U.S. patent application number 09/371150 was filed with the patent office on 2001-07-19 for systems and methods for preparing and analyzing low volume analyte array elements.
Invention is credited to KOSTER, HUBERT, LITTLE, DANIEL P..
Application Number | 20010008615 09/371150 |
Document ID | / |
Family ID | 25142121 |
Filed Date | 2001-07-19 |
United States Patent
Application |
20010008615 |
Kind Code |
A1 |
LITTLE, DANIEL P. ; et
al. |
July 19, 2001 |
SYSTEMS AND METHODS FOR PREPARING AND ANALYZING LOW VOLUME ANALYTE
ARRAY ELEMENTS
Abstract
Serial and parallel dispensing tools that can deliver defined
and controlled volumes of fluid to generate multi-element arrays of
sample material on a substrate surface are provided. The substrates
surfaces can be flat or geometrically altered to include wells of
receiving material. Also provided are tools that allow the parallel
development of a sample array. To this end, the tool can be
understood as an assembly of vesicle elements, or pins, where each
of the pins can include a narrow interior chamber suitable for
holding nanoliter volumes of fluid. Each of the pins can fit inside
a housing that forms an interior chamber. The interior chamber can
be connected to a pressure source that will control the pressure
within the interior chamber to regulate the flow of fluid within
the interior chamber of the pins. The prepared sample arrays can
then be passed to a plate assembly that disposes the sample arrays
for analysis by mass spectrometry.
Inventors: |
LITTLE, DANIEL P.; (BOSTON,
MA) ; KOSTER, HUBERT; (LA JOLLA, CA) |
Correspondence
Address: |
HELLER EHRMAN WHITE & MCAULIFFE LLP
4250 EXECUTIVE SQ
7TH FLOOR
LA JOLLA
CA
92037
US
|
Family ID: |
25142121 |
Appl. No.: |
09/371150 |
Filed: |
August 9, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09371150 |
Aug 9, 1999 |
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08787639 |
Jan 23, 1997 |
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6024925 |
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Current U.S.
Class: |
422/400 ;
422/68.1; 436/174; 436/175 |
Current CPC
Class: |
B01J 2219/00605
20130101; C07F 9/2429 20130101; C40B 40/06 20130101; B01J
2219/00504 20130101; B01J 2219/00621 20130101; G01N 35/1067
20130101; B01J 2219/00648 20130101; B01J 2219/00722 20130101; B01J
2219/00387 20130101; C40B 40/10 20130101; B01J 2219/00317 20130101;
B01J 2219/00527 20130101; B01J 2219/0052 20130101; C40B 60/14
20130101; B01J 2219/00725 20130101; C07B 2200/11 20130101; B01J
2219/00596 20130101; Y10T 436/2575 20150115; B01J 2219/00612
20130101; B01J 2219/00659 20130101; B01L 3/0268 20130101; C07H
21/00 20130101; B01J 2219/00511 20130101; G01N 2035/1069 20130101;
B01J 2219/00315 20130101; B01J 2219/005 20130101; G01N 2035/1037
20130101; B01J 2219/00585 20130101; C07F 9/2408 20130101; G01N
35/1074 20130101; B01L 3/0262 20130101; B01J 19/0046 20130101; Y10T
436/25 20150115; B01J 2219/00468 20130101; Y10T 436/25125 20150115;
B01J 2219/0061 20130101; B01J 2219/0072 20130101; G01N 2035/00237
20130101; B01J 2219/00497 20130101 |
Class at
Publication: |
422/102 ;
422/68.1; 422/99; 422/100; 422/101; 436/174; 436/175 |
International
Class: |
G01N 021/31; G01N
001/00 |
Claims
We claim:
1. A dispensing apparatus for dispensing nanovolumes of fluid in
chemical or biological procedures onto the surface of a substrate,
comprising a housing having a plurality of sides and a bottom
portion having formed therein a plurality of apertures, said walls
and bottom portion of said housing defining an interior volume, one
or more fluid transmitting vesicles, mounted within said apertures,
having a nanovolume sized fluid holding chamber for holding
nanovolumes of fluid, said fluid holding chamber being disposed in
fluid communication with said interior volume of said housing, and
dispensing means in communication with said interior volume of said
housing for selectively dispensing nanovoluments of fluid from said
nanovolume sized fluid transmitting vesicles when the fluid is
loaded with said fluid holding chambers of said vesicles, whereby
said dispensing means dispenses nanovolumes of the fluid onto the
surface of the substrate when the apparatus is disposed over and in
registration with the substrate.
2. The apparatus of claim 1, wherein each said fluid transmitting
vesicle has an open proximal end and a distal tip portion that
extends beyond said housing bottom portion when mounted within said
apertures, said open proximal end disposing said fluid holding
chamber in fluid communication with said interior volume when
mounted with the apertures.
3. The apparatus of claim 1, wherein said plurality of fluid
transmitting vesicles are removably and replaceably mounted within
said apertures of said housing.
4. The apparatus of claim 1, wherein said plurality of fluid
transmitting vesicles include a glue seal for fixedly mounting said
vesicles within said housing.
5. The apparatus of claim 1, wherein said fluid holding chamber
includes a narrow bore dimensionally adapted for being filled with
the fluid through capillary action.
6. The apparatus of claim 1, wherein each said fluid holding
chamber of said plurality of fluid transmitting vesicles are sized
to fill substantially completely with the fluid through capillary
action.
7. The apparatus of claim 1, wherein said plurality of fluid
transmitting vesicles comprise an array of fluid delivering
needles.
8. The apparatus of claim 7, wherein said fluid delivering needles
are formed of metal.
9. The apparatus of claim 7, wherein said fluid delivering needles
are formed of glass.
10. The apparatus of claim 7, wherein said fluid delivering needles
are formed of silica.
11. The apparatus of claim 7, wherein said fluid delivering needles
are formed of polymeric material.
12. The apparatus of claim 1, wherein the number of said plurality
of fluid transmitting vesicles is less than or equal to the number
of wells of a multi-well substrate.
13. The apparatus of claim 1, wherein said housing further includes
a top portion, an further comprising mechanical biasing means of
mechanically biasing said plurality of fluid transmitting vesicles
into sealing contact with said housing bottom portion.
14. The apparatus of claim 13, wherein each said fluid transmitting
vesicle has a proximal end portion that includes a flange, and
further comprising a sealer element disposed between the flange and
an inner surface of the housing bottom portion for forming a seal
between the interior volume and an external environment.
15. The apparatus of claim 14, wherein said mechanical biasing
means includes a plurality of spring elements each of which are
coupled at one end to said proximal end of each said plurality of
fluid transmitting vesicles, and at another end to an inner surface
of said housing top portion, said spring element applying a
mechanical biasing force to said vesicle proximal end to form said
seal.
16. The apparatus of claim 1, wherein said housing further includes
a top portion, and further comprising securing means for securing
said housing top portion to said housing bottom portion.
17. The apparatus of claim 16, wherein said securing means
comprises a plurality of fastner-receiving apertures formed within
one of said top and bottom portions or said housing, and a
plurality of fastners for mounting within said apertures for
securing together said housing top and bottom portions.
18. The apparatus of claim 1, wherein said dispensing mens
comprises a pressure source fluidly coupled to said interior volume
of said housing for disposing said interior volume at a selected
pressure condition.
19. The apparatus of claim 18, wherein said fluid transmitting
vesicles are filled through capillary action, and wherein said
dispensing means further comprises means for varying said pressure
source to dispose said interior volume of said housing at varying
pressure conditions, said means for varying disposing said interior
volume at a selected pressure condition sufficient to offset said
capillary action to fill the fluid holding chamber of each vesicle
to a predetermined height corresponding to a predetermined fluid
amount.
20. The apparatus of claim 1 9, wherein said means for varying
further comprises fluid selection means for selectively discharging
a selected nanovolume fluid amount from said chamber of each said
vesicle.
21. The apparatus of claim 1, wherein said fluid transmitting
vesicle has a proximal end that opens onto said interior volume of
sid housing, and wherein said fluid holding chamber of said
vesicles are sized to substantially completely fill with the fluid
through capillary action without forming a meniscus at said
proximal open end.
22. The apparatus of claim 1, wherein said dispensing means
comprises fluid selection means for selectively varying the amount
of fluid dispensed from said fluid holding chamber of each
vesicle.
