 20130101; B01J 2219/00725 20130101; B01J 2219/00626
20130101; B01J 2219/00621 20130101; B01J 2219/0061 20130101; C40B
40/06 20130101; B01J 2219/00596 20130101 |
Class at
Publication: |
435/7.1 ; 435/6;
436/518; 427/2.11 |
International
Class: |
C12Q 001/68; G01N
033/53; G01N 033/543; B05D 003/00 |
Claims
We claim:
1. A method for generating an array of molecular moieties on a
porous substrate surface divided into a plurality of discrete
surface sites, the method comprising successively applying focused
acoustic energy to each of a plurality of reservoirs each
containing a molecular moiety in a fluid, wherein the focused
acoustic energy is applied by (a) acoustically coupling an acoustic
ejector comprised of an acoustic radiation generator and a focusing
means to one of the reservoirs and then (b) activating the acoustic
ejector in a manner effective to eject a droplet from the reservoir
toward the porous substrate surface, and repeating (a) and (b) with
each of the reservoirs in succession such that the molecular moiety
in each droplet attaches to a localized region within a discrete
surface site, wherein no longer than about 1 second elapses between
each repetition of (a).
2. The method of claim 1, wherein each molecular moiety is
different.
3. The method of claim 2, wherein a droplet is ejected toward each
surface site, such that every surface site has a molecular moiety
attached thereto.
4. The method of claim 3, wherein each molecular moiety is
different.
5. The method of claim 1, wherein the molecular moieties are
biomolecules.
6. The method of claim 5, wherein the biomolecules are
nucleotidic.
7. The method of claim 6, wherein the biomolecules are
oligonucleotides.
8. The method of claim 7, wherein the biomolecules are nucleotidic
monomers, and the method further comprises stepwise synthesis of an
oligonucleotide within each surface site by repeated deposition of
individual nucleotidic monomers at each site using focused acoustic
energy.
9. The method of claim 5, wherein the biomolecules are
peptidic.
10. The method of claim 3, wherein the porous substrate surface is
comprised of at least 62,500 discrete surface sites.
11. The method of claim 10, wherein the porous substrate surface is
comprised of at least 250,000 discrete surface sites.
12. The method of claim 11, wherein the porous substrate surface is
comprised of at least 1,000,000 discrete surface sites.
13. The method of claim 12, wherein the porous substrate surface is
comprised of at least 1,500,000 discrete surface sites.
14. The method of claim 1, wherein no longer than about 0.1 seconds
elapses between each repetition of (a).
15. The method of claim 14, wherein no longer than about 0.001
seconds elapses between each repetition of (a).
16. The method of claim 1, wherein the acoustic ejector and the
reservoirs move continuously throughout the method until the array
is generated.
17. The method of claim 16, wherein the acoustic ejector and the
reservoirs are moved at a rate effective to provide reservoir
transitions of over 10 Hz.
18. The method of claim 17, wherein the acoustic ejector and the
reservoirs are moved at a rate effective to provide reservoir
transitions of over 100 Hz.
19. A method for generating an array of molecular moieties on a
porous substrate surface divided into a plurality of discrete
surface sites, the method comprising applying focused acoustic
energy to each of a plurality of reservoirs each containing a
molecular moiety in a fluid, wherein the distance between the
centers of any two adjacent reservoirs is less than about 1
centimeter, and further wherein the focused acoustic energy is
applied using an acoustic ejector comprised of an acoustic
radiation generator and a focusing means in a manner effective to
eject a droplet from each reservoir toward the substrate surface
such that the molecular moiety in each droplet attaches to a
localized region within a discrete surface site.
20. The method of claim 19, wherein each molecular moiety is
different.
21. The method of claim 20, wherein a droplet is ejected toward
each surface site, such that every surface site has a molecular
moiety attached thereto.
22. The method of claim 21, wherein each molecular moiety is
different.
23. The method of claim 19, wherein the molecular moieties are
biomolecules.
24. The method of claim 23, wherein the biomolecules are
nucleotidic.
25. The method of claim 24, wherein the biomolecules are
oligonucleotides.
26. The method of claim 25, wherein the biomolecules are
nucleotidic monomers, and the method further comprises stepwise
synthesis of an oligonucleotide within each surface site by
repeated deposition of individual nucleotidic monomers at each site
using focused acoustic energy.
27. The method of claim 23, wherein the biomolecules are
peptidic.
28. The method of claim 21, wherein the porous substrate surface is
comprised of at least 62,500 discrete surface sites.
29. The method of claim 28, wherein the porous substrate surface is
comprised of at least 250,000 discrete surface sites.
30. The method of claim 29, wherein the porous substrate surface is
comprised of at least 1,000,000 discrete surface sites.
31. The method of claim 30, wherein the porous substrate surface is
comprised of at least 1,500,000 discrete surface sites.
32. The method of claim 19, wherein the distance between the
centers of any two adjacent reservoirs is less than about 1
millimeter.
33. The method of claim 32, wherein the distance between the
centers of any two adjacent reservoirs is less than about 0.5
millimeter.
34. The method of claim 33, wherein at least one of the reservoirs
is adapted to contain more than about 100 nanoliters of fluid.
35. The method of claim 34, wherein at least one of the reservoirs
is adapted to contain more than about 10 nanoliters of fluid.
36. The method of claim 34, wherein the reservoirs are adapted to
contain more than about 100 nanoliters of fluid.
37. The method of claim 36, wherein the reservoirs are adapted to
contain more than about 10 nanoliters of fluid.
38. The method of claim 37, wherein each of the ejected droplets
has a volume of about 1 pL or less.
39. The method of claim 38, wherein each of the ejected droplets
has a volume in the range of about 0.025 pL to about 1 pL.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of U.S. patent application Ser. No.
09/964,215, filed Sep. 25, 2001, which is a continuation-in-part of
U.S. patent application Ser. No. 09/727,392, filed Nov. 29, 2000,
which is a continuation-in-part of U.S. patent application Ser. No.
09/669,996, filed Sep. 25, 2000, the disclosures of which are
incorporated by reference herein.
TECHNICAL FIELD
[0002] This invention relates generally to the use of focused
acoustic energy in the preparation of molecular arrays, and more
particularly relates to acoustic ejection of fluid droplets to
prepare high density molecular arrays.
BACKGROUND
[0003] The discovery of novel materials having useful biological,
chemical and/or physical properties often leads to emergence of
useful products and technologies. Extensive research in recent
years has focused on the development and implementation of new
methods and systems for evaluating potentially useful chemical
compounds. In the biomacromolecule arena, for example, much recent
research has been devoted to potential methods for rapidly and
accurately identifying the properties of various oligomers of
specific monomer sequences, including ligand and receptor
interactions, by screening high density arrays of biopolymers
including nucleotidic, peptidic and saccharidic polymers.
[0004] For biological molecules, the complexity and variability of
biological interactions and the physical interactions that
determine, for example, protein conformation or structure other
than primary structure, preclude predictability of biological,
material, physical and/or chemical properties from theoretical
considerations at this time. For non-biological materials,
including bulk liquids and solids, despite much inquiry and vast
advances in understanding, a theoretical framework permitting
sufficiently accurate prediction de novo of composition, structure
and synthetic preparation of novel materials is still lacking.
[0005] Consequently, the discovery of novel useful materials
depends largely on the capacity to make and characterize new
compositions of matter. Of the elements in the periodic table that
can be used to make multi-elemental compounds, relatively few of
the practically inexhaustible possible compounds have been made or
characterized. A general need in the art consequently exists for a
more systematic, efficient and economical method for synthesizing
novel materials and screening them for useful properties. Further,
a need exists for a flexible method to make compositions of matter
of various material types and combinations of material types,
including molecular materials, crystalline covalent and ionic
materials, alloys, and combinations thereof such as crystalline
ionic and alloy mixtures, or crystalline ionic and alloy layered
materials.
[0006] The immune system is an example of systematic protein and
nucleic acid macromolecular combinatorial chemistry that is
performed in nature. Both the humoral and cell-mediated immune
systems produce molecules having novel functions by generating vast
libraries of molecules that are systematically screened for a
desired property. For example, the humoral immune system is capable
of determining which of 10.sup.12 B-lymphocyte clones that make
different antibody molecules bind to a specific epitope or
immunogenic locale, in order to find those clones that specifically
bind various epitopes of an immunogen and stimulate their
proliferation and maturation into plasma cells that make the
antibodies. Because T cells, responsible for cell-mediated
immunity, include regulatory classes of cells and killer T cells,
and the regulatory T cell classes are also involved in controlling
both the humoral and cellular response, more clones of T cells
exist than of B cells, and must be screened and selected for
appropriate immune response. Moreover, the embryological
development of both T and B cells is a systematic and essentially
combinatorial DNA splicing process for both heavy and light chains.
See, e.g., Therapeutic Immunology, Eds. Austen et al. (Blackwell
Science, Cambridge Mass., 1996).
[0007] Recently, the combinatorial prowess of the immune system has
been harnessed to select for antibodies against small organic
molecules such as haptens; some of these antibodies have been shown
to have catalytic activity akin to enzymatic activity with the
small organic molecules as substrate, termed "catalytic antibodies"
(Hsieh et al. (1993) Science 260(5106):337-339). The proposed
mechanism of catalytic antibodies is a distortion of the molecular
conformation of the substrate towards the transition state for the
reaction and additionally involves electrostatic stabilization.
Synthesizing and screening large libraries of molecules has, not
unexpectedly, also been employed for drug discovery. Proteins are
known to form an induced fit for a bound molecule such as a
substrate or ligand (Stryer, Biochemistry, 4.sup.th Ed. (1999) W.
H. Freeman & Co., New York), with the bound molecule fitting
into the site much like a hand fits into a glove, requiring some
basic structure for the glove that is then shaped into the bound
structure with the help of a substrate or ligand.
[0008] Geysen et al. (1987) J. Immun. Meth. 102:259-274 have
developed a combinatorial peptide synthesis in parallel on rods or
pins involving functionalizing the ends of polymeric rods to
potentiate covalent attachment of a first amino acid, and
sequentially immersing the ends in solutions of individual amino
acids. In addition to the Geysen et al. method, techniques have
recently been introduced for synthesizing large arrays of different
peptides and other polymers on solid surfaces. Arrays may be
readily appreciated as additionally being efficient screening
tools. Miniaturization of arrays saves synthetic reagents and
conserves sample, a useful improvement in both biological and
non-biological contexts. See, for example, U.S. Pat. Nos. 5,700,637
and 6,054,270 to Southern et al., which describe a method for
chemically synthesizing a high density array of oligonucleotides of
chosen monomeric unit length within discrete cells or regions of a
support material, wherein the method employs an inkjet printer to
deposit individual monomers on the support. So far, however,
miniaturized arrays have been costly to make and contain
significant amounts of undesired products at sites where a desired
product is made. Thus, even in the biological arena, where a given
sample might be unique and therefore priceless, use of high density
biomacromolecule microarrays has met resistance by the academic
community as being too costly, as yet insufficiently reliable
compared to arrays made by lab personnel.
[0009] Arrays of thousands or even millions of different
compositions of the elements may be formed by such methods. Various
solid phase microelectronic fabrication derived polymer synthetic
techniques have been termed "Very Large Scale Immobilized Polymer
Synthesis," or "VLSIPS" technology. Such methods have been
successful in screening potential peptide and oligonucleotide
ligands for determining relative binding affinity of the ligand for
receptors.
[0010] The solid phase parallel, spatially directed synthetic
techniques currently used to prepare combinatorial biomolecule
libraries require stepwise, or sequential, coupling of monomers.
U.S. Pat. No. 5,143,854 to Pirrung et al. describes synthesis of
polypeptide arrays, and U.S. Pat. No. 5,744,305 to Fodor et al.
describes an analogous method of synthesizing oligo- and
poly-nucleotides in situ on a substrate by covalently bonding
photoremovable groups to the surface of the substrate. Selected
substrate surface locales are exposed to light to activate them, by
use of a mask. An amino acid or nucleotide monomer with a
photoremovable group is then attached to the activated region. The
steps of activation and attachment are repeated to make
polynucleotides and polypeptides of desired length and sequence.
Other synthetic techniques, exemplified by U.S. Pat. Nos. 5,700,637
and 6,054,270 to Southern et al., teach the use of inkjet printers,
which are also substantially parallel synthesis because the
synthetic pattern must be predefined prior to beginning to "print"
the pattern. These solid phase synthesis techniques, which involve
the sequential coupling of building blocks (e.g., amino acids) to
form the compounds of interest, cannot readily be used to prepare
many inorganic and organic compounds.
[0011] U.S. Pat. No. 5,985,356 to Schultz et al. teaches
combinatorial chemistry techniques in the field of materials
science, providing methods and a device for synthesis and use of an
array of diverse materials in predefined regions of a substrate. An
array of different materials on a substrate is prepared by
delivering components of various compositions of matter to
predefined substrate surface locales. This synthetic technique
permits many classes of materials to be made by systematic
combinatorial methods. Examples of the types of materials include,
but are not limited to, inorganic materials, including ionic and
covalent crystalline materials, intermetallic materials, metal
alloys and composite materials including ceramics. Such materials
can be screened for useful bulk and surface properties as the
synthesized array, for example, electrical properties, including
super- and semi-conductivity, and thermal, mechanical,
thermoelectric, optical, optoelectronic, fluorescent and/or
biological properties, including immunogenicity.
[0012] Discovery and characterization of materials often requires
combinatorial deposition onto substrates of thin films of precisely
known chemical composition, concentration, stoichiometry, area
and/or thickness. Devices and methods for making arrays of
different materials, each with differing composition,
concentration, stoichiometry and thin-layer thickness at known
substrate locales, permitting systematic combinatorial array based
synthesis and analysis that utilize thin layer deposition methods,
are already known. Although existing thin-layer methods have
effected the precision of reagent delivery required to make arrays
of different materials, the predefinition required in these
synthetic techniques is inflexible, and the techniques are slow and
thus relatively costly. Additionally, thin-layer techniques are
inherently less suited to creating experimental materials under
conditions that deviate drastically from conditions that are
thermodynamically reversible or nearly so. Thus, a need exists for
more efficient and rapid delivery of precise amounts of reagents
needed for materials array preparation, with more flexibility as to
predetermination and conditions of formation than attainable by
thin-layer methods.
[0013] In combinatorial synthesis of biomacromolecules, U.S. Pat.
Nos. 5,700,637 and 6,054,270 to Southern et al., as noted
previously, describe a method for generating an array of
oligonucleotides of chosen monomeric unit length within discrete
cells or regions of a support material. The in situ method
generally described for oligo- or polynucleotide synthesis
involves: coupling a nucleotide precursor to a discrete
predetermined set of cell locations or regions; coupling a
nucleotide precursor to a second set of cell locations or regions;
coupling a nucleotide precursor to a third set of cell locations or
regions; and continuing the sequence of coupling steps until the
desired array has been generated. Covalent linking is effected at
each location either to the surface of the support or to a
nucleotide coupled in a previous step.
[0014] The '637 and '270 patents also teach that impermeable
substrates are preferable to permeable substrates, such as paper,
for effecting high combinatorial site densities, because the fluid
volumes required will result in migration or wicking through a
permeable substrate, precluding attainment of the small feature
sizes required for high densities (such as those that are
attainable by parallel photolithographic synthesis, which requires
a substrate that is optically smooth and generally also
impermeable; see U.S. Pat. No. 5,744,305 to Fodor et al.). As the
inkjet printing method is a parallel synthesis technique that
requires the array to be "predetermined" in nature, and therefore
inflexible, and does not enable feature sites in the micron range
or smaller, there remains a need in the art for a
non-photolithographic in situ combinatorial array preparation
method that can provide the high densities attainable by
photolithographic arrays, a feat that requires small volumes of
reagents and a highly accurate deposition method, without the
inflexibility of a highly parallel process that requires a
predetermined site sequence. Also, as permeable substrates offer a
greater surface area for localization of array constituents, a
method of effecting combinatorial high density arrays
non-photolithographically by delivery of sufficiently small volumes
to permit use of permeable substrates is also an advance over the
current state of the art of array making.
[0015] As explained above, the parallel photolithographic in situ
formation of biomolecular arrays of high density, e.g.,
oligonucleotide or polynucleotide arrays, is also known in the art.
