U.S. patent application number 10/037356 was filed with the patent office on 2002-10-03 for method and apparatus for delivery of submicroliter volumes onto a substrate.
This patent application is currently assigned to SEQUENOM, INC.. Invention is credited to Becker, Thomas, Hanson, Aaron, Heaney, Paul, Lin, Chao, Willis, Michael C., Yao, Xian-Wei.
Application Number | 20020142483 10/037356 |
Document ID | / |
Family ID | 22922607 |
Filed Date | 2002-10-03 |
United States Patent
Application |
20020142483 |
Kind Code |
A1 |
Yao, Xian-Wei ; et
al. |
October 3, 2002 |
Method and apparatus for delivery of submicroliter volumes onto a
substrate
Abstract
A slotted pin tool, a delivery system containing the pin tool, a
substrate for use in the system and methods using the pin tool and
system are provided. The slotted pin tool contains a plurality of
pins having slotted ends designed to fit around each loci of
material deposited on a surface, such as a microarray, without
contacting any of the deposited material. Sample is delivered by
contacting the pin tool with the surface; the amount delivered is
proportional to the velocity of the pin tool as it contacts the
surface or the velocity of the liquid when movement of the pin is
halted.
Inventors: |
Yao, Xian-Wei; (San Diego,
CA) ; Lin, Chao; (San Diego, CA) ; Heaney,
Paul; (Solana Beach, CA) ; Becker, Thomas; (La
Jolla, CA) ; Hanson, Aaron; (San Diego, CA) ;
Willis, Michael C.; (La Jolla, CA) |
Correspondence
Address: |
HELLER EHRMAN WHITE & MCAULIFFE LLP
4250 EXECUTIVE SQ
7TH FLOOR
LA JOLLA
CA
92037
US
|
Assignee: |
SEQUENOM, INC.
|
Family ID: |
22922607 |
Appl. No.: |
10/037356 |
Filed: |
October 24, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60244404 |
Oct 30, 2000 |
|
|
|
Current U.S.
Class: |
436/180 ;
422/400; 422/63; 422/67; 436/173; 436/43; 436/46; 436/54 |
Current CPC
Class: |
Y10T 436/11 20150115;
B01J 2219/00725 20130101; B01J 2219/00619 20130101; B01J 2219/00596
20130101; G01N 1/10 20130101; B01L 3/0244 20130101; G01N 2035/1037
20130101; B01J 2219/00612 20130101; B01J 2219/00677 20130101; G01N
2035/00158 20130101; Y10T 436/2575 20150115; B01J 2219/00608
20130101; B01J 2219/00387 20130101; B01J 2219/0059 20130101; B01J
2219/00691 20130101; B01J 2219/00686 20130101; B01L 3/0248
20130101; G01N 35/1065 20130101; B01J 2219/00529 20130101; B01J
2219/00605 20130101; G01N 35/10 20130101; Y10T 436/112499 20150115;
Y10T 436/24 20150115; C40B 60/14 20130101; B01J 2219/00689
20130101; C40B 40/10 20130101; B01J 19/0046 20130101; B01J
2219/00527 20130101; B01J 2219/00585 20130101; G01N 35/1016
20130101; G01N 35/028 20130101; C40B 40/06 20130101; B01L 2200/0657
20130101; G01N 35/0099 20130101; B01J 2219/00722 20130101; Y10T
436/119163 20150115; B01J 2219/00659 20130101; B82Y 30/00
20130101 |
Class at
Publication: |
436/180 ;
436/173; 436/43; 436/46; 436/54; 422/63; 422/67; 422/100 |
International
Class: |
G01N 001/10 |
Claims
1. A method of delivering liquid samples to a substrate, the method
comprising: dipping a slotted pin tool having an open tip into a
sample reservoir containing a liquid sample to be delivered onto
the substrate, thereby drawing a volume of liquid sample up into
the pin tool; moving the slotted pin tool from the sample reservoir
to an elevated position above the substrate such that the pin tool
is above a target location that is to receive the liquid sample;
and lowering the slotted pin tool toward the substrate at a
predetermined speed and then halting the movement of the pin tool
toward the substrate, thereby expelling the liquid sample from the
slotted pin tool onto the target location of the substrate.
2. A method of claim 1, wherein halting the movement of the slotted
pin tool toward the substrate occurs substantially when the open
tip of the slotted pin tool contacts the substrate.
3. A method of claim 2, wherein the open tip of the slotted pin
tool is adapted to fit around material deposited at the target
location on the substrate without making contact with any portion
of the material.
4. A method of claim 1, wherein the slotted pin tool has a
substantially cylindrical tip having a lateral slot forming a
cavity that fits around a material at the target location on the
substrate.
5. A method of claim 1, wherein the slotted pin tool has a
substantially cylindrical tip having a lateral slot forming a
cavity with a width of greater than approximately 75 .mu.m.
6. A method of claim 5, wherein the cavity of the cylindrical tip
has a width between about 100 .mu.m up to approximately 300
.mu.m.
7. A method of claim 5, wherein the cavity of the cylindrical tip
has a width between about 30 .mu.m up to approximately 1000
.mu.m.
8. A method of claim 5, wherein the cavity of the cylindrical tip
has a height greater than approximately 100 .mu.m
9. A method of claim 1, wherein dipping the pin tool comprises
dipping the tip into the reservoir, whereby liquid is drawn into
the slot.
10. A method of claim 1, wherein dipping the pin tool comprises
dipping the tip into the reservoir such that the sample fluid level
has a depth at least equal to the height of the slot in the tip,
and the dipping halts when the pin tool has been dipped to a depth
equal to the pin tool slot.
11. A method of claim 1, wherein positioning of the slotted pin
tool is effected by pattern recognition to determine correct
positioning above the substrate and, then moving the slotted pin
tool to the determined position.
12. A method of claim 1, wherein moving the slotted pin tool
comprises identifying an orientation mark on the substrate that
indicates an appropriate location of the substrate.
13. A method of claim 1, wherein lowering the slotted pin tool
comprises moving the slotted pin tool at a predetermined speed of
lowing.
14. A method of claim 1, further comprising changing the volume of
liquid sample delivered onto the substrate by changing the speed of
lowering.
15. A method of claim 14, wherein the speed of lowering is changed
in response to known composition of the liquid sample.
16. A method of claim 1, further comprising: cleaning the slotted
pin tool in a liquid bath after a first liquid sample has been
delivered; and drying slotted pin tool prior to dipping the pin
tool in the liquid sample.
17. A method of claim 16, wherein cleaning and drying occur after
the first liquid sample has been delivered and before a second
liquid sample is delivered.
18. A method of claim 16, wherein drying comprises moving an air
flow air over the slotted pin tool.
19. A method of claim 16, wherein the liquid bath comprises an
ultrasonic bath.
20. The method of claim 1, wherein the sample comprises matrix
material for mass spectrometric analyses.
21. A method of claim 5, wherein the cavity of the cylindrical tip
has a width between about 100 .mu.m up to approximately 500
.mu.m.
22. The method of claim 12, wherein the orientation mark comprises
a symbology, or an electronic tag or a physical deformation of the
substrate for identification of orientation and/or identification
of the substrate from among a plurality thereof.
23. A system for delivery of liquid samples from one or more pin
tools to target locations on a substrate, the system comprising: a
plurality of processing stations, each of which performs a
procedure on the pin tool, wherein the pin tool includes a lower
tip having a slot into which a liquid sample may be drawn; a
transport system that transports the slotted pin tool from
processing station to processing station; and a control system that
loads the slotted pin tool by moving it to a loading station at
which the slotted pin tool is loaded with the liquid sample, then
moves the slotted pin tool to an elevated position above the
substrate such that the pin tool is above a target location that is
to receive the liquid sample, and then dispenses the liquid sample
by lowering the slotted pin tool toward the substrate at a
predetermined speed and then halts the movement of the pin tool
toward the substrate, thereby expelling the liquid sample from the
slotted pin tool onto the target location of the substrate.
24. A system of claim 23, wherein the control system halts the
movement of the slotted pin tool toward the substrate substantially
when the open tip of the slotted pin tool contacts the
substrate.
25 A system of claim 24, wherein the open tip of the slotted pin
tool is adapted to fit around a material at the target location on
the substrate without making contact with any portion of the
material.
26. A system of claim 23, wherein the slotted pin tool has a
substantially cylindrical tip having a lateral slot forming a
cavity that fits around a material at the target location on the
substrate.
27. A system of claim 23, wherein the slotted pin tool has a
substantially cylindrical tip having a lateral slot forming a
cavity with a width of greater than approximately 100 .mu.m.
28. A system of claim 27, wherein the cavity of the cylindrical tip
has a width of approximately 300 .mu.m.
29. A system of claim 27, wherein the cavity of the cylindrical tip
has a height greater than approximately 100 .mu.m.
30. A system of claim 23, wherein the control system loads the
slotted pin tool by dipping the tip of the pin tool into a
sample-containing reservoir at the loading station; and the control
system halts the dipping substantially when the pin tool has been
dipped to a depth equal to the pin tool slot.
31. A system of claim 23, wherein the control system moves the
slotted pin tool to the elevated position using pattern recognition
techniques to determine correct positioning above the
substrate.
32. A system of claim 23, wherein the control system moves the
slotted pin tool to the elevated position by identifying an
orientation mark on the substrate that indicates an appropriate
location of the substrate.
33. A system of claim 23, wherein the control system lowers the
slotted pin tool by moving the slotted pin tool at a predetermined
speed of lowering.
34. A system of claim 33, wherein the control system changes the
volume of liquid sample delivered onto the substrate by changing
the speed of lowering.
35. A system of claim 33, wherein the control system changes the
speed of lowering in accordance with known composition of the
liquid sample.
36. A system of claim 23, wherein the control system further moves
the slotted pin tool by cleaning the slotted pin tool in a liquid
bath after a first liquid sample has been delivered and drying the
slotted pin tool prior to dipping the pin tool in the liquid
sample.
37. A system of claim 36, wherein the control system performs
cleaning and drying after the first liquid sample has been
delivered and before it delivers a second liquid sample.
38. A system of claim 36, wherein drying comprises moving an air
flow air over the slotted pin tool.
39. A system of claim 36, wherein the liquid bath comprises an
ultrasonic bath.