23. The apparatus according to claim 1, having plural vesicles,
wherein a first portion of said plural vesicles include fluid
holding chambers of a first size and a second portion including
fluid holding chambers of a second size, whereby plural fluid
volumes can be dispensed.
24. The apparatus of claim 22, wherein said fluid selection means
comprises a pressure source coupled to said housing and in
communications with said interior volume for disposing said
interior volume at a selected pressure condition, and adjustment
means coupled to said pressure source for varying said pressure
within said interior volume of said housing to apply a positive
pressure in said fluid chamber of each said fluid transmitting
vesicle to vary the amount of fluid dispensed therefrom.
25. A fluid dispensing apparatus for dispensing a fluid in chemical
or biological procedures into one or more wells of a multi-well
substrate, comprising a housing having a plurality of sides and a
bottom portion having formed therein a plurality of apertures, said
walls and bottom portion defining an interior volume, a plurality
of fluid transmitting vesicles, mounted within said apertures
having a fluid holding chamber disposed in communication with said
interior volume of said housing, a fluid selection and dispensing
means in communication with said interior volume of said housing
for variably selecting an amount of the fluid loaded with said
fluid holding chambers of said vesicles to be dispensed from a
single set of plurality of fluid transmitting vesicles, and whereby
said dispensing means dispenses a selected amount of the fluid into
the wells of the multi-well substrate when the apparatus is
disposed over and in registration with the substrate.
26. The fluid dispensing apparatus of claim 25, wherein said fluid
selection and dispensing means is adapted to select various amounts
of fluid to be dispensed from said single set of vesicles.
27. The fluid dispensing apparatus of claim 25, wherein said fluid
selection and dispensing means comprises a pressure source fluidly
coupled to said interior volume of said housing for disposing said
interior volume at a selected pressure condition.
28. The fluid dispensing apparatus of claim 27, further
compromising means for varying the pressure within the interior
volume of the housing to select the amount of fluid to dispense
from said fluid transmitting vesicles.
29. The fluid dispensing apparatus of claim 27, wherein said fluid
transmitting vesicles are filled with the fluid through capillary
action, and further comprising means for varying said pressure
source to dispose said interior volume of said housing at varying
pressure conditions, said means for varying disposing said interior
volume at a pressure condition sufficient to offset said capillary
action to fill the fluid holding chamber of each vesicle to a
predetermined height corresponding to a predetermined fluid
amount.
30. The fluid dispensing apparatus of claim 25, wherein said fluid
selection means comprises a pressure source coupled to said housing
and in communication with said interior volume for disposing said
interior volume at a selected pressure condition, and adjustment
means coupled to said pressure source for varying said pressure
within said interior volume of said housing to apply a positive
pressure in said fluid chamber of each said fluid transmitting
vesicle to vary the amount of fluid dispensed therefrom.
31. A fluid dispensing apparatus for dispensing fluid in chemical
or biological procedures into one or more wells of a multi-well
substrate, said apparatus comprising a housing having a plurality
of sides and top and bottom portions of said bottom portion having
formed therein a plurality of apertures, said walls and top and
bottom portions of said housing defining an interior volume, a
plurality of fluid transmitting vesicles, mounted within said
apertures having a fluid holding chamber sized to hold nanovolumes
of the fluid, said fluid holding chamber being disposed in fluid
communication with said volume of said housing and mechanical
biasing means for mechanically biasing said plurality of said
transmitting vesicles into sealing contact with said housing bottom
portion.
32. The fluid dispensing apparatus of claim 31, wherein each said
fluid transmitting vesicle has a proximal end portion that includes
a flange, and further comprising a sealer element disposed between
the flange and an inner surface of the housing bottom portion for
forming a pressure and fluid seal between the internal and external
environment.
33. The fluid dispensing apparatus of claim 31, wherein said
mechanical biasing means includes a plurality of spring elements
each of which are coupled at one end to said means includes a
plurality of spring elements each of which are coupled at one end
to said proximal end of said fluid transmitting vesicle, and at
another end to an inner surface of said housing top portion, said
spring elements applying a mechanical biasing force to said vesicle
proximal end to form said fluid and pressure seal.
34. The fluid dispensing apparatus of claim 31, further comprising
securing means for securing said housing top portion to said
housing bottom portion.
35. The fluid dispensing apparatus of claim 34, wherein said
securing means comprises a plurality of fastener-receiving
apertures formed within one of said top and bottom portions of said
housing, and a plurality of fasteners for mounting within said
apertures for securing said housing top and bottom portions
together.
36. The fluid dispensing apparatus of claim 31, further comprising
dispensing means in communication with said interior volume of said
housing for selectively dispensing the fluid from said fluid
transmitting vesicles when the fluid is loaded within said fluid
holding chambers of said vesicles, whereby said dispensing means
dispenses the fluid into the wells of the multi-well substrate when
the apparatus is disposed over an in registration with the
substrate.
37. The fluid dispensing apparatus of claim 36, wherein said
dispensing means comprises a pressure source fluidly coupled to
said interior volume of said housing for disposing said interior
volume at a selected pressure condition.
38. The fluid dispensing apparatus of claim 31, wherein said
plurality of fluid transmitting vesicles are removably and
replaceably mounted within said apertures of said housing.
39. The fluid dispensing apparatus of claim 31, wherein said
plurality of fluid transmitting vesicles comprises an array of
fluid delivering needles.
40. The fluid dispensing apparatus of claim 36, wherein said fluid
transmitting vesicles are filled with the fluid through capillary
action, and wherein said dispensing means further comprises means
for varying said pressure source to dispose said interior volume of
said housing at varying pressure conditions, said means for varying
disposing said interior volumes at a selected pressure condition
sufficient to offset said capillary action to fill the fluid
holding chamber of each vesicle to a predetermined height
corresponding to a predetermined fluid amount.
41. The fluid dispensing apparatus of claim 36, wherein said
dispensing means comprises fluid selection means for selectively
varying the amount of fluid dispensed from said fluid holding
chamber of each vesicle.
42. The fluid dispensing apparatus of claim 31, further comprising
a pressure source coupled to the housing and in communication with
the interior volume for disposing the interior volume at a selected
pressure condition, and adjustment means coupled to the pressure
source for varying the pressure within the interior volume of the
housing to apply a positive pressure to the fluid chamber of each
the fluid transmitting vesicle to vary the amount of fluid
dispensed therefrom.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 08/787,639 to Little et al., entitled SYSTEMS AND METHODS FOR
PREPARING AND ANALYZING LOW VOLUME ANALYTE ARRAY ELEMENTS, filed
Jan. 23, 1997. The subject matter of U.S. application Ser. No.
08/787,639 is incorporated herein in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to systems and methods for preparing a
sample for analysis, and more specifically to systems and methods
for dispensing low volumes of fluid material onto a substrate
surface for generating an array of samples for diagnostic
analysis.
BACKGROUND OF THE INVENTION
[0003] In recent years, developments in the field of life sciences
have proceeded at a breathtaking rate. Universities, hospitals and
newly formed companies have made groundbreaking scientific
discoveries and advances that promise to reshape the fields of
medicine, agriculture, and environmental science. However, the
success of these efforts depends, in part, on the development of
sophisticated laboratory tools that will automate and expedite the
testing and analysis of biological samples. Only upon the
development of such tools can the benefits of these recent
scientific discoveries be achieved fully.
[0004] At the forefront of these efforts to develop better
analytical tools is a push to expedite the analysis of complex
biochemical structures. This is particularly true for human genomic
DNA, which is comprised of at least about one hundred thousand
genes located on twenty four chromosomes. Each gene codes for a
specific protein, which fulfills a specific biochemical function
within a living cell. Changes in a DNA sequence are known as
mutations and can result in proteins with altered or in some cases
even lost biochemical activities; this in turn can cause a genetic
disease. More than 3,000 genetic diseases are currently known. In
addition, growing evidence indicates that certain DNA sequences may
predispose an individual to any of a number of genetic diseases,
such as diabetes, arteriosclerosis, obesity, certain autoimmune
diseases and cancer. Accordingly, the analysis of DNA is a
difficult but worthy pursuit that promises to yield information
fundamental to the treatment of many life threatening diseases.
[0005] Unfortunately, the analysis of DNA is made particularly
cumbersome due to size and the fact that genomic DNA includes both
coding and non-coding sequences (e.g., exons and introns). As such,
traditional techniques for analyzing chemical structures, such as
the manual pipeting of source material to create samples for
analysis, are of little value. To address the scale of the
necessary analysis, scientist have developed parallel processing
protocols for DNA diagnostics.
[0006] For example, scientists have developed robotic devices that
eliminate the need for manual pipeting and spotting by providing a
robotic arm that carries at its proximal end a pin tool device that
includes a matrix of pin elements. The individual pins of the
matrix are spaced apart from each other to allow each pin be dipped
within a well of a microtiter plate. The robotic arm dips the pins
into the wells of the microtiter plate thereby wetting each of the
pin elements with sample material. The robotic arm then moves the
pin tool device to a position above a target surface and lowers the
pin tool to the surface contacting the pins against the target to
form a matrix of spots thereon. Accordingly, the pin tool expedites
the production of samples by dispensing sample material in
parallel.
[0007] Although this pin tool technique works well to expedite the
production of sample arrays, it suffers from several drawbacks.
First during the spotting operation, the pin tool actually contacts
the surface of the substrate. Given that each pin tool requires a
fine point in order that a small spot size is printed onto the
target, the continuous contact of the pin tool against the target
surface will wear and deform the fine and delicate points of the
pin tool. This leads to errors which reduce accuracy and
productivity.
[0008] An alternative technique developed by scientists employs
chemical attachment of sample material to the substrate surface. In
one particular process, DNA is synthesized in situ on a substrate
surface to produce a set of spatially distinct and diverse chemical
products. Such techniques are essentially photolithographic in that
they combine solid phase chemistry, photolabile protecting groups
and photo activated lithography. Although these systems work well
to generate arrays of sample material, they are chemically
intensive, time consuming, and expensive.
[0009] It is further troubling that neither of the above techniques
provide sufficient control over the volume of sample material that
is dispensed onto the surface of the substrate. Consequently, error
can arise from the failure of these techniques to provide sample
arrays with well controlled and accurately reproduced sample
volumes. In an attempt to circumvent this problem, the preparation
process will often dispense generous amounts of reagent materials.
Although this can ensure sufficient sample volumes, it is wasteful
of sample materials, which are often expensive and of limited
availability.
[0010] Even after the samples are prepared, scientists still must
confront the need for sophisticated diagnostic methods to analyze
the prepared samples. To this end, scientists employ several
techniques for identifying materials such as DNA. For example,
nucleic acid sequences can be identified by hybridization with a
probe which is complementary to the sequence to be identified.
Typically, the nucleic acid fragment is labeled with a sensitive
reporter function that can be radioactive, fluorescent, or
chemiluminescent. Although these techniques can work well, they do
suffer from certain drawbacks. Radioactive labels can be hazardous
and the signals they produce decay over time. Nonisotopic (e.g.
fluorescent) labels suffer from a lack of sensitivity and fading of
the signal with high intensity lasers are employed during the
identification process. In addition, labeling is a laborious and
time consuming error prone procedure.
[0011] Consequently, the process of preparing and analyzing arrays
of a biochemical sample material is complex and error prone.
SUMMARY OF THE INVENTION
[0012] Accordingly, it is an object herein to provide improved
systems and methods for preparing arrays of sample material.
[0013] It is a further object to provide systems that allow for the
rapid production of sample arrays.
[0014] It is yet another object to provide systems and methods for
preparing arrays of sample material that are less expensive to
employ and that conserve reagent materials.
[0015] It is a further object to provide systems and methods for
preparing arrays of sample material that provide high
reproducibility of the arrays generated.
[0016] Other objects of the apparatus and methods provided herein
will be apparent from the description also disclosed in the
following.
[0017] Serial and parallel dispensing tools that can be employed to
generate multi-element arrays of sample material on a substrate
surface are provided. The substrates surfaces can be flat or
geometrically altered to include wells of receiving material. In
one embodiment, a tool that allows the parallel development of a
sample array is provided. To this end, the tool can be understood
as an assembly of vesicle elements, or pins, wherein each of the
pins can include a narrow interior chamber suitable for holding
nano liter volumes of fluid. Each of the pins can fit inside a
housing that itself has in interior chamber. The interior housing
can be connected to a pressure source that will control the
pressure within the interior housing chamber to regulate the flow
of fluid through the interior chamber of the pins. This allows for
the controlled dispensing of defined volumes of fluid from the
vesicles. In an alternative embodiment, the invention provides a
tool that includes a jet assembly that can include a capillary pin
having an interior chamber, and a transducer element mounted to the
pin and capable of driving fluid through the interior chamber of
the pin to eject fluid from the pin. In this way, the tool can
dispense a spot of fluid to a substrate surface by spraying the
fluid from the pin. Alternatively, the transducer can cause a drop
of fluid to extend from the capillary so that fluid can be passed
to the substrate by contacting the drop to the surface of the
substrate. Further, the tool can form an array of sample material
by dispensing sample material in a series of steps, while moving
the pin to different locations above the substrate surface to form
the sample array. In a further embodiment, the invention then
passes the prepared sample arrays to a plate assembly that disposes
the sample arrays for analysis by mass spectrometry. To this end, a
mass spectrometer is provided that generates a set of spectra
signal which can be understood as indicative of the composition of
the sample material under analysis.
[0018] To this end a dispensing apparatus for dispensing defined
volumes of fluid, including nano and sub-nano volumes of fluid, in
chemical or biological procedures onto the surface of a substrate
is provided. The apparatus provided herein can include a housing
having a plurality of sides and a bottom portion having formed
therein a plurality of apertures, the walls and bottom portion of
the housing defining an interior volume; one or more fluid
transmitting vesicles, or pins, mounted within the apertures,
having a nanovolume sized fluid holding chamber for holding
nanovolumes of fluid, the fluid holding chamber being disposed in
fluid communication with the interior volume of the housing, and a
dispensing element that is in communication with the interior
volume of the housing for selectively dispensing nanovolumes of
fluid form the nanovolume sized fluid transmitting vesicles when
the fluid is loaded with the fluid holding chambers of the
vesicles. As described herein, this allows the dispensing element
to dispense nanovolumes of the fluid onto the surface of the
substrate when the apparatus is disposed over and in registration
with the substrate.
[0019] In one embodiment the fluid transmitting vesicle has an open
proximal end and a distal tip portion that extends beyond the
housing bottom portion when mounted within the apertures. In this
way the open proximal end can dispose the fluid holding chamber in
fluid communication with the interior volume when mounted with the
apertures. Optionally, the plurality of fluid transmitting vesicles
are removably and replaceably mounted within the apertures of the
housing, or alternatively can include a glue seal for fixedly
mounting the vesicles within the housing.
[0020] In one embodiment the fluid holding chamber includes a
narrow bore dimensionally adapted for being filled with the fluid
through capillary action, and can be sized to fill substantially
completely with the fluid through capillary action.
[0021] In one embodiment, the plurality of fluid transmitting
vesicles comprise an array of fluid delivering needles, which can
be formed of metal, glass, silica, polymeric material, or any other
suitable material.
[0022] In one embodiment the housing can include a top portion, and
mechanical biasing elements for mechanically biasing the plurality
of fluid transmitting vesicles into sealing contact with the
housing bottom portion. In one particular embodiment, each fluid
transmitting vesicle has a proximal end portion that includes a
flange, and further includes a seal element disposed between the
flange and an inner surface of the housing bottom portion for
forming a seal between the interior volume and an external
environment. The biasing elements can be mechanical and can include
a plurality of spring elements each of which are coupled at one end
to the proximal end of each the plurality of fluid transmitting
vesicles, and at another end to an inner surface of the housing top
portion. The springs can apply a mechanical biasing force to the
vesicle proximal end to form the seal.
[0023] In a further embodiment, the housing further includes a top
portion, and securing element for securing the housing top portion
to the housing bottom portion. The securing element can comprise a
plurality of fastener- receiving apertures formed within one of the
top and bottom portions of the housing, and a plurality of
fasteners for mounting within the apertures for securing together
the housing top and bottom portions.
[0024] In one embodiment the dispensing element can comprise a
pressure source fluidly coupled to the interior volume of the
housing for disposing the interior volume at a selected pressure
condition. Moreover, in an embodiment wherein the fluid
transmitting vesicles are filled through capillary action, the
dispensing element can include a pressure controller than can vary
the pressure source to dispose the interior volume of the housing
at varying pressure conditions. This allows the controller varying
element to dispose the interior volume at a selected pressure
condition sufficient to offset the capillary action to fill the
fluid holding chamber of each vesicle to a predetermined height
corresponding to a predetermined fluid amount. Additionally, the
controller can further include a fluid selection element for
selectively discharging a selected nanovolume fluid amount from the
chamber of each the vesicle. In one particular embodiment, the
apparatus includes a pressure controller that operates under the
controller of a computer program operating on a data processing
system to provide variable control over the pressure applied to the
interior chamber of the housing.
[0025] In one embodiment the fluid transmitting vesicle can have a
proximal end that opens onto the interior volume of the housing,
and the fluid holding chamber of the vesicles are sized to
substantially completely fill with the fluid through capillary
action without forming a meniscus at the proximal open end.
Optionally, the apparatus can have plural vesicles, wherein a first
portion of the plural vesicles include fluid holding chambers of a
first size and a second portion including fluid holding chambers of
a second size, whereby plural fluid volumes can be dispensed.
[0026] In another embodiment the apparatus can include, a fluid
selection element that has a pressure source coupled to the housing
and in communication with the interior volume for disposing the
interior volume at a selected pressure condition, and an adjustment
element that couples to the pressure source for varying the
pressure within the interior volume of the housing to apply a
positive pressure in the fluid chamber of each the fluid
transmitting vesicle to vary the amount of fluid dispensed
therefrom. The selection element and adjustment element can be
computer programs operating on a data processing system that
directs the operation of a pressure controller connected to the
interior chamber.
[0027] In a further alternative embodiment, an apparatus for
dispensing a fluid in chemical or biological procedures into one or
more wells of a multi-well substrate is provided. The apparatus can
include a housing having a plurality of sides and a bottom portion
having formed therein a plurality of apertures, the walls and
bottom portion defining an interior volume, a plurality of fluid
transmitting vesicles, mounted within the apertures, having a fluid
holding chamber disposed in communication with the interior volume
of the housing, and a fluid selection and dispensing means in
communication with the interior volume of the housing for variably
selecting am amount of the fluid loaded within the fluid holding
chambers of the vesicles to be dispensed from a single set of the
plurality of fluid transmitting vesicles. Accordingly, the
dispensing means dispenses a selected amount of the fluid into the
wells of the multi-well substrate when the apparatus is disposed
over and in registration with the substrate.
[0028] In yet another embodiment, a fluid dispensing apparatus for
dispensing fluid in chemical or biological procedures into one or
more wells of a multi-well substrate, that includes a housing
having a plurality of sides and top and bottom portions, the bottom
portion having formed therein a plurality of apertures, the walls
and top and bottom portions of the housing defining an interior
volume, a plurality of fluid transmitting vesicles, mounted within
the apertures, having a fluid holding chamber sized to hold
nanovolumes of the fluid, the fluid holding chamber being disposed
in fluid communication with the volume of the housing, and
mechanical biasing element for mechanically biasing the plurality
of fluid transmitting vesicles into sealing contact with the
housing bottom portion is provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 illustrates one system provided herein for preparing
arrays of a sample material for analysis;
[0030] FIG. 2 illustrates a pin assembly suitable for use with the
system depicted in FIG. 1 for implementing a parallel process of
dispensing material to a surface of a substrate;
[0031] FIG. 3 depicts a bottom portion of the assembly shown in
FIG. 2;
[0032] FIG. 4 depicts an alternative view of the bottom portion of
the pin assembly depicted in FIG. 2;
[0033] FIGS. 5A-5D depict one method provided herein for preparing
an array of sample material;
[0034] FIGS. 6A-6B depict an alternative assembly for dispensing
material to the surface of a substrate.
[0035] FIG. 7 depicts one embodiment of a substrate having wells
etched therein that are suitable for receiving material for
analysis.
[0036] FIG. 8 depicts one example of spectra obtained from a linear
time of flight mass spectrometer instrument and representative of
the material composition of the sample material on the surface of
the substrate depicted in FIG. 7; and
[0037] FIG. 9 depicts molecular weights determined for the sample
material having spectra identified in FIG. 8.
DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS
[0038] FIG. 1 illustrates one system provided herein for preparing
arrays of sample material for analysis by a diagnostic tool. FIG. 1
depicts a system that includes a data processor 12, a motion
controller 14, a robotic arm assembly 1 6, a monitor element 18A, a
central processing unit 18B, a microliter plate of source material
20, a stage housing 22, a robotic arm 24, a stage 26, a pressure
controller 28, a conduit 30, a mounting assembly 32, a pin assembly
38, and substrate elements 34. In the view shown by FIG. 1, it is
also illustrated that the robotic assembly 16 can include a
moveable mount element 40 and a horizontal slide groove 42. The
robotic arm 24 can optionally pivot about a pin 36 to increase the
travel range of the arm 24 so that arm 24 can disposes the pin
assembly 38 above the source plate 20.
[0039] The data processor 12 depicted in FIG. 1 can be a
conventional digital data processing system such as an IBM PC
compatible computer system that is suitable for processing data and
for executing program instructions that will provide information
for controlling the movement and operation of the robotic assembly
16. It will be apparent to one skilled in the art that the data
processor unit 12 can be any type of system suitable for processing
a program of instructions signals that will operate the robotic
assembly that is integrated into the robotic housing 16. Optionally
the data processor 12 can be a micro-controlled assembly that is
integrated into robotic housing 16. In further alternative
embodiments, the system 10 need not be programmable and can be a
singleboard computer having a firmware memory for storing
instructions for operating the robotic assembly 16.
[0040] In the embodiment depicted in FIG. 1, there is a controller
14 that electronically couples between the data processor 12 and
the robotic assembly 16. The depicted controller 14 is a motion
controller that drives the motor elements of the robotic assembly
16 for positioning the robotic arm 24 at a selected location.
Additionally, the controller 14 can provide instructions to the
robotic assembly 16 to direct the pressure controller 28 to control
the volume of fluid ejected from the individual pin elements of the
depicted pin assembly 38. The design and construction of the
depicted motion controller 14 follows from principles well known in
the art of electrical engineering, and any controller element
suitable for driving the robotic assembly 16 can be used.
[0041] The robotic assembly 16 depicted in FIG. 1 electronically
couples to the controller 14. The depicted robotic assembly 16 is a
gantry system that includes an XY table for moving the robotic arm
about a XY plane, and further includes a Z axis actuator for moving
the robotic arm orthogonally to that XY plane. The robotic assembly
16 depicted in FIG. 1 includes an arm 24 that mounts to the XY
stage which moves the arm within a plane defined by the XY access.
In the depicted embodiment, the XY table is mounted to the Z
actuator to move the entire table along the Z axis orthogonal to
the XY plane. In this way, the robotic assembly provides three
degrees of freedom that allows the pin assembly 38 to be disposed
to any location above the substrates 34 and the source plate 20
which are shown in FIG. 1 as sitting on the stage 26 mounted to the
robotic assembly 16.
[0042] The depicted robotic assembly 16 follows from principles
well known in the art of electrical engineering and is just one
example of a robotic assembly suitable for moving a pin assembly to
locations adjacent a substrate and source plate such as the
depicted substrate 34. Accordingly, it will be apparent to one of
skill in the art that alternative robotic systems can be used.
[0043] FIG. 1 depicts an embodiment of a robotic assembly 16 that
includes a pressure controller 28 that connects via a conduit 30 to
the mount 32 that connects to the pin assembly 38. In this
embodiment the mount 32 has an interior channel for fluidicly
coupling the conduit 30 to the pin assembly 38. Accordingly, the
pressure controller 28 is fluidicly coupled by the conduit 30 and
the mount 32 to the pin assembly 38. In this way the controller 1 4
can send signals to the pressure controller 28 to control
selectively a fluid pressure delivered to the pin assembly 38.
[0044] FIG. 2 depicts one embodiment of a pin assembly 50 suitable
for practice with the system depicted in FIG. 1 which includes the
pressure controller 28. In the depicted embodiment, the pin
assembly 50 includes a housing formed from an upper portion 52 and
a lower portion 54 that are joined together by the crews 56A and
56B to define an interior chamber volume 58. FIG. 2 further depicts
that to fluidicly seal the interior chamber volume 58 the housing
can include a seal element depicted in FIG. 2 as an O-ring gasket
60 that sites between the upper block and the lower block 54 and
surrounds completely the perimeter of the interior chamber volume
58. FIG. 2 further depicts that the pin assembly 50 includes a
plurality of vesicles 62A-62D, each of which include an axial bore
extending therethrough to form the depicted holding chambers
64A-64D. Each of the depicted vesicles extends through a respective
aperture 68A-68D disposed within the lower block 54 of the
housing.
[0045] As further shown in the depicted embodiment, each of the
vesicles 62A-62D has an upper flange portion that sits against a
seal element 70A-70D to form a fluid-tight seal between the vesicle
and the lower block 54 to prevent fluid from passing through the
apertures 68A-68D. To keep the seal tight, the depicted pin
assembly 50 further includes a set of biasing elements 74A-74D
depicted in FIG. 2 as springs which, in the depicted embodiments,
are in a compressed state to force the flange element of the
vesicles 62A-62D against their respective seal elements 70A-70D. As
shown in FIG. 2, the biasing elements 74A-74D extend between the
vesicles and the upper block 52. Each of the springs 74A-74D can be
fixedly mounted to a mounting pad 76A-76D where the spring elements
can attach to the upper block 52. The upper block 52 further
includes an aperture 78 depicted in FIG. 2 as a centrally disposed
aperture that includes a threaded bore for receiving a swagelok 80
that can be rotatably mounted within the aperture 78.
[0046] As further depicted in FIG. 2, the swagelok 80 attaches by a
conduit to a valve 82 than can connect the swagelok 80 to a conduit
84 that can be coupled to a pressure source, or alternatively can
couple the swagelok 80 to a conduit 86 that provides for venting of
the interior chamber 58. A central bore 88 extends through the
swagelok 80 and couples to the tubing element which further
connects to the valve 82 to thereby fluidicly and selectively
couple the interior chamber volume 58 to either a pressure source,
or a venting outlet.
[0047] The pin assembly 50 described above and depicted in FIG. 2
disposed above a substrate element 90 that includes a plurality of
wells 92 that are etched into the upper surface of the substrate
90. As illustrated by FIG. 2, the pitch of the vesicles 62A-62D is
such that each vesicle is spaced from the adjacent vesicles by a
distance that is an integral multiple of the pitch distance between
wells 92 etched into the upper surface of the substrate 90. As will
be seen from the following description, this spacing facilitates
the parallel dispensing of fluid, such that fluid can be dispensed
into a plurality of wells in a single operation. Each of the
vesicles can be made from stainless steel, silica, polymeric
material or any other material suitable for holding fluid sample.
In one example, 16 vesicles are employed in the assembly, which are
made of hardened beryllium copper, gold plated over nickel plate.
They are 43.2 mm long and the shaft of the vesicle is graduated to
0.46 mm outer diameter with a concave tip. Such a pin was chosen
since the pointing accuracy (distance between the center of
adjacent tips) can be approximately 501 micrometers. However, it
will be apparent that any suitable pin style can be employed
for-the device, including but not limited to flat, star-shaped,
concave, pointed solid, pointed semi-hollow, angled on one or both
sides, or other such geometries.
[0048] FIG. 3 shows from a side perspective the lower block 54 of
the pin assembly 50 depicted in FIG. 2. FIG. 3 shows approximate
dimensions for one pin assembly suited for use in the methods and
with the apparatus provided herein. As shown, the lower block 54
has a bottom plate 98 and a surrounding shoulder 100. The bottom
plate 98 is approximately 3 mm in thickness and the shoulder 100 is
approximately 5 mm in thickness.
[0049] FIG. 4 shows from an overhead perspective the general
structure and dimensions for one lower block 54 suitable for use
with the pin assembly for use with the pin assembly 50 shown in
FIG. 2. As shown in FIG. 4, the lower block 54 includes a
four-by-four matrix of apertures 68 to provide 16 apertures each
suitable for receiving a vesicle. As described above with reference
to FIG. 2, the spacing between the aperture 68 is typically an
integral multiple of the distance between wells on a substrate
surface as well as the wells of a source plate. Accordingly, a pin
assembly having the lower block 54 as depicted in FIG. 4 can
dispense fluid in up to 16 wells simultaneously. FIG. 4 also shows
general dimensions of one lower block 54 such that each side of
block 54 is generally 22 mm in length and the pitch between
aperture 68 is approximately 4.5 mm. Such a pitch is suitable for
use with a substrate where fluid is to be dispensed at locations
approximately 500 .mu.m apart, as exemplified by the substrate 90
of FIG. 2. FIG. 4 also shows that the lower block 54 can include an
optional O-ring groove 94 adapted for receiving an O-ring seal
element, such as the seal element 60 depicted in FIG. 2. It is
understood that such a groove element 94 can enhance and improve
the fluid seal formed by the seal element 60.
[0050] The pinblock can be manufactured of stainless steel as this
material can be drilled accurately to about +25 .mu.m, but a
variety of probe materials can also be used, such as G10 laminate,
PMMA or other suitable material. The pin block can contain any
number of apertures and is shown with 16 receptacles which hold the
16 pins in place. To increase the pointing accuracy of each pin, an
optional alignment place can be placed below the block so that
about 6 mm of the pin tip is left exposed to enable dipping into
the wells of a microtiter plate. The layout of the probes in the
depicted tool is designed to coordinate with a 384-well microtiter
plate, thus the center-to-center spacing of the probes in 4.5 mm.
An array of 4.times.4 probes was chosen since it would produce an
array that would fit in less than one square inch, which is the
travel range of an xy stage of a MALDI TOF MS employed by the
assignee. The pintool assembly is completed with a stainless steel
cover on the top side of the device which is then attached onto the
Z-arm of the robot.
[0051] With references to FIG. 5, the operation of one embodiment
can be explained. In this exemplary embodiment, the robotic
assembly 16 employs a pin tool assembly 38 that is configured
similarly as the pin tool assembly 50 depicted in FIG. 2. The
pressure controller 28 selectively controls the pressure within
chamber 58. With this embodiment, a control program operates on the
data processor 12 to control the robotic assembly 16 in a way that
the assembly 16 prints an array of elements on the substrates
34.
[0052] In a first step, FIG. 5A, the program can direct the robotic
assembly 16 to move the pin assembly 38 to be disposed above the
source plate 20. The robotic assembly 16 will then dip the pin
assembly into the source plate 20 which can be a 384 well DNA
source plate. As shown in FIG. 4 the pin assembly can include 16
different pins such that the pin assembly 50 will dip 16 pins into
different 16 wells of the 384 well DNA source plate 20. Next the
data processor 12 will direct the motion controller 14 to operate
the robotic assembly 16 to move the pin assembly to a position
above the surface of the substrate 34. The substrate 34 can be any
substrate suitable for receiving a sample of material and can be
formed of silicon, plastic, metal, or any other such suitable
material. Optionally the substrate will have a flat surface, but
can alternatively include a pitted surface, a surface etched with
wells or any other suitable surface typography. The program
operating on data processor 12 can then direct the robotic
assembly, through the motion controller 14, to direct the pressure
controller 28 to generate a positive pressure within the interior
chamber volume 58. In this practice, the positive interior pressure
will force fluid from the holding chambers of vesicles 62 to eject
fluid from the vesicles and into a respective well 92 of the
substrate 90.
[0053] In this practice of the methods and using the apparatus
provided herein, the program operating on data processor 12 can
also direct the controller 14 to control the pressure controller 28
to control filling the holding chambers with source material from
the source plate 20. The pressure controller 28 can generate a
negative pressure within the interior chamber volume 58 of the pin
assembly. This will cause fluid to be drawn up into the holding
chambers of the vesicles 62A-62D. The pressure controller 28 can
regulate the pressure either by open-loop or closed-loop control to
avoid having fluid overdrawn through the holding chambers and
spilled into the interior chamber volume 58. Loop control systems
for controlling pressure are well known in the art and any suitable
controller can be employed. Such spillage could cause
cross-contamination, particularly if the source material drawn from
the source plate 20 varies from well to well.
[0054] In an alternative embodiment, each of the holding chambers
64A-64D is sufficiently small to allow the chambers to be filled by
capillary action. In such a practice, the pin assembly can include
an array of narrow bore needles, such as stainless steel needles,
that extend through the apertures of the lower block 54. The
needles that are dipped into source solutions will be filled by
capillary action. In one embodiment, the length of capillary which
is to be filled at atmospheric pressure is determined approximately
by: 1 H = 2 PGR
[0055] where H equals Height, gamma equals surface tension, P
equals solution density, G equals gravitational force and R equals
needle radius. Thus the volume of fluid held by each vesicle can be
controlled by selecting the dimensions of the interior bore. It is
understood that at room temperature water will fill a 15 cm length
of 100 .mu.m radius capillary. Thus, a short bore nanoliter volume
needle will fill to full capacity, but should not overflow because
the capillary force is understood to be too small to form a
meniscus at the top of the needle orifice. This prevents
cross-contamination due to spillage. In one embodiment, the
vesicles of the pin assembly can be provided with different sized
interior chambers for holding and dispensing different volumes of
fluid.
[0056] In an alternative practice, to decrease the volume of liquid
that is drawn into the holding chambers of the vesicles, a small
positive pressure can be provided within the interior chamber
volume 58 by the pressure controller 28. The downward force created
by the positive pressure can be used to counter the upward
capillary force. In this way, the volume of fluid that is drawn by
capillary force into the holding chambers of the vesicles can be
controlled.
[0057] FIG. 5B, shows that fluid within the holding chambers of the
needle can be dispensed by a small positive pressure introduced
through the central bore 88 extending through a swagelok 80. By
regulating the pressure pulse that is introduced into the interior
chamber volume 58, fluid can be ejected either as a spray or by
droplet formation at the needle tip. It is understood that the rate
of dispensing, droplet versus spray, depends in part upon the
pressure applied by the pressure controller 28. In one practice,
pressure is applied in the range of between 10 and 1,000 Torr of
atmospheric pressure.
[0058] To this end the data processor 12 can run a computer program
that controls and regulates the volume of fluid dispensed. The
program can direct the controller 28 to eject a defined volume of
fluid, either by generating a spray or by forming a drop that sits
at the end of the vesicle, and can be contacted with the substrate
surface for dispensing the fluid thereto.
[0059] FIGS. 5C and 5D show the earlier steps shown in FIGS. 5A-5B
can again be performed, this time at a position on the substrate
surface that is offset from the earlier position. In the depicted
process, the pin tool is offset by a distance equal to the distance
between two wells 92. However, it will be apparent that other
offset printing techniques can be employed.
[0060] It will be understood that several advantages of the pin
assembly depicted in FIG. 2 are achieved. For example, rinsing
between dispensing events is straightforward, requiring only single
or multiple pin fillings and emptying events with a rinse solution.
Moreover, since all holding chambers fill to full capacity, the
accuracy of the volumes dispensed varies only according to needle
inner dimensions which can be carefully controlled during pin
production. Further the device is cost effective, with the greatest
expense attributed to the needles, however because no contact with
a surface is required, the needles are exposed to little physical
strain or stress, making replacement rare and providing long
life.
[0061] Alternatively, deposition of sample material onto substrate
surface can include techniques that employ pin tool assemblies that
have solid pin elements extending from a block wherein a robotic
assembly dips the solid pin elements of the pin assembly into a
source of sample material to wet the distal ends of the pins with
the sample materials. Subsequently the robotic assembly can move
the pin assembly to a location above the substrate and then lower
the pin assembly against the surface of the substrate to contact
the individual wetted pins against the surface for spotting
material of the substrate surface.
[0062] FIGS. 6A and 6B depict another alternative system for
dispensing material on or to the surface of the substrate. In
particular, FIG. 6A depicts a jet printing device 110 which
includes a capillary element 112, a transducer element 114 and
orifice (not shown) 118, a fluid conduit 122, and a mount 124
connecting to a robotic arm assembly, such as the robotic arm 24
depicted in FIG. 1. As further shown in FIG. 6A the jet assembly
110 is suitable for ejecting from the orifice 118 a series of drops
120 of a sample material for dispensing sample material onto the
surface 128.
[0063] The capillary 112 of the jet assembly 110 can be a glass
capillary, a plastic capillary, or any other suitable housing that
can carry a fluid sample and that will allow the fluid sample to be
ejected by the action of a transducer element, such as the
transducer element 114. The transducer element 114 depicted in FIG.
6A is a piezo electric transducer element which forms around the
parameter of the capillary 112 and can transform an electrical
pulse received from the pulse generator within a robotic assembly
16 to cause fluid to eject from the orifice 118 of the capillary
112. One such jet assembly having a piezoelectric transducer
element is manufactured by MicroFab Technology, Inc., of Germany.
Any jet assembly that is suitable for dispensing defined and
controlled the volumes of fluid can be used, including those that
use piezoelectric transducers, electric transducers,
electrorestrictive transducers, magnetorestrictive transducers,
electromechanical transducers, or any other suitable transducer
element. In the depicted embodiment, the capillary 112 has a fluid
conduit 122 for receiving fluid material. In an optional
embodiment, fluid can be drawn into the capillary by action of a
vacuum pressure that will draw fluid through the orifice 118 when
the orifice 118 is submerged in a source of fluid material. Other
embodiments of the jet assembly 110 can be employed.
[0064] FIG. 6B illustrates a further alternative assembly suitable
for use herein, and suitable for being carried on the robotic arm
of a robotic assembly, such as the assembly 1 6 depicted in FIG. 1.
FIG. 6B illustrates four jet assemblies connected together,
130A-130D. Similar to the pin assembly in FIG. 2, the jet assembly
depicted in FIG. 6B can be employed for the parallel dispensing of
fluid material. It will be understood by the skilled artisan in the
art of electrical engineering, that each of the jet assemblies
130A-130D can be operated independently of the others, for allowing
the selective dispensing of fluid from select ones of the jet
assemblies. Moreover, each of the jet assemblies 130A-130D can be
independently controlled to select the volume of fluid that is
dispensed from each respected one of the assembly 130A-130D. Other
modifications and alterations can be made to the assembly depicted
in FIG. 6B.
[0065] In another aspect, methods for rapidly analyzing sample
materials are provided. To this end sample arrays can be formed on
a substrate surface according to any of the techniques discussed
above. The sample arrays are then analyzed by mass spectrometry to
collect spectra data that is representative of the composition of
the samples in the array. It is understood that the above methods
provide processes that allow for rapidly dispensing definite and
controlled volumes of analyte material. In particular these
processes allow for dispensing sub to low nanoliter volumes of
fluid. These low volume deposition techniques generate sample
arrays well suited for analysis by mass spectrometry. For example,
the low volumes yield reproducibility of spot characteristics, such
as evaporation rates and reduced dependence on atmospheric
conditions such as ambient temperature and light.
[0066] Continuing with the example showing in FIG. 1, the arrays
can be prepared by loading oligonucleotides (0.1-50 ng/lll) of
different sequences or concentrations into the wells of a 96 well
microtiter source plate 20; the first well can be reserved for
holding a matrix solution. A substrate 34, such as a pitted silicon
chip substrate, can be placed on the stage 26 of the robotics
assembly 16 and can be aligned manually to orient the matrix of
wells about a set of reference axes. The control program executing
on the data processor 12 can receive the coordinates of the first
well of the source plate 20. The robotic arm 24 can dip the pin
assembly 38 into source plate 20 such that each of the 16 pins is
dipped into one of the wells. Each vesicle can fill by capillary
action so that the full volume of the holding chamber contains
fluid. Optionally, the program executing on the data processor 12
can direct the pressure controller to fill the interior chamber 58
of the pin assembly 38 with a positive bias pressure that will
counteract, in part, the force of the capillary action to limit or
reduce the volume of fluid that is drawn into the holding
chamber.
[0067] Optionally, the pin assembly 38 can be dipped into the same
16 wells of the source plate 20 and spotted on a second target
substrate. This cycle can be repeated on as many target substrates
as desired. Next the robotic arm 24 can dip the pin assembly 38 in
a washing solution, and then dip the pin assembly into 16 different
wells of the source plate 20, and spot onto the substrate target
offset a distance from the initial set of 16 spots. Again this can
be repeated for as many target substrates as desired. The entire
cycle can be repeated to make a 2.times.2 array from each vesicle
to produce an 8.times.8 array of spots (2.times.2
elements/vesicle.times.16 vesicles=64 total elements spotted).
However, it will be apparent to one of skill in the art that any
process suitable for forming arrays can be used with the methods
herein.
[0068] In an alternative embodiment, oligonucleotides of different
sequences or concentrations can be loaded into the wells of up to
three different 384-well microtiter source plates; one set of 16
wells can be reserved for matrix solution. The wells of two plates
are filled with washing solution. Five microtiter plates can be
loaded onto the stage of the robotic assembly 16. A plurality of
target substrates can be placed abutting an optional set of banking
or registration pins disposed on the stage 26 and provided for
aligning the target substrates along a set of reference axes. If
the matrix and oligonucleotide are not pre-mixed, the pin assembly
can be employed to first spot matrix solution on all desired target
substrates. In a subsequent step the oligonucleotide solution can
be spotted in the same pattern as the matrix material to
re-dissolve the matrix. Alternatively, a sample array can be made
by placing the oligonucleotide solution on the wafer first,
followed by the matrix solution, or by pre-mixing the matrix and
oligonucleotide solutions.
[0069] After depositing the sample arrays onto the surface of the
substrate, the arrays can be analyzed using any of a variety of
means (e.g., spectrometric techniques, such as UV/VIS, IR,
fluorescence, chemiluminescence, NMR spectrometry or mass
spectrometry. For example, subsequent to either dispensing process,
sample loaded substrates can be placed onto a MALDI-TOF source
plate and held there with a set of beveled screw mounted
polycarbonate supports. In one practice, the plate can be
transferred on the end of a probe to be held onto a 1 .mu.m
resolution, 1" travel xy stage (Newport) in the source region of a
time-of-flight mass spectrometer. It will be apparent to one of
skill in the art that any suitable mass spectrometry tool can be
employed in the methods and with the apparatus and systems provided
herein.
[0070] Preferred mass spectrometer formats include, but are not
limited to, ionization (I) techniques including but not limited to
matrix assisted laser desorption (MALDI), continuous or pulsed
electrospray (ESI) and related methods (e.g. lonspray or
Thermospray), or massive cluster impact (MCI); those ion sources
can be matched with detection formats including linear or
non-linear reflectron time-of-flight (TOF), single or multiple
quadruple, single or multiple magnetic sector, Fourier Transform
ion cyclotron resonance (FTICR), ion trap, and combinations thereof
(e.g., ion-trap/time-of-flight). For ionization, numerous
matrix/wavelength combinations (MALDI) or solvent combinations
(ESI) can be employed. Subattomole levels of protein have been
detected for example, using ESI (Valaskovic, G. A. et al., (1996)
Science 273: 1199-1202) or MALDI (Li, L. et al., (1996) J. Am.
Chem. Soc 118: 1662-1663) mass spectrometry.
[0071] Thus, that in the processes provided herein a completely
non- contact, high-pressure spray or partial-contact, low pressure
droplet formation mode can be employed. In the latter, the only
contact that will occur is between the droplet and the walls of the
well or a hydrophilic flat surface of the substrate 34. However, in
neither practice need there be any contact between the needle tip
and the surface.
[0072] Definitions
[0073] As used herein the following terms and phrases shall have
the meanings set forth below:
[0074] As used herein, the term "nucleic acid" refers to
oligonucleotides or polynucleotides such as deoxyribonucleic acid
DNA) and ribonucleic acid (RNA) as well as analogs of either RNA or
DNA, for example made from nucleotide analog, any of which are in
single or double stranded form, Nucleic acid molecules can by
synthetic or can be isolated from a particular biological sample
using any of a number or procedures which are well-known in the
art, the particular isolation procedure chosen being appropriate
for the particular biological sample. For example, freeze-thaw and
alkaline lysis procedures can be useful for obtaining nucleic acid
molecules from solid materials; heat and alkaline lysis procedures
can be useful for obtaining nucleic acid molecules for urine; and
proteinase K extraction can be used to obtain nucleic acid from
blood (Rolff, A. et al. PCR: Clinical Diagnostics and Research,
Springer (1994)).
[0075] The terms "protein", "polypeptide" and "peptide" are used
interchangeably herein when referring to a translated nucleic acid
(e.g. a gene product).
[0076] "Sample" as used herein, shall refer 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.
[0077] "Substrate" shall mean an insoluble support onto which a
sample is deposited. Examples of appropriate substrates include
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.
EXAMPLES
[0078] Robot-driven serial and parallel pL-nL dispensing tools were
used to generate 10-10.sup.3 element DNA arrays on <1" square
chips with flat or geometrically altered (e.g. with wells) surfaces
for matrix assisted laser desorption ionization mass spectrometry
analysis. In the former, a `piezoelectric pipette` (70 .mu.m id
capillary) dispenses single or multiple-0.2 nL droplets of matrix,
and then analyte, onto the chip; spectra from as low as 0.2 fmol of
a 36-mer DNA have been acquired using this procedure. Despite the
fast (<5 sec) evaporation, micro-crystals of 3-hydroxypicolinic
acid matrix containing the analyte are routinely produced resulting
in higher reproducibility than routinely obtained with larger
volume preparations; all of 1 00 five fmol sports of a 23-mer in
800 .mu.m wells yielded easily interpreted mass spectra, with
99/100 parent ion signals having signal to noise ration of >5.
In a second approach, probes from 384 well microtiter plate are
dispensed 1 6 at a time into chip wells or onto flat surfaces using
an array of spring loaded pins which transfer-20 nL to the chip by
surface contact; MS analysis of array elements deposited with the
parallel method are comparable in terms of sensitivity and
resolution to those made with the serial method.
[0079] I. Description of the Piezoelectric Serial Dispenser.
[0080] The experimental system built on a system purchased from
Microdrop GmbH, Norderstedt Germany and includes a piezoelectric
element driver which sends a pulsed signal to a piezoelectric
element bonded to and surrounding a glass capillary which holds the
solution to be dispensed; a pressure transducer to load (by
negative pressure) or empty (by positive pressure) the capillary; a
robotic xyz stage and robot driver to maneuver the capillary for
loading, unloading, dispensing, and cleaning, a stroboscope and
driver pulsed at the frequency of the piezo element to enable
viewing of `suspended` droplet characteristics; separate stages for
source and designation plates or sample targets (i.e. Si chip); a
camera mounted to the robotic arm to view loading to designation
plate; and a data station which controls the pressure unit, xyz
robot, and piezoelectric driver.
[0081] II. Description of the Parallel Dispenser.
[0082] The robotic pintool includes 16 probes housed in a probe
block and mounted on an X Y, Z robotic stage. The robotic stage was
a gantry system which enables the placement of sample trays below
the arms of the robot. The gantry unit itself is composed of X and
Y arms which move 250 and 400 mm, respectively, guided by brushless
linear servo motors with positional feedback provided by linear
optical encoders. A lead screw driven Z axis (50 mm vertical
travel) is mounted to the xy axis slide of the gantry unit and is
controlled by an in-line rotary servo motor with positional
feedback by a motor-mounted rotary optical encoder. The work area
of the system is equipped with a slide-out tooling plate that holds
five microtiter plates (most often, 2 plates of wash solution and 3
plates of sample for a maximum of 1152 different oligonucleotide
solutions) and up to ten 20.times.20 mm wafers. The wafers are
placed precisely in the plate against two banking pins and held
secure by vacuum. The entire system is enclosed in plexi-glass
housing for safety and mounted onto a steel support frame for
thermal and vibrational damping. Motion control is accomplished by
employing a commercial motion controller which was a 3-axis servo
controller and is integrated to a computer; programming code for
specific applications is written as needed.
[0083] Samples were dispensed with the serial system onto several
surfaces which served as targets in the MALDI TOF analysis
including [1] A flat stainless steel sample target as supplied for
routine use in a Thermo Bioanalysis Vision 2000; [2] the same
design stainless steel target with micromachined nonpits; [3] flat
silicon (Si) wafers; [4] polished flat Si wafers; [5] Si wafers
with rough (3-6 pLm features) pits; [6](a) 12.times.12 or ((b)
18.times.18) mm Si chips with (a) 10.times.10 (or (b) 16.times.16)
arrays of chemically etched wells, each 800.times.8001 lm on a side
with depths ranging from 99-400 (or(b) 120) micrometer, pitch (a)
1.0 (or(b) 1.125 mm); [7] 15.times.15 mm Si chips with 28.times.28
arrays of chemically etched wells, each 450.times.450 micrometer on
a side with depths ranging from 48-300 micrometer, pitch 0.5 mm;
[8]flat polycarbonate or other plastics; [9] gold and other metals;
[10] membranes; [11] plastic surfaces sputtered with gold or other
conducting materials. The dispensed volume is controlled from
10.sup.-10 to 10.sup.-6 L by adjusting the number of droplets
dispensed.
[0084] Sample Preparation and Dispensing: Serial Oligonucleotides
(0.1-50 ng/microliter of different sequence or concentrations were
loaded into wells of a 96 well microtiter plate; the first well was
reserved for matrix solution. A pitted chip (target 6a in MALDI
targets' section) was placed on the stage and aligned manually.
Into the (Windows-based) robot control software were entered the
coordinates of the first well, the array size (ie number of spots
in x and y) and spacing between elements, and the number of 0.2 nL
drops per array element. The capillary was filled with .about.10
.mu.l rinse H.sub.2O, automatically moved in view of a strobe
light-illuminated camera for checking tip integrity and cleanliness
while in continuous pulse mode, and emptied. The capillary was then
filled with matrix solution, again checked at the stroboscope, and
then used to spot an array onto flat or pitted surfaces. For
reproducibilty studies in different MS modes, typically a
101.times.10 array of 0.2-20 nL droplets were dispensed. The
capillary was emptied by application of positive pressure,
optionally rinsed with H.sub.2O, and let to the source oligo plate
where .about.5 .mu.L of 0.05-2.0 .mu.M synthetic oligo were drawn.
The capillary was then rastered in a series over each of the matrix
spots with 0.2-20 nL aqueous solution added to each.
[0085] Sample Preparation and Dispensing:
[0086] Parallel Programs were written to control array making by
offset printing; to make an array of 64 elements on 10 wafers, for
example, the tool was dipped into 16 wells of a 384 well DNA source
plate, moved to the target (e.g. Si, plastic, metal), and the
sample spotted by surface contact. The tool was then dipped into
the same 16 wells and spotted on the second target; this cycle was
repeated on all ten wafers. Next the tool was dipped in washing
solution, then dipped into 16 different wells of the source plate,
and spotted onto the target 2.25 mm offset from the initial set of
16 spots; again this as repeated on all 10 wafers; the entire cycle
was repeated to make a 2.times.2 array from each pin to produce an
8.times.8 array of spots (2.times.2 elements/pin X 16 pins=64 total
elements spotted).
[0087] To make arrays for MS analysis, olegonucleotides of
different sequences or concentrations were loaded into the wells of
up to three different 384-well microtiter plates, one set of 16
wells was reserved for matrix solution. The wells of two plates
were filled with washing solution. The five microtiter plates were
loaded onto the slide-out tooling plate. Ten wafers were placed
abutting the banking pins on the tooling plate, and the vacuum
turned on. In cases where matrix and oligonucleotide were not
pre-mixed, the pintool was used to spot matrix solution first on
all desired array elements of the ten wafers. For this example, a
16.times.16 array was created, thus the tool must spot each of the
ten wafers 16 times, with an offset of 1.125 mm. Next, the
oligonucleotide solution was spotted in the same pattern to
re-dissolve the matrix, Similarly, an array could be made by
placing the oligonucleotide solution on the wafer first, followed
by the matrix solution, or by pre-mixing the matrix and
oligonucleotide solutions.
[0088] Mass spectrometry.
[0089] Subsequent to either dispensing scheme, loaded chips were
held onto a MALDI-TOF source plate with a set of beveled screw
mounted polycarbonated supports. The plate was transferred on the
end of a probe to be held onto a 1 .mu.m resolution, 1" travel xy
stage (Newport) in the source region of a time-of-flight mass
spectrometer. The instrument, normally operated with 18-26 kV
extraction, could be operated in linear or curved field reflectron
mode, and in continuous or delayed extraction mode.
[0090] Observations
[0091] I. Serial dispensing with the piezoelectric pipette. While
delivery of a saturated 3HPA solution can result in tip clogging as
the solvent at the capillary-air interface evaporates, pre-mixing
DNA and matrix sufficiently dilutes the matrix such that it remains
in solution while stable sprays which could be maintained until the
capillary was emptied were obtained; with 1:1 diluted (in H.sub.2O)
matrix solution, continuous spraying for >>10 minutes was
possible. Turning off the piezo element so that the capillary sat
inactive for >5 minutes, and reactivating the piezo element also
did not result in a clogged capillary.
[0092] Initial experiments using stainless steel sample targets as
provided by Finnigan Vision 2000 MALDI-TOF system run in reflectron
mode utilized a pre-mixed solution of the matrix and DNA prior to
dispensing onto the sample target. In a single microtiter well,
50,uL saturated matrix solution, 25 .mu.L of a 51 .mu.L solution of
the 12-mer (ATCG)3, and 25 .mu.L of a 51 .mu.L solution of the
28-mer (ATCG)7 were mixed. A set of 10.times.10 arrays of 0.6 .mu.L
drops was dispensed directly onto a Finnigan Vision 2000 sample
target disk; MALDI-TOF mass spectrum was obtained from a single
array element which contained 750 attomoles of each of the two
oligonucleotides. Interpretable mass spectra has been obtained for
DNAs as large as a 53-mer (350 amol loaded, not shown) using this
method.
[0093] Mass spectra were also obtained from DNAs microdispensed
into the wells of a silicon chip. FIG. 7 shows a 12.times.12mm
silicon chip with 100 chemically etched wells; mask dimensions and
etch time were set such that fustum (i.e., inverted flat top
pyramidal) geometry wells with 800.times.800 .mu.m (top surface)
and 100 .mu.m depth were obtained. Optionally, the wells can be
roughed or pitted. As described above, the chip edge was aligned
against a raised surface on the stage to define the x and y
coordinate systems with respect to the capillary. (Alternatives
include optical alignment, artificial intelligence pattern
recognition routines, and dowel-pin based manual alignment). Into
each well was dispensed 20 droplets (.about.5 nL) of 3-HPA matrix
solution without analyte; for the 50% CH.sub.3CN solution employed,
evaporation times for each droplet were on the order of 5-10
seconds. Upon solvent evaporation, each microdispensed matrix
droplet as viewed under a 120.times. stereomicroscope generally
appeared as an amorphous and `milky` flat disk; such appearances
are consistent with those of droplets from which the FIG. 3b
spectrum was obtained. Upon tip emptying, rinsing, and refilling
with a 1.4 .mu.m aqueous solution of a 23-mer DNA (Mr(calc)=6967
Da), the capillary was directed above each of the 100 spots of
matrix where 5 nL of the aqueous DNA solution was dispensed
directly on top of the matrix droplets. Employing visualization via
a CCD camera, it appeared that the aqueous analyte solution mixed
with and re-dissolved the matrix (complete evaporation took -10 sec
at ambient temperature and humidity). The amorphous matrix surfaces
were converted to true micro-crystalline surfaces, with crystalline
features on the order of <1 .mu.m.
[0094] Consistent with the improved crystallization afforded by the
matrix re-dissolving method, mass spectrum acquisition appeared
more reproducible than with pre-mixed matrix plus analyte
solutions; each of the 100 five fmol spots of the 23-mer yielded
interpreted mass spectra (FIG. 8), with 99/100 parent ion signals
having signal to noise rations of >5; such reproducibility was
also obtained with the flat silicon and metallic surfaces tried
(not shown). The FIG. 8 spectra were obtained on a linear TOF
instrument operated at 26 kV. Upon internal calibration of the top
left spectrum (well `k1`) using the singly and doubly charged
molecular ions, and application of this calibration file to all
other spectra as an external calibration (FIG. 9), a standard
deviation of <9 Da from the average molecular weight was
obtained, corresponding to a relative standard deviation of
.about.0.1%.
[0095] II. Parallel Dispensing with robotic pintool. Arrays were
made with offset printing as described above. The velocity of the X
and Y stages are 35 inches/sec, and the velocity of the Z stage is
5.5 inches/sec. It is possible to move the X and Y stages at
maximum velocity to decrease the cycle times, however the speed of
the Z stage is to be decreased prior to surface contact with the
wafer to avoid damaging it. At such axes speeds, the approximate
cycle time to spot 16 elements (one tool impression of the same
solutions) on all ten wafers is 20 seconds, so to make an array of
256 elements would take .about.5.3 minutes. When placing different
oligonucleotide solutions on the array, an additional washing step
much be incorporated to clean the pin tip prior to dipping in
another solution, thus the cycle time would increase to 25 seconds
or 6.7 minutes to make 10 wafers.
[0096] Sample delivery by the tool was examined using radio-labeled
solutions and the phosphorimager as described previously; it was
determined that each pin delivers approximately 1 nL of liquid. The
spot-to-spot reproducibility is high. An array of 256
oligonucleotide elements of varying sequence and concentration was
made on flat silicon wafers using the pintool, and the wafer was
analyzed by MALDI-TOF MS.
[0097] It will be understood that the above-described examples and
illustrated embodiments are provided for describing the invention
set forth herein and are not to be taken as limiting in any way,
and the scope of the invention is to understood by the claims.
* * * * *