For example, U.S. Pat. Nos. 5,744,305 and 5,445,934 to Fodor et al.
describe arrays of oligonucleotides and polynucleotides attached to
a surface of a planar non-porous solid support at a density
exceeding 400 and 1000 different oligonucleotides/cm.sup.2
respectively. The arrays are generated using light-directed,
spatially addressable synthesis techniques (see also U.S. Pat. Nos.
5,143,854 and 5,405,783, and International Patent Publication No.
WO 90/15070). With respect to these photolithographic parallel in
situ synthesized microarrays, Fodor et al. have developed
photolabile nucleoside and peptide protecting groups, and masking
and automation techniques; see U.S. Pat. No. 5,489,678 and
International Patent Publication No. WO 92/10092).
[0016] The aforementioned patents disclose that photolithographic
techniques commonly used in semiconductor fabrication may be
applied in the fabrication of arrays of high density.
Photolithographic in situ synthesis is best for parallel synthesis,
requiring an inordinate number of masking steps to effect a
sequential in situ combinatorial array synthesis. Even the parallel
combinatorial array synthesis that involves a minimized number of
masking steps still employs a significant number of such steps,
which increases for each monomeric unit added in the synthesis.
Further, the parallel photolithographic in situ array synthesis is
inflexible and requires a predetermined mask sequence.
[0017] Because photolithographic fabrication requires a large
number of masking steps, the yield for this process is lowered
relative to a non-photolithographic in situ synthesis by the
failure to block and/or inappropriate photo-deblocking by some of
the photolabile protective groups. These problems with photolabile
protective groups compound the practical yield problem for
multi-step in situ syntheses in general by adding photochemical
steps to the synthetic process. The problems have not been
addressed by the advances made in the art of making and using such
photolabile blockers for in situ synthesis, in part because some
photolabile blocking groups are shielded from the light or "buried"
by the polymer on which they reside, an effect exacerbated with
increasing polymer length. Therefore, the purity of the desired
product is low, as the array will contain significant impurities of
undesired products that can reduce both sensitivity and
selectivity.
[0018] As the photolithographic process for in situ synthesis
defines site edges with mask lines, mask imperfections and
misalignment, diffractive effects and perturbations of the optical
smoothness of the substrate can combine to reduce purity by
generating polymers similar in sequence and/or structure to the
desired polymer as impurities, a problem that becomes more
pronounced at the site edges. This is exacerbated when
photolithographic protocols attempt to maximize site density by
creating arrays that have abutting sites. Because the likelihood of
a mask imperfection or misalignment increases with the number of
masking steps and the associated number of masks, these edge
effects are worsened by an increased number of masking steps and
utilization of more mask patterns to fabricate a particular array.
Site impurity, i.e., generation of polymers similar in sequence
and/or structure to the desired polymer, leads to reduced
sensitivity and selectivity for arrays designed to analyze a
nucleotide sequence.
[0019] Some efforts have been directed to adapting printing
technologies, particularly, inkjet printing technologies, to form
biomolecular arrays. For example, U.S. Pat. No. 6,015,880 to
Baldeschwieler et al. is directed to array preparation using a
multistep in situ synthesis. A liquid microdrop containing a first
reagent is applied by a single jet of a multiple jet reagent
dispenser to a locus on the surface chemically prepared to permit
covalent attachment of the reagent. The reagent dispenser is then
displaced relative to the surface, or the surface is displaced with
respect to the dispenser, and at least one microdrop containing
either the first reagent or a second reagent from another dispenser
jet is applied to a second substrate locale, which is also
chemically activated to be reactive for covalent attachment of the
second reagent. Optionally, the second step is repeated using
either the first or second reagents, or different liquid-borne
reagents from different dispenser jets, wherein each reagent
covalently attaches to the substrate surface. The patent discloses
that inkjet technology may be used to apply the microdrops.
[0020] Ordinary inkjet technology, however, suffers from a number
of drawbacks. Often, inkjet technology involves heating or using a
piezoelectric element to force a fluid through a nozzle in order to
direct the ejected fluid onto a surface. Thus, the fluid may be
exposed to a surface exceeding 200.degree. C. before being ejected,
and most, if not all, peptidic molecules, including proteins,
degrade under such extreme temperatures. In addition, forcing
peptidic molecules through nozzles creates shear forces that can
alter molecular structure. Nozzles are subject to clogging,
especially when used to eject a macromolecule-containing fluid, and
the use of elevated temperatures exacerbates the problem because
liquid evaporation results in deposition of precipitated solids on
the nozzles. Clogged nozzles, in turn, can result in misdirected
fluid or ejection of improperly sized droplets. Finally, ordinary
inkjet technology employing a nozzle for fluid ejection generally
cannot be used to deposit arrays with feature densities comparable
to those obtainable using photolithography or other techniques
commonly used in semiconductor processing.
[0021] A number of patents have described the use of acoustic
energy in printing. For example, U.S. Pat. No. 4,308,547 to
Lovelady et al. describes a liquid drop emitter that utilizes
acoustic principles in ejecting droplets from a body of liquid onto
a moving document to form characters or bar codes thereon. A
nozzleless inkjet printing apparatus is used wherein controlled
drops of ink are propelled by an acoustical force produced by a
curved transducer at or below the surface of the ink. In contrast
to inkjet printing devices, nozzleless fluid ejection devices
described in the aforementioned patent are not subject to clogging
and the disadvantages associated therewith, e.g., misdirected fluid
or improperly sized droplets.
[0022] The applicability of nozzleless fluid ejection has generally
been appreciated for ink printing applications. Development of ink
printing applications is primarily economically driven by printing
cost and speed for acceptable text. For acoustic printing,
development efforts have therefore focused on reducing printing
costs rather than improving quality, and on increasing printing
speed rather than accuracy. For example, U.S. Pat. No. 5,087,931 to
Rawson is directed to a system for transporting ink under constant
flow to an acoustic ink printer having a plurality of ejectors
aligned along an axis, each ejector associated with a free surface
of liquid ink. When a plurality of ejectors is used instead of a
single ejector, printing speed generally increases, but controlling
fluid ejection, specifically droplet placement, becomes more
difficult.
[0023] U.S. Pat. No. 4,797,693 to Quate describes an acoustic ink
printer for printing polychromatic images on a recording medium.
The printer is described as comprising a combination of a carrier
containing a plurality of differently colored liquid inks, a single
acoustic printhead acoustically coupled to the carrier for
launching converging acoustic waves into the carrier, an ink
transport means to position the carrier to sequentially align the
differently colored inks with the printhead, and a controller to
modulate the radiation pressure used to eject ink droplets. This
printer is described as designed for the realization of cost
savings. Because two droplets of primary color, e.g., cyan and
yellow, deposited in sufficient proximity will appear as a
composite or secondary color, the level of accuracy required is
fairly low and inadequate for biomolecular array formation. Such a
printer is particularly unsuitable for in situ synthesis requiring
precise droplet deposition and consistent placement, so that the
proper chemical reactions occur. That is, the drop placement
accuracy needed to effect perception of a composite secondary color
is much lower than is required for chemical synthesis at
photolithographic density levels. Consequently, an acoustic
printing device that is suitable for printing visually
ascertainable material is inadequate for microarray preparation.
Also, this device can eject only a limited quantity of ink from the
carrier before the liquid meniscus moves out of acoustic focus and
drop ejection ceases. This is a significant limitation with
biological fluids, which are typically far more costly and rare
than ink. The Quate et al. patent does not address how to use most
of the fluid in a closed reservoir without adding additional liquid
from an external source.
[0024] Thus, there is a general need in the art for improved array
preparation methodology. An ideal array preparation technique would
provide for highly accurate deposition of minute volumes of fluids
on a substrate surface, wherein droplet volume--and thus "spot"
size on the substrate surface--can be carefully controlled and
droplets can be precisely directed to particular sites on a
substrate surface. It would also be optimal if such a technique
could be used with porous or even permeable surfaces, as such
surfaces can provide substantially greater surface area on which to
attach molecular moieties that serve as array elements, and would
enable preparation of higher density arrays. To date, as alluded to
above, high density arrays have been prepared only on nonporous,
impermeable surfaces, and only low density arrays could be prepared
on porous surfaces.
SUMMARY OF THE INVENTION
[0025] Accordingly, it is an object of the present invention to
provide methods and molecular arrays that address the
aforementioned need in the art. It has now been discovered that
focused acoustic energy can be advantageously used with very small
fluid volumes, on the order of 1 picoliter or less, to prepare high
density arrays on substrates having a porous or even a permeable
surface. Prior array fabrication methods have not enabled
preparation of high density arrays on porous or permeable surfaces
because prior spotting processes are nowhere near as accurate as
the present acoustic deposition method, and prior processes have
also required larger droplet volumes. In contrast to prior methods
of manufacturing arrays, then, the present acoustic ejection
process enables highly accurate deposition of extremely small
liquid droplets, such that diffusion of a deposited droplet into
neighboring cells is not a problem, and ultra-high array densities
can now be achieved with high porosity, permeable surfaces. Porous,
permeable surfaces can provide substantially more surface area on
which to attach molecules within an array, as the biomolecules can
penetrate the surface of the substrate and can thus generate a
substantially larger signal per unit of projected area due to the
resulting non-planar distribution of the molecules on and within
the substrate surface.
[0026] The present invention overcomes a significant disadvantage
of the prior art, since prior methods could not constrain the
formation or deposition of biomolecules to a small region, due to
the inability to localize the synthesis process or to restrict the
migration of materials to prevent adjacent "spots" from diffusing
into each other. The accurate placement of extremely small volumes
of fluids can enable both multistep in situ synthesis on a
substrate surface and deposition of an intact molecule within an
extraordinarily small zone without the need to mask, pre-activate
surface sites, or otherwise modify a predetermined region of a
surface.
[0027] The invention makes use of a focused acoustic energy device
as described in U.S. patent application Ser. No. U09/669,996
("Acoustic Ejection of Fluids from a Plurality of Reservoirs"),
inventors Ellson, Foote and Mutz, filed on Sep. 25, 2000, and
assigned to Picoliter, Inc. (Mountain View, Calif.). As described
in the aforementioned patent application, the device enables
acoustic ejection of a plurality of fluid droplets toward
designated sites on a substrate surface for deposition thereon, and
comprises: a plurality of reservoirs or other fluid-containing
means, each adapted to contain a fluid; an acoustic ejector that
includes an acoustic radiation generator and a focusing means for
focusing the generated acoustic radiation at a focal point
sufficiently near the fluid surface in each of the reservoirs such
that droplets are ejected therefrom; and a means for positioning
the ejector in acoustic coupling relationship to each of the
reservoirs. Preferably, each of the reservoirs is removable,
comprised of an individual well in a well plate, and/or arranged in
an array. In addition, it is preferred that the reservoirs are
substantially acoustically indistinguishable from one another.
[0028] Focused acoustic ejection is carried out by positioning the
acoustic ejector so as to be in acoustically coupled relationship
with a first fluid-containing reservoir, and then activating the
ejector to generate and direct acoustic radiation into the fluid so
as to eject a fluid droplet toward a site on a porous substrate
surface. Then, the ejector is repositioned so as to be in
acoustically coupled relationship with a second fluid-containing
reservoir and activated again as above to eject a droplet of the
second fluid toward a second site on the porous surface. The method
may be repeated with a plurality of fluid reservoirs each
containing a fluid, with each reservoir generally although not
necessarily containing a different fluid. The acoustic ejector is
thus repeatedly repositioned so as to eject a droplet from each
reservoir toward a different site on a substrate surface, or toward
sites that already have a droplet "spot" thereon. In such a way,
the method is readily adapted for use in generating an array of
molecular moieties on a porous surface.
[0029] The invention also provides high density arrays of various
molecular moieties, typically biomolecules, on a porous substrate
surface. The arrays provided by the instant invention do not
possess the edge effects that result from optical and alignment
effects of photolithographic masking, nor are they subject to
imperfect spotting alignment from inkjet nozzle-directed deposition
of reagents. Focused acoustic ejection can be used to prepare
molecular arrays on porous surfaces that have a density of greater
than about 62,500 molecular moieties, or array elements, per square
centimeter of substrate surface, preferably a density of greater
than about 250,000, more preferably greater than about 1,000,000,
still more preferably greater than about 1,500,000, and most
preferably in the range of about 1,500,000 to about 4,000,000
molecular moieties per square centimeter of substrate surface.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIGS. 1A and 1B, collectively referred to as FIG. 1,
schematically illustrate in simplified cross-sectional view an
embodiment of the inventive device comprising first and second
reservoirs, an acoustic ejector, and an ejector positioning means.
FIG. 1A shows the acoustic ejector acoustically coupled to the
first reservoir and having been activated in order to eject a
droplet of fluid from within the first reservoir toward a site on a
substrate surface. FIG. 1B shows the acoustic ejector acoustically
coupled to a second reservoir.
[0031] FIGS. 2A, 2B, 2C and 2D, collectively referred to as FIG. 2,
schematically illustrate in simplified cross-sectional view an
embodiment of the inventive method in which a dimer is synthesized
in situ on a substrate using the device of FIG. 1. FIG. 2A
illustrates the ejection of a droplet of surface modification fluid
onto a site of a substrate surface. FIG. 2B illustrates the
ejection of a droplet of a first fluid containing a first molecular
moiety adapted for attachment to the modified surface of the
substrate. FIG. 2C illustrates the ejection of a droplet of second
fluid containing a second molecular moiety adapted for attachment
to the first molecule. FIG. 2D illustrates the substrate and the
dimer synthesized in situ by the process illustrated in FIGS. 2A,
2B and 2C.
DETAILED DESCRIPTION OF THE INVENTION
[0032] Before describing the present invention in detail, it is to
be understood that this invention is not limited to specific
fluids, biomolecules or device structures, as such may vary. It is
also to be understood that the terminology used herein is for the
purpose of describing particular embodiments only, and is not
intended to be limiting.
[0033] It must be noted that, as used in this specification and the
appended claims, the singular forms "a," "an" and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "a reservoir" includes a plurality
of reservoirs, reference to "a fluid" includes a plurality of
fluids, reference to "a biomolecule" includes a, combination of
biomolecules, and the like.
[0034] In describing and claiming the present invention, the
following terminology will be used in accordance with the
definitions set out below.
[0035] The terms "acoustic coupling" and "acoustically coupled"
used herein refer to a state wherein an object is placed in direct
or indirect contact with another object so as to allow acoustic
radiation to be transferred between the objects without substantial
loss of acoustic energy. When two entities are indirectly
acoustically coupled, an "acoustic coupling medium" is needed to
provide an intermediary through which acoustic radiation may be
transmitted. Thus, an ejector may be acoustically coupled to a
fluid, e.g., by immersing the ejector in the fluid or by
interposing an acoustic coupling medium between the ejector and the
fluid to transfer acoustic radiation generated by the ejector
through the acoustic coupling medium and into the fluid.
[0036] The term "adsorb" as used herein refers to the noncovalent
retention of a molecule by a substrate surface. That is, adsorption
occurs as a result of noncovalent interaction between a substrate
surface and adsorbing moieties present on the molecule that is
adsorbed. Adsorption may occur through hydrogen bonding, van der
Waal's forces, polar attraction or electrostatic forces (i.e.,
through ionic bonding). Examples of adsorbing moieties include, but
are not limited to, amine groups, carboxylic acid moieties,
hydroxyl groups, nitroso groups, sulfones and the like. Often the
substrate may be functionalized with adsorbent moieties to interact
in a certain manner, as when the surface is functionalized with
amino groups to render it positively charged in a pH neutral
aqueous environment. Likewise, adsorbate moieties may be added in
some cases to effect adsorption, as when a basic protein is fused
with an acidic peptide sequence to render adsorbate moieties that
can interact electrostatically with a positively charged adsorbent
moiety.
[0037] The term "attached," as in, for example, a substrate surface
having a moiety "attached" thereto, includes covalent binding,
adsorption, and physical immobilization. The terms "binding" and
"bound" are identical in meaning to the term "attached."
[0038] The term "array" used herein refers to a two-dimensional
arrangement of features such as an arrangement of reservoirs (e.g.,
wells in a well plate) or an arrangement of different materials
including ionic, metallic or covalent crystalline, including
molecular crystalline, composite or ceramic, glassine, amorphous,
fluidic or molecular materials on a substrate surface (as in an
oligonucleotide or peptidic array). Different materials in the
context of molecular materials includes chemical isomers, including
constitutional, geometric and stereoisomers, and in the context of
polymeric molecules constitutional isomers having different monomer
sequences. Arrays are generally comprised of regular, ordered
features, as in, for example, a rectilinear grid, parallel stripes,
spirals, and the like, but non-ordered arrays may be advantageously
used as well. The arrays or patterns formed using the devices and
methods of the invention generally have no optical significance to
the unaided human eye. For example, the invention does not involve
ink printing on paper or other substrates in order to form letters,
numbers, bar codes, figures, or other inscriptions that have
optical significance to the unaided human eye. In addition, arrays
and patterns formed by the deposition of ejected droplets on a
porous surface as provided herein are preferably substantially
invisible to the unaided human eye.
[0039] The terms "biomolecule" and "biological molecule" are used
interchangeably herein to refer to any organic molecule, whether
naturally occurring, recombinantly produced, or chemically
synthesized in whole or in part, that is, was or can be a part of a
living organism. The terms encompass, for example, nucleotides,
amino acids and monosaccharides, as well as oligomeric and
polymeric species such as oligonucleotides and polynucleotides,
peptidic molecules such as oligopeptides, polypeptides and
proteins, saccharides such as disaccharides, oligosaccharides,
polysaccharides, mucopolysaccharides or peptidoglycans
(peptido-polysaccharides) and the like. The term also encompasses
ribosomes, enzyme cofactors, pharmacologically active agents, and
the like.
[0040] The terms "library" and "combinatorial library" are used
interchangeably herein to refer to a plurality of chemical or
biological moieties present on the surface of a substrate, wherein
each moiety is different from each other moiety. The moieties may
be, e.g., peptidic molecules and/or oligonucleotides.
[0041] The term "moiety" refers to any particular composition of
matter, e.g., a molecular fragment, an intact molecule (including a
monomeric molecule, an oligomeric molecule, and a polymer), or a
mixture of materials (for example, an alloy or a laminate).
[0042] It will be appreciated that, as used herein, the terms
"nucleoside" and "nucleotide" refer to nucleosides and nucleotides
containing not only the conventional purine and pyrimidine bases,
i.e., adenine (A), thymine (T), cytosine (C), guanine (G) and
uracil (U), but also protected forms thereof, e.g., wherein the
base is protected with a protecting group such as acetyl,
difluoroacetyl, trifluoroacetyl, isobutyryl or benzoyl, and purine
and pyrimidine analogs. Suitable analogs will be known to those
skilled in the art and are described in the pertinent texts and
literature. Common analogs include, but are not limited to,
1-methyladenine, 2-methyladenine, N.sup.6-methyladenine,
N.sup.6-isopentyladenine, 2-methylthio-N.sup.6-isopentyladenine,
N,N-dimethyladenine, 8-bromoadenine, 2-thiocytosine,
3-methylcytosine, 5-methylcytosine, 5-ethylcytosine,
4-acetylcytosine, 1-methylguanine, 2-methylguanine,
7-methylguanine, 2,2-dimethylguanine, 8-bromoguanine,
8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8-thioguanine,
5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
5-ethyluracil, 5-propyluracil, 5-methoxyuracil,
5-hydroxymethyluracil, 5-(carboxyhydroxymethyl)uracil,
5-(methylaminomethyl)uracil, 5-(carboxymethylaminomethyl)-uracil,
2-thiouracil, 5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil,
uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester,
pseudouracil, 1-methylpseudouracil, queosine, inosine,
1-methylinosine, hypoxanthine, xanthine, 2-aminopurine,
6-hydroxyaminopurine, 6-thiopurine and 2,6-diaminopurine. In
addition, the terms "nucleoside" and "nucleotide" include those
moieties that contain not only conventional ribose and deoxyribose
sugars, but other sugars as well. Modified nucleosides or
nucleotides also include modifications on the sugar moiety, e.g.,
wherein one or more of the hydroxyl groups are replaced with
halogen atoms or aliphatic groups, or are functionalized as ethers,
amines, or the like.
[0043] As used herein, the term "oligonucleotide" shall be generic
to polydeoxynucleotides (containing 2-deoxy-D-ribose), to
polyribonucleotides (containing D-ribose), to any other type of
polynucleotide that is an N-glycoside of a purine or pyrimidine
base, and to other polymers containing nonnucleotidic backbones
(for example PNAs), providing that the polymers contain nucleobases
in a configuration that allows for base pairing and base stacking,
such as is found in DNA and RNA. Thus, these terms include known
types of oligonucleotide modifications, for example, substitution
of one or more of the naturally occurring nucleotides with an
analog, inter-nucleotide modifications such as, for example, those
with uncharged linkages (e.g., methyl phosphonates,
phosphotriesters, phosphoramidates, carbamates, etc.), with
negatively charged linkages (e.g., phosphorothioates,
phosphorodithioates, etc.), and with positively charged linkages
(e.g., aminoalklyphosphoramidates, aminoalkylphosphotriesters),
those containing pendant moieties, such as, for example, proteins
(including nucleases, toxins, antibodies, signal peptides,
poly-L-lysine, etc.), those with intercalators (e.g., acridine,
psoralen, etc.), those containing chelators (e.g., metals,
radioactive metals, boron, oxidative metals, etc.). There is no
intended distinction in length between the term "polynucleotide"
and "oligonucleotide," and these terms will be used
interchangeably. These terms refer only to the primary structure of
the molecule. As used herein the symbols for nucleotides and
polynucleotides are according to the IUPAC-IUB Commission of
Biochemical Nomenclature recommendations (Biochemistry 9:4022,
1970).
[0044] The terms "peptide," "peptidyl" and "peptidic" as used
throughout the specification and claims are intended to include any
structure comprised of two or more amino acids. For the most part,
the peptides in the present arrays comprise about 5 to 10,000 amino
acids, preferably about 5 to 1000 amino acids. The amino acids
forming all or a part of a peptide may be any of the twenty
conventional, naturally occurring amino acids, i.e., alanine (A),
cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine
(F), glycine (G), histidine (H), isoleucine (I), lysine (K),
leucine (L), methionine (M), asparagine (N), proline (P), glutamine
(Q), arginine (R), serine (S), threonine (T), valine (V),
tryptophan (W), and tyrosine (Y). Any of the amino acids in the
peptidic molecules forming the present arrays may be replaced by a
non-conventional amino acid. In general, conservative replacements
are preferred. Conservative replacements substitute the original
amino acid with a non-conventional amino acid that resembles the
original in one or more of its characteristic properties (e.g.,
charge, hydrophobicity, stearic bulk; for example, one may replace
Val with Nval). The term "non-conventional amino acid" refers to
amino acids other than conventional amino acids, and include, for
example, isomers and modifications of the conventional amino acids
(e.g., D-amino acids), non-protein amino acids,
post-translationally modified amino acids, enzymatically modified
amino acids, constructs or structures designed to mimic amino acids
(e.g., .alpha.,.alpha.-disubstituted amino acids, N-alkyl amino
acids, lactic acid, .beta.-alanine, naphthylalanine,
3-pyridylalanine, 4-hydroxyproline, O-phosphoserine,
N-acetylserine, N-formylmethionine, 3-methylhistidine,
5-hydroxylysine, and nor-leucine), and peptides having the
naturally occurring amide --CONH-- linkage replaced at one or more
sites within the peptide backbone with a non-conventional linkage
such as N-substituted amide, ester, thioamide, retropeptide
(--NHCO--), retrothioamide (--NHCS--), sulfonamido
(--SO.sub.2NH--), and/or peptoid (N-substituted glycine) linkages.
Accordingly, the peptidic molecules of the array include
pseudopeptides and peptidomimetics. The peptides of this invention
can be (a) naturally occurring, (b) produced by chemical synthesis,
(c) produced by recombinant DNA technology, (d) produced by
biochemical or enzymatic fragmentation of larger molecules, (e)
produced by methods resulting from a combination of methods (a)
through (d) listed above, or (f) produced by any other means for
producing peptides.
[0045] Peptidic compounds include any pharmacologically active
peptide, polypeptide or protein, such as, but not limited to
enzymes, monoclonal and polyclonal antibodies, antigens,
coagulation modulators, cytokines, endorphins, peptidyl hormones,
kinins, and structurally similar bioactive equivalents thereof. By
a "structurally similar bioactive equivalent" is meant a peptidyl
compound with structure sufficiently similar to that of an
identified bioactive peptidyl compound to produce substantially
equivalent therapeutic effects. As used herein and in the appended
claims, the terms "protein", "peptide" and "polypeptide" refer to
both the specific peptidic compound(s) identified as well as
structurally similar bioactive equivalents thereof.
[0046] Examples of various peptidyl compounds include, but are not
limited to, the following:
[0047] coagulation modulators, such as .alpha..sub.1-antitrypsin,
.alpha..sub.2-macroglobulin, antithrombin III, factor I
(fibrinogen), factor II (prothrombin), factor III (tissue
prothrombin), factor V (proaccelerin), factor VII (proconvertin),
factor VIII (antihemophilic globulin or AHG), factor IX (Christmas
factor, plasma thromboplastin component or PTC), factor X
(Stuart-Power factor), factor XI (plasma thromboplastin antecedent
or PTA), factor XII (Hageman factor), heparin cofactor II,
kallikrein, plasmin, plasminogen, prekallikrein, protein C, protein
S, thrombomodulin and combinations thereof;.
[0048] cytokines, such as: transforming growth factors (TGFs),
including TGF-.beta.1, TGF-.beta.2, and TGF-.beta.3; bone
morphogenetic proteins (for example, BMP-1, BMP-2, BMP-3, BMP-4,
BMP-5, BMP-6, BMP-7, BMP-8, BMP-9); heparin-binding growth factors
(for example, fibroblast growth factor (FGF), epidermal growth
factor (EGF), platelet-derived growth factor (PDGF),
heparin-binding neurotrophic factor (HBNF), and insulin-like growth
factor (IGF)); connective tissue activated peptides (CTAPs),
osteogenic factors; colony stimulating factor; interferons,
including interferon-.alpha., interferon .alpha.-2a, interferon
.alpha.-2b, interferon .alpha.-n3, interferon-.beta., and
interferon-.gamma.; interleukins, including interleukin-1,
interleukin-2, interleukin-3, interleukin-4, interleukin-5,
interleukin-6, interleukin-7, interleukin-8, interleukin-9,
interleukin-10, interleukin-11, interleukin-12, interleukin-13,
interleukin-14, interleukin-15, interleukin-16, and interleukin-17;
tumor necrosis factor; tumor necrosis factor-.alpha.; granuloycte
colony-stimulating factor (G-CSF); granulocyte-macrophage
colony-stimulating factor (GM-CSF); macrophage colony-stimulating
factor; Inhibins (e.g., Inhibin A and Inhibin B); growth
differentiating factors (e.g., GDF-1); Activins (e.g., Activin A,
Activin B, and Activin AB); midkine (MD); and thymopoietin.
[0049] endorphins, i.e., peptides that activate opiate receptors,
including pharmacologically active endorphin derivatives such as
dermorphin, dynorphin, .alpha.-endorphin, .beta.-endorphin,
.gamma.-endorphin, .sigma.-endorphin [Leu.sup.5]enkephalin,
[Met.sup.5]enkephalin, substance P, and combinations thereof;
[0050] peptidyl hormones, such as activin, amylin, angiotensin,
atrial natriuretic peptide (ANP), calcitonin (derived from chicken,
eel, human, pig, rat, salmon, etc.), calcitonin gene-related
peptide, calcitonin N-terminal flanking peptide, cholecystokinin
(CCK), ciliary neurotrophic factor (CNTF), corticotropin
(adrenocorticotropin hormone, ACTH), corticotropin-releasing factor
(CRF or CRH), follicle-stimulating hormone (FSH), gastrin, gastrin
inhibitory peptide (GIP), gastrin-releasing peptide, glucagon,
gonadotropin-releasing factor (GnRF or GNRH), growth hormone
releasing factor (GRF, GRH), human chorionic gonadotropin (hCH),
inhibin A, inhibin B, insulin (derived from beef, human, pig,
etc.), leptin, lipotropin (LPH), luteinizing hormone (LH),
luteinizing hormone-releasing hormone (LHRH), lypressin,
.alpha.-melanocyte-stimulati- ng hormone,
.beta.-melanocyte-stimulating hormone, .gamma.-melanocyte-stim-
ulating hormone, melatonin, motilin, oxytocin (pitocin), pancreatic
polypeptide, parathyroid hormone (PTH), placental lactogen,
prolactin (PRL), prolactin-release inhibiting factor (PIF),
prolactin-releasing factor (PRF), secretin, somatostatin,
somatotropin (growth hormone, GH), somatostatin (SIF, growth
hormone-release inhibiting factor, GIF), thyrotropin
(thyroid-stimulating hormone, TSH), thyrotropin-releasing factor
(TRH or TRF), thyroxine, triiodothyronine, vasoactive intestinal
peptide (VIP), and vasopressin (antidiuretic hormone, ADH);.
[0051] analogues of LHRH, such as buserelin, deslorelin,
fertirelin, goserelin, histrelin, leuprolide (leuprorelin),
lutrelin, nafarelin, tryptorelin and combinations thereof,
[0052] kinins, such as bradykinin, potentiator B, bradykinin
potentiator C, and kallidin and combinations thereof.
[0053] enzymes, such as transferases, hydrolases, isomerases,
proteases, ligases and oxidoreductases such as esterases,
phosphatases, glycosidases and peptidases. Specific examples of
enzymes include super oxide dismutase (SOD), tissue plasminogen
activator (TPA), renin, adenosine deaminase,
.beta.-glucocerebrosidase, asparaginase, dornase-.alpha.,
hyaluronidase, elastase, trypsin, thymidin kinase (TK), tryptophan
hydroxylase, urokinase, and kallikrein;
[0054] enzyme inhibitors, such as leupeptin, chymostatin,
pepslatin, renin inhibitors, angiotensin converting enzyme (ACE)
inhibitors, and the like;
[0055] antibodies, including both monoclonal and polyclonal
antibodies, as well as antibody fragments, such as the
F(ab').sub.2, Fab, Fv and Fc fragments of monoclonal antibodies;
and
[0056] other peptidyl drugs, such as abarelix, anakinra, ancestim,
bivalirudin, bleomycin, bombesin, desmopressin acetate,
des-Q14-ghrelin, enterostatin, erythropoeitin, exendin-4,
filgrastim, gonadorelin, insulinotropin, lepirudin, magainin I,
magainin II, nerve growth factor, pentigetide, thrombopoietin,
thymosin .alpha.-1, and urotensin II and combinations thereof.
[0057] The term "fluid" as used herein refers to matter that is
nonsolid or at least partially gaseous and/or liquid. A fluid may
contain a solid that is minimally, partially or fully solvated,
dispersed or suspended. Examples of fluids include, without
limitation, aqueous liquids (including water per se and salt water)
and nonaqueous liquids such as organic solvents and the like. As
used herein, the term "fluid" is not synonymous with the term "ink"
in that an ink must contain a colorant and may not be gaseous.
[0058] The term "near" is used to refer to the distance from the
focal point of the focused acoustic radiation to the surface of the
fluid from which a droplet is to be ejected. The distance should be
such that the focused acoustic radiation directed into the fluid
results in droplet ejection from the fluid surface, and one of
ordinary skill in the art will be
[0059] able to select an appropriate distance for any given fluid
using straightforward and routine experimentation. Generally,
however, a suitable distance between the focal point of the
acoustic radiation and the fluid surface is in the range of about 1
to about 15 times the wavelength of the speed of sound in the
fluid, more typically in the range of about 1 to about 10 times
that wavelength, preferably in the range of about 1 to about 5
times that wavelength.
[0060] The terms "focusing means" and "acoustic focusing means"
refer to a means for causing acoustic waves to converge at a focal
point by either a device separate from the acoustic energy source
that acts like an optical lens, or by the spatial arrangement of
acoustic energy sources to effect convergence of acoustic energy at
a focal point by constructive and destructive interference. A
focusing means may be as simple as a solid member having a curved
surface, or it may include complex structures such as those found
in Fresnel lenses, which employ diffraction in order to direct
acoustic radiation. Suitable focusing means also include phased
array methods as known in the art and described, for example, in
U.S. Pat. No. 5,798,779 to Nakayasu et al. and Amemiya et al.
(1997) Proceedings of the 1997 IS&T NIP13 International
Conference on Digital Printing Technologies Proceedings, at pp.
698-702.
[0061] The term "reservoir" as used herein refers to a receptacle
or chamber for holding or containing a fluid. Thus, a fluid in a
reservoir necessarily has a free surface, i.e., a surface that
allows a droplet to be ejected therefrom. A reservoir may also be a
locus on a substrate surface within which a fluid is
constrained.
[0062] The term "substrate" as used herein refers to a substrate
having a porous surface. The entire substrate may be porous, in
which case the porous surface is the surface of the substrate
itself. Alternatively, a nonporous substrate may be used providing
that a porous surface layer is present that provides the porous
surface. The substrate may be constructed in any of a number of
forms such as wafers, slides, well plates, membranes, etc. Suitable
substrates include uncoated porous glass slides, including
controlled pore glass (CPG) slides; porous glass slides coated with
a polymeric coating, e.g., an aminosilane or poly-L-lysine coating,
thus having a porous polymeric surface; and nonporous glass slides
coated with a porous coating, such as may be comprised of a
cellulosic polymer (e.g., nitrocellulose), polyacrylamide, or a
porous metal (for example, comprised of microporous aluminum).
Examples of commercially available substrates having porous
surfaces include the Fluorescent Array Surface Technology
(FAST.TM.) slides available from Schleicher & Schuell, Inc.
(Keene, N.H.), which are coated with a 10-30 .mu.m thick porous,
fluid-permeable nitrocellulose layer that substantially increases
the available binding area per unit area of surface. Other
commercially available porous substrates include the
CREATIVECHIP.RTM. permeable slides currently available from
Eppendorf AG (Hamburg, Germany), and substrates having
"three-dimensional" geometry, by virtue of an ordered, highly
porous structure that enables reagents to flow into and penetrate
through the pores and channels of the entire structure. Such
substrates are available from Gene Logic, Inc. under the tradename
"Flow-Thru Chip," and are described by Steel et al. in Chapter 5 of
Microarray Biochip Technology (BioTechniques Books, Natick, Mass.,
2000).
[0063] The term "porous" as in a "porous substrate" or a "substrate
having a porous surface," refers to a substrate or surface,
respectively, having a porosity (void percentage) in the range of
about 1% to about 99%, preferably about 5% to about 99%, more
preferably in the range of about 15% to about 95%, and an average
pore size of about 100 .ANG. to about 1 mm, typically about 500
.ANG. to about 0.5 mm.
[0064] The term "impermeable" is used in the conventional sense to
mean not permitting water or other fluid to pass through. The term
"permeable" as used herein means not "impermeable." Thus, a
"permeable substrate" and a "substrate having a permeable surface"
refer to a substrate or surface, respectively, which can be
permeated with water or other fluid.
[0065] While the foregoing support materials are representative of
conventionally used substrates, it is to be understood that a
substrate may in fact comprise any biological, nonbiological,
organic and/or inorganic material so long as a porous surface is
provided, and may be in any of a variety of physical forms, e.g.,
particles, strands, precipitates, gels, sheets, tubing, spheres,
containers, capillaries, pads, slices, films, plates, and the like,
and may further have any desired shape, such as a disc, square,
sphere, circle, etc. The substrate surface may or may not be flat,
e.g., the surface may contain raised or depressed regions. A
substrate may additionally contain or be derivatized to contain
reactive functionalities that covalently link a compound to the
substrate surface. These are widely known and include, for example,
silicon dioxide supports containing reactive Si--OH groups,
polyacrylamide supports, polystyrene supports, polyethylene glycol
supports, and the like.
[0066] The term "surface modification" as used herein refers to the
chemical and/or physical alteration of a surface by an additive or
subtractive process to change one or more chemical and/or physical
properties of a substrate surface or a selected site or region of a
substrate surface. For example, surface modification may involve
(1) changing the wetting properties of a surface, (2)
functionalizing a surface, i.e., providing, modifying or
substituting surface functional groups, (3) defunctionalizing a
surface, i.e., removing surface functional groups, (4) otherwise
altering the chemical composition of a surface, e.g., through
etching, (5) increasing or decreasing surface roughness, (6)
providing a coating on a surface, e.g., a coating that exhibits
wetting properties that are different from the wetting properties
of the surface, and/or (7) depositing particulates on a
surface.
[0067] "Optional" or "optionally" means that the subsequently
described circumstance may or may not occur, so that the
description includes instances where the circumstance occurs and
instances where it does not.
[0068] The term "substantially" as in, for example, the phrase
"substantially all molecules of an array," refers to at least 90%,
preferably at least 95%, more preferably at least 99%, and most
preferably at least 99.9%, of the molecules of an array. Other uses
of the term "substantially" involve an analogous definition.
[0069] The invention accordingly provides a method for acoustically
generating fluid droplets from a plurality of individual reservoirs
to form a high density molecular array on a porous substrate
surface. That is, focused acoustic energy is used to eject single
fluid droplets from the free surface of a fluid (e.g., in a
reservoir or well plate), generally toward discrete sites on a
porous substrate surface, enabling extraordinarily precise
deposition and consistent droplet size and velocity. The device
comprises a plurality of reservoirs, each adapted to contain a
fluid, an ejector comprising an acoustic radiation generator for
generating acoustic radiation and a focusing means for focusing the
acoustic radiation generated (at a focal point within and
sufficiently near the fluid surface in each of the reservoirs to
result in the ejection of droplets therefrom), and a means for
positioning the ejector in acoustic coupling relationship to each
of the reservoirs.
[0070] The use of such a focused acoustic ejection system enables
preparation of arrays on porous surfaces that will generally have a
density in the range of at least 62,500 molecular moieties as array
"elements" (e.g., surface-bound oligomers) per square centimeter of
substrate surface, preferably at least 250,000 molecular moieties
per square centimeter of substrate surface, more preferably at
least 1,000,000, most preferably at least 1,500,000, and optimally
in the range of about 1,500,000 to about 4,000,000 molecular
moieties per square centimeter of substrate surface. Each molecular
moiety may be different from every other molecular moiety within
the array, such that the array is a "combinatorial" library of the
different molecular moieties.
[0071] A significant advantage of using focused acoustic energy
technology in the manufacture of arrays is that the aforementioned
array densities may be achieved on substrates with porous surfaces,
and even permeable surfaces. More specifically, the present
acoustic ejection method can be used to manufacture high density
arrays that can be read with a high precision digitizing scanner
capable of 2 .mu.m resolution, by depositing droplets having a
volume on the order of 1 pL, resulting in deposited spots about 18
.mu.m in diameter. For ultra-high density arrays, a smaller droplet
volume is necessary, typically less than about 0.03 pL (deposition
of droplets having a volume on the order of 0.025 pL will result in
deposited spots about 4.5 .mu.m in diameter). Localization of
deposited droplets using chemical or physical means, such as
described in U.S. Pat. No. 6,054,270 to Southern et al., is
unnecessary because acoustic ejection enables precisely directed
minute droplets to be deposited with extraordinary accuracy at a
particular site.
[0072] FIG. 1 illustrates a focused acoustic ejection device in
simplified cross-sectional view. As with all figures referenced
herein, in which like parts are referenced by like numerals, FIG. 1
is not to scale, and certain dimensions may be exaggerated for
clarity of presentation. The device 11 includes a plurality of
reservoirs, i.e., at least two reservoirs, with a first reservoir
indicated at 13 and a second reservoir indicated at 15, each
adapted to contain a fluid having a fluid surface, e.g., a first
fluid 14 and a second fluid 16 having fluid surfaces respectively
indicated at 17 and 19. Fluids 14 and 16 may the same or different.
As shown, the reservoirs are of substantially identical
construction so as to be substantially acoustically
indistinguishable, but identical construction is not a requirement.
The reservoirs are shown as separate removable components but may,
if desired, be fixed within a plate or other substrate. For
example, the plurality of reservoirs may comprise individual wells
in a well plate, optimally although not necessarily arranged in an
array. Each of the reservoirs 13 and 15 is preferably axially
symmetric as shown, having vertical walls 21 and 23 extending
upward from circular reservoir bases 25 and 27 and terminating at
openings 29 and 31, respectively, although other reservoir shapes
may be used. The material and thickness of each reservoir base
should be such that acoustic radiation may be transmitted
therethrough and into the fluid contained within the
reservoirs.
[0073] The device also includes an acoustic ejector 33 comprised of
an acoustic radiation generator 35 for generating acoustic
radiation and a focusing means 37 for focusing the acoustic
radiation at a focal point within the fluid from which a droplet is
to be ejected, near the fluid surface. As shown in FIG. 1, the
focusing means 37 may comprise a single solid piece having a
concave surface 39 for focusing acoustic radiation, but the
focusing means may be constructed in other ways as discussed below.
The acoustic ejector 33 is thus adapted to generate and focus
acoustic radiation so as to eject a droplet of fluid from each of
the fluid surfaces 17 and 19 when acoustically coupled to
reservoirs 13 and 15 and thus to fluids 14 and 16, respectively.
The acoustic radiation generator 35 and the focusing means 37 may
function as a single unit controlled by a single controller, or
they may be independently controlled, depending on the desired
performance of the device. Typically, single ejector designs are
preferred over multiple ejector designs because accuracy of droplet
placement and consistency in droplet size and velocity are more
easily achieved with a single ejector.
[0074] As will be appreciated by those skilled in the art, any of a
variety of focusing means may be employed in conjunction with the
present invention. For example, one or more curved surfaces may be
used to direct acoustic radiation to a focal point near a fluid
surface. One such technique is described in U.S. Pat. No. 4,308,547
to Lovelady et al. Focusing means with a curved surface have been
incorporated into the construction of commercially available
acoustic transducers such as those manufactured by Panametrics Inc.
(Waltham, Mass.). In addition, Fresnel lenses are known in the art
for directing acoustic energy at a predetermined focal distance
from an object plane. See, e.g., U.S. Pat. No. 5,041,849 to Quate
et al. Fresnel lenses may have a radial phase profile that
diffracts a substantial portion of acoustic energy into a
predetermined diffraction order at diffraction angles that vary
radially with respect to the lens. The diffraction angles should be
selected to focus the acoustic energy within the diffraction order
on a desired object plane.
[0075] There are also a number of ways to acoustically couple the
ejector 33 to each individual reservoir and thus to the fluid
therein. One such approach is through direct contact as is
described, for example, in U.S. Pat. No. 4,308,547 to Lovelady et
al., wherein a focusing means constructed from a hemispherical
crystal having segmented electrodes is submerged in a liquid to be
ejected. The aforementioned patent further discloses that the
focusing means may be positioned at or below the surface of the
liquid. However, this approach for acoustically coupling the
focusing means to a fluid is undesirable when the ejector is used
to eject different fluids in a plurality of containers or
reservoirs, as repeated cleaning of the focusing means would be
required in order to avoid cross-contamination. The cleaning
process would necessarily lengthen the transition time between each
droplet ejection event. In addition, in such a method, fluid would
adhere to the ejector as it is removed from each container, wasting
material that may be costly or rare.
[0076] Thus, a preferred approach would be to acoustically couple
the ejector to the reservoirs and reservoir fluids without
contacting any portion of the ejector, e.g., the focusing means,
with any of the fluids to be ejected. To this end, the present
invention provides an ejector positioning means for positioning the
ejector in controlled and repeatable acoustic coupling with each of
the fluids in the reservoirs to eject droplets therefrom without
submerging the ejector therein. This typically involves direct or
indirect contact between the ejector and the external surface of
each reservoir. When direct contact is used in order to
acoustically couple the ejector to each reservoir, it is preferred
that the direct contact is wholly conformal to ensure efficient
acoustic energy transfer. That is, the ejector and the reservoir
should have corresponding surfaces adapted for mating contact.
Thus, if acoustic coupling is achieved between the ejector and
reservoir through the focusing means, it is desirable for the
reservoir to have an outside surface that corresponds to the
surface profile of the focusing means. Without conformal contact,
efficiency and accuracy of acoustic energy transfer may be
compromised. In addition, since many focusing means have a curved
surface, the direct contact approach may necessitate the use of
reservoirs having a specially formed inverse surface.
[0077] Optimally, acoustic coupling is achieved between the ejector
and each of the reservoirs through indirect contact, as illustrated
in FIG. 1A. In the figure, an acoustic coupling medium 41 is placed
between the ejector 33 and the base 25 of reservoir 13, with the
ejector and reservoir located at a predetermined distance from each
other. The acoustic coupling medium may be an acoustic coupling
fluid, preferably an acoustically homogeneous material in conformal
contact with both the acoustic focusing means 37 and each
reservoir. In addition, it is important to ensure that the fluid
medium is substantially free of material having different acoustic
properties than the fluid medium itself. As shown, the first
reservoir 13 is acoustically coupled to the acoustic focusing means
37 such that an acoustic wave is generated by the acoustic
radiation generator and directed by the focusing means 37 into the
acoustic coupling medium 41, which then transmits the acoustic
radiation into the reservoir 13.
[0078] In operation, reservoirs 13 and 15 of the device are each
filled with first and second fluids 14 and 16, respectively, as
shown in FIG. 1. The acoustic ejector 33 is positionable by means
of ejector positioning means 43, shown below reservoir 13, in order
to achieve acoustic coupling between the ejector and the reservoir
through acoustic coupling medium 41. Substrate 45 is positioned
above and in proximity to the first reservoir 13 such that one
surface of the substrate, shown in FIG. 1 as underside surface 51,
faces the reservoir and is substantially parallel to the surface 17
of the fluid 14 therein. Once the ejector, the reservoir and the
substrate are in proper alignment, the acoustic radiation generator
35 is activated to produce acoustic radiation that is directed by
the focusing means 37 to a focal point 47 near the fluid surface 17
of the first reservoir. As a result, droplet 49 is ejected from the
fluid surface 17 onto a designated site on the underside surface 51
of the substrate. The ejected droplet may be retained on the
substrate surface by solidifying thereon after contact; in such an
embodiment, it is necessary to maintain the substrate at a low
temperature, i.e., a temperature that results in droplet
solidification after contact. Alternatively, or in addition, a
molecular moiety within the droplet attaches to the substrate
surface after contract, through adsorption, physical
immobilization, or covalent binding.
[0079] Then, as shown in FIG. 1B, a substrate positioning means 50
repositions the substrate 45 over reservoir 15 in order to receive
a droplet therefrom at a second designated site. FIG. 1B also shows
that the ejector 33 has been repositioned by the ejector
positioning means 43 below reservoir 15 and in acoustically coupled
relationship thereto by virtue of acoustic coupling medium 41. Once
properly aligned as shown in FIG. 1B, the acoustic radiation
generator 35 of ejector 33 is activated to produce acoustic
radiation that is then directed by focusing means 37 to a focal
point within fluid 16 near the fluid surface 19, thereby ejecting
droplet 53 onto the substrate. It should be evident that such
operation is illustrative of how the inventive device may be used
to eject a plurality of fluids from reservoirs in order to form a
pattern, e.g., an array, on the substrate surface 51. It should be
similarly evident that the device may be adapted to eject a
plurality of droplets from one or more reservoirs onto the same
site of the substrate surface.
[0080] As discussed above, either individual, e.g., removable,
reservoirs or well plates may be used to contain fluids that are to
be ejected, wherein the reservoirs or the wells of the well plate
are preferably substantially acoustically indistinguishable from
one another. Also, unless it is intended that the ejector is to be
submerged in the fluid to be ejected, the reservoirs or well plates
must have acoustic transmission properties sufficient to allow
acoustic radiation from the ejector to be conveyed to the surfaces
of the fluids to be ejected. Typically, this involves providing
reservoir or well bases that are sufficiently thin to allow
acoustic radiation to travel therethrough without unacceptable
dissipation. In addition, the material used in the construction of
reservoirs must be compatible with the fluids contained therein.
Thus, if it is intended that the reservoirs or wells contain an
organic solvent such as acetonitrile, polymers that dissolve or
swell in acetonitrile would be unsuitable for use in forming the
reservoirs or well plates. For water-based fluids, a number of
materials are suitable for the construction of reservoirs and
include, but are not limited to, ceramics such as silicon oxide and
aluminum oxide, metals such as stainless steel and platinum, and
polymers such as polyester and polytetrafluoroethylene. Many well
plates suitable for use with the employed device are commercially
available and may contain, for example, 96, 384 or 1536 wells per
well plate. Manufactures of suitable well plates for use in the
employed device include Corning Inc. (Corning, N.Y.) and Greiner
America, Inc. (Lake Mary, Fla.). However, the availability of such
commercially available well plates does not preclude manufacture
and use of custom-made well plates containing at least about 10,000
wells, or as many as 100,000 wells or more. For array forming
applications, it is expected that about 100,000 to about 4,000,000
or more reservoirs may be employed. In addition, to reduce the
amount of movement and time needed to align the ejector with each
reservoir or reservoir well, it is preferable that the center of
each reservoir is located not more than about 1 centimeter,
preferably not more than about 1 millimeter and optimally not more
than about 0.5 millimeter from a neighboring reservoir center.
[0081] Moreover, the present methodology may be adapted to eject
fluids of virtually any type and amount desired. The fluid may be
aqueous and/or nonaqueous. Examples of fluids include, but are not
limited to, aqueous fluids such as water per se and water-solvated
ionic and non-ionic solutions, organic solvents, and lipidic
liquids, suspensions of immiscible fluids and suspensions or
slurries of solids in liquids. Because the invention is readily
adapted for use with high temperatures, fluids such as liquid
metals, ceramic materials, and glasses may be used; see, e.g.,
co-pending patent application U.S. Ser. No. 09/669/194 ("Method and
Apparatus for Generating Droplets of Immiscible Fluids"), inventors
Ellson and Mutz, filed on Sep. 25, 2000, and assigned to Picoliter,
Inc. (Mountain View, Calif.). U.S. Pat. Nos. 5,520,715 and
5,722,479 to Oeftering describe the use of acoustic ejection for
liquid metal for forming structures using a single reservoir and
adding fluid to maintain focus. U.S. Pat. No. 6,007,183 to Horine
is another patent that pertains to the use of acoustic energy to
eject droplets of liquid metal. The capability of producing fine
droplets of such materials is in sharp contrast to piezoelectric
technology, insofar as piezoelectric systems perform suboptimally
at elevated temperatures. Furthermore, because of the precision
that is possible using the inventive technology, the device may be
used to eject droplets from a reservoir adapted to contain no more
than about 100 nanoliters of fluid, preferably no more than 10
nanoliters of fluid. In certain cases, the ejector may be adapted
to eject a droplet from a reservoir adapted to contain about 1 to
about 100 nanoliters of fluid. This is particularly useful when the
fluid to be ejected contains rare or expensive biomolecules,
wherein it may be desirable to eject droplets having a volume of
about 1 picoliter or less, e.g., having a volume in the range of
about 0.025 pL to about 1 pL.
[0082] It will be appreciated that various components of a suitable
acoustic ejection device may require individual control or
synchronization to form an array on a substrate. For example, the
ejector positioning means may be adapted to eject droplets from
each reservoir in a predetermined sequence associated with an array
to be prepared on a substrate surface. Similarly, the substrate
positioning means for positioning the substrate surface with
respect to the ejector may be adapted to position the substrate
surface to receive droplets in a pattern or array thereon. Either
or both positioning means, i.e., the ejector positioning means and
the substrate positioning means, may be constructed from, for
example, motors, levers, pulleys, gears, a combination thereof, or
other electromechanical or mechanical means known to one of
ordinary skill in the art. It is preferable to ensure that there is
a correspondence between the movement of the substrate, the
movement of the ejector and the activation of the ejector to ensure
proper array formation.
[0083] The device may also include certain performance-enhancing
features. For example, the device may include a cooling means for
lowering the temperature of the substrate surface to ensure, for
example, that the ejected droplets adhere to the substrate. The
cooling means may be adapted to maintain the substrate surface at a
temperature that allows fluid to partially or preferably
substantially solidify after the fluid comes into contact
therewith. In the case of aqueous fluids, the cooling means should
have the capacity to maintain the substrate surface at about
0.degree. C. In addition, repeated application of acoustic energy
to a reservoir of fluid may result in heating of the fluid. Heating
can of course result in unwanted changes in fluid properties such
as viscosity, surface tension and density. Thus, the device may
further comprise means for maintaining fluid in the reservoirs at a
constant temperature. Design and construction of such temperature
maintaining means are known to one of ordinary skill in the art and
may comprise, e.g., components such a heating element, a cooling
element, or a combination thereof. For many biomolecular deposition
applications, it is generally desired that the fluid containing the
biomolecule is kept at a constant temperature without deviating
more than about 1.degree. C. or 2.degree. C. therefrom. In
addition, for a biomolecular fluid that is particularly heat
sensitive, it is preferred that the fluid be kept at a temperature
that does not exceed about 10.degree. C. above the melting point of
the fluid, preferably at a temperature that does not exceed about
5.degree. C. above the melting point of the fluid. Thus, for
example, when the biomolecule-containing fluid is aqueous, it may
be optimal to keep the fluid at about 4.degree. C. during
ejection.
[0084] For some applications, especially those involving acoustic
deposition of molten metals or other materials, a heating element
may be provided for maintaining the substrate at a temperature
below the melting point of the molten material, but above ambient
temperature so that control of the rapidity of cooling may be
effected. The rapidity of cooling may thus be controlled, to permit
experimentation regarding the properties of combinatorial
compositions such as molten deposited alloys cooled at different
temperatures. For example, it is known that metastable materials
are generally more likely to be formed with rapid cooling, and
other strongly irreversible conditions. The approach of generating
materials by different cooling or quenching rates my be termed
combinatorial quenching, and could be effected by changing the
substrate temperature between acoustic ejections of the molten
material. A more convenient method of evaluating combinatorial
compositions solidified from the molten state at different rates is
by generating multiple arrays having the same pattern of nominal
compositions ejected acoustically in the molten state onto
substrates maintained at different temperatures.
[0085] In some cases, a substrate surface may be modified prior to
formation of an array thereon. Surface modification may involve
functionalization or defunctionalization, smoothing or roughening,
changing surface conductivity, coating, degradation, passivation,
or otherwise altering the surface's chemical composition or
physical properties. A preferred surface modification method
involves altering the wetting properties of the surface, for
example to facilitate confinement of a droplet ejected on the
surface within a designated area or enhancement of the kinetics for
the surface attachment of molecular moieties contained in the
ejected droplet. A preferred method for altering the wetting
properties of the substrate surface involves deposition of droplets
of a suitable surface modification fluid at each designated site of
the substrate surface prior to acoustic ejection of fluids to
form-an array thereon. In this way, the "spread" of the
acoustically ejected droplets may be optimized and consistency in
spot size (i.e., diameter, height and overall shape) ensured. One
way to implement the method involves acoustically coupling the
ejector to a modifier reservoir containing a surface modification
fluid and then activating the ejector, as described in detail
above, to produce and eject a droplet of surface modification fluid
toward a designated site on the substrate surface. The method is
repeated as desired to deposit surface modification fluid at
additional designated sites. This method is useful in a number of
applications including, but not limited to, spotting oligomers to
form an array on a substrate surface or synthesizing array
oligomers in situ. As noted above, other physical properties of the
surface that may be modified include thermal properties and
electrical conductivity.
[0086] FIG. 2 schematically illustrates in simplified
cross-sectional view a specific embodiment of the aforementioned
method in which a dimer is synthesized on a substrate using a
device similar to that illustrated in FIG. 1, but including a
modifier reservoir 59 containing a surface modification fluid 60
having a fluid surface 61. FIG. 2A illustrates the ejection of a
droplet 63 of surface modification fluid 60 selected to alter the
wetting properties of a designated site on surface 51 of the
substrate 45 where the dimer is to be synthesized. The ejector 33
is positioned by the ejector positioning means 43 below modifier
reservoir 59 in order to achieve acoustic coupling therewith
through acoustic coupling medium 41. Substrate 45 is positioned
above the modifier reservoir 19 at a location that enables acoustic
deposition of a droplet of surface modification fluid 60 at a
designated site. Once the ejector 33, the modifier reservoir 59 and
the substrate 45 are in proper alignment, the acoustic radiation
generator 35 is activated to produce acoustic radiation that is
directed by the focusing means 37 in a manner that enables ejection
of droplet 63 of the surface modification fluid 60 from the fluid
surface 61 onto a designated site on the underside surface 51 of
the substrate. Once the droplet 63 contacts the substrate surface
51, the droplet modifies an area of the substrate surface to result
in an increase or decrease in the surface energy of the area with
respect to deposited fluids.
[0087] Then, as shown in FIG. 2B, the substrate 45 is repositioned
by the substrate positioning means 50 such that the region of the
substrate surface modified by droplet 63 is located directly over
reservoir 13. FIG. 3B also shows that the ejector 33 is positioned
by the ejector positioning means below reservoir 13 to acoustically
couple the ejector and the reservoir through acoustic coupling
medium 41. Once properly aligned, the ejector 33 is again activated
so as to eject droplet 49 onto substrate. Droplet 49 contains a
first monomeric moiety 65, preferably a biomolecule such as a
protected nucleoside or amino acid, which after contact with the
substrate surface attaches thereto by covalent bonding or
adsorption.
[0088] Then, as shown in FIG. 2C, the substrate 45 is again
repositioned by the substrate positioning means 50 such that the
site having the first monomeric moiety 65 attached thereto is
located directly over reservoir 15 in order to receive a droplet
therefrom. FIG. 3B also shows that the ejector 33 is positioned by
the ejector positioning means below reservoir 15 to acoustically
couple the ejector and the reservoir through acoustic coupling
medium 41. Once properly aligned, the ejector 33 is again activated
so as to eject droplet 53 is ejected onto substrate. Droplet 53
contains a second monomeric moiety 67, adapted for attachment to
the first monomeric moiety 65, typically involving formation of a
covalent bond so as to generate a dimer as illustrated in FIG. 3D.
The aforementioned steps may be repeated to generate an oligomer,
e.g., an oligonucleotide, of a desired length.
[0089] The chemistry employed in synthesizing substrate-bound
oligonucleotides in this way will generally involve
now-conventional techniques known to those skilled in the art of
nucleic acid chemistry and/or described in the pertinent literature
and texts. See, for example, DNA Microarrays: A Practical Approach,
M. Schena, Ed. (Oxford University Press, 1999). That is, the
individual coupling reactions are conducted under standard
conditions used for the synthesis of oligonucleotides and
conventionally employed with automated oligonucleotide
synthesizers. Such methodology is described, for example, in D. M.
Matteuci et al. (1980) Tet. Lett. 521:719, U.S. Pat. No. 4,500,707
to Caruthers et al., and U.S. Pat. Nos. 5,436,327 and 5,700,637 to
Southern et al.
[0090] Alternatively, an oligomer may be synthesized prior to
attachment to the substrate surface and then "spotted" onto a
particular locus on the surface using the focused acoustic ejection
methodology described in detail above. Again, the oligomer may be
an oligonucleotide, an oligopeptide, or any other biomolecular (or
nonbiomolecular) oligomer moiety. Preparation of substrate-bound
peptidic molecules, e.g., in the formation of peptide arrays and
protein arrays, is described in co-pending patent application U.S.
Ser. No. 09/669,997 ("Focused Acoustic Energy in the Preparation of
Peptidic Arrays"), inventors Mutz and Ellson, filed Sep. 25, 2000
and assigned to Picoliter, Inc. (Mountain View, Calif.).
Preparation of substrate-bound oligonucleotides, particularly
arrays of oligonucleotides wherein at least one of the
oligonucleotides contains one or more partially nonhybridizing
segments, is described in co-pending patent application U.S. Ser.
No. 09/699,267 ("Arrays of Oligonucleotides Containing
Nonhybridizing Segments"), inventor Ellson, also filed on Sep. 25,
2000 and assigned to Picoliter, Inc. Preparation of other types of
arrays using focused acoustic energy is described in co-pending
patent application U.S. Ser. No. 09//727/392, filed on Nov. 29,
2000 and also assigned to Picoliter, Inc.
[0091] It will be appreciated by those in the art that the
invention is useful in the preparation of high density
combinatorial libraries containing a variety of synthetic,
semi-synthetic or naturally occurring molecular moieties, insofar
as focused acoustic energy makes possible the use and manipulation
of extremely small volumes of fluids with extraordinary accuracy.
This is in sharp contrast to prior techniques for preparing
combinatorial libraries, with which effective spot-to-spot binding
cannot be guaranteed. Furthermore, piezoelectric jet technologies
are limited with respect to the fluids that may be used since high
temperatures are required, while the present invention does not
require high temperatures (although heat may be necessary in some
cases, i.e., with fluids having high melting points) and thus
allows for the possibility of using fluids that may be
heat-sensitive or even flammable.
[0092] It should be evident, then, that many variations of the
invention are possible. For example, each of the ejected droplets
may be deposited as an isolated and "final" feature, e.g., in
spotting oligonucleotides, as mentioned above. Alternatively, or in
addition, a plurality of ejected droplets may be deposited on the
same location of a substrate surface in order to synthesize a
biomolecular array in situ, as described above. For array
fabrication, it is expected that various washing steps may be used
between droplet ejection steps. Such wash steps may involve, e.g.,
submerging the entire substrate surface on which features have been
deposited in a washing fluid. In a modification of this process,
the substrate surface may be deposited on a fluid containing a
reagent that chemically alters all features at substantially the
same time, e.g., to activate and/or deprotect biomolecular features
already deposited on the substrate surface to provide sites on
which additional coupling reactions may occur.
[0093] The focused acoustic energy methodology described herein
enables ejection of droplets at a rate of at least about 1,000,000
droplets per minute from the same reservoir, and at a rate of at
least about 100,000 drops per minute from different reservoirs. In
addition, current positioning technology allows for the ejector
positioning means to move from one reservoir to another quickly and
in a controlled manner, thereby allowing fast and controlled
ejection of different fluids. That is, current commercially
available technology allows the ejector to be moved from one
reservoir to another, with repeatable and controlled acoustic
coupling at each reservoir, in less than about 0.1 second for high
performance positioning means and in less than about 1 second for
ordinary positioning means. A custom designed system will allow the
ejector to be moved from one reservoir to another with repeatable
and controlled acoustic coupling in less than about 0.001 second.
In order to provide a custom designed system, it is important to
keep in mind that there are two basic kinds of motion: pulse and
continuous. Pulse motion involves the discrete steps of moving an
ejector into position, emitting acoustic energy, and moving the
ejector to the next position; again, using a high performance
positioning means with such a method allows repeatable and
controlled acoustic coupling at each reservoir in less than 0.1
second. A continuous motion design, on the other hand, moves the
ejector and the reservoirs continuously, although not at the same
speed, and provides for ejection during movement. Since the pulse
width is very short, this type of process enables over 10 Hz
reservoir transitions, and even over 1000 Hz reservoir
transitions.
[0094] In order to ensure the accuracy of fluid ejection, it is
important to determine the location and the orientation of the
fluid surface from which a droplet is to be ejected
[0095] with respect to the ejector. Otherwise, ejected droplets may
be improperly sized or travel in an improper trajectory. Thus,
another embodiment of the invention relates to a method for
determining the height of a fluid surface in a reservoir between
ejection events. The method involves acoustically coupling a
fluid-containing reservoir to an acoustic radiation generator and
activating the generator to produce a detection acoustic wave that
travels to the fluid surface and is reflected thereby as a
reflected acoustic wave. Parameters of the reflected acoustic
radiation are then analyzed in order to assess the spatial
relationship between the acoustic radiation generator and the fluid
surface. Such an analysis will involve the determination of the
distance between the acoustic radiation generator and the fluid
surface and/or the orientation of the fluid surface in relationship
to the acoustic radiation generator.
[0096] More particularly, the acoustic radiation generator may be
activated so as to generate low energy acoustic radiation that is
insufficiently energetic to eject a droplet from the fluid surface.
This is typically done by using an extremely short pulse (on the
order of tens of nanoseconds) relative to that normally required
for droplet ejection (on the order of microseconds). By determining
the time it takes for the acoustic radiation to be reflected by the
fluid surface back to the acoustic radiation generator and then
correlating that time with the speed of sound in the fluid, the
distance--and thus the fluid height--may be calculated. Of course,
care must be taken in order to ensure that acoustic radiation
reflected by the interface between the reservoir base and the fluid
is discounted. It will be appreciated by those of ordinary skill in
the art that such a method employs conventional or modified sonar
techniques.
[0097] Once the analysis has been performed, an ejection acoustic
wave having a focal point near the fluid surface is generated in
order to eject at least one droplet of the fluid, wherein the
optimum intensity and directionality of the ejection acoustic wave
is determined using the aforementioned analysis optionally in
combination with additional data. The "optimum" intensity and
directionality are generally selected to produce droplets of
consistent size and velocity. For example, the desired intensity
and directionality of the ejection acoustic wave may be determined
by using not only the spatial relationship assessed as above, but
also geometric data associated with the reservoir, fluid property
data associated with the fluid to be ejected, and/or by using
historical droplet ejection data associated with the ejection
sequence. In addition, the data may show the need to reposition the
ejector so as to reposition the acoustic radiation generator with
respect to the fluid surface, in order to ensure that the focal
point of the ejection acoustic wave is near the fluid surface,
where desired. For example, if analysis reveals that the acoustic
radiation generator is positioned such that the ejection acoustic
wave cannot be focused near the fluid surface, the acoustic
radiation generator is repositioned using vertical, horizontal
and/or rotational movement to allow appropriate focusing of the
ejection acoustic wave.
[0098] In general, screening for the properties of the array
constituents will be performed in a manner appropriate to the type
of array generated. Screening for biological properties such as
ligand binding or hybridization may be generally performed in the
manner described in U.S. Pat. Nos. 5,744,305 and 5,445,934 to Fodor
et al. 5,143,854 and 5,405,783 to Pirrung et al., and 5,700,637 and
6,054,270 to Southern et al.
[0099] Screening a substrate for material properties may be
effected by measuring physical and chemical properties by routine
methods easily adaptable to microarrays. In addition to bulk
material characteristics or properties, surface specific properties
may be measured by surface specific physical techniques and
physical techniques that are adapted to surface characterization.
Macroscopic surface phenomena including adsorption, catalysis,
surface reactions including oxidation, hardness, lubrication and
friction, may be examined on a molecular scale using such
characterization techniques. Various physical surface
characterization techniques include without limitation diffractive
techniques, spectroscopic techniques, microscopic surface imaging
techniques, surface ionization mass spectroscopic techniques,
thermal desorption techniques and ellipsometry. It should be
appreciated that these classifications are arbitrary made for
purposes of explication, and some overlap may exist.
[0100] It is to be understood that while the invention has been
described in conjunction with the preferred specific embodiments
thereof, the foregoing description is intended to illustrate and
not limit the scope of the invention. Other aspects, advantages and
modifications will be apparent to those skilled in the art to which
the invention pertains.
[0101] All patents, patent applications, journal articles and other
references cited herein are incorporated by reference in their
entireties.
[0102] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to implement the invention, and are not intended
to limit the scope of what the inventors regard as their invention.
Efforts have been made to ensure accuracy with respect to numbers
(e.g., amounts, temperature, etc.) but some errors and deviations
should be accounted for. Unless indicated otherwise, parts are
parts by weight, temperature is in .degree. C. and pressure is at
or near atmospheric.
EXAMPLE 1
[0103] This example describes preparation of an array of
oligonucleotides in the form of a library, and demonstrates the use
of focused acoustic energy in the solid phase synthesis of
oligonucleotides.
[0104] Microporous glass, preferably controlled pore size glass
(CPG), is sintered onto the surface of a glass plate to form a CPG
layer having a thickness sufficient to enable permeation to both
the downward flow and the lateral wicking of fluids. Generally, a
sufficient thickness is greater than about 10 .mu.m.
[0105] Accordingly, the CPG is applied to the glass surface at a
thickness of about 20 .mu.m and the glass with powdered CPG
resident thereon is heated at 750.degree. C. for about 20 minutes
then cooled. Commercially available microscope slides (BDH Super
Premium 76.times.26.times.1 mm) are used as supports. Depending on
the specific glass substrate and CPG material used the sintering
temperature and time may be adjusted to obtain a permeable and
porous layer that is adequately attached to the glass beneath while
substantially maintaining the permeability to fluids and thickness
of the microporous glass layer. The slides heated for 20 minutes
with a 1 cm square patch of microporous glass applied at a
pre-heating thickness of about 20 .mu.m yield a sintered layer of
substantially the same depth as pre-heating, namely 20 .mu.m.
[0106] The microporous glass layer is derivatized with a long
aliphatic linker that can withstand conditions required to
deprotect the aromatic heterocyclic bases, i.e. 30% NH.sub.3 at
55.degree. C. for 10 hours. The linker, which bears a hydroxyl
moiety, the starting point for the sequential formation of the
oligonucleotide from nucleotide precursors, is synthesized in two
steps. First, the sintered microporous glass layer is treated with
a 25% solution of 3-glycidoxypropyltriethoxysilane in xylene
containing several drops of Hunig's base as a catalyst in a
staining jar fitted with a drying tube, for 20 hours at 90.degree.
C. The slides are then washed with MeOH, Et.sub.2O and air dried.
Neat hexaethylene glycol and a trace amount of concentrated
H.sub.2SO.sub.4 acid are then added and the mixture is kept at
80.degree. C. for 20 hours. The slides are washed with MeOH,
Et.sub.2O, air dried and stored desiccated at -20.degree. C. until
use. (Preparative technique generally described in British Patent
Application 8822228.6 filed Sep. 21, 1988.)
[0107] Focused acoustic ejection of about 0.24 pL of anhydrous
acetonitrile (the primary coupling solvent) containing a
fluorescent marker onto the microporous substrate is then shown to
obtain a circular patch of about 5.6 .mu.m diameter on the
permeable sintered microporous glass substrate. The amount of
acoustic energy applied at the fluid surface may be adjusted to
ensure an appropriate diameter of chemical synthesis for the
desired site density. 5.6 .mu.m diameter circular patches are
suitable for preparing an array having a site density of 10.sup.6
sites/cm.sup.2 with the circular synthetic patches spaced 10 .mu.m
apart center to center, and the synthetic patches therefore spaced
edge to edge at least 4 .mu.m apart at the region of closest
proximity. All subsequent spatially directed acoustically ejected
volumes in this example are of about 0.24 pL; it will be readily
appreciated that the ejection volumes can be adjusted for solutions
other than pure acetonitrile by adjusting the acoustic energy as
necessary for delivery of an appropriately sized droplet after
spreading on the substrate (here about a 5 .mu.m radius).
[0108] The oligonucleotide synthesis cycle is performed using a
coupling solution prepared by mixing equal volumes of 0.5M
tetrazole in anhydrous acetonitrile with a 0.2M solution of the
required .beta.-cyanoethylphosph- oramidite, e.g.
A-.beta.-cyanoethyl-phosphoramidite,
C-.beta.-cyanoethylphosphoramidite,
G-.beta.-cyanoethylphosphoramidite, T(or
U)-.beta.-cyanoethylphosphoramidite. Coupling time is three
minutes. Oxidation with a 0.1M solution of I.sub.2 in
THF/pyridine/H.sub.2O yields a stable phosphotriester bond.
Detritylation of the 5' end with 3% trichloroacetic acid (TCA) in
dichloromethane allows further extension of the oligonucleotide
chain. No capping step is required because the excess of
phosphoramidites used over reactive sites on the substrate is large
enough to drive coupling to completion. After coupling the slide
the subsequent chemical reactions (oxidation with I.sub.2, and
detritylation by TCA) are performed by dipping the slide into
staining jars. Alternatively the focused acoustic delivery of
I.sub.2 in THF/pyridine/H.sub.2O and/or 3% TCA in dichloromethane
to effect the oxidation and tritylation steps only at selected
sites may be performed if sufficient time transpires to permit
evaporation of substantially all the solvent from the previous step
so that the synthetic patch edges do not move outwards and closer
to the neighboring synthetic patches, and further to provide an
anhydrous environment for subsequent coupling steps if I.sub.2 in
THF/pyridine/H.sub.2O is delivered within the reaction chamber.
[0109] After the synthesis is complete, the oligonucleotide is
deprotected in 30% NH.sub.3 for 10 hours at 55.degree. C . Because
the coupling reagents are moisture-sensitive, and the coupling step
must be performed under anhydrous conditions in a sealed chamber or
container. This may be accomplished by performing the acoustic
spotting in a chamber of desiccated gas obtained by evacuating a
chamber that contains the acoustic ejection device and synthetic
substrate and replacing the evacuated atmospheric gas with
desiccated N.sub.2 by routine methods; washing steps may be
performed in the chamber or by removing the slide and washing it in
an appropriate environment, for example, by a staining jar fitted
with a drying tube. Because washing and other steps such as
detritylation may be more conveniently carried out outside the
chamber, the synthesis may also be performed in a controlled
humidity room that contains the controlled atmosphere chamber in
which the spotting is done, with the other steps carried out in the
room outside the chamber. Alternatively, a controlled humidity room
may be used for spotting with other steps carried out in less
controlled environment by use of, for example, a staining jar
fitted with a drying tube.
EXAMPLE 2
[0110] This example describes preparation of a peptide array in the
form of a combinatorial library, and demonstrates the use of
focused acoustic energy in the combinatorial solid phase synthesis
of all tetramers that can be made from the 20 naturally occurring
amino acids (20.sup.4 or =160,000 amino acid sequences in all) in a
quadruplicate array format. Four identical copies of the
combinatorial array to be prepared are contained in a 1 cm.times.1
cm area nominally divided into four quadrants, each quadrant
containing 250,000 synthesized sites of size 10 .mu.m.times.10
.mu.m arrayed in 500 rows and 500 columns. Only 400 rows and
columns are used in each quadrant; the first and last 50 rows and
columns are not used for synthesis, and function to space the four
identical arrays from each other and the edges of the area,
although alternative arrangement of the four identical arrays can
obtain greater distance between arrays by moving each array closer
to the corners of the square area. In addition to systematically
generating the combinatorial sequences, deposition of the monomers
employs a systematic method of ensuring that similar amino acid
sequences are less likely to be spatially close. Although many such
methods exist, with some requiring sophisticated computation, and
can take into account side chain similarities in addition to
identity, e.g. hydrophobic Val, Leu, Ile the scheme used relies on
a basic sequential list of amino acids which is phase shifted as
the row number increases. For example the 20 natural amino acids
can be listed sequentially based on the alphabetic order of their
single letter abbreviations, in which case: Ala (A) is "1"; Cys (C)
is "2"; Asp (D) is 3; . . . Val (V) is "19"; and Trp (W) is
"20".
[0111] For the first monomer deposited, in the first row in a given
quadrant in which a peptide is synthesized, which is the 51.sup.st
nominal row in that quadrant, beginning with the first synthetic
column (51.sup.st nominal column) amino acids (as activated for the
synthesis described in more detail below) are deposited as the
basic sequential list from 1 to 20 in alphabetical order of the one
letter abbreviations. Beginning with the second synthetic row
(52.sup.nd nominal row), the order is shifted by one position
starting at "2" and returning to "1" after "20" (2, 3, 4, 5 . . .
19, 20, 1); thus for the quadruplicate spaced array arrangement
being made, in the 52.sup.nd nominal row (second synthetic row) of
a given quadrant, the first amino acid deposited in the 51.sup.st
and 431.sub.st nominal column of the 52.sup.nd nominal row is "2"
or Cys, and the amino acids deposited in the 68.sup.th and
448.sup.th, 69.sup.th and 449.sup.th, and 70.sup.th and 450.sup.th
nominal columns of this row are 19, 20 and 1 respectively (V, W,
A).
[0112] Additional monomers are added in the quadrants as follows,
although numerous alternatives exist. For the second monomer in the
first synthetic row (51.sup.st nominal row) the monomer deposition
order for the second monomer is the same as for the first monomer
in the first 20 synthetic columns (nominal 51-70) of this row, and
the order is shifted by one for each successive group of 20
synthetic columns, thus the order is 2, 3 . . . 19, 20, 1 for
nominal columns 71-90 (hereinafter denoted [71-90]-{2, 3 . . . 19,
20, 1}) and according to this notation: [91-110]-{3, 4 . . . 20, 1,
2}; [111-130]-{4, 5 . . . 1, 2, 3} . . . [431-450]-{20, 1 . . . 17,
18, 19}. For the second and third monomers in the second synthetic
row (52.sup.nd nominal row) the monomer deposition order is shifted
by one relative to the order for the underlying monomer in the
first 20 synthetic columns (nominal 51-70) of this row, and the
order is shifted by one for each successive group of 20 synthetic
columns, thus for the second monomer the order is 3, 4 . . . 20, 1,
2 for nominal columns 51-70 and: [71-90]-{4, 5 . . . 1, 2, 3}
[91-110]-{5, 6 . . . 2, 3, 4}; [111-130]-6, 7 . . . 3, 4, 5} . . .
[431-450]-{2, 3 . . . 19, 20, 1}. Note that for the second monomer
of the second synthetic row, the shift relative to the order of the
first monomer in the first monomer in the first 20 columns of the
first row ({1, 2 . . . 18, 19, 20}), is 2 because one is the shift
between subsequent monomers (1.sup.st.fwdarw.2.sub.nd;
2.sup.nd.fwdarw.3.sup.rd) and the first monomer of the second
synthetic row is shifted by one relative to the first monomer of
the first synthetic row. For the second and third monomers in the
third synthetic row (53.sup.rd nominal row) the monomer deposition
order is shifted by two relative to the order for the underlying
monomer in the first 20 synthetic columns (nominal 51-70) of this
row, and the order is shifted by one for each successive group of
20 synthetic columns, thus the order for the second monomer is 5 .
. . 20, 1, 2, 3, 4 for nominal columns 51-70 and: [71-90]-{6 . . .
1, 2,3, 4, 5}, [91-110]-{7, . . . 2, 3, 4, 5, 6}, [111-130]-{8, . .
. 4, 5, 6, 6, 7} . . . [431-450]-{4, . . . 19, 20, 1, 2, 3}. For
the second monomer in the Nth synthetic row (nominal row=50+N) the
monomer deposition order for the second monomer is shifted by (N-1)
relative to the order for the first monomer in the first 20
synthetic columns (nominal 51-70) of this row, and the order is
shifted by one for each successive group of 20 synthetic columns,
thus (for (k*N+a)>20, (k*N+a) is shifted as beginning with
N+a-20*1, where I is the integer dividend of the quotient of
(k*N+a) and 20, representing number of cycles with each integral
multiple of 20 representing unshifted) the order for the second
monomer is (2*N-1), 2*N . . . (2*N-3), (2*N-2) for nominal columns
51-70 and: [71-90]-{(2*N . . . (2*N-2), (2*N-1)},
[91-110]-{(2*N+1), (2*N+2) . . . (2*N-1), 2*N}, [111-130]-{(2*N+2),
(2*N+3) . . . 2*N, (2*N+1)} . . . [431-450]-{(2*N-2), (2*N-1) . . .
(2*N-4), (2*N-3)}. Thus for the second monomer in the 400.sup.th
synthetic row (450.sup.th nominal row) the monomer deposition order
for the second monomer begins with 19 (799-780) is circularly
shifted by 18 relative to the order for the first monomer in the
first 20 synthetic columns (nominal 51-70) of the first row, and
the order is shifted by one for each successive group of 20
synthetic columns, thus the order is 19, 20 . . . (17), (18) for
nominal columns 51-70 and: [71-90]-{20, 1 . . . 17, 18, 19},
[91-110]-{1, 2 . . . 18, 19, 20}, [111-130]-{2, 3 . . . 19, 20, 1}
. . . [431-450]-{20, 1 . . . 17, 18, 19}. Note that for the second
monomer of the Nth synthetic row, the shift relative to the order
of the first monomer in the in the first 20 synthetic columns of
the first row ({1, 2 . . . 18, 19, 20}), is 2*(N-1) because (N-1)
is the shift between subsequent monomers (1.sup.st.fwdarw.2.sup.nd;
2.sup.nd.fwdarw.3.sup.rd) and the first monomer of a synthetic row
N is shifted by (N-1) relative to the first monomer of the first
synthetic row.
[0113] The synthetic chemical steps are modified from known solid
phase synthetic techniques (as described, for example, in Geysen et
al., International Patent Application PCT/AU84/00039, published as
WO 84/83564) that are adapted from the pioneering solid phase
peptide synthesis of Merrifield et al. ((1965) Nature
207:(996):522-23; (1965) Science 150(693)178-85; (1966) Anal. Chem.
38(13):1905-14; (1967) Recent. Prog. Horm. Res. 23:451-82). The
conventional methods of solid phase peptide synthesis as taught in
these seminal papers are described in detail in Ericksen, B. W. and
Merrifield, R. B. (1973) The Proteins 2:255-57 Academic Press, New
York, and Meinhofer, J. (1976) The Proteins 2:45-267 Academic
Press, New York. Briefly, all these methods add amino acid monomers
protected by tert-butoxycarbonyl (t-butoxycarbonyl, t-Boc) at their
amino groups, including their alpha amino groups (N.sup..alpha.) to
a nascent peptide that is attached to the substrate at the
carboxy-terminal (C-terminal). The carbonyl moiety of the
N.sup..alpha.-t-Boc amino acid to be added to the peptide is
activated to convert the hydroxyl group of the carboxylic moiety
into an effective leaving group, resembling an acid anhydride in
reactivity, using dicyclohexylcarbodiimide (DCC) to permit
nucleophilic displacement by the terminal N of the nascent peptide
to form a peptide bond that adds the monomer to the forming
peptide. The newly added monomer has an N-terminus protected from
further reaction by t-Boc, which is removed with trifluoroacetic
acid (TFA), rendering the terminal amino group protonated, followed
by deprotonation of the terminal amino group with triethylamine
(TEA) to yield the reactive free amino group suitable for addition
of another monomer.
[0114] The substrate employed is polyethylene, although the classic
substrate for solid phase peptide synthesis, divinylbenzene
cross-linked polystyrene chloromethylated by Friedel-Crafts
reaction of the polystyrene resin on approximately one in four
aromatic rings, could also be employed. Preparation of the
polyethylene substrate, described in Geysen et al., International
Patent Application PCT/AU84/00039, published as WO 84/83564,
involves .gamma.-ray irradiation (1 mrad dose) of polyethylene
immersed in aqueous acrylic acid (6% v/v) to yield reactive
polyethylene polyacrylic acid (PPA), according to the method of
Muller-Schulte et al. (1982) Polymer Bulletin 7:77-81.
N.sup..alpha.-t-Boc-Lysine methyl ester is then coupled to the PPA
by the Lysine .epsilon.-amino side chain. After deprotection of the
N.sup..alpha. by removal of the t-Boc with TFA followed by TEA,
DCC/N.sup..alpha.-t-Boc-Alanine is added to couple t-Boc-Ala to the
N.sup..alpha. of the Lys, thereby forming a peptide like
N.sup..alpha.-t-Boc-Ala-Lys-.epsilon.-N-PPA linker to which the DCC
activated N.sup..alpha.-t-Boc-amino acid monomers can be
sequentially added to form the desired polymers upon deprotection
of the N.sup..alpha. group of the N.sup..alpha.-t-Boc-Ala.
[0115] For an array format, and to increase the effective surface
area for polymer formation and enhance adhesion of acoustically
ejected reagent droplets to the synthetic substrate, polyethylene
fiber sheet material, approximate thickness 25 .mu.m, available
commercially and prepared by conventional methods is heat or fusion
bonded according to routine methods to a smooth polyethylene
backing approximately 0.15 cm thick to form a polyethylene fiber
coated rough permeable substrate. The fiber coated sheet s cut into
strips having the approximate dimensions of a commercial slide, and
.gamma.-irradiated (1 mrad) in 6% v/v aqueous acrylic acid to form
the PPA activated substrate. The substrate must be adequately dried
because the t-Boc protected and DCC activated reagents are water
sensitive, and water contamination of acids applied to the
synthetic sites, such as TFA application can hydrolyze the peptide
bond. Thus anhydrous synthetic conditions are required throughout.
Conventional drying of the substrate is effected with warm dry air
at atmospheric or subatmospheric pressure by routine methods,
specifically, the slides are washed with MeOH, Et.sub.2O, air dried
and stored desiccated at -20.degree. C. until use.
[0116] The sequential combinatorial addition of monomers is
performed as described above with all sites spotted with the
appropriate DCC/N.sup..alpha.-t-Boc-amino acid. The appropriate
volume for acoustic ejection is as above. This yields a
quasi-parallel synthesis because the spotting of different sites is
not simultaneous, but the can be modified to synthesize the desired
peptides only at some sites and synthesize at other sites later.
The actual synthesis requires anhydrous organic solvent washing
steps to remove unreacted activated amino acids or TFA or TEA, for
a total of 11 steps per monomer addition. Thus a completely
sequential synthesis would increase the number of steps performed
for synthesizing an array drastically, but, for example
synthesizing only at every other site in a first synthetic round
and then synthesizing in a second session would improve array
quality and only double the number of steps. To ensure that
peptides are only formed at the chosen sites, the
N.sup..alpha.-t-Boc-Ala-Lys-.epsilon.-N-PPA linker can be
selectively deprotected to expose the N.sup..alpha. of Ala only at
chosen sites, by selective acoustic energy directed ejection of TFA
onto the desired sites, followed by washing and selective
application of TEA, followed by washing to effect, for example,
selective deprotection of every other site.
[0117] The basic quasi-parallel combinatorial synthesis of all
tetra-peptides that can be made from the naturally occurring amino
acids may be performed in 44 steps excluding substrate preparation.
As no selective linker deprotection is required, the substrate is
immersed in TFA in a staining jar fitted with a drying tube, then
washed, and immersed in TEA, and washed again, all under anhydrous
conditions. The synthesis must be carried so that ejection of the
fluid droplets occurs in a controlled atmosphere that is at minimum
dry, and inert to the reagents used. This may be obtained by
performing the acoustic spotting in a chamber of desiccated gas
obtained by evacuating a chamber that contains the acoustic
ejection device and synthetic substrate and replacing the evacuated
atmospheric gas with desiccated N.sub.2 by routine methods; washing
steps may be performed in the chamber or by removing the slide and
washing it in an appropriate environment, for example, by a
staining jar fitted with a drying tube. Because washing and other
steps such as detritylation may be more conveniently carried out
outside the chamber, the synthesis may also be performed in a
controlled humidity room that contains the controlled atmosphere
chamber in which the spotting is done, with the other steps carried
out in the room outside the chamber. Alternatively, a controlled
humidity room may be used for spotting with other steps carried out
in less controlled environment by use of, for example, a staining
jar fitted with a drying tube.
[0118] Use of pre-synthesized short oligopeptides can also be used
in lieu of amino acid monomers. Since focused acoustic ejection
enables the rapid transition from the ejection of one fluid to
another, many oligopeptides can be provided in small volumes on a
single substrate (such as a microtiter plate) to enable faster
assembly of amino acid chains. For example, all possible peptide
dimers may be synthesized and stored in a well plate of over 400
wells. Construction of the tetramers can than be accomplished by
deposition of only two dimers per site and a single linking step.
Extending this further, a well plate with at least 8000 wells can
be used to construct peptides with trimers.
EXAMPLE 3
[0119] Combinatorial methods of the preceding Examples 1 and 2 can
be adapted to form combinatorial arrays of polysaccharides
according to the instant invention. In oligosaccharides, the
monosaccharide groups are normally linked via oxy-ether linkages.
Polysaccharide ether linkages are difficult to construct chemically
because linking methods are specific for each sugar employed. The
ether oxygen linking group is also susceptible to hydrolysis by
non-enzymatic chemical hydrolysis. Thus, there are no known methods
of automated syntheses for ether linked carbohydrates, and
conventional methods of making combinatorial arrays are not
sufficiently flexible to permit combinatorial arrays of
polysaccharides. The flexibility of acoustic spotting can be
adapted to form oxy-ether linkage based combinatorial arrays by
analogy to the alternative method of selective deblocking that may
be employed for making the arrays of Examples 1 and 2. That is, the
specific chemical methods for forming the linkage between any pair
of sugars may be conveniently selected so that a different solution
is ejected for adding a glucose to a specific terminal sugar of the
forming polysaccharide, such as fructose, than is ejected for
adding glucose to a different terminal sugar, such as ribose,
without increasing the number of steps involved as would be the
case with photolithographic synthesis, and might be the case with
parallel printing of multiple reagents through conventional multi
nozzle ink-jet type printers. The resulting polysaccharides remain
susceptible to hydrolysis.
[0120] Polysaccharides may be synthesized in solution rather than
the solid phase, as can the biomolecules made in the preceding
examples, and the acoustic ejection of droplets can effect the
solution syntheses of arrayed polysaccharides at high density on a
substrate without any attachment during polymer formation by
selective application of deblocking reagents to different sites. In
situ solid phase synthesis is more readily adaptable to automation
of even oxy-ether linkage based polysaccharides because at least
the deblocking steps may be done simultaneously for all sites,
although the susceptibility of the different linkages to hydrolysis
may affect overall yield for different monomer sequences
differently. Recently, methods of replacing the oxy-ether with a
thio-ether linkage (U.S. Pat. Nos. 5,780,603 and 5,965,719) and
with an amide linkage with the N atom linked to the anomeric C of
the sugar (U.S. Pat. No. 5,756,712) have been introduced. The solid
phase synthetic methods of the thioether linkage methods may be
directly adapted to form high density combinatorial arrays in an
analogous manner as techniques for the Merrifield peptide
synthesis. Similarly, the amide linkage based polysaccharides may
be adapted for solid phase high density array formation by
employing, for example the thioether based substrate linkage taught
in U.S. Pat. Nos. 5,780,603 and 5,965,719, or an amide linkage to
an appropriate moiety functionalized surface by analogy to the
linkage of U.S. Pat. No. 5,756,712.
[0121] Only the thioether based substrate linkage will be
exemplified in detail, and this linkage will be used to make
thioether (amide based oligosaccharides may be made analogously by
reference to U.S. Pat. No. 5,756,712; with a thioether, or other,
substrate linkage) based combinatorial array of oligosaccharides.
The classic substrate for solid phase peptide synthesis,
divinylbenzene cross-linked polystyrene chloromethylated by
Friedel-Crafts reaction of the polystyrene resin on approximately
one in four aromatic rings is employed, although a polyethylene
substrate may be substituted.
[0122] Spun polystyrene sheet made by conventional methods or
obtained commercially is heat or fusion bonded to a polystyrene
backing to yield a porous permeable layer of spun polystyrene of
approximately 25 .mu.m thickness. The appropriate extent of cross-
linking and chloromethylation is effected by conventional chemical
synthetic methods as required. The thickness of the permeable layer
will be appreciated to affect the dimensions of the area of actual
chemical synthesis, as more vertical wicking room will result in
less lateral spread of the acoustically deposited reagents. It also
will be appreciated that the extent of crosslinking may be adjusted
to control the degree of swelling, and softening upon application
of organic solvents, and that the fibrous nature of the porous,
permeable layer of spun polystyrene provides relatively more
synthetic surface per nominal surface area of the substrate than
provided by beads, thus less swelling is required to expand
synthetic area to polymer sites inside the fibers. The substrate is
aminated by conventional chemical synthetic methods, washed and
stored desiccated at -20.degree. C. until use.
[0123] The linking of a sugar to this substrate is first effected.
Succinic anhydride (1.2 equivalents) is added to a solution of
1,2:3,4-di-O-isopropylidene-D-galactopyranose (1 equivalent) in
pyridine at room temperature. The reaction is stirred overnight
then concentrated in vacuo to yield
1,2:3,4-di-O-isopropylidene-6-O-(3-carboxy)propanoyl-D--
galactopyranose. 80% aqueous acetic acid is added to the residue to
remove the isopropylidene groups. When this reaction is complete,
the reaction mixture is concentrated in vacuo. Excess 1:1 acetic
anhydride/pyridine is then added to the residue to form
1,2,3,4-O-acetyl-6-O-(3-carboxy)propano- yl-D-galactopyranose, to
which excess thiolacetic acid in dry dichloromethane under argon at
0.degree. C. and BF.sub.3 etherate is then added. The cold-bath is
removed after 10 minutes. After 24 h the mixture is diluted with
dichloromethane, washed with saturated sodium bicarbonate, dried
over sodium sulfate, and concentrated to yield
1-S-acetyl-2,3,4-tri-O-acetyl-6-O-(3-carboxy)propanoyl-1-thio-.alpha.-D-g-
alactopyranose. The aminated polystyrene (Merrifield resin)
substrate is contacted with the
1-S-acetyl-2,3,4-tri-O-acetyl-6-O-(3-carboxy)propanoyl-
-1-thio-.alpha.-D-galactopyranose and a carbodiimide coupling
reagent to afford the O,S-protected galactopyranose coupled to the
substrate through the 6-O-(3-carboxy)propanoyl group.
[0124] The preceding substrate is used for combinatorial synthesis
of thioether linked polysaccharides based on thiogalactose
derivatives. Nine copies of the combinatorial array of all possible
trimers of four monomeric 1-thiogalactose derivatives (4.sup.3=64
in all) are synthesized on a total substrate surface area of 1
cm.sup.2 divided into square synthetic sites 333 .mu.m.times.333
.mu.m, corresponding to a site density of 1000 sites/cm.sup.2. This
arrangement permits a 3 site or 999 .mu.m spacing between each copy
of the array in each axis of the array plane. A 25 pL droplet of
fluorescent solvent deposited on the described porous permeable
spun polystyrene on polystyrene substrate yields a spot of about 56
.mu.m diameter, and a 100 pL droplet yields a spot of about 112
.mu.m diameter (cylindrical shaped spot wicked into depth of porous
substrate with about 1/2 of porous layer occupied by solid
polystyrene and little swelling thereof).
[0125] Step A--Synthesis of 1-Dithioethyl-2,3,4,6;
-tetra-O-acetyl-galacto- pyranoside:
1-Thio-2,3,4,6-tetra-O-acetyl-galactopyranoside (500 mg, 1.37 mmol)
and diethyl- N-ethyl-sulfenylhydrazidodicarboxylate (360 mg, 2.0
mmol) (prepared by known methods as described by Mukaiyama et al.
(1968) Tetrahedron Letters 56:5907-8) are dissolved in
dichloromethane (14 mL) and stirred at room temperature. After 10
min, the solution is concentrated and column chromatography
(SiO.sub.2, hexane/ethylacetate 2:1) yields
1-dithioethyl-2,3,4,6-tetra-O-acetyl-galactopyranoside (580 mg,
quant) as a white solid (R.sub.f0.27 in hexanes/ethyl acetate
(2:1)). .sup.1H-NMR (360 MHz, CHCl.sub.3): ..delta.1.30 (dd, 3H,
J=7.4 Hz, CH.sub.3), 1.96, 2.02, 2.03, 2.13 (4 s, 12H,
4CH.sub.3CO), 2.79 (ddd, 2H, J=7.4 Hz, J=7.4 Hz, J=1.3 Hz,
CH.sub.2), 3.94 (ddd, 1H, J.sub.4,5=1.0 Hz, J.sub.5,6a=6.6 Hz,
J.sub.5,6b=7.6 Hz, 5-H), 4.10 ddd, 2H, 61-H, 6b-H), 4.51 (d, 1H,
J.sub.1,2=10.0 Hz, 1-H), 5.05 (dd, 1H, J.sub.2,3=10.0 Hz, J.sub.3,
4=3.3 Hz, 3-H)), 5.38 (dd, 1H, J.sub.1,2=10.0 Hz, J.sub.3,3=10.0
Hz, 2-H), 5.40 (dd, 1H, J.sub.3,4=3.3 Hz, J.sub.4,5=1.0 Hz, 4-H);
m/z calculated for C.sub.16H.sub.24O.sub.9S.sub.2 (M+Na) 447.1,
found 447.0.
[0126] Step B--Synthesis of
1-dithioethyl-.beta.-D-galactopyranoside:
1-Dithioethyl-2,3,4,6-tetra-O-acetyl-galactopyranoside from Step A
(500 mg, 1.18 mmol) is dissolved in dry methanol (10 mL) and
treated with methanolic sodium methoxide (1 M, 150 .mu.L). After 2
h, the solution is neutralized with Amberlite 1R-120 (H.sup.+)
resin, filtered and concentrated to give
1-dithioethyl-6-.beta.-D-galactopyranoside as a white solid (300
mg, quant).
[0127] Step C--Coupling of 1-Dithioethyl-.beta.-D-galactopyranoside
to the Substrate: 1-Dithioethyl-6-.beta.-D-galactopyranoside (200
mg, 780 .mu.mol) is dissolved in dry pyridine (8 mL), and DMAP (5
mg) is added to the mixture, which is maintained at 60.degree. C.
throughout.
[0128] Of the total (9.times.64=576) sites used to form the 9
duplicate arrays, and in each duplicate array of 64 sites of actual
synthesis, 1/4 (16 per array, 144 total) of the array sites are
patterned with the 1-dithioethyl-6-.beta.-D-galactopyranoside/DMAP.
in dry pyridine. This solution is acoustically ejected onto the
substrate at the desired locations. Dry controlled atmospheric
conditions, namely a dry inert gas environment, are also used for
this oligosaccharide synthesis. The appropriate volume deposited at
each site is determined by test deposition at some of the array
sites, taking into consideration that the synthetic area should be
wholly contained in the synthetic site, and too much dead space is
preferably avoided. About 10 to 100 pL droplet volumes are found to
be appropriate, and 100 pL is spotted onto the sites where the
first monomer is desired to be
1-dithioethyl-6-.beta.-D-galactopyrano- side. The substrate is as
described, spun polystyrene resin on a polystyrene backing (trityl
chloride-resin, loading 0.95 mmol/g of active chlorine, polymer
matrix: copolystyrene-1% DVB) is heated for 24 h at 60.degree. C.
The resin is filtered off, and washed successively with methanol,
tetrahydrofuran, dichloromethane and diethyl ether (10 mL each) to
afford 1-dithioethyl-6-.beta.-D-galactopyranoside covalently linked
to the trityl resin through the hydroxyl group in the 6-position at
the desired sites.
[0129] Step D--Patterning Additional 1-Dithioethyl-6-pyranosides:
It will be readily appreciated that this step can be practiced with
other 1-dithioethyl-6-pyranosides as desired to be linked to the
substrate. 1/4 of the sites of each of the duplicate arrays are
spotted with a solution for linking
1-dithioethyl-6-.beta.-D-glucopyranoside in about the same volume
as deposited in Step C, 1/4 are spotted to yield the
1-dithioethyl-6-.beta.-D-mannopyranoside, and the remaining 1/4 are
spotted to yield the 1-dithioethyl-6-.beta.-D-allopyranoside.
[0130] Step E--Generation of the Free Thiol on the Substrate: The
substrate sites from Step C spotted with dry tetrahydrofuran (THF)
in the area of 1-dithioethyl-6-pyranoside deposition (about 4 pL
per pL deposited in Step C). Dry methanol (about 3/4 pL per pL
deposited in Step C), dithiothreitol (about 185 picograms) and
triethylamine (about 1/2 pL per pL deposited in Step C) are
deposited at desired synthetic areas of the combinatorial sites by
acoustic deposition and the sites are allowed to react under the
specified controlled atmosphere conditions for about 10 minutes to
an hour at room temperature. The entire substrate is washed by
immersion in an adequate volume, successively, of methanol,
tetrahydrofuran, dichloromethane and diethyl ether. Micro-FTIR (of
substrate deposition sites): 2565 cm.sup.-1 (SH stretch).
Alternatively, if selective generation of the free thiol is not
desired, the substrate may be treated on the whole of the surface
as follows: 8 ml dry THF is applied to the surface of the substrate
which is placed in a shallow container just large enough to contain
the substrate, 1.2 ml dry ethanol, 256 mg dithiothreitol, and 0.8
ml triethylamine are added to the THF and the container is shaken
for about 10 hours at room temperature under the described
conditions.
[0131] Step F--Michael Addition Reaction: The substrate from Step E
is again placed in the shallow container of Step E and swollen in
dry N,N-dimethylformamide (4 mL) and then cyclohept-2-en-1-one (280
.mu.l, 252 .mu.mol) is added and the container is shaken at room
temperature. After 2 hours, the liquid is removed and the substrate
is washed successively with methanol, tetrahydrofuran,
dichloromethane and diethyl ether (40 mL each). Alternatively if
selective Michael addition is desired, the desired sites may be
selectively spotted in the area of synthesis: N,N-dimethylformamide
(about 2.5 pL per pL deposited in Step C); cyclohept-2-en-1-one
(about 0.2 pL, 0.2 picomole per pL deposited in Step C). The
selectively spotted sites are allowed to react under the specified
controlled atmosphere conditions for about 10 minutes to an hour at
room temperature prior to the specified washing steps.
[0132] Step G--Reductive Amination with an Amino Acid: The
substrate from Step F is again placed in the shallow container of
preceding steps and swollen in dichloromethane (4 mL). Glycine
tert-butyl ester hydrochloride (150 mg, 1,788 .mu.mol), sodium
sulfate (400 mg), sodium triacetoxyborohydride (252 mg, 1188
.mu.mol) and acetic acid (40 .mu.L) are added at room temperature
under argon atmosphere and the container shaken for 24 hours. The
liquid is removed and the substrate is washed successively with
washed successively with water, methanol, tetrahydrofuran and
dichloromethane.
[0133] Additional monomers may be added by repetition of the
preceding steps with the desired 1-dithioethyl-6-pyranosides. It
will be readily appreciated that this step can be practiced with
1-dithioethyl-6-.beta.-D- -galactopyranoside/DMAP and the other
1-dithioethyl-6-pyranoside/DMAP desired for linking to the
substrate. The desired sites of each of the duplicate arrays are
selectively spotted with the appropriate
1-dithioethyl-6-pyranoside/DMAP solution for linking in about the
same volume as deposited in Step C
(1-dithioethyl-6-.beta.-D-mannopyranoside/D- MAP,
1-dithioethyl-6-.beta.-D-allopyranoside/ DMAP, and
1-dithioethyl-6-.beta.-D-glucopyranoside/DMAP).
EXAMPLE 4
[0134] Combinatorial arrays of alloys can readily be prepared using
the methodology of the invention. Molten metals are acoustically
ejected onto array sites on a substrate. No monomer sequence exists
for metals, but the composition of the alloys may be altered by
deposition of more of a given metal at a certain site without
problems associated with polymer elongation; the problem with
deposition of more metal droplets of the same volume to form
different compositions is that array density must be decreased to
accommodate the most voluminous composition made, as the size of
droplets is not conveniently adjusted over wide ranges of droplet
volume. An additional reason to reduce array density in alloy
formation is that with alloys it is often desirable to form a
material that has a bulk and surface, rather than a film which has
a surface but not a bulk and therefore the properties of the
thin-layer "surface" are not the same as the surface of the bulk
material (see generally Somorjai, Surface Chemistry and Catalysis,
supra).
[0135] As may be readily appreciated, an infinite number of
compositions of any two metals exist. Composition in terms of
combinatorial synthesis of arrays of alloys by acoustic ejection of
fluid is complicated by the volumetric acoustic ejection being
different for different molten metals having different densities
and interatomic interactions, but the different stoichiometric
compositions generated correspond to different combinations of
metal and number of droplets deposited are reproducible, e.g. an
alloy of 5 droplets of Sn ejected at an energy, E.sub.1 and five
droplets of Cu ejected at E.sub.1 or E.sub.2 will have the same
compositions when duplicated under the same conditions, and the
stoichiometric composition of alloys of interest can always be
determined by SIMS. To promote uniform alloy formation it is
desirable to spot all the droplets of molten metal to be deposited
onto a site in rapid succession rather than waiting for a droplet
to solidify before depositing another, although such combinatorial
"stacks" are also of potential interest. As it is most convenient
not to change acoustic energy between deposition of droplets, the
same energy is most conveniently used for ejecting different
metals, and the stoichiometric and other, including surface
properties of the material so generated may be determined later and
reproduced by exact duplication of the synthetic process. The
molten metals must be at an appropriate temperature (T) above its
melting point to ensure that the droplet is still molten when it
reaches the substrate. In addition to an inert gas environment,
which may be appreciated to be important if making alloys rather
than stacks of oxidized metal salts is desired, to prevent
oxidation of the metals especially at the surface of the droplets,
a gas with low heat capacity is preferable to high heat capacity
gases. In addition, the temperature of the substrate and the
distance between the substrate and the fluid meniscus may be
adjusted to ensure molten material reaching the substrate and
remaining molten for sufficient time to permit alloying with
subsequently deposited droplets. Furthermore, after a given alloy
composition is made at a given array site, both the ejection energy
and the meniscus to substrate distance may require adjustment in
light of the foregoing considerations, as is readily
appreciated.
[0136] A convenient systematic combinatorial approach involves
selecting a number of molten compositions for ejection and a total
number of droplets deposited at each site. Array density of
10.sup.5 sites/cm.sup.2 is convenient as each site is conveniently
a 100 .mu.m square, an area which can be easily appreciated to
accommodate 10, approximately picoliter (pL) sized, droplets,
because 10 pL spread uniformly over the area of the site would be
only 1 .mu.m, deep, and gravity prevents such complete spreading
and low surface angle.
[0137] For 4 different molten metallic compositions available for
ejection and 10 droplets, it may easily be demonstrated that 342
possible compositions exist, and likewise for 15 droplets, 820
possible compositions exist in terms of droplet number. For d
droplet compositions with m ejected metals (although the molten
ejection vessel contents need not be a pure metal, and may
themselves be an alloy):
.sup.dQ.sub.m=n=1.fwdarw.m.SIGMA.(S(m).sub.n)*(Z(m,d).sub.n)
[0138] .sup.dQ.sub.m is defined as # metal compositions for d=#
droplets, m=# of molten compositions available to be ejected;
S(m).sub.n is the # of unique sets having n members of the m
available molten compositions; Z(n,d).sub.n is # of d droplet
combinations of n used of the m available for deposition,
corresponding to S(m).sub.n. Further:
Z(m,d).sub.n=i=1.fwdarw.C(n,d).SIGMA.O(n,d).sub.i
[0139] CS(n,d).sub.,i denotes ith set of coefficients for n
components that add to d droplets, with C(n,d), representing the
total number of coefficient sets satisfying this requirement;
O(n,d).sub.i is the number of possible orderings of the ith set of
n coefficients for d droplets corresponding to CS(n,d).sub.,i.
[0140] For example, for d=10, m=4, let the 4 vessels contain,
respectively, Sn, In, Cd and Zn.
[0141] 1 metal compositions (n=1):
[0142]
Z(4,10).sub.1=i=.sub.1.fwdarw.C(1,10).SIGMA.O(1,10).sub.i=1*1,
because the only possible coefficient is 10, and it can be ordered
in only one way. The corresponding S(4).sub.1 is 4, as 4 unique
sets of 1 metal can be chosen for ejection.
[0143] 2 metal compositions (n=2):
[0144] The corresponding S(4).sub.2 is 6, as [4!/2!]/2! unique sets
of 2 metals can be chosen for ejection. The C(2,10) unique sets of
2 non-negative, nonzero coefficients that add to 10, such as (9,1)
and the corresponding O (2,10).sub.i are [denoted by the notation
{CS(2,10).sub.1:O(2,10).sub.1, CS(2,10).sub.2,1:O(2,10).sub.2 . . .
CS(2,10).sub.C(n,d):O(2,10).sub.C(n,d)}]:
{(9,1):2, (8,2):2, (7,3):2, (6,4):2,
(5,5):1};.fwdarw.Z(4,10).sub.2=.sub.i-
=1.fwdarw.C(2,10).SIGMA.O(2,10).sub.i=2+2+2+2+1=9.
[0145] 3 metal compositions:
[0146] The corresponding S(4).sub.3 is 4 ([4!/1!]/3!), 4 unique
sets of 3 metals can be chosen for ejection. The C(3,10) unique
sets of 3 non-negative, nonzero coefficients that add to 10
are:
[0147] {(8,1,1):3, (7,2,1):6, (6,3,1):6, (6,2,2):3, (5,4,1):6,
(5,3,2):6, (4,4,2):3, (4,3,3):3};.fwdarw.
Z(4,10).sub.3=.sub.i=1.fwdarw.C(3,10).SIGMA.O(3,10)i=3+6+6+3+6+6+3+3=36.
[0148] 4 metal compositions:
[0149] The corresponding S(4).sub.4 is 1 (4!/4!), as 1 unique sets
of 4 metals can be chosen for ejection. The C(4,10) unique sets of
4 non-negative, nonzero coefficients that add to 10 are:
[0150] {(7,1,1,1):4, (6,2,1,1):12, (5,3,1,1):12, (5,2,2,1):12,
(4,4,1,1):6, (4,3,2,1):24, (4,4,2,2):6, (3,3,3,1):4,
(3,3,2,2):6};.fwdarw.
Z(4,10).sub.4=i=.sub.1.fwdarw.C(4,10).SIGMA.O(4,10)i=4+12+12+12+6+24+6+4+6-
=86.
[0151] From the preceding:
.sup.10Q.sub.4=.sub.n=1.fwdarw.4.SIGMA.(S(4).sub.n)*(Z(4,10).sub.n)=4*1+6*-
9+4*36+1*86=288.
[0152] An appropriate substrate for the alloy array of acoustically
deposited molten metallic compositions is made of sintered alumina
by conventional methods or obtained commercially. An array of Sn
(mp=281.8.degree. C.), In (mp=156.6.degree. C.), Cd
(mp=320.9.degree. C.) and Zn (mp=419.6.degree. C.) components (e.g.
pure ejected molten metal compositions) is formed by acoustic
deposition of 15 droplets/array site on a sintered alumina
substrate. Thickness of the substrate is about 0.25 cm, to
withstand the heat. The site density is chosen to allow all
possible droplet compositions that can be made from four metals
with 15 droplets, 820 possible compositions including, for example
(in droplets): 14(Sn), 1*(In); 12Sn, 1In, 1Cd, 1Zn; 1Sn, 12In, 1Cd,
1Zn. These compositions and the 901 remaining compositions may be
obtained as above demonstrated for 10 droplet compositions of four
components. The chosen density is 1000 sites/cm2, corresponding to
a nominal site size of 333.times.333 .mu.m, and permitting the
complete collection of compositions to be made on a 1 cm.sup.2
area. Duplicate copies of the array are made on a commercial
microscope slide sized strip of substrate, separated by 1/2 cm to
permit the convenient separation of the two identical arrays.
[0153] The acoustic energy is adjusted to yield an average droplet
volume of about 1 pL, and 15 droplet ejection that does not exceed
the 333.times.333 .mu.m square area provided for the site, under
the desired conditions, including atmosphere pressure and
composition, length of droplet flight, substrate temperature. After
the average droplet size is adjusted to about one pL, 15 droplets
of each metal are acoustically ejected onto a site and the ejection
energy is adjusted downwards if any of these pure sites exceed the
margins of the site. Enough sites exist for all 820 possible
compositions to be ejected onto each 1 cm square array after using
up to 96 of the available 1000, sites for calibration, but the
single ejected component sites so created may function as the
single composition sites if sufficiently the localized region
within which the alloy resides similar to the other sites in
dimension, as dimensions affect cooling and a substantially
different geometry would not be precisely the same material.
[0154] Although the actual volumes ejected of the different molten
components may be adjusted to be equal by using a different
acoustic energy of ejection, more rapid ejection is possible if the
ejection energy is held constant. It is readily apprehended that if
too wide a discrepancy exists between the droplet volumes ejected
for each component, that the overall geometry of the cooling
composition could vary widely depending on its makeup, but this is
not the case for the metals being deposited here, because both
their densities and factors determining interatomic interactions in
the molten state, such as polarizability, are sufficiently similar.
In all cases the conditions for the formation of the alloy at a
given site are always reproducible, and the actual composition and
other physical properties of the composition may be ascertained by
physical methods including all described surface physical
characterization methods.
[0155] Because of the toxicity of Cd, the acoustic deposition of
the molten metals is carried out in a separate atmospherically
controlled low humidity chamber under Ar gas to reduce undesired
reactions and cooling. Higher heat capacity inert gases and more
reactive gases, such as O.sub.2, and O.sub.2/hydrocarbons may be
used for experiments under different conditions, but may require
adjustment of the distance between the fluid meniscus and substrate
or the temperature of the molten reagent to be ejected or both to
ensure that the droplet reaches the substrate in a molten
state.
[0156] After calibration the first duplicate array is spotted by
acoustic ejection as described onto a substrate maintained at a
temperature of 125.degree. C. Each of the 820 possible 15 droplet
compositions is made by sequentially depositing fifteen droplets at
each site, the 15 droplets deposited according to the different
coefficient arrangements described above. The metals are maintained
at a known temperature that is sufficiently greater than the mp of
the metal that the ejected droplet arrives at the substrate surface
molten under the conditions, including distance of flight and
pressure, temperature and heat capacity of the atmosphere. The
droplets are deposited at each site lowest melting metal first in
order of increasing melting temperature with the highest melting
temperature metal deposited last, e.g., In, Sn, Cd, Zn, so that
successive droplets of higher melting temperature metal will melt
any solidified material. The procedure is repeated at different
substrate temperatures at 5 degree intervals until arrays formed
with substrate temperature ranging from 40.degree. C. to
425.degree. C. are formed.
* * * * *