40. A pin tool for use in a sample delivery system, the pin tool
comprising one or more slotted pins each having an open tip adapted
to fit around a material at a target location on a substrate
without making contact with any portion of the material, wherein:
the slotted pin tool is adapted to be dipped into a sample
reservoir containing a liquid sample to be delivered onto the
substrate, thereby drawing a volume of liquid sample up into the
slotted pins in the pin tool.
41. A pin tool of claim 40, wherein a pin in the pin tool has a
substantially cylindrical tip having a lateral slot forming a
cavity with a width of greater than approximately 75 .mu.m.
42. A pin tool of claim 41, wherein the cavity of the cylindrical
tip has a width up to approximately 500 .mu.m.
43. A pin tool of any of claims 40, wherein the cavity of the
cylindrical tip has a height greater than approximately 100
.mu.m.
44. A combination of a pin tool of claim 41 and a substrate
comprising target loci for deposition of sample material, wherein
the array of pins in the pin tool is matched to the array of loci
on the substrate.
45. A substrate for use in mass spectrometric analyses, comprising
target locations defined by application of photoresist materials
and photolithographic deposition; wherein the resulting array of
target locations on the substrate are less hydrophobic than the
surrounding areas.
46. A substrate of claim 45, wherein a substrate starting surface
is comprised of a material that has an available --OH or primary
amine.
47. The substrate of claim 45, wherein: the substrate comprises two
materials such that the one material has a contact angle that
differs by at least about 20 degrees from the second material.
48. The substrate of claim 47, wherein the first material is
polytetrafluoroethylene or a derivative thereof or is
dimethyidichlorosilane (DMDCS); and the second material is silicon
or silicon dioxide, which form the target loci.
49. A combination of a pin tool and the substrate of claim 48,
wherein the pin tool comprises at least one pin that is
slotted.
50. A combination comprising: a substrate, comprising an array of
target locations on a surfaces, wherein the target locations less
hydrophobic than the surrounding areas; and a pin tool comprising
at least one pin having a substantially cylindrical tip with a
lateral slot forming a cavity that fits around a material deposited
at a target location on the substrate.
51. A combination of claim 50, wherein the pin has a substantially
cylindrical tip having a lateral slot forming a cavity with a width
of greater than approximately 75 .mu.m, preferably greater than 100
.mu.m.
52. A combination of claim 50, wherein the cavity of the
cylindrical tip has a width between about 100 .mu.m up to
approximately 300 .mu.m.
53. A combination of claim 50, wherein the cavity of the
cylindrical tip in a pin has a height greater than approximately
100 .mu.m.
54. A combination of claim 50, wherein: the slotted pin tool is
adapted to be dipped into a sample reservoir containing a liquid
sample to be delivered onto the substrate, thereby drawing a volume
of liquid sample up into the slotted pins in the pin tool, and then
is moved to an elevated position above the substrate such that the
pin tool is above the target location(s), and then is lowered
toward the substrate such that upon halting the movement thereof
the liquid sample is expelled from the slotted pin tool onto the
target location of the substrate.
55. A method of mass spectrometric analysis, comprising: depositing
matrix material and sample on the target loci of a substrate of
claim 45; introducing the substrate into a mass spectrometer for
analysis of the samples; and analyzing the samples by mass
spectrometry.
56. The method of claim 55, wherein the sample comprises nucleic
acids or proteins.
57. A method of preparing a substrate, comprising: coating a
surface of a substrate with a photoresist material and solidifying
it on the surface; exposing the surface through a mask to light of
a wavelength that permits removal of the photoresist from exposed
surface; washing the photoresist off with a developer leaving an
array of photoresist pads having on the surface; baking the
resulting substrate.
58. The method of claim 57, wherein the substrate is baked at
between about 190-200.degree. C. for a period of about 50-70
minutes.
59. The method of claim 57, wherein the photoresist contains a
diazonaphthoquinone sensitizer and a phenolic resin.
60. The pin tool of claim 40, wherein a pin in the tool is tapered.
Description
RELATED APPLICATIONS
[0001] Benefit of priority under 35 U.S.C. .sctn.119(e) to U.S.
provisional application Serial No. 60/244,404, filed Oct. 30, 2000,
to Chao Lin et al., entitled "METHOD AND APPARATUS FOR DELIVERY OF
SUBMICROLITER VOLUMES ONTO A SUBSTRATE" is claimed herein. The
subject matter of the provisional application is incorporated by
reference in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to sample dispensing systems and, more
particularly, to the delivery of liquid samples onto substrate,
such as a microarray, for laboratory analysis.
BACKGROUND
[0003] Description of the Background Art
[0004] Genetic sequencing efforts, such as the Human Genome
project, have produced vast amounts of information for basic
genetic research that have proven useful in developing advances in
health care and drug research. These advances are possible because
of improvements in engineering and instrumentation that provide
advanced tools for the biotechnology community to continue with
basic genetic research. With these advances, scientists can move
from basic genomic discoveries to associating specific phenotypes
and diseases, and can thereby better identify targets for drug
development.
[0005] Nucleic acid sequencing and diagnostic methods often analyze
samples deposited onto target locations on substrates microarrays,
such has microplates, silicon chips and other such supports capable
of retaining biological molecules or samples at discrete loci.
Microarrays have been used to execute tests on large batches of
genetic samples to generate phenotype associations and improve
interpretation of the large data sets that result from such tests.
A typical microarray, referred to as a chip, includes a substrate,
such as a silicon or silicon-coated substrate, on which a large
number of reactive points receive samples for testing. Microarray
chips provide a technology that permits operators to increase
sample throughput, allowing the screening of large numbers of
samples and reducing reagent costs by using submicroliter sample
volumes. Preparation of such arrays employs a variety of
methodologies, including printed arrays and spotted arrays, with a
wide variety of substrate surfaces and different modes of
quantification. The resulting microarrays are used as substrates
for a variety of biochemical applications.
[0006] Among the ways for delivery of multiple samples to loci on
microarray surfaces are solid pins. The solid pins typically are
dipped into a liquid sample, which coats the tip of each pin,
holding a sample droplet by surface tension. The coated pins are
then touched to a target surface on a microarray substrate, so that
the sample is transferred to the target by contact printing. The
size and taper of the pin tool tip can affect the volume of liquid
sample that is picked up during dipping. The amount of liquid
sample transferred on contact will vary with the surface tension of
the liquid. Pin tools also can be problematic for high throughput
systems because the pins may have to be changed if different sample
volumes are desired, or if the nature of the liquid sample is
changed to avoid sample contamination. In addition, pin tools
cannot be used in situations where contact dispensing where there
is a risk of damage to a fragile preloaded sample, such as for mass
spectrometric analyses in which samples are deposited on loci that
have preloaded material, such as matrix material for
matrix-assisted laser desorption (MALDI).
[0007] Some mass spectrometry formats, such as MALDI formats,
combine the sample to be tested with a matrix material, such as an
inorganic acid, which when dried forms a crystal structure. Matrix
material can be preloaded on a mass spectrometry substrate and the
sample can be added at a later time, using an appropriate liquid
dispensing apparatus. When a sample target is preloaded or
prespotted with the porous matrix material required for mass
spectrometry, direct contact by the solid pin with the matrix
material can crush the material.
[0008] Other liquid samples dispensing apparatus rely on
piezoelectric mechanisms, sometimes using quill-type pin tools that
hold the samples in a cut-out at the lower tool tip. Such
piezoelectric delivery systems are susceptible to dispensing
satellite droplets on a target location because of surface tension
effects. Piezoelectric systems also may be prone to variations in
voltage and frequency among different tips, which results in
variation between the volume of liquid sample dispensed from
different individual tips.
[0009] From the discussion above, it is apparent that there is a
need for a dispensing systems that can accurately deposit precise
amounts of liquid sample on target locations on a substrate, with a
high throughput rate, without risk of cross contamination of
samples or damage to the deposited material. Therefore, it is an
object herein to provide apparatus, methods and substrates for
fulfilling these and other needs.
SUMMARY OF THE INVENTION
[0010] A delivery system for delivery of precise amounts small
volumes, particularly submicroliter and smaller volumes is
provided. Also provided are pin tools for use in the system and
substrates for retaining samples, particularly substrates for use
in the systems provided herein.
[0011] One delivery system with pin tool as constructed as provided
herein, accurately delivers small volumes, typically submicroliter
or nanoliter or picoliter volumes, of liquid samples onto a
substrates, such as a microarray substrate, at high throughput
rates by dipping a slotted pin tool (a pin tool having one or more
pins with slotted ends) having an open tip into a sample reservoir
or well containing a liquid sample to be delivered onto the
substrate, thereby drawing a volume of liquid sample up into the
pin tool. The slotted pin tool is then moved toward the substrate
at a predetermined rate and then is halted, thereby expelling the
liquid sample from the slotted pin tool onto the reaction location
of the substrate. Thus, the sample fluid is expelled from the
slotted pin tool by the force of momentum. The volume of liquid
sample expelled is proportional to the momentum of the moving pin
tool (i.e., the amount delivered is proportional to the velocity of
the pin tool as it contacts the surface or to the velocity of the
liquid in a pin when movement of the pin tool is halted). Hence
volume delivered is a function of the speed of moving the pin tool
toward the microarray, which provides a way to accurately control
and deliver desired sample volumes. For each pin tool size there is
a range of volumes in which the amount of volume delivered is a
linearly of the velocity of the pin tool. Sample volume delivered
is not dependent on tip surface areal, thereby providing for
flexibility in use since it is not necessary to change pins to
dispense different volumes.
[0012] The system uses the slotted pin tool provided herein. The
pin tool has a slot that is sufficiently large to contain the
volume of sample liquid desired for delivery. The slotted pin tool
and system are provided herein. In one aspect, the pin tool slot
may be sized to fit around target locations, such as loci on which
material has been deposited on a substrate, such as a microarray
substrate, to prevent contact between the pin tool and the
material. The slotted pins can be mounted in a holding block so as
to move up and down in the block; the positions of each pin in the
block are selected to match the target loci on the substrate. The
slotted pins in the pin tool have a substantially cylindrical tip
having a lateral slot forming a cavity with a width of greater than
at least about 10, 30, 50, 75 or 100 .mu.m, and can be of a size up
to about 300 .mu.m or 500 .mu.m or 1000 .mu.m and having a height
of at least about, 25, 50 .mu.m, 100 .mu.m or greater. The selected
size is a function of the delivered volume of liquid. Such pins can
deliver samples of as low or lower about 1 nanoliter and higher,
and can be as low as about 3-10 picoliters.
[0013] For example, a pin tool provided herein with a 300 .mu.m
slot permits delivery of volumes of as low as about 1 nanoliter to
30 nanoliters. For this pin tool and for delivery of volumes in
this range the volume delivered is linearly related to the velocity
of the tool prior to halting it. Varying the size of the slot
permits greater variation in volume delivered.
[0014] The particular geometry of the slot in the pin tool is
selected as a function of the size of the loci on the target array.
In some embodiments, system is designed so that the pin tool halts
prior to contacting the surface. In other embodiments it contacts
the surface. For embodiments in which the halting of the movement
of the pin tool results from or includes contact with the
substrate, the slots fit around each locus.
[0015] Generally the sample selected to be delivered, when intended
for mass spectrometric analysis by MALDI, results in a spot on the
substrate surface that is at least the size of the laser spot but
can be smaller or larger as desired. A typical laser spot is about
30-50 .mu.m. Delivery of about 5 nanoliters results in a spot of
about 100 .mu.m. The precise size of the spot varies depending upon
the surface on which it is delivered.
[0016] To move the pin tool towards the substrate, the holding
block can be moved toward the substrate, such as a microarray
substrate, until the slotted pins on the tool make contact with the
microarray, whereupon the pin tool tips fit around the loci, such
as spots of matrix material, without contacting any deposited
material on the surface. The pin tool then moves upward in the pin
tool holding block, which is then moved away from the microarray.
Because it is designed to fit around each locus, the pin tool does
not contact any material, such as matrix material for MALDI, cells,
protein crystals or other materials, on the substrate. In this way,
the dispensing system accurately deposits precise amounts of liquid
sample on target locations, such as on a microarray substrate, with
a high throughput rate, without contacting or damaging any
material, such as matrix material, deposited on a substrate.
[0017] A microarray substrate that can be used with the system is
also provided. This microarray is constructed using
photolithographic techniques and hydrophobic materials. Target
locations on the microarray are defined with the application of
photoresist materials and photolithographic deposition to create an
array of locations on the chip that are less hydrophobic than the
surrounding areas. The differential hydrophobicity confines the
droplets to a desired locus. The microarrays can contain any
desired number of loci from 1 to 1000, to 2000 or more, and
typically have 96-, 384-, 1536-loci. Higher densities are also
contemplated. The pins in the pin tools are in a pattern that
matches a selected array.
[0018] By virtue of the pin tool design herein, it is possible to
transfer the sample to a pre-determined locus on a substrate that
already has pre-deposited material, such as matrix, cells, such as
bacterial or mammalian cells, protein crystals and other materials
sensitive to contact. Since the instant tools provided herein rely
on inertial forces for delivery, delivery of liquids is primarily
dependent upon the momentum of the liquid in the slotted tool, not
on the relative surface tensions of the pin and the substrate for
the liquid. As one result, the pin tools provided herein permit
accurate and controlled delivery of defined volumes by selection of
the velocity of the tool at impact or as it reaches it the
substrate and is stopped prior to contact.
[0019] Substrates that contain two materials, a photoresist
material treated to render it resistant to chemical treatments such
as silation used mass spectrometry and other synthetic procedures,
and a second more hydrophobic material are provided. Unlike most
substrates that employ photolithographic methods, the photoresist
is not removed from the surface, but includes the target loci of
the surface. This is achieved by baking the substrate. Hence a
substrate that contains photoresist material as the target loci are
provided.
[0020] Also provided are combinations of pin tools that contain
slotted pins and substrates, where the number and arrangement of
pins and size of the slots is designed to match the arrayed loci
and, preferably, the slots are of a size that is greater than each
locus, or each locus with loaded or preloaded material, such as
matrix material.
[0021] Other features and advantages of the apparatus and methods
provided herein should be apparent from the following description
of preferred embodiments, which illustrate, by way of example, the
principles of the methods and apparatus and substrates.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 shows a sample delivery system constructed as
provided herein.
[0023] FIG. 2 is a perspective view of a pin tool block and its use
in the delivery system shown in FIG. 1.
[0024] FIG. 3 is a block diagram illustrating the primary
components of a delivery system shown in FIG. 1.
[0025] FIG. 4 is a side phantom view of a slotted pin tool in the
sample delivery system illustrated in FIG. 1, illustrating a
"floating pin" configuration.
[0026] FIG. 5 is a detail side view of a slotted pin tool of the
FIG. 1 system.
[0027] FIG. 6 is a plan view of the slotted pin tool of FIG. 5,
looking down through the pin tool toward the microarray
substrate.
[0028] FIGS. 7A, 7B, and 7C are side views of the slotted pin tool
showing the liquid sample as drawn into the pin tool and deposited
onto the substrate.
[0029] FIG. 8 is a side view of an alternative embodiment of a
slotted pin tool for the FIG. 1 system, illustrating a
spring-loaded configuration.
[0030] FIGS. 9A, 9B, and 9C are side views of an alternative
embodiment of a pin tool for the FIG. 1 system, illustrating a
solenoid-activated hollow pin tool.
[0031] FIG. 10 is a plan view of a microarray substrate for use in
the FIG. 1 sample delivery system.
[0032] FIG. 11 is a side view of an alternative embodiment of a
slotted pin tool having a tapered end permitting entry into very
small wells.
[0033] FIG. 12 is an enlarged view of the tip of the pin tool
illustrated in FIG. 11.
DETAILED DESCRIPTION
[0034] Definitions
[0035] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which this invention belongs. In the event
there are different definitions for terms herein, the definitions
in this section control. Where permitted, all patents,
applications, published applications and other publications and
sequences from GenBank and other data bases referred to throughout
in the disclosure herein are incorporated by reference in their
entirety.
[0036] Among the issued patents and published international
applications incorporated by reference are: U.S. Pat. Nos.
5,807,522, 6,110,426, 6,024,925, 6,133,436, 5,900,481, 6,043,031,
5,605,798, 5,691,141, 5,547,835, 5,872,003, 5,851,765, 5,622,824,
6,074,823, 6,022,688, 6,111,251, 5,777,324, 5,928,906, 6,225,450,
6,146,854, 6,207,370, International PCT application Nos. WO
99/12040, WO 97/42348, WO 98/20020, WO 98/20019, WO 99/57318, WO
00/56446 and WO 00/60361. These patents and publications describe a
variety of mass spectrometric analytical methods, substrates and
matrices used in mass spectrometric analyses, and related methods
and apparatus, including pin tools and other dispensing systems. It
is intended that the methods, substrates, pin tools and delivery
systems provided herein are for use in place or addition to the
delivery methods, apparatus and substrates described and used in
these patents and patent applications. Other intended uses include
any methods and assays that use microarrays and other such
substrates for syntheses and screening, including sequencing,
oligonucleotide and protein syntheses and diagnostic assays, and
are particularly suitable for use in high throughput formats.
[0037] As used herein, a molecule refers to any molecule or
compound that is linked to a substrate. Typically such molecules
are macromolecules or components or precursors thereof, such as
peptides, proteins, small organics, oligonucleotides or monomeric
units of the peptides, organics, nucleic acids and other
macromolecules. A monomeric unit refers to one of the constituents
from which the resulting compound is built. Thus, monomeric units
include, nucleotides, amino acids, and pharmacophores from which
small organic molecules are synthesized.
[0038] As used herein, macromolecule refers to any molecule having
a molecular weight from the hundreds up to the millions.
Macromolecules include peptides, proteins, nucleotides, nucleic
acids, and other such molecules that are generally synthesized by
biological organisms, but can be prepared synthetically or using
recombinant molecular biology methods.
[0039] As used herein, the term "biopolymer" is used to mean a
biological molecule, including macromolecules, composed of two or
more monomeric subunits, or derivatives thereof, which are linked
by a bond or a macromolecule. A biopolymer can be, for example, a
polynucleotide, a polypeptide, a carbohydrate, or a lipid, or
derivatives or combinations thereof, for example, a nucleic acid
molecule containing a peptide nucleic acid portion or a
glycoprotein, respectively. The methods and systems herein, though
described with reference to biopolymers, can be adapted for use
with other synthetic schemes and assays, such as organic syntheses
of pharmaceuticals, or inorganics and any other reaction or assay
performed on a solid support or in a well in nanoliter or smaller
volumes.
[0040] As used herein, a biological particle refers to a virus,
such as a viral vector or viral capsid with or without packaged
nucleic acid, phage, including a phage vector or phage capsid, with
or without encapsulated nucleotide acid, a single cell, including
eukaryotic and prokaryotic cells or fragments thereof, a liposome
or micellar agent or other packaging particle, and other such
biological materials. For purposes herein, biological particles
include molecules that are not typically considered macromolecules
because they are not generally synthesized, but are derived from
cells and viruses.
[0041] As used herein, the term "nucleic acid" refers to
single-stranded and/or double-stranded polynucleotides such as
deoxyribonucleic acid (DNA), and ribonucleic acid (RNA) as well as
analogs or derivatives of either RNA or DNA. Also included in the
term "nucleic acid" are analogs of nucleic acids such as peptide
nucleic acid (PNA), phosphorothioate DNA, and other such analogs
and derivatives or combinations thereof.
[0042] As used herein, the term "polynucleotide" refers to an
oligomer or polymer containing at least two linked nucleotides or
nucleotide derivatives, including a deoxyribonucleic acid (DNA), a
ribonucleic acid (RNA), and a DNA or RNA derivative containing, for
example, a nucleotide analog or a "backbone" bond other than a
phosphodiester bond, for example, a phosphotriester bond, a
phosphoramidate bond, a phophorothioate bond, a thioester bond, or
a peptide bond (peptide nucleic acid). The term "oligonucleotide"
also is used herein essentially synonymously with "polynucleotide,"
although those in the art recognize that oligonucleotides, for
example, PCR primers, generally are less than about fifty to one
hundred nucleotides in length.
[0043] Nucleotide analogs contained in a polynucleotide can be, for
example, mass modified nucleotides, which allows for mass
differentiation of polynucleotides; nucleotides containing a
detectable label such as a fluorescent, radioactive, luminescent or
chemiluminescent label, which allows for detection of a
polynucleotide; or nucleotides containing a reactive group such as
biotin or a thiol group, which facilitates immobilization of a
polynucleotide to a solid support. A polynucleotide also can
contain one or more backbone bonds that are selectively cleavable,
for example, chemically, enzymatically or photolytically. For
example, a polynucleotide can include one or more
deoxyribonucleotides, followed by one or more ribonucleotides,
which can be followed by one or more deoxyribonucleotides, such a
sequence being cleavable at the ribonucleotide sequence by base
hydrolysis. A polynucleotide also can contain one or more bonds
that are relatively resistant to cleavage, for example, a chimeric
oligonucleotide primer, which can include nucleotides linked by
peptide nucleic acid bonds and at least one nucleotide at the 3'
end, which is linked by a phosphodiester bond, or the like, and is
capable of being extended by a polymerase. Peptide nucleic acid
sequences can be prepared using well known methods (see, for
example, Weiler et al., Nucleic acids Res. 25:2792-2799
(1997)).
[0044] A polynucleotide can be a portion of a larger nucleic acid
molecule, for example, a portion of a gene, which can contain a
polymorphic region, or a portion of an extragenic region of a
chromosome, for example, a portion of a region of nucleotide
repeats such as a short tandem repeat (STR) locus, a variable
number of tandem repeats (VNTR) locus, a microsatellite locus or a
minisatellite locus. A polynucleotide also can be single stranded
or double stranded, including, for example, a DNA-RNA hybrid, or
can be triple stranded or four stranded. Where the polynucleotide
is double stranded DNA, it can be in an A, B, L or Z configuration,
and a single polynucleotide can contain combinations of such
configurations.
[0045] As used herein, the term "polypeptide," means at least two
amino acids, or amino acid derivatives, including mass modified
amino acids and amino acid analogs, that are linked by a peptide
bond, which can be a modified peptide bond. A polypeptide can be
translated from a polynucleotide, which can include at least a
portion of a coding sequence, or a portion of a nucleotide sequence
that is not naturally translated due, for example, to its location
in a reading frame other than a coding frame, or its location in an
intron sequence, a 3' or 5' untranslated sequence, a regulatory
sequence such as a promoter. A polypeptide also can be chemically
synthesized and can be modified by chemical or enzymatic methods
following translation or chemical synthesis. The terms
"polypeptide," "peptide" and "protein" are used essentially
synonymously herein, although the skilled artisan recognizes that
peptides generally contain fewer than about fifty to one hundred
amino acid residues, and that proteins often are obtained from a
natural source and can contain, for example, post-translational
modifications. A polypeptide can be post-translationally modified
by, for example, phosphorylation (phosphoproteins), glycosylation
(glycoproteins, proteoglycans), which can be performed in a cell or
in a reaction in vitro.
[0046] As used herein, the term "conjugated" refers stable
attachment, typically by virtue of a chemical interaction,
including ionic and/or covalent attachment. Among preferred
conjugation means are: streptavidin- or avidin- to biotin
interaction; hydrophobic interaction; magnetic interaction (e.g.,
using functionalized magnetic beads, such as DYNABEADS, which are
streptavidin-coated magnetic beads sold by Dynal, Inc. Great Neck,
N.Y. and Oslo Norway); polar interactions, such as "wetting"
associations between two polar surfaces or between
oligo/polyethylene glycol; formation of a covalent bond, such as an
amide bond, disulfide bond, thioether bond, or via crosslinking
agents; and via an acid-labile or photocleavable linker.
[0047] As used herein, "sample" refers to a composition containing
a material to be detected. In a preferred embodiment, the sample is
a "biological sample" (i.e., any material obtained from a living
source (e.g. human, animal, plant, bacteria, fungi, protist,
virus). The biological sample can be in any form, including solid
materials (e.g. tissue, cell pellets and biopsies) and biological
fluids (e.g. urine, blood, saliva, amniotic fluid and mouth wash
(containing buccal cells)). Preferably solid materials are mixed
with a fluid. In particular, herein, the sample refers to a mixture
of matrix used or mass spectrometric analyses and biological
material such as nucleic acids. The pin tools and systems provided
herein are designed to dispense nucleic acid compositions into
matrix that has been deposited on a substrate or to dispense
compositions containing matrix material and biological material
such as nucleic acids onto a selected locus or plurality of loci on
a substrate.
[0048] As used herein, a composition refers to any mixture. It may
be a solution, a suspension, liquid, powder, a paste, aqueous,
non-aqueous or any combination thereof.
[0049] As used herein, a combination refers to any association
between among two or more items. The combination can be two or more
separate items, such as two compositions or two collections, can be
a mixture thereof, such as a single mixture of the two or more
items, or any variation thereof.
[0050] As used herein, fluid refers to any composition that can
flow. Fluids thus encompass compositions that are in the form of
semi-solids, pastes, solutions, aqueous mixtures, gels, lotions,
creams and other such compositions.
[0051] As used herein, the term "solid support" means a
non-gaseous, non-liquid material having a surface. Thus, a solid
support can be a flat surface constructed, for example, of glass,
silicon, metal, plastic or a composite; or can be in the form of a
bead such as a silica gel, a controlled pore glass, a magnetic or
cellulose bead; or can be a pin, including an array of pins
suitable for combinatorial synthesis or analysis.
[0052] As used herein, "substrate" refers to an insoluble support
onto which a sample and/or matrix is deposited. Support can be
fabricated from virtually any insoluble or solid material. For
example, silica gel, glass (e.g. controlled-pore glass (CPG)),
nylon, Wang resin, Merrifield resin, Sephadex, Sepharose,
cellulose, magnetic beads, Dynabeads, a metal surface (e.g. steel,
gold, silver, aluminum, silicon and copper), a plastic material
(e.g., polyethylene, polypropylene, polyamide, polyester,
polyvinylidenedifluoride (PVDF)). Exemplary substrate include, but
are not limited to, beads (e.g., silica gel, controlled pore glass,
magnetic, Sephadex/Sepharose, cellulose), capillaries, flat
supports such as glass fiber filters, glass surfaces, metal
surfaces (steel, gold, silver, aluminum, copper and silicon),
plastic materials including multiwell plates or membranes (e.g., of
polyethylene, polypropylene, polyamide, polyvinylidenedifluoride),
pins (e.g., arrays of pins suitable for combinatorial synthesis or
analysis or beads in pits of flat surfaces such as wafers (e.g.,
silicon wafers) with or without plates. The solid support is in any
desired form, including, but not limited to: a bead, capillary,
plate, membrane, wafer, comb, pin, a wafer with pits, an array of
pits or nanoliter wells and other geometries and forms known to
those of skill in the art. Preferred support are flat surfaces
designed to receive or link samples at discrete loci. Most
preferred as flat surfaces with hydrophobic regions surrounding
hydrophilic loci for receiving, containing or binding a sample.
[0053] As used herein, the term "target site" refers to a specific
locus on a solid support upon which material, such as matrix
material, matrix material with sample, and sample, can be deposited
and retained. A solid support contains one or more target sites,
which can be arranged randomly or in ordered array or other
pattern. When used for mass spectrometric analyses, such as MALDI
analyses, a target site or the resulting site with deposited
material, is preferably equal to or less than the size of the laser
spot that will be focussed on the substrate to effect desorption.
Thus, a target site can be, for example, a well or pit, a pin or
bead, or a physical barrier that is positioned on a surface of the
solid support, or combinations thereof such as a beads on a chip,
chips in wells, or the like. A target site can be physically placed
onto the support, can be etched on a surface of the support, can be
a "tower" that remains following etching around a locus, or can be
defined by physico-chemical parameters such as relative
hydrophilicity, hydrophobicity, or any other surface chemistry that
retains a liquid therein or thereon. A solid support can have a
single target site, or can contain a number of target sites, which
can be the same or different, and where the solid support contains
more than one target site, the target sites can be arranged in any
pattern, including, for example, an array, in which the location of
each target site is defined. The pin tools provided herein contain
blocks that hold the pins in a pattern that matches the pattern of
target sites on a the support, such that upon contacting the
support, the ends of the pins surround, but do not touch each loci
nor any of the loci.
[0054] As used herein, the term "predetermined volume" is used to
mean any desired volume of a liquid. For example, where it is
desirable to perform a reaction in a 5 microliter volume, 5
microliters is the predetermined volume. Similarly, where it is
desired to deposit 200 nanoliters at a target site, 200 nanoliters
is the predetermined volume.
[0055] As used herein, the term "liquid dispensing system" means a
device that can transfer a predetermined amount of liquid to a
target site. The amount of liquid dispensed and the rate at which
the liquid dispensing system dispenses the liquid to a target
site.
[0056] As used herein, the term "liquid" is used broadly to mean a
non-solid, non-gaseous material, which can be homogeneous or
heterogeneous, and can contain one or more solid or gaseous
materials dissolved or suspended therein.
[0057] As used herein, the term "reaction mixture" refers to any
solution in which a chemical, physical or biological change is
effected. In general, a change to a molecule is effected, although
changes to cells also are contemplated. A reaction mixture can
contain a solvent, which provides, in part, appropriate conditions
for the change to be effected, and a substrate, upon which the
change is effected. A reaction mixture also can contain various
reagents, including buffers, salts, and metal cofactors, and can
contain reagents specific to a reaction, for example, enzymes,
nucleoside triphosphates, amino acids, and the like. For
convenience, reference is made herein generally to a "component" of
a reaction, wherein the component can be a cell or molecule present
in a reaction mixture, including, for example, a biopolymer or a
product thereof.
[0058] As, used herein, submicroliter volume, refers to a volume
conveniently measured in nanoliters or smaller and encompasses, for
example, about 500 nanoliters or less, or 50 nanoliters or less or
10 nanoliters or less, or can be measured in picoliters, for
example, about 500 picoliters or less or about 50 picoliters or
less. For convenience of discussion, the term "submicroliter" is
used herein to refer to a reaction volume less than about one
microliter, although it will be readily apparent to those in the
art that the systems and methods disclosed herein are applicable to
subnanoliter reaction volumes as well.
[0059] As used herein, nanoliter volumes generally refer to volumes
between about 1 nanoliter up to less than about 100, generally
about 50 or 10 nanoliters.
[0060] As used herein, with respect to the supports provided
herein, an element is defined as less hydrophobic than another by
the relative "wettability" of the element or contact angles, where
the contact angle of an element is less than the surrounding
surface. The contact angle is the angle the breaks the surface
tension when a liquid is delivered. A hydrophilic substrate
requires a relatively lower contact angle than a more hydrophobic
material. Hence contact angle refers to relative hydrophobicity
between or among surfaces.
[0061] As used herein, high-throughput screening (HTS) refers to
processes that test a large number of samples, such as samples of
diverse chemical structures against disease targets to identify
"hits" (see, e.g., Broach et al. High throughput screening for drug
discovery, Nature, 384:14-16 (1996); Janzen, et al. High throughput
screening as a discovery tool in the pharmaceutical industry, Lab
Robotics Automation: 8261-265 (1996); Fernandes, P. B., Letter from
the society president, J. Biomol. Screening, 2:1 (1997); Burbaum,
et al., New technologies for high-throughput screening, Curr. Opin.
Chem. Biol., 1:72-78 (1997)]. HTS operations are highly automated
and computerized to handle sample preparation, assay procedures and
the subsequent processing of large volumes of data.
[0062] As used herein, a photoresist refers to a photoresist
obtained by polymerization of a diazo photosensitive material with
a phenol resin. These photoresists are generally called positive
type photoresists.
[0063] As used herein, symbology refers to a code, such as a bar
code or other symbol, that is engraved, stamped or imprinted on a
substrate. The symbology is any code known or designed by the user.
In general, the symbols are identifiable to the user or are
associated with information stored in a computer or memory and
associated with identifying information.
[0064] As used herein, the abbreviations for amino acids and
protective groups and other abbreviations are in accord with their
common usage and, if appropriate, the IUPAC-IUB Commission on
Biochemical Nomenclature (see, (1972) Biochem. 11: 942-944).
[0065] Delivery System
[0066] As noted the delivery system provided herein delivers small
volumes, typically submicroliter volumes, of liquid samples onto a
substrate at high throughput rates by dipping a slotted pin tool
having an open tip into a sample reservoir or well containing a
liquid sample to be delivered onto a substrate, thereby drawing a
volume of liquid sample up into the pins in the pin tool. The pin
tool with slotted pin(s) is moved from the sample well to an
elevated position above a reaction location on the microarray that
is to receive the liquid sample, is lowered toward the substrate at
a predetermined speed, and then the movement of the pin tool toward
the substrate is halted, thereby expelling the liquid sample from
the slotted pin tool onto the reaction location of the substrate,
such that the sample fluid is expelled from the slotted pin tool by
the force of momentum. The volume of liquid sample expelled is
determined by the speed of moving the pin tool toward the
microarray. Typically the pin tool contains a plurality of slotted
pins. In certain embodiments, the outer surface of the pin is
rendered hydrophobic, such as by silanation or other chemical
means, relative to the inner surface to thereby reduce or eliminate
any satellite drops that adhere to the outer surface.
[0067] FIG. 1 depicts an exemplary instrument for pin tool based
dispensing. Any such instrument may be adapted for use with the pin
tool provided herein. FIG. 1 shows a sample delivery system 100
constructed as described herein. The system 100 includes a base
table 102 on which is mounted a transport system 104 having rails
or runners that can move a sample in x, y, and z planar coordinates
relative to the table and to microtiter plates containing reagents.
The x-direction (which will also referred to as left-right) is
indicated in FIG. 1 by the arrows 106, the y-direction (also
referred to as top-bottom) is indicated by the arrows 108, and the
z-direction (also referred to as up-down) is indicated by the
arrows 110.
[0068] A pin tool holding block 112 is mounted to the transport
system 104 for movement in the y-direction along a y-axis rail 114
and for movement in the z-axis direction along a z-axis rail 116.
Microtiter plates 118 mounted on an MTP table 120 are moved in the
x-axis direction along an MTP rail 122, to move back and forth
relative to the pin tool holding block 112. The target microarray
chips 124 are mounted on a target table 126 and are moved in the
x-axis direction along a target rail 128 for delivery of liquid
samples, as described further below.
[0069] The pin tool holding block 112 is illustrated in FIG. 2,
which shows that the holding block holds a plurality of pin tools
202. The number of pin tools held in the holding block will
typically correspond to the number of target locations on the
microarray chip 124 (FIG. 1) that will receive samples. In genomic
research, for example, a microarray substrate chip may typically
contain ninety-six or more target locations. Microarray chips with
other numbers of target locations may also be used, such as
384-target arrays or even 1536-target arrays.
[0070] The pin tool holding block 112 may also include a single-pin
holding station 204, where a single pin tool may be attached. The
single holding station permits one pin tool to be easily attached
for single-point processing or other special operations involving a
single pin tool. The single pin holding station 204 is preferably
located on the block 112 such that the single pin tool may be
easily accessed for attachment and removal from the pin tool block
112 during normal system operation.
[0071] As described further below, the sample delivery system 100
can deliver accurately-controlled volumes of liquid samples onto
target locations of a microarray substrate at high throughput rates
by dipping a slotted pin tool into a reservoir of liquid sample,
thereby drawing a volume of liquid sample up into the pin tool,
then moving the slotted pin tool to a position above the substrate,
lowering the slotted pin tool toward the substrate at a
predetermined speed, and then abruptly halting the movement of the
pin tool and expelling the liquid sample from the pin tool onto the
substrate. The sample is expelled due to the momentum of the
liquid, which is still traveling at the speed of the pin tool when
the pin tool is halted. Thus, using the momentum delivery technique
described herein, the liquid sample can be deposited onto the
microarray substrate without making extended contact with the
substrate and without contact between the pin tools and any
material at a locus or loci on the substrate.
[0072] As described further below, the movement of a pin tool
toward the microarray 124 may be halted either because the slotted
pin tool makes contact with the microarray, or because the pin tool
reaches the limit of travel relative to the pin tool holding block
112. If the pin tool makes contact with the microarray during
downward movement of the holding block, then the pin tool is
preferably mounted in the block so as to move upwardly independent
of the block, so that the pin tool can move up while the block
itself is moving downward. In such an arrangement, the pin tool
contacts the microarray and the block is stopped in its downward
movement substantially simultaneously, followed by lifting up the
holding block, carrying the pin tools with it. Alternatively, the
pin tool may be moved independently from the block 112 toward the
microarray by an actuating mechanism such as a solenoid, halting
when the pin tool reaches the limit of travel in the actuating
mechanism. These alternatives are described further below.
[0073] It has been found that the speed of lowering the pin tool
precisely determines the volume of the liquid sample that is
expelled. That is, the slot of the pin tool is loaded with liquid
sample by dipping, and the portion of that loaded sample that will
be expelled is determined by the speed of lowering the pin tool.
The momentum delivery technique of the provided herein utilizes any
pin tool lowering speed that will impart a momentum force to the
liquid sample that is greater than the surface tension of the
liquid. The delivery system 100 permits precise control over the
speed of moving the pin tool toward the microarray substrate 124.
The delivery system thereby carefully controls delivery of the
liquid sample, and expels the samples from the pin tools with
reduced contamination problems and with increased efficiency and
throughput.
[0074] Process Steps for Sample Delivery
[0075] To deliver the samples to the microarray 124, the pin tool
holding block 112 is moved across the table 102 and the pin tools
are moved vertically in the z-direction 110 as described above. The
pin tool holding block 112 is mounted to an arm 130 of the
transport system 104. As the arm 130 moves along the y and z rails
114, 116, the holding block 112 is moved as well, and can thereby
be moved across the stations of the table 102 for sample delivery
onto microarray substrate chips.
[0076] The pin tools are moved first to an ultrasonic cleaning
station 132 that includes a cleaning solution bath 134 into which
the tips of the pin tools are dipped. The ultrasonic cleaning will
typically require approximately five to ten seconds to sufficiently
clean the pin tool tips of any remaining samples from prior system
operation. When a pin tool is being used on an initial operation, a
longer cleaning time is usually necessary, to remove contaminants
from the manufacturing process. Such initial cleanings are referred
to as preconditioning, and typically require approximately thirty
seconds in the ultrasonic bath 134.
[0077] The next station of the system 100 is a rinsing and drying
station 136. This station includes an empty recess into which the
pin tools are lowered, whereupon the recess is filled with a rinse
solution, such as distilled water. The rinse bath maintains the pin
tool tips in a submerged state for a duration of one to ten
seconds, as required for sufficient cleaning of the sample fluid
being processed. At the conclusion of the submerged time, the rinse
solution is drained, and an air bath is begun, for drying the pin
tools. In the preferred embodiment, a vacuum is used in the bottom
of the recess to draw off any excess rinse solution and empty the
recess. The vacuum drying time is typically on the order of one to
ten seconds duration.
[0078] Next, the pin tool holding block 112 is moved to a
microtiter plate that includes wells containing the liquid sample
to be delivered. The holding block is moved from the rinsing and
drying station 136 to a position over the MTP table 120. The MTP
table may include multiple microtiter plates; only one such
microtiter plate 118 is shown in FIG. 1 for simplicity of
illustration. Each microtiter plate will preferably contain as many
sample wells as there are target locations on the destination
microarray 124. The illustrated system 100 has a capacity of ten
microtiter plates, as well as a capacity of ten microarray chips,
but it should be apparent that different capacities can be easily
provided, as desired. A system operator can specify the location of
the source microtiter plate 118 in x-y coordinates of the MTP table
120, and also can specify the x-y location of the destination chip
124, with a user control interface described further below, or
automatic modes of operation can be implemented to move the pin
tools from the various preparatory stations 132, 134 and from one
microtiter to the next for continuous processing, as desired. The
MTP table 120 will move along the MTP rail 122 and the holding
block 112 will move along the y-rail 114 in cooperation to position
the desired microtiter plate beneath the holding block.
[0079] When the pin tool holding block 112 is located over the
appropriate microtiter plate on the MTP table 120, the holding
block will be lowered so that the pin tool tips are dipped into the
liquid sample contained in the microtiter wells. The volume of
liquid sample that will be drawn into the slots of the pin tools
will be determined by the size of the slots and the surface tension
of the liquid sample. The duration or holding time of the pin tools
in the microtiter wells, as well as the speed of lowering, can be
selected by the system operator. The duration and speed of dipping
will be selected by the operator for the desired sample volume, in
accordance with the nature of the sample and factors such as sample
viscosity, temperature, and the like.
[0080] After the liquid sample has been drawn into each pin tool
slot and the pin tool holding block 112 has been raised up from the
MTP table 120, the holding block will be moved over an appropriate
microarray chip 124 for sample delivery. The chip is located along
the microarray chip rail 128, on which multiple chips may be
located, and will be moved into proper position for receiving
liquid samples. Only one microarray chip 124 is shown in FIG. 1 for
simplicity of illustration, but the preferred embodiment permits as
many chips to be located on the chip rail 128 as there are
microtiter plates 118 on the MTP table 120.
[0081] The proper alignment of the pin tools over target locations
of the appropriate chip is a critical process, and can be
accomplished for example, by the system 100 with a robotic vision
unit 140. Initial alignment for a particular pin tool can be
accomplished in a number of ways, For example, a camera is mounted
on the machine that seeks the target. To align the pin tool with
the target loci it is necessary to locate pin(s) relative to the
target and/or the pins. To locate the pins, the pins are, for
example, dipped into a dye or ink and then contacted with a blank
substrate. The camera and software therefor the "learns" or images
the locations of the spots and can then direct the pin tool to the
corresponding positions on the actual substrate. Alternatively,
other marks can be used. Transparent sticky tape can be placed on
the surface of the blank substrate and the pin touched thereon to
imprint its image on the tape. The camera with software can then
learn the locations of the pins. This procedure can also be
automated. Such procedure should be performed for each pin tool to
create an image thereof so that the loci on the substrate and the
pins can be properly aligned.
[0082] The robotic vision unit includes a camera 142 that is
mounted above the pin tool holding block 112, having a field of
view that encompasses at least one corner of the microarray chip
that is beneath the holding block. The image that should be
observed in the camera field of view when the holding block is
properly positioned is known (i.e., see, for example, above).
Therefore, the system 100 can confirm proper positioning by
comparing the image being received from the camera 142 with the
known image that should be obtained.
[0083] The system 100, for example, can check for the appearance of
a known registration mark that is imprinted on the microarray chip
124, to confirm that the mark is in the expected location, or the
system can check for the presence of a target location on the chip
that should be in a known position in the camera field of view when
there is proper registration of the pin tool block. If the expected
registration mark or target location does not appear in the
expected position, then the system will issue a warning to the
operator and will halt sample processing.
[0084] The system may perform a pattern recognition operation to
check for proper positioning of the microarray chip and also check
for proper chip composition. The image that is obtained in the
camera field of view can be compared with the image that should be
obtained with a properly produced and aligned chip. Any anomalies
in the obtained image may indicate a defective chip, or may
indicate that the chip is misaligned. In either case, a warning may
be provided and operation of the system may be automatically
halted. After the situation with the defective or improperly
positioned chip has been corrected, the system operator can
indicate to the system that it should continue with normal
processing.
[0085] If the pin tool holding block 112 is properly positioned, as
confirmed by the robotic vision unit 140, then the pin tools are
moved toward the microarray chip 124 at a predetermined speed for a
time sufficient to impart downward momentum to the liquid sample
volumes contained within the slots of the pin tools. The pin tools
are then halted in their travel and, because the liquid samples
still have downward momentum, the liquid samples keep moving at
their imparted speed and therefore are expelled from the pin tools.
The liquid samples therefore are simultaneously deposited on the
target locations of the chip 124.
[0086] After the samples have been deposited on the microarray, the
pin tool holding block 112 is moved from its position over the chip
124 and is moved back to the starting point for operations, which
is the cleaning station 132. Alternatively, the holding block may
be moved to a home position, as commanded by the system operator.
The process of cleaning, rinsing and drying, dipping, and expelling
may then be repeated, with corresponding movement of the microtiter
plates 118 and microarray chips 124 for dipping and expelling,
respectively.
[0087] System Control
[0088] FIG. 3 is a schematic block diagram illustrating the primary
components of the delivery system shown in FIG. 1. A controller 302
contains a computer processor and associated software and circuitry
to communicate with and control the mechanisms of the sample table
304. The sample table 304 as illustrated in FIG. 3 represents the
table mechanisms shown in FIG. 1 for moving the pin tools along
their respective rails, and for moving the microarray chips along
the microtiter rail. The table 304 also includes the mechanisms for
controlling the operation of the cleaning station, rinsing and
drying station, dipping station, and robotic vision unit described
above.
[0089] The controller 302 is itself controlled by a user interface
device 306, such as a conventional laptop or desktop Personal
Computer with application software to provide a graphical user
interface. The system operator may adjust the speed of downward pin
tool movement and may specify target microarray chips and
microtiter plates, as well as other operational parameters, through
the user interface device 306.
[0090] Pin Tools
[0091] The pin tools provided herein are include pins that are
slotted such that upon contacting the surface of a substrate with
deposited material or other loci, the pin tool contacts the surface
of the substrate, but does not touch any of the loci or material
deposited thereon. The slotted pins are also provided, as is a
block containing or other support containing a plurality of pins in
an array or arrangement that matches the array of loci on a target
surface.
[0092] The individual pins in the pin tools 202 (FIG. 2) are of
slotted construction to work with the momentum delivery technique
of the system 100. A pin tool 202 is shown in side section in FIG.
4, illustrating a "floating" pin arrangement. A detail side view of
a pin tool tip is shown in FIG. 5 and a plan view through the pin
tool tip is shown in FIG. 6.
[0093] FIG. 4 shows a pin tool 202 in the pin tool holding block
112 and indicates that the holding block is hollow, having a top
wall 402 and bottom wall 404 joined by a side wall 406. It should
be understood that only one pin tool is shown for simplicity of
illustration, but that the holding block has a greater holding
capacity, as described above. The pin tool 202 slides up and down
relative to the holding block 112 through an upper guide hole 408
in the top wall 402 and a lower guide hole 410 in the bottom wall
404. A clip 412 attached to the pin tool prevents the pin tool from
falling out through the bottom wall. Thus, the pin tool is free to
move upwardly in the holding block 112. When the holding block is
moved toward the microarray 124, the pin tool is free to make
contact with the surface of the microarray without suffering damage
and without damaging any material on the microarray, and the
holding block 112 can be set to move upward at that point, through
the user interface. By permitting upward movement of the pin tool
202 relative to the holding block 112, there is a greater operating
margin for setting the point at which the holding block will be
moved back upward away from the microarray. This reduces the risk
of damage. Moreover, this construction permits the pin tool to be
abruptly halted in downward movement (by making contact with the
microarray), thereby expelling the liquid sample, without damaging
the pin tools or microarray.
[0094] When a pin tool 202 makes contact with the microarray, no
material deposited on the microarray will be damaged, because the
tip of the pin tool has a slotted construction. FIG. 4 shows a slot
420 in the tip of the pin tool. The slot is sized to fit around
material deposited at a target location on the microarray.
[0095] FIG. 5 shows side detail of a slotted pin tool that is
constructed as described herein, and FIG. 6 is a plan view. FIG. 5
and FIG. 6 illustrate how the pin tool preferably fits around the
deposited material. In FIG. 5, the pin tool 202 is shown with the
slot 420 having sufficient width to permit a mound of material 502
on a microarray 124 to fit within the slot. Other than the slot
420, the pin tool 202 is a solid core construction. FIG. 6 is a
plan view, showing that the slot 420 is cut through the pin tool
202 so that the material 502 fits within the slot. Alternatively,
the pin tool may be constructed with a hollow core.
[0096] For a conventional microarray, the matrix material spots on
the microarray surface are typically greater than 100 .mu.m in
diameter, and frequently approximately 200 .mu.m in diameter. The
distance from the center of one target location (or spot with
deposited material, such as matrix for MALDI) to the center of an
adjacent target location (or matrix material spot) on the
microarray is typically approximately 4.5 mm. Other target spacing
may be used as well, including more dense spacing of 2.25 mm from
center to center or less dense spacing of 9 mm. Accordingly, the
slot 420 preferably has a width of approximately 300 .mu.m, which
is a width that provides a sufficient margin of error in
positioning of the pin tool and production of the material so that
the pin tool safely fits around the typical material spot. The
outer diameter of the pin tool is typically approximately 600
.mu.m.
[0097] The height of the slot 420 is approximately 5 mm, a height
that results in a desired volume of liquid sample being drawn into
the slot by capillary action when the pin tool is immersed in the
well of a microtiter plate 124 at the MTP table 120 of the system.
The system operator will adjust the dipping control mechanism, in
concert with the expected depth of reagent in the microtiter wells,
through the user interface so that the pin tool is lowered into the
microtiter well just below the height of the slot 420 and is raised
out of the well before a bubble of air can form in the top of the
slot and become trapped. If a bubble is trapped, the volume of
liquid sample drawn into the slot may be imprecise, and the volume
that is expelled may likewise be imprecise. Thus, FIG. 5 and FIG. 6
show a slotted pin tool 202 that has a slot volume for containing
liquid sample of approximate dimensions 300 .mu.m.times.600
.mu.m.times.5 mm. The volume of liquid sample drawn into the slot
typically contains a volume of between 50 nl and 100 .mu.l. Those
skilled in the art will appreciate that this volume represents a
larger dispensing volume than obtainable with conventional pin tool
delivery systems, thus eliminating evaporation problems that might
otherwise arise with smaller sample volumes.
[0098] FIGS. 7A, 7B, and 7C are side views of the slotted pin tool
202 showing an operational sequence of the liquid sample after it
is drawn into the pin tool and then as it is deposited onto the
microarray 124. Only one pin tool is illustrated, but it should be
understood that the sequence depicted applies to all the pin tools
mounted in the pin tool holding block 112. In FIG. 7A, the pin tool
has been dipped into the microtiter well and liquid sample has been
drawn into the slot, and the holding block 112 is moving toward the
microarray 124.
[0099] In FIG. 7B, the pin tool holding block 112 has been lowered
sufficiently such that the slotted end of the pin tool has made
contact with the microarray surface, fitting around the material as
described above. Thus, the downward movement of the pin tool
(depicted in FIG. 7A) has been abruptly halted in FIG. 7B. The pin
tool floats in the holding block, and therefore the position of the
pin tool in FIG. 7B relative to the holding block is somewhat
elevated. Due to the momentum of the liquid sample, the liquid
continues in the downward direction when the pin tool is halted,
expelling the liquid sample out of the pin tool slot and onto the
microarray. At approximately the same time when the pin tool makes
contact with the microarray, the holding block is moved upward and
away from the microarray, thereby eliminating any significant delay
in processing. Moving the pin tool block without significant delay
increases the system throughput and maintains efficiency. FIG. 7C
shows the holding block 112 lifted upward, away from the microarray
124, with the liquid sample remaining behind on the microarray.
[0100] FIG. 8 is a side view of an alternative embodiment of a pin
tool mounting block 800 and pin tool 802 for the FIG. 1 system,
illustrating a spring-loaded configuration. As with the FIG. 4
configuration, the pin tool holding block 800 has a hollow
construction, with an upper wall 804 and a bottom wall 806 through
which the pin tool 802 may slide up and down. In FIG. 8, however, a
spring 808 is attached around the pin tool, between the upper wall
and bottom wall. A spring clip 810 fixes the bottom of the spring
to a point on the shaft of the pin tool. When the pin tool makes
contact with the surface of the microarray 124, the pin tool will
begin to move upward in the holding block 800. Because the spring
808 is fixed to the pin tool, the top of the spring is compressed
against the upper wall 804, thereby cushioning the upward movement
of the pin tool. The spring constant of the spring 808 may be
selected for the desired operation and action.
[0101] FIG. 8 also shows that a slotted pin tool constructed as
described herein may have a tip having a taper, rather than the
cylindrical construction of the FIG. 4 embodiment. The FIG. 8 pin
tool embodiment, for example, has an outside diameter at the tip of
approximately 600 .mu.m, and has an outside diameter at the full
extent of the pin tool shaft of approximately 1.5 mm. The greater
shaft diameter away from the pin tool tip provides a more durable
construction and easier handling, while the narrower tip diameter
permits dispensing of small, microliter sample volumes and also
denser packing of the pin tool tips. As with the FIG. 4 embodiment,
the volume of liquid sample carried by the pin tool 802 will be
determined by the volume of the pin tool slot 812. In both the FIG.
4 and FIG. 8 embodiments, the pin tool slot preferably has a width
that is typically greater than 200 .mu.m, to fit around material on
the microarray, and has a slot height of approximately 100 .mu.m
(0.1 mm) to 5 mm, depending on the sample volume desired. The
desired volume of liquid sample to be drawn in for delivery will
typically be between 50 nl and 100 .mu.l.
[0102] In any of the pin tool embodiments described herein, the
interior of the pin tool slot may be coated with an ion exchange
resin or other resin to assist with cleaning the slot or condition
the liquid sample prior to dispensing. The increased size of the
pin tool slot illustrated above compared with conventional
quill-type pin tools permits reduced manufacturing costs and easier
slot inspection for signs of wear. The pin tools provided herein
are configured, through controlled downward speed and through
spring loading (FIG. 8), such that the pin tools make contact with
the microarray surface at a carefully controlled speed, thereby
reducing the amount of wear typically experienced by other pin tool
configurations.
[0103] FIGS. 9A, 9B, and 9C are side views of an alternative
embodiment of a pin tool for the FIG. 1 system, illustrating a
solenoid-activated hollow pin tool. A metal pin tool 902 or array
of pin tools are held in a pin tool holding block 904 by metal
e-clips 906. The movement of the pin tool is limited to a vertical
direction. The holding block itself can be mounted to any x-y-z
station. As shown in FIGS. 9A, 9B, and 9C, the pin tool is spring
loaded. A spring 908 having a spring constant "D" is connected to
the pin tool and to the holding block, so that the spring can be
loaded by pulling the pin tool in the vertical (z) direction.
Because both ends of the spring are connected to the holding block
and to the pin tool, the pin tool reaches a potential energy
E.sub.pot(spring) defined by:
E.sub.pot(spring)=1/2 D s.sup.2,
[0104] for a spring constant "D" when shortened by the distance
"s". The pin tool itself does not contain a slot, such as
illustrated in FIG. 4, but rather has a hollowed opening at its
lower tip, similar to the end of a capillary tube. A volume of
liquid sample is aspirated into the pin tool using capillary
action, when the pin tool is dipped into a microtiter plate or
other sample well. The size of the hollowed opening defines the
amount of liquid sample that will eventually be dispensed. The
volume of sample that fills the hollowed opening of the pin tool by
capillary action will be the same as the dispensed volume. This
provides an important means of controlling the volume of liquid
sample that is dispensed.
[0105] Samples are dispensed by loading the spring 908 and bringing
the spring to the potential energy level indicated by the equation
above. The spring is loaded (FIG. 9B) by mechanical compression or
by electromagnetic force such as supplied by a solenoid. In FIG.
9A, FIG. 9B, and FIG. 9C, a solenoid 910 is shown at the top end of
the pin tool, but other configurations for loading the spring 908
will occur to those skilled in the art. When the spring is
released, the pin tool is moved toward the microarray 124 at a
predetermined velocity, given by the equation above, carrying the
liquid sample in the hollowed opening and imparting it with the
same velocity as the pin tool. When the pin tool reaches the end of
its travel, at FIG. 9C, the pin tool stops in the holding block,
but the liquid sample continues moving, due to the imparted
momentum. Therefore, the liquid sample is expelled from the
hollowed opening and is delivered to the microarray via momentum
force.
[0106] FIG. 11 is a side view of an alternative embodiment of a
slotted pin tool 1100 constructed as described herein, having a
tapered end. As illustrated in FIG. 11, the upper portion 1102 of
the pin tool toward the holding block has a nominal outer diameter
of approximately 1.6 mm and a length of 63.5 mm. The outer diameter
of the pin tool shaft begins to taper from the nominal diameter at
a location 1104 approximately 12 mm from the tip of the pin tool.
The dimensions of the tapered end, described further below, have
been found to provide accurate dispensing of nanoliter and
subnanoliter volumes. The taper of the pin tool permits dipping the
pin tool into relatively small volume wells without contacting the
sidewalls of the wells, thereby permitting small volumes of liquid
to be drawn into the pin tool. The taper of the pin tool also
decreases the importance of pin tool alignment with the wells, as
there is increased clearance between the pint tool and the
sidewalls of the wells. The decreased time used for pin tool
alignment increases the efficiency of the dispensing system.
[0107] FIG. 12 shows an enlarged view of the slotted pin tool 1100
illustrated in FIG. 11, showing details of the pin tool tip. The
upper location where the taper begins is again indicated by the
arrow 1104, approximately 12.0 mm above the pin tool tip. The slot
of the pin tool extends from the tip to a location 1120 that may be
from approximately 0.1 mm to 5.0 mm above the tip, depending on the
sample volume that is to be dispensed. The greater the height of
the slot, the greater the volume of sample liquid that can be drawn
into the pin tool and dispensed therefrom.
[0108] At the pin tool tip 1122, the outer diameter of the pin tool
is approximately 0.4 mm to 0.6 mm. The width of the slot is
preferably approximately 0.3 mm. These dimensions permit convenient
use with sample wells of decreased diameter, and provides increased
tolerances for misalignment. The taper from the upper taper
location 1104 to the tip 1122 is generally a linear taper, from
about 1.6 mm diameter to about 0.6-0.4 mm diameter, such that half
of the taper diameter occurs approximately half way between the
upper taper point 1104 and the lower taper point 1122. It should be
understood, however, that different dimensions and taper
configurations may be employed, and are a function of various
parameters, including the configuration of sample wells being used
and sample volumes that are desired.
[0109] Substrates
[0110] Any substrate suitable for biological and chemical reactions
and assays, such as diagnostic and hybridization assays in which
samples are deposited at discrete loci is contemplated for use
herein. The loci on the substrates and pin tools are matched so
that the pattern of pins and size of the slots matches the
arrangement and size of loci with preloaded material thereon.
Preferably, the number of pins is the same as the number of loci or
the loci are a multiple thereof to permit deposition of material at
a plurality of loci. Combinations of the substrates and the pin
tools are also provided.
[0111] Substrates with microarrays in which a relatively
hydrophilic region or contact region is surrounded by a more
hydrophobic area and methods for preparation of such substrates are
provided herein. The substrate surface is any surface that has an
available reactive group, such as --OH or a primary amine, or is
derivatized to have such group. Surfaces include but not limited to
TEFLON.RTM. (polytetrafluoroethylene (PFTE); Trademark, E. I.
DuPont), glass, derivatized glass, plastics, silicon, silicon
dioxide (SiO.sub.2) and any other such materials known to those of
skill in the art.
[0112] Also provided are methods of producing substrates and the
resulting substrates that have contact angles that result in
hydrophobic focusing of hydrophilic liquids on loci formed from
photoresist materials. The resulting substrates include elements
(loci) on a surface that are less hydrophobic than the surrounding
surface, where hydrophobicity is measured by the relative
wettability (relative contact angel) of the surrounding area
compared to each locus (element). The contact angel of each element
is less than that of the surrounding surface. To produce such
arrays, a surface, such as any of those described herein or known
to those of skill in the art to be suitable for linking or
retaining macromolecules, including biopolymers, such as silicon or
SiO.sub.2 is coated with photoresist, covered with a mask that
blocks light as loci on the surface, and exposed to light, the
photoresist in the unmasked portions is washed off. The resulting
surface is baked to render the photoresist stable to chemical
treatments such as silation. The surface is then silated. Since the
silane does not stick to photoresist, the resulting surface has
silated regions that surround the photoresist elements at the loci.
Examples 1 and 3 exemplify this process and the resulting
substrates with patterned microarrays. The substrates are
preferably about 3000 mm.times.2000 mm, such as 3068 mm.times.1960
mm, or can be smaller or larger. The number of elements (loci) on
each substrate can be any desired number, such as, 8, 16, 24, 96,
384, 1536, higher densities or any convenient number. Other
combinations of surface materials in which the contact angel
between the two surfaces is less than or equal to 20.degree. C. are
contemplated.
[0113] The step of baking the photoresist on the target loci is
renders the surface resistant to chemical treatments, such as
silation. The selection of the temperature and time is selected so
that the photoresist does not become too hydrophobic relative to
the rest of the surface for liquid to be focussed at the target
loci. Baking should be performed at temperatures of about
190-200.degree. C. for at least about 50-70 minutes. The
temperature and time are variable and can be empirically determined
for the particular photoresist materials and time of baking to
obtain the requisite chemical resistance and stability to treatment
and hydrophobicity. Both parameters are important for production of
surface with the requisite properties so that the surface can be
treated and used in analyses, such as mass spectrometry, and the
two materials have the appropriate relative
hydrophobicity/hydrophilicity to achieve hydrophobic focussing of
the droplets on the target loci.
[0114] Photoresist Materials for Preparation of the Substrates
[0115] Many photoresist materials are known to those of skill in
the art and are readily available. For use herein, selection among
such material is made and the materials are tested. Select from
among the available those that when spun or coated on a surface and
baked as described herein have a contact angle of no greater than
70.degree. C. where the surrounding area is about 90.degree. C., or
that have a relative contact angle that is less than the
surrounding surface that results in a hydrophobic/hydrophilic
focussing of sample material on the loci. For example, a
differential of at least about 20.degree. C. is suitable for use in
substrates intended for mass spectrometric analysis. The
differential is such that it provides a wettable surface.
[0116] The photoresist is from the class of commercially available
diazoquinone containing positive photoresists (see U.S. Pat. Nos.
3,402,044, 2,797,213, 3,148,983, 3,046,118, 3,201,239, 3,046,120,
3,184,310, 3,567,453, 4,550,069, 5,607,816, 5,567,569, 5,561,029,
5,558,983, 5,550,004, 4,491,629, 4,458,994 and many others).
Suitable photoresists can be selected by preparing coated
substrates as described herein and assessing hydrophobic focusing
of a hydrophilic liquid onto the resulting hydrophilic loci, such
as by visual analysis of 3-hydroxypicolinic acid (3-HPA) crystals.
To make such assessment an aqueous formulation (14.5 nano liters)
of 3-HPA is dispensed on and overlapping the loci on the substrate.
As the aqueous solvent evaporates it leaves 3-HPA crystals.
Successful focusing of the hydrophilic liquid results in a crystal
that conforms to the shape of the hydrophilic loci. If the focusing
is not successful the crystallization will occur at the site of
dispensing, consequently overlapping the loci.
[0117] Suitable photoresist compositions for use herein are
coatable liquids containing at a diazo photoactive compound with a
resin, such as a novolak (phenolic) base resin for increased
viscosity, suspended in an organic solvent. The diazonapthaquinone
(DNQ) sensitized phenolic resin (known as novolak resin) are widely
available for wafer photolithography processes. Such photoresist
compositions are well known (see, e.g., U.S. Patents cited above;
such resins are commercially available from, for example, Shipley
Co., Marlboro, Mass. and Clarian Corp., Charlotte, N.C. and others)
and any may be employed in the methods herein to produce the
substrates provided herein. The most suitable are those that yield
the best hydrophobic focusing as described above. For example,
AZ111XFS available from Clariant Corp., Charlotte, N.C., contains
cresol novolak resin (117520-84-0), 2, 1, 4-diazonaphthoquinone
ester with cumyl phenol, polyvinyl methyl ether, styrene/acrylic
polymer in propylene glycol monomethyl ether acetate.
[0118] Exemplary Substrate
[0119] FIG. 10 is a plan view of a microarray substrate 1000 for
use in the FIG. 1 sample delivery system. An exemplary substrate
1000 can be constructed using photolithographic techniques and
hydrophobic materials as described herein to form the target
locations (loci) at which, for example, material will be applied
and at which liquid samples will be deposited. The target locations
on the microarray are defined with the application of photoresist
materials and photolithographic deposition such that the target
locations on the chip are less hydrophobic than the surrounding
areas. This differential hydrophobicity reduces the occurrence of
satellite droplets that might otherwise extend from the liquid
sample and adhere to the microarray surface and permits deposition
of small sample amounts.
[0120] FIG. 10 shows a 12.times.8 grid of target locations that
have been formed on a 3068 mm.times.1960 mm surface. Other
densities of target locations may be obtained, as desired. The
starting surface may be any material that has an available --OH or
primary amine, including SiO.sub.2 and other forms of glass,
plastic, and "TEFLON"-brand materials (Trademark, E. I. DuPont for
polytetrafluoroethylene), such as any other material to which the
samples, matrix, molecules or biological particles of interest do
not adhere, and include any other such materials that are commonly
used in the electronics industry to passivate electronic components
and circuit boards, and materials used as a coating for medical
devices, especially implants, catheters, probes and surfaces of
needles. As noted, an element is defined as less hydrophobic than
another by the relative "wettability" of the element or contact
angles, where the contact angle of an element is less than the
surrounding surface. For example, exemplary microarrays with
differential hydrophobicity provided herein have SiO.sub.2 surfaces
with contact angles of 50-55 degrees and contain arrays of target
photoresist elements having contact angles of 65 degrees. To create
a less hydrophobic environment at each target site, the starting
surface was treated with a 3.5% solution of DiMethylDiChloroSilane
(DMDCS) from United Chemical Technology of Bristol, Pa., USA in 95%
Hexanes from EM Industries, Inc. of Hawthorne, N.Y., USA. The DMDCS
does not stick to photoresist and provides a surrounding
environment on the substrate whose contact angle is 90 degrees.
[0121] With the techniques exemplified below, a target location on
the microarray is formed such that the outer area surrounding the
target location has a greater hydrophobicity than the inner target
area. This increases the accuracy of liquid sample dispensing by
reducing or substantially eliminating any satellite droplets from
the liquid sample that might adhere to the outer area.
[0122] The following examples are included for illustrative
purposes only and are not intended to limit the scope of the
invention.
EXAMPLE 1
[0123] A flat substrate containing an array of less hydrophobic
elements surrounded by more hydrophobic elements, was prepared with
an array of photoresist elements. To prepare the array, silicon
dioxide (SiO.sub.2) was grown on silicon wafers to a height of 3025
angstroms, .+-.5%. Alternatively, the SiO.sub.2 can be grown to a
height of about 1050 angstroms. This process is performed by a "wet
oxidation" method in which H.sub.2 and O.sub.2 gases are used in
converting the Si to SiO.sub.2.
[0124] A photoresist material (such as "AZ 111 XFS" photoresist
from Clariant Corporation of Charlotte, N.C., USA) was spun onto
the SiO.sub.2 to a thickness of 0.2 .mu.m to 1.22 .mu.m, with a
height of about 1.0 .mu.m. The photoresist was solidified by baking
at 65 degrees Celsius for two to three minutes. The surface was
then exposed to light of 365 nm wavelength through a mask that
blocked light at the target locations. The photoresist that was
exposed to light in the unmasked portions of the substrate was then
washed off with a phosphoric acid-based developer, leaving an array
of photoresist pads having dimensions of approximately 200
.mu.m.sup.2.times.1.0 .mu.m. The wafer was then baked at
195.degree. C. for 60 minutes to remove any remaining solvents. The
substrate was then silated with DMDCS. The microarrays can contain
any desired number of loci, and typically have 96-, 384-,
1536-loci. Higher densities of loci and densities that are
multiples of other than 96 also are contemplated.
[0125] In this example, the surface is silicon; any surface that
has an available reactive group, such as --OH or a primary amine,
including but not limited to TEFLON (polytetrafluoroethylene
(PFTE)), glass, derivatized glass, plastics and other such
materials may be used.
EXAMPLE 2
[0126] In another process provided herein, a microarray was
produced with a flat starting substrate having an array of
SiO.sub.2 elements surrounded by a silane surface, thereby creating
an array of elements less hydrophobic than the surrounding area.
The resulting substrate has target locations that are bare silicon
dioxide, and the surrounding regions are silated with DMDCS.
[0127] Silicon dioxide was grown on silicon wafers to a height of
3025 Angstroms.+-.5%. Alternatively, the SiO.sub.2 can be grown to
a height of about 1050 angstroms. This process is performed by a
"wet oxidation" method in which H.sub.2 and O.sub.2 gases are used
in converting the Si to SiO.sub.2.
[0128] The resulting substrate was patterned with "MEGAPOSIT" SPR
900-0.8 photoresist from Shipley Company, L.L.C. of Marlborough,
Mass., USA in the manner described above in Example 1. The wafer
was then baked at 70.degree. C. for 30 minutes to remove any
remaining solvents. The patterned substrate was silated with 3.5%
DMDCS for twenty minutes, as described above for Example 1. The
photoresist pads were then removed by washing the substrate in
acetone for eight minutes at room temperature, thereby dissolving
the photoresist and exposing the SiO.sub.2. The contact angles of
the two materials DMDCS and the bare SiO.sub.2 creates a less
hydrophobic environment at each target location.
EXAMPLE 3
[0129] In another process, a microarray was produced using a flat
substrate having an array of SiO.sub.2 elements surrounded by a
TEFLON.RTM. (polytetrafluoroethylene (PFTE)) surface, to create an
array of target elements less hydrophobic than the surrounding
area.
[0130] Silicon dioxide was grown on silicon wafers to a height of
3025 Angstroms.+-.5%. Alternatively, the SiO.sub.2 can be grown to
a height of about 1050 angstroms. This process is performed by a
"wet oxidation" method in which H.sub.2 and O.sub.2 gases are used
in converting the Si to SiO.sub.2.
[0131] The resulting substrate was patterned with "MEGAPOSIT" SPR
900-0.8 photoresist from Shipley Company, L.L.C. of Marlborough,
Mass., USA as described above for Example 1. The resulting
substrate was baked as in Example 2.
[0132] The patterned substrate was coated with a TEFLON.RTM.
(polytetrafluoroethylene (PFTE)) coating, such as "PerFluoroCoat"
from Cytonix Company of Beltsville, Md., USA, to a height of 148 to
1200 Angstroms. The photoresist pads were removed by washing the
substrate in acetone for eight minutes at room temperature, thereby
dissolving the photoresist and exposing the SiO.sub.2. The contact
angles of the two materials TEFLON.RTM. and SiO.sub.2 create
microarray target locations with a less hydrophobic environment
than the surrounding area. For the microarray of Example 3, the
TEFLON.RTM. (polytetrafluoroethylene (PFTE)) material has a contact
angle of 110 degrees, and the SiO.sub.2 material has a contact
angle of 55 degrees.
[0133] In these examples, the areas of differential hydrophobicity
on the produced microarray reduces the occurrence of satellite
droplets that might otherwise extend from the liquid sample and
adhere to the microarray surface, thereby increasing the volume
dispensing accuracy.
[0134] The methods and apparatus provided herein have been
described above in terms of presently preferred embodiments so that
an understanding of the present invention can be conveyed. There
are, however, many configurations for sample delivery processes and
systems not specifically described herein, but with which the
present methods and apparatus and disclosure herein is applicable.
The present invention should therefore not be seen as limited to
the particular embodiments described herein, but rather, it should
be understood that the present invention has wide applicability
with respect to sample delivery processes and systems generally.
All modifications, variations, or equivalent arrangements and
implementations that are within the scope of the attached claims
should therefore be considered within the scope of the
invention.
[0135] Since modifications will be apparent to those of skill in
this art, it is intended that this invention be limited only by the
scope of the appended claims.
* * * * *