U.S. patent application number 12/174824 was filed with the patent office on 2008-11-06 for acoustic radiation for ejecting and monitoring pathogenic fluids.
This patent application is currently assigned to Picoliter Inc.. Invention is credited to Richard N. Ellson, Mitchell W. Mutz.
Application Number | 20080274538 12/174824 |
Document ID | / |
Family ID | 30443439 |
Filed Date | 2008-11-06 |
United States Patent
Application |
20080274538 |
Kind Code |
A1 |
Mutz; Mitchell W. ; et
al. |
November 6, 2008 |
Acoustic Radiation for Ejecting and Monitoring Pathogenic
Fluids
Abstract
The invention relates to methods and devices that use focused
radiation to handle and/or monitor pathogenic fluids. In
particular, a method is provided for dispensing one or more
droplets of a fluid containing a pathogen. The method involves
providing the pathogen-containing fluid in a reservoir and applying
focused radiation to the pathogen-containing fluid in the reservoir
in a manner effective to eject a droplet of the fluid therefrom.
Often, a pathogen-impermeable enclosure is used.
Inventors: |
Mutz; Mitchell W.; (La
Jolla, CA) ; Ellson; Richard N.; (Palo Alto,
CA) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS, GLOVSKY AND POPEO, P.C
5 Palo Alto Square - 6th Floor, 3000 El Camino Real
PALO ALTO
CA
94306-2155
US
|
Assignee: |
Picoliter Inc.
Sunnyvale
CA
|
Family ID: |
30443439 |
Appl. No.: |
12/174824 |
Filed: |
July 17, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10199907 |
Jul 18, 2002 |
7405072 |
|
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12174824 |
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Current U.S.
Class: |
435/286.3 ;
427/2.11; 427/2.14; 427/457; 427/458; 427/466; 427/472; 435/173.1;
435/174; 435/235.1; 435/243; 435/283.1; 435/286.1; 435/30;
435/307.1; 435/309.1; 435/40.5; 435/7.1; 436/57 |
Current CPC
Class: |
B01J 2219/00722
20130101; B01L 2400/0436 20130101; B01L 3/0293 20130101; C40B 40/10
20130101; B01L 2200/0647 20130101; C40B 40/06 20130101; B01J
2219/00378 20130101; B01J 2219/00725 20130101; B01L 3/0268
20130101; C12Q 1/22 20130101; C40B 60/14 20130101; B01J 2219/00362
20130101; B01L 3/508 20130101; B01J 2219/00659 20130101 |
Class at
Publication: |
435/286.3 ;
427/2.11; 427/2.14; 427/457; 427/458; 427/466; 427/472; 435/173.1;
435/174; 435/235.1; 435/243; 435/283.1; 435/286.1; 435/30;
435/307.1; 435/309.1; 435/40.5; 435/7.1; 436/57 |
International
Class: |
C12P 1/00 20060101
C12P001/00; C12N 11/00 20060101 C12N011/00; C12N 11/02 20060101
C12N011/02; C12N 15/09 20060101 C12N015/09 |
Claims
1. A method for sealing a fluid in a pathogen-impermeable
enclosure, comprising: (a) providing the fluid in a reservoir; (b)
positioning a substrate so that a designated site on a surface
thereof is in droplet-receiving relationship with respect to the
reservoir; (c) applying focused radiation to the fluid in the
reservoir in a manner effective to eject a droplet of the fluid
therefrom onto the substrate surface at the designated site; and
(d) sealing the fluid droplet at the designated site in the
pathogen-impermeable enclosure.
2. The method of claim 1, wherein the fluid is a bodily fluid.
3. The method of claim 1, wherein the focused radiation is focused
acoustic radiation.
4. The method of claim 3, further comprising, before (d),
(d.degree.) depositing a pathogen-containing fluid at the
designated site.
5. The method of claim 4, wherein the pathogenic fluid is a bodily
fluid.
6. The method of claim 4, wherein the pathogen-containing fluid is
comprised of a discrete pathogenic particle in a carrier fluid.
7. The method of claim 6, wherein the pathogen is a virus.
8. The method of claim 6, wherein the pathogen is bacterial.
9. The method of claim 4, wherein the pathogen is a toxin.
10. The method of claim 4, wherein (d.degree.) is carried out after
(c).
11. The method of claim 4, wherein (d.degree.) is carried out
during (c).
12. The method of claim 4, wherein (d.degree.) is carried out
before (c).
13. The method of claim 3, wherein (b) comprises (b') opening the
pathogen-impermeable enclosure so as to expose the designated site
within the enclosure to the reservoir.
14. The method of claim 10, wherein the pathogen-impermeable
enclosure comprises a cover and the substrate, and (b') comprises
positioning the cover to expose the designated site within the
enclosure to the reservoir.
15. The method of claim 11, wherein (d) comprises positioning the
cover in sealing contact with the substrate.
16. The method of claim 15, further comprising, after (d), (e)
subjecting the pathogen impermeable enclosure to temperatures
associated with PCR thermal cycling.
17. The method of claim 3, wherein steps (b) and (c) are repeated
such that a plurality of droplets is deposited on the substrate
surface at different designated sites, and wherein the fluid
droplets at each designated site are sealed in a
pathogen-impermeable enclosure.
18. The method of claim 17, wherein the different designated sites
form an array of sites.
19. The method of claim 17, wherein (d) comprises individually
isolating each of the fluid droplets in a compartment of the
pathogen-impermeable enclosure.
20. The method of claim 3, wherein (a) comprises providing a
plurality of reservoirs each containing a different fluid, and
steps (b) and (c) are repeated such that a droplet from each
reservoir is deposited on the substrate surface.
21. The method of claim 20, wherein the droplets are deposited on
the substrate surface at the same designated site.
22. The method of claim 20, wherein the droplets are deposited on
the substrate surface at different designated sites.
23. A method for monitoring for a change in the amount and/or
concentration of a pathogen in a pathogenic fluid, comprising: (a)
providing a pathogen-impermeable enclosure enclosing the pathogenic
fluid, wherein the pathogenic fluid comprises a pathogen and a
carrier fluid; and (b) acoustically monitoring for a change in the
amount and/or concentration of the pathogen enclosed in the
pathogen-impermeable enclosure.
24. The method of claim 23, wherein the change is an increase in
pathogen content.
25. The method of claim 23, wherein the change is a decrease in
pathogen content.
26. The method of claim 23, further comprising, before (b), (a')
introducing additional material into the reservoir.
27. The method of claim 23, further comprising, after (a) and
before (b), (a') subjecting the enclosure to a temperature
change.
28. The method of claim 27, wherein (a') comprises raising the
temperature of the enclosure.
29. The method of claim 27, wherein (a') comprises subjecting the
enclosure to a temperature selected to facilitate an increase in
the amount and/or concentration of the pathogen in the
enclosure.
30. The method of claim 27, wherein (a') comprises subjecting the
enclosure to a temperature selected to facilitate a decrease in the
amount and/or concentration of the pathogen in the enclosure.
31. A method for selecting a localized volume in a pathogenic fluid
for removal from a reservoir, comprising: (a) providing the
pathogen-containing fluid in a reservoir comprised of plurality of
particles and a carrier fluid; and (b) acoustically locating the
localized volume within the pathogen-containing fluid.
32. The method of claim 31, further comprising, after (b), (c)
removing the localized volume from the fluid reservoir.
33. The method of claim 32, wherein (c) is carried out by applying
focused radiation to eject a droplet containing the localized
volume from the reservoir.
34. The method of claim 33, wherein the focused radiation applied
is focused acoustic radiation.
35. The method of claim 31, wherein at least some of the particles
are pathogenic particles.
36. The method of claim 31, wherein at least some of the particles
are nonpathogenic particles.
37. The method of claim 31, wherein the plurality comprises at
least one pathogenic particle and at least one nonpathogenic
particle.
38. The method of claim 31, wherein the localized volume contains
at least one particle.
39. The method of claim 37, wherein the localized volume contains a
single particle.
40. The method of claim 31, wherein the localized volume contains
no particle.
41. The method of claim 31, wherein the localized volume contains a
pathogen.
42. The method of claim 31, wherein the localized volume contains
no pathogen.
43. A method for detecting for an interaction between a fluid and a
compound, wherein the fluid and/or compound are pathogenic,
comprising: (a) providing a reservoir containing the fluid; (b)
depositing the compound onto a designated site on a surface of a
substrate; (c) positioning the substrate so that the designated
site is in droplet-receiving relationship with respect to the
reservoir; (d) applying focused radiation to the fluid in the
reservoir in a manner effective to eject a droplet of the fluid
therefrom onto the substrate surface at the designated site; (e)
sealing the fluid droplet and the compound at the designated site
in a pathogen-impermeable enclosure; and (f) detecting for an
interaction between the fluid and the compound.
44. The method of claim 43, wherein the compound is pathogenic.
45. The method of claim 43, wherein the fluid is pathogenic.
46. The method of claim 43, wherein the interaction is acoustically
detected.
47. The method of claim 43, wherein the interaction is optically
detected.
48. The method of claim 43, wherein the interaction is
fluorescently detected.
49. The method of claim 43, wherein the interaction is magnetically
detected.
50. The method of claim 43, wherein the interaction is electrically
detected.
51. The method of claim 43, further comprising, after (e), (e')
subjecting the pathogen-impermeable enclosure to temperatures
associated with PCR thermal cycling.
52. A method for transferring one or more droplets of a
pathogen-containing fluid, comprising: (a) providing the
pathogen-containing fluid in a reservoir which lies within a sealed
enclosure; and (b) applying focused acoustic radiation to the
pathogen-containing fluid in the reservoir in a manner effective to
transfer a droplet of the fluid onto a surface within the sealed
enclosure by means of an acoustic radiation generator external to
the sealed enclosure, wherein the acoustic radiation generation is
coupled to the reservoir by means of an acoustic coupling
fluid.
53. The method of claim 52, further comprising the steps of (c)
providing a second pathogen-containing fluid in a second reservoir
within the sealed enclosure, (d) moving the acoustic radiation
generator so that it is able to direct focused acoustic radiation
to the second pathogen-containing fluid, and (e) applying focused
acoustic radiation to the second pathogen-containing fluid within
the sealed enclosure by means of the acoustic radiation generator
in a manner effective to transfer a droplet of the fluid onto a
surface within the sealed enclosure, wherein the acoustic radiation
generator is coupled to the second reservoir by means of an
acoustic coupling fluid.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/199,907, filed Jul. 18, 2002, which is hereby
incorporated by reference in its entirety.
TECHNICAL FIELD
[0002] The invention relates generally to the use of acoustic
radiation in conjunction with pathogenic fluids. In particular, the
invention relates to the acoustic monitoring of the pathogenic
contents within a reservoir, as well as to the acoustic ejection of
pathogenic fluid droplets. The invention also relates to the use of
acoustic radiation in conjunction with pathogen-impermeable
enclosures.
BACKGROUND
[0003] Cultures containing cellular matter may be employed to study
pathogenic material such as bacteria and viruses. For example,
pathogen-impermeable containers having an interior surface coated
with a layer of solid or semisolid medium within which cells are
grown may be inoculated with the desired type of cells. After the
cells are subjected to conditions appropriate for cultivation, they
may be removed from the containers as a suspension and may
optionally be concentrated. Also, if desired, viral matter may be
extracted from the cells after removal from the containers.
[0004] Pathogenic substances, however, including viruses (such as
the human immunodeficiency virus (HIV), rabies, and herpes) and
bacteria (such as bacillus anthracis, yersinia pestis, and those of
the streptococcus genus), must be handled with extreme care to
prevent release of the pathogen. In addition, there exists a need
in pharmaceutical, biotechnological, and other scientific
industries to quickly screen, identify, and/or process large
numbers or varieties of fluids, pathogenic or otherwise. As a
result, much attention has been focused on developing efficient,
precise, and accurate fluid handling methods that may be used, for
example, to carry out screening assays and/or combinatorial
techniques. Since fluids used in pharmaceutical, biotechnological,
and other scientific industries may be rare and/or expensive,
techniques capable of handling small volumes of fluids provide
readily apparent advantages over those requiring relatively larger
volumes. Furthermore, as pathogenic fluids represent a potential
safety hazard, it is also desirable to reduce the quantities used
to carry out studies or investigations involving such
substances.
[0005] Typically, fluids for use in combinatorial methods are
provided as a collection or library of organic and/or biological
compounds. In many instances, such libraries and collections are
provided in a well plate format for screening and/or processing.
Well plates are typically single-piece in construction and comprise
a plurality of identical wells, wherein each well is adapted to
contain a small volume of fluid. Such well plates are commercially
available in standardized formats and sizes, and may contain, for
example, 96, 384, 1536, or 3456 wells per well plate. Fluids are
typically transferred from such well plates, e.g., during
formatting and reformatting procedures, using devices that require
contact between the fluid to be transferred and a solid surface of
a device. For example, capillaries (Eppendorf-type or otherwise)
having small interior channels are commonly employed for sample
fluid handling by submerging their ends into a pool of sample.
Pipetting systems, whether automated, robotic, or otherwise, that
have submergible tips may be employed as well. Contact between the
solid surface and the fluid to be transferred typically results in
surface wetting that represents a source of unavoidable fluid waste
as well as a source of potential pathogenic contamination. In
addition, if more than one fluid is to contact an interior or
exterior solid surface of a non-disposable capillary or pipette
tip, the surface must be washed between sample transfers in order
to eliminate cross contamination and sample carry-over. The liquid
biohazard waste created from this wash process must then be
disposed of and rendered harmless. It would be desirable to avoid
liquid waste generation from repeated wash processes and eliminate
additional storage and disposal costs. Disposable pipette tips or
capillaries may be used to avoid the generation of liquid waste.
However, disposal of solid waste also incurs storage and disposal
costs.
[0006] Thus, there is a need for fluid handling systems that enable
safe and convenient handling, formatting, and reformatting of
potentially dangerous bacterial, viral, and other pathogenic
specimens. Such fluid handling systems may be used, for example, to
perform clinical diagnostic tests, engage in high-throughput drug
screening, and carry out growth inhibition studies. In order to
ensure that pathogens are not released during fluid handling
procedures, pathogen-impermeable enclosures such as glove boxes may
be used to contain the pathogenic specimens. Small volume
pathogenic cultures, however, often require complicated manual
manipulations, which are not easily carried out using glove boxes;
thus, performing such procedures in a glove box would likely
introduce error during handling and result in possible unwarranted
experimental conclusions. Various automated devices to control
fluid transfer in closed systems for culturing living pathogens
have been developed. U.S. Pat. No. 6,022,742 to Kopf, for example,
describes one such automated device.
[0007] The use of acoustic energy in printing technology is also
known. For example, U.S. Pat. No. 4,308,547 to Lovelady et al.
describes a liquid drop emitter that utilizes acoustic principles
to eject liquid from a body of liquid onto a moving document in
order to form characters or barcodes thereon. Lovelady et al. is
directed to a nozzleless inkjet printing apparatus, wherein
controlled drops of ink are propelled by an acoustical force
produced by a curved transducer at or below the surface of the ink.
In contrast to capillaries, syringes, pipettes, inkjet print heads,
and other such fluid dispensing devices that employ a nozzle, tip,
or tubing for fluid transfer, nozzleless fluid ejection devices as
described in the aforementioned patent do not contain components
requiring cleaning and/or disposal after use. In addition,
disadvantages associated with nozzles or tips in fluid dispensing
systems, including clogging, misdirected fluid, improperly sized
droplet formation, and the like, are avoided. More recently,
acoustic ejection has been employed in contexts other than in ink
printing applications. For example, U.S. Patent Application
Publication No. 20020037579 to Ellson et al. describes the use of
focused acoustic radiation to dispense fluids with sufficient
accuracy and precision to prepare biomolecular arrays from a
plurality of reservoirs.
[0008] Acoustic radiation has also been used to assess the contents
of a container adapted to contain a liquid. Traditionally, the
contents may be assessed by contacting a sensor with the liquid
(see U.S. Pat. No. 5,507,178 to Dam), or by transmitting acoustic
radiation through an open top of a container and detecting
radiation reflected from an air-liquid interface of the container
back to the sensor (see U.S. Pat. No. 5,880,364 to Dam). More
recently, U.S. patent application Ser. No. 10/010,972, Publication
No. 20030101819, "Acoustic Assessment of Fluids in a Plurality of
Reservoirs," inventors Mutz, Ellson, and Foote, filed on Dec. 7,
2001, describes an improved acoustic assessment technique that
involves the transmission of acoustic radiation through a reservoir
to assess the fluid contents within the reservoir without requiring
direct contact with the fluid contents therein. By analyzing a
characteristic of the acoustic radiation transmitted through the
fluid, various properties of the fluid within the reservoir may be
determined. This type of acoustic monitoring may be used
advantageously in conjunction with optically opaque reservoirs.
[0009] Similarly, focused acoustic energy recently has been used in
applications involving biological matter such as living cells. For
example, a number of U.S. patent applications describe the use of
focused acoustic radiation to manipulate and sort cells. See U.S.
Patent Application Publication No. 20020064808 to Mutz et al.; U.S.
patent application Ser. No. 09/999,166, Publication No.
20020142286, filed Nov. 29, 2001, for "Focused Acoustic Energy for
Ejection Cells from a Fluid," inventors Mutz and Ellson, assigned
to Picoliter, Inc. (Mountain View, Calif.); U.S. Patent Application
Publication No. 20020064809 to Mutz et al.; and U.S. patent
application Ser. No. 10/040,926, Publication No. 20020090720, filed
Dec. 28, 2001, for "Focused Acoustic Ejection Cell Sorting System
and Method," inventors Mutz, Ellson, and Lee, assigned to
Picoliter, Inc. (Mountain View, Calif.). Furthermore, the use of
focused acoustic radiation has been described for preparing and
analyzing a cellular sample surface. (See U.S. patent application
Ser. No. 10/087,372, Publication No. 20020171037, filed Mar. 1,
2002, entitled "Method and System Using Acoustic Ejection for
Preparing and Analyzing a Cellular Sample Surface," inventors
Ellson, Mutz, and Caprioli.)
[0010] The use of focused acoustic energy in the context of
applications involving pathogenic fluids, however, has previously
been unknown in the art. Thus, through the use of focused acoustic
radiation, the invention provides previously unrealized
opportunities in pathogenic studies.
SUMMARY OF THE INVENTION
[0011] In a first embodiment, the invention relates to a method for
dispensing one or more droplets of a fluid containing a pathogen.
The method involves providing the pathogen-containing fluid in a
reservoir and applying focused radiation to the pathogen-containing
fluid in the reservoir in a manner effective to eject a droplet of
the fluid therefrom. Typically, focused acoustic radiation is
employed to carry out the invention. In addition, the invention may
be used in conjunction with any of a number of different types of
pathogens. The pathogen may be a toxin, virus, and/or
bacterium.
[0012] In some instances, the pathogen-containing fluid may be
comprised of a carrier fluid and a plurality of discrete pathogenic
particles. In addition, a plurality of discrete nonpathogenic
particles may be present in the carrier fluid as well. Accordingly,
the inventive method may involve locating a discrete pathogenic
particle in the carrier fluid using focused radiation. When focused
acoustic radiation is employed, the location of the pathogenic
particle may be detected by virtue of one or more acoustic
properties, such as acoustic impedance, which ensures that the
ejected droplet contains the pathogenic particle. In some cases,
however, the invention may be used to eject droplets containing
nonpathogenic particles or no particles at all.
[0013] Thus, the invention also provides a method for selecting a
localized volume in a pathogenic fluid for removal from a
reservoir. When a pathogen-containing fluid is provided in a
reservoir and is comprised of a plurality of particles and a
carrier fluid, the localized volume may be acoustically located and
optionally removed. The localized volume may contain zero, one, or
more particles. Furthermore, the localized volume may or may not be
pathogenic. In other words, the invention may be used to sort
pathogenic from nonpathogenic fluids and particles and vice
versa.
[0014] The invention may be used to deposit a droplet of fluid on a
designated site of a substrate surface, typically by positioning
the substrate so that the designated site is in droplet-receiving
relationship with respect to the reservoir. In some instances,
additional droplets of fluid are deposited on the substrate surface
from the same reservoir, or from different reservoirs. When the
invention provides a plurality of reservoirs, each reservoir
typically contains a different fluid. In any case, droplets may be
deposited on the substrate surface at the same site or at different
designated sites. When the droplets are deposited at different
designated sites, the sites may form an array of sites.
[0015] Thus, in some instances, the method may be used to determine
whether the pathogen-containing fluid droplet interacts with a
compound of interest. This may be carried out by either ensuring
that a compound of interest is present at the designated site prior
to the deposition of the droplet, or by depositing a compound of
interest at the designated site after the deposition of the
droplet. The compound of interest, for example, may be deposited on
the designated site using focused radiation as well.
[0016] Once deposited, a droplet on the substrate surface may be
isolated in a pathogen-impermeable enclosure. When a plurality of
droplets is deposited, the droplets may be either individually
isolated in a plurality of enclosures, or collectively isolated in
the same enclosure. In either case, the pathogen-impermeable
enclosure or enclosures may be formed by placing a
pathogen-impermeable cover in sealing contact with the
substrate.
[0017] In another embodiment, the invention relates to a method for
sealing a fluid in a pathogen-impermeable enclosure. The method
involves first providing the fluid in a reservoir and positioning a
substrate so that a designated site on a surface thereof is in
droplet-receiving relationship with respect to the reservoir. Then,
focused radiation is applied to the fluid in the reservoir in a
manner effective to eject a droplet of the fluid therefrom onto the
substrate surface at the designated site. The fluid droplet at the
designated site is then sealed in the pathogen-impermeable
enclosure. As before, this method typically employs focused
acoustic radiation.
[0018] The enclosure is typically sealed after introducing a
pathogenic fluid therein, to ensure that the pathogen is not
released. The pathogenic fluid droplet may be ejected from the
reservoir or dispensed from elsewhere. Thus, the pathogenic fluid
droplet may be deposited at the designated site before, during, or
after focused radiation is applied to the reservoir to eject a
droplet of fluid therefrom. In some instances, however, the
enclosure is sealed to ensure that no pathogen is introduced
therein. That is, the pathogen may be controllably sealed in or
sealed out of the enclosure. In either case, the
pathogen-impermeable enclosure is typically opened so as to expose
the designated site within the enclosure to the reservoir, and
sealed after a fluid droplet has been placed in the enclosure. In
some instances, the pathogen-impermeable enclosure may be formed
from a cover and the substrate. In such a case, sealing the
enclosure may involve placing the cover and the substrate in
sealing contact with each other.
[0019] Often, droplets from the reservoir are deposited on the
substrate surface. In some instances, however, a plurality of
reservoirs each containing a different fluid is provided and a
droplet from each reservoir is deposited on the substrate surface.
In some instances, droplets are deposited at the same designated
site. In other instances, the droplets are deposited at different
designated sites. The different designated sites may form an array
of sites. When fluid droplets are deposited at different designated
sites, the droplets may all be sealed in a pathogen-impermeable
enclosure, either together in the same compartment, or isolated in
separate compartments of the pathogen-impermeable enclosure.
[0020] In a further embodiment, the invention provides a device for
dispensing one or more droplets of fluid. The device includes a
reservoir adapted to contain a fluid, an ejector for applying
focused radiation to the reservoir in a manner effective to eject a
droplet of fluid from the reservoir, a means for positioning a
substrate to receive a droplet of fluid from the reservoir, and a
pathogen-impermeable enclosure for isolating the reservoir and
substrate therein. Typically, the ejector is an acoustic ejector.
Although ordinary inkjet technologies may be employed, it is
preferred that the ejector is a nozzleless acoustic device that
employs an acoustic generator and a focusing means for focusing the
acoustic radiation generated thereby.
[0021] The inventive device may include additional features that
serve to enhance the performance of the device. For example, the
device may further include a means for manually manipulating items
within the enclosure without compromising the pathogenic
impermeability of the enclosure. In addition, a locating means may
be provided for locating a discrete particle in the pathogenic
fluid. When the device includes an acoustic generator, the locating
means may include an analyzer for analyzing acoustic radiation
generated by the acoustic generator. Such an analyzer is typically
positioned to receive acoustic radiation generated by the acoustic
generator and transmitted through fluid contained in the reservoir.
In some instances, the analyzer is positioned to receive acoustic
radiation reflected by a free fluid surface contained in the
reservoir. In such a case, the analyzer may include a component
common to the acoustic generator, e.g., a piezoelectric
element.
[0022] Typically, the reservoir is detachable from the device and
may be adapted for single use. In addition, the device may further
include a pathogen-impermeable cover. Such a cover may be adapted
to make sealing contact with the reservoir in order to contain a
pathogenic fluid therein.
[0023] Optionally, the device includes a plurality of reservoirs.
In some instances, the reservoirs are provided in a single-piece
unit, such as when the reservoirs represent wells of a well plate.
The reservoirs are preferably substantially acoustically
indistinguishable from each other. In addition, the device may
further include a means for successively positioning the acoustic
device in an acoustically coupled relationship with each of the
reservoirs.
[0024] In still another embodiment, the invention relates to a
method for monitoring a change in the amount and/or concentration
of a pathogen in a pathogenic fluid. The method involves providing
a pathogen-impermeable enclosure that encloses a pathogenic fluid
comprising a pathogen and a carrier fluid, and acoustically
monitoring for a change in the amount and/or concentration of the
pathogen enclosed in the pathogen-impermeable enclosure. The method
may be used to measure either an increase or a decrease in pathogen
content. Thus, the method is particularly suited for carrying out
processes and/or assays in which pathogen content and/or
concentration is altered. For example, additional material, e.g.,
nutrients in a culturing solution, may be introduced into the
enclosure, which then may be subjected to a temperature change. The
temperature may be selected to facilitate an increase or decrease
in the amount and/or concentration of the pathogen in the
enclosure.
[0025] In a further embodiment, the invention relates to a method
for detecting for an interaction between a fluid and a compound.
The method involves: (a) providing a reservoir containing the
fluid; (b) depositing the compound onto a designated site on a
surface of a substrate; (c) positioning the substrate so that the
designated site is in droplet-receiving relationship with respect
to the reservoir; (d) applying focused radiation to the fluid in
the reservoir in a manner effective to eject a droplet of the fluid
therefrom onto the substrate surface at the designated site; (e)
sealing the fluid droplet and the compound at the designated site
in a pathogen-impermeable enclosure; and (f) detecting for an
interaction between the fluid and the compound. Either the
compound, fluid, or both may be pathogenic. The interaction may be
detected through various means such as acoustic, optic,
fluorescence, magnetic and/or electrical means.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIGS. 1A-1D, collectively referred to as FIG. 1,
schematically illustrate in simplified cross-sectional view the
operation of an enclosed system that uses focused acoustic
radiation to study the interaction between a candidate compound and
a bacterial pathogen. FIG. 1A shows the use of acoustic radiation
to locate a pathogenic bacterial particle near the surface of a
fluid in a reservoir. FIG. 1B shows the ejection of a droplet
containing a bacterial pathogen from the reservoir onto a
designated site of a substrate surface. FIG. 1C illustrates the
placement of the substrate in acoustically coupled relationship
with an acoustic analyzer and an initial acoustic assessment of the
pathogenic contents of the well. FIG. 1D illustrates a subsequent
assessment of the pathogenic contents of the well after exposure to
culturing conditions.
[0027] FIG. 2 schematically illustrates in simplified
cross-sectional view the acoustic assessment of the pathogenic
contents of pathogen-impermeable enclosure in transmissive
mode.
DETAILED DESCRIPTION OF THE INVENTION
[0028] Before describing the present invention in detail, it is to
be understood that, unless otherwise indicated, this invention is
not limited to specific fluids, acoustic devices, substrates, or
the like, as such may vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to be limiting.
[0029] It must be noted that, as used in this specification and the
appended claims, the singular forms "a," "an," and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, the term "a fluid" is intended to mean a single
fluid or a mixture of fluids, "a reservoir" is intended to mean one
or more reservoirs, "a pathogen" refers to a single pathogen as
well as a plurality of pathogens.
[0030] In describing and claiming the present invention, the
following terminology will be used in accordance with the
definitions set out below.
[0031] The terms "acoustic coupling" and "acoustically coupled" as
used herein refer to a state wherein an object is placed in direct
or indirect contact with another object so as to allow acoustic
radiation to be transferred between the objects without substantial
loss of acoustic energy. When two entities are indirectly
acoustically coupled, an "acoustic coupling medium" is needed to
provide an intermediary through which acoustic radiation may be
transmitted. Thus, an acoustic device may be acoustically coupled
to a fluid, such as by immersing the acoustic device in the fluid,
or by interposing an acoustic coupling medium between the acoustic
device and the fluid, in order to transfer acoustic radiation
generated by the acoustic device through the acoustic coupling
medium and into the fluid.
[0032] The terms "acoustic radiation" and "acoustic energy" are
used interchangeably herein and refer to the emission and
propagation of energy in the form of sound waves. As with other
waveforms, acoustic radiation may be focused using a focusing
means, as discussed below.
[0033] The term "array" as used herein refers to a two-dimensional
arrangement of features, such as an arrangement of reservoirs
(e.g., wells in a well plate) or an arrangement of different
moieties, including ionic, metallic, or covalent crystalline (e.g.,
molecular crystalline), composite, ceramic, vitreous, amorphous,
fluidic, or molecular materials on a substrate surface (as in an
oligonucleotide or peptidic array). Arrays are generally comprised
of regular features ordered in, for example, a rectilinear grid,
parallel stripes, spirals, and the like, but nonordered arrays may
be advantageously used as well. An array is distinguished from the
more general term "pattern" in that patterns do not necessarily
contain regular and ordered features.
[0034] The terms "biomolecule" and "biological molecule" are used
interchangeably herein to refer to any organic molecule that is,
was, or can be a part of a living organism, regardless of whether
the molecule is naturally occurring, recombinantly produced, or
chemically synthesized in whole or in part. The terms encompass,
for example, monomeric molecules, such as nucleotides, amino acids,
and monosaccharides, oligomeric and polymeric species, such as
oligonucleotides and polynucleotides, peptidic molecules, such as
oligopeptides, polypeptides, and proteins, saccharides, such as
disaccharides, oligosaccharides, polysaccharides,
mucopolysaccharides, and peptidoglycans (peptido-polysaccharides),
and the like. The terms also encompass ribosomes, enzyme cofactors,
pharmacologically active agents, and the like. Additional
information relating to the term "biomolecule" can be found in U.S.
Patent Application Publication No. 20020037579 by Ellson et al.
[0035] The term "enclosure" is used herein in its ordinary sense
and refers to anything that encloses. Examples of enclosures
include, but are not limited to, bottles, boxes, canisters, cans,
cartons, cartridges, containers, drums, jars, and vials.
[0036] The term "fluid" as used herein refers to matter that is
nonsolid, or at least partially gaseous and/or liquid, but not
entirely gaseous. A fluid may contain a solid that is minimally,
partially, or fully solvated, dispersed, or suspended. Examples of
fluids include, without limitation, aqueous liquids (including
water per se and salt water) and nonaqueous liquids such as organic
solvents and the like. As used herein, the term "fluid" is not
synonymous with the term "ink," in that ink must contain a colorant
and may not be gaseous. Thus, the term "bodily fluid" as used
herein refers to any fluid that can be extracted from an
individual's body, pathogenic or nonpathogenic. When the individual
is a mammal, e.g., human, the term includes fluids such as blood,
plasma, serum, interstitial fluid, lymph, bile, spinal fluid,
amnionic fluid, urine, saliva, vaginal fluid, and etc.
[0037] The term "focusing means" refers to a means for causing
waves to converge at a focal point. When acoustic radiation is
involved, an "acoustic focusing means" causes acoustic radiation to
converge at a focal point either by a device separate from the
acoustic energy source that acts like an optical lens, or by the
spatial arrangement of acoustic energy sources to effect
convergence of acoustic energy at a focal point by constructive and
destructive interference. An acoustic focusing means may be as
simple as a solid member having a curved surface, or it may include
complex structures such as those found in Fresnel lenses, which
employ diffraction in order to direct acoustic radiation. Suitable
focusing means also include phased array methods as are known in
the art and described, for example, in U.S. Pat. No. 5,798,779 to
Nakayasu et al. and Amemiya et al. (1997) Proceedings of the 1997
IS&T NIP13 International Conference on Digital Printing
Technologies, pp. 698-702.
[0038] The term "impermeable" is used in its ordinary sense to mean
not permitting something to pass through. Similarly, the term
"permeable" is used herein in its ordinary sense and means "not
impermeable." Typically, the term "impermeable" is used to describe
certain enclosures, and the term "permeable" is used to describe
certain "substrates" or "surfaces." Thus, a "pathogen-impermeable
enclosure" refers to an enclosure that does not allow a pathogen to
pass through, and a "permeable substrate" and a "substrate having a
permeable surface" refer to a substrate or surface, respectively,
that can be permeated with water or other fluid.
[0039] The terms "library" and "combinatorial library" are used
interchangeably herein to refer to a plurality of chemical or
biological moieties arranged in a pattern or an array such that the
moieties are individually addressable. In some instances, the
plurality of chemical or biological moieties is present on the
surface of a substrate, and in other instances, the plurality of
moieties represents the contents of a plurality of reservoirs.
Preferably, but not necessarily, each moiety is different from each
of the other moieties. The moieties may be, for example, peptidic
molecules and/or oligonucleotides.
[0040] The term "moiety" refers to any particular composition of
matter, e.g., a molecular fragment, an intact molecule (including a
monomeric molecule, an oligomeric molecule, and a polymer), or a
mixture of materials (for example, an alloy or a laminate).
[0041] "Optional" or "optionally" means that the subsequently
described circumstance may or may not occur, so that the
description includes instances where the circumstance occurs and
instances where it does not.
[0042] The terms "pathogen" and "pathogenic" as used herein refer
to any agent that is capable of causing disease and/or a toxic
response in an individual. The individual may be a human, an animal
(mammalian or otherwise), or on occasion, a plant. Typically, a
pathogen referred to herein is a bacterium or virus, but may also
be an organic toxin such as strychnine or botulinum, or an
inorganic toxin such as arsenic or sodium cyanide. Often, pathogens
are biomolecular in nature. Thus, exemplary bacterial pathogens
include, but are not limited to, bacteria of the following genera,
Campylobactera, Bacteroides, Bordetella, Haemophilus, Pasteurella,
Francisella, Actinobacillus, Klebisella, Moraxella, Pseudomonas,
pneumococci, Proteus, Ornithobacterium, Staphylococci and
Streptococci. Salmonella is another exemplary genus of pathogenic
bacteria and includes species such as Salmonella typhimurium,
Salmonella enteriditis, Salmonella gallinarum, Salmonella pullorum,
Salmonella arizona, Salmonella heidelberg, Salmonella anatum,
Salmonella hadar, Salmonella agana, Salmonella montevideo,
Salmonella kentucky, Salmonella infantis, Salmonella
schwarzengrund, Salmonella saintpaul, Salmonella brandenburg,
Salmonella instanbul, Salmonella cubana, Salmonella bredeney,
Salmonella braenderup, Salmonella livingstone, Salmonella berta,
Salmonella california, Salmonella senfenberg, and Salmonella
mbandaka. Mycobacterium is another type of pathogenic bacteria that
is particularly harmful to humans and includes species such as
Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium
paratuberculosis, Mycobacterium bovis and Mycobacterium leprae.
[0043] Anaerobic bacterial pathogens include, for example, those in
the genera Peptostreptococci, Actinomyces, Clostridium,
Anaerobiospirillum, Fusobacterium, and Bilophila. Thus, exemplary
anaerobic bacterial pathogens include, for example,
Peptostreptococci asaccharolyticus, Peptostreptococci magnus,
Peptostreptococci micros, Peptostreptococci prevotii, Porphyromonas
asaccharolytica, a Porphyromonas canoris, Porphyromonas gingivalis,
Porphyromonas macaccae, Actinomyces israelii, Actinomyces
odontolyticus, Clostridium innocuum, Clostridium clostridioforme,
Clostridium difficile, Bacteroides tectum, Bacteroides ureolyticus,
Bacteroides gracilis (Campylobacter gracilis), Prevotella
intermedia, Prevotella heparinolytica, Prevotella oris-buccae,
Prevotella bivia, Prevotella melaninogenica, Fusobacterium
naviforme, Fusobacterium necrophorum, Fusobacterium varium,
Fusobacterium ulcerans, Fusobacterium russii, and Bilophila
wadsworthia.
[0044] Exemplary upper respiratory pathogenic bacteria include, for
example, those in the genera Pseudomonas and Legionella. Thus
exemplary upper respiratory upper respiratory pathogens include,
Pseudomonas aeruginosa, Legionella dumoffli, Legionella
longbeacheae, Legionella micdadei, Legionella oakridgensis,
Legionella feelei, Legionella anisa, Legionella sainthelensi,
Legionella bozemanii, Legionella gormanii, Legionella wadsworthii,
and Legionella jordanis.
[0045] Nonbacterial pathogens include, but are not limited to
viruses and fungi and prions. Exemplary viral pathogens include,
generally, those of classes I-VI, and more specifically, hepatitis
viruses types A-E, ebola viruses, human papilloma viruses,
keratoconjunctivitis viruses, Parvoviruses, erythroviruses,
dependoviruses, echo viruses, enteroviruses, Epstein-Barr viruses,
equine arteritis virus, equine coital exanthema virus, equine
encephalosis virus, feline sarcoma viruses, hantaviruses, herpes
viruses, human inmmunodeficiency viruses, human T-cell leukaemia
viruses, influenza viruses types A-C, JC viruses, Kirsten sarcoma
viruses, Lassa viruses, Machupo viruses, Marburg viruses,
mastadenoviruses, measles virus, Mengo viruses, Moloney murine
leukemia viruses, Newcastle Disease virus, orbiviruses, polio
viruses, retroviruses, simian immunodeficiency viruses, small pox
viruses, Tamiami viruses, and tobacco mosaic viruses. Fungal
pathogens include, for example, Pyrenophora tritici-repentis,
Drechslera sorokiniana, Rhizoctonia cerealis, Fusarium graminearum,
Fusarium culmorum, Microdochium nivale, Pseudocercosporella
herpotrichoides, Pseudocercosporella herpotrichoides, Septoria
nodorum, Septoria tritici, Cladosporium herbarum, Cercospora
arachidicola, Helminthosporium sativum, Pyrenophora teres, and
Pyrenophora tritici-repentis. It should be noted that these
pathogens are enumerated in no particular order and some overlap
may occur. Other pathogens are known in the art and identified, for
example, in Sherris Medical Microbiology: An Introduction to
Infectious Diseases, 3rd Ed. (Appleton & Lange, Stamford,
Conn., 1994).
[0046] Thus, the term "pathogen-containing fluid" refers to
nonsolid matter that is completely or partially pathogenic in
nature. Such a fluid, for example, may be comprised of liquid that
contains a pathogen minimally, partially, or fully solvated,
dispersed, or suspended therein. Examples of pathogen-containing
fluids include, without limitation, a culturing medium containing
bacterial or viral infectious agents.
[0047] Similarly, the "nonpathogenic" refers to matter that is not
pathogenic, i.e., any agent that is not likely to cause disease or
a toxic response. Nonpathogenic particles, for example, include,
without limitation, beneficial cellular matter such as
lactobacilli, yeast, epidermal cells, beads and the like.
Nonpathogenic fluids include, for example, sterile saline, glucose
solutions, and the like.
[0048] The term "radiation" is used in its ordinary sense and
refers to emission and propagation of energy in the form of a
waveform disturbance traveling through a medium such that energy is
transferred from one particle of the medium to another without
causing any permanent displacement of the medium itself. Thus,
radiation may refer, for example, to electromagnetic waveforms as
well as acoustic vibrations.
[0049] The term "reservoir" as used herein refers to a receptacle
or chamber for containing a fluid. In some instances, a fluid
contained in a reservoir necessarily will have a free surface,
e.g., a surface that allows acoustic radiation to be reflected
therefrom or a surface from which a droplet may be acoustically
ejected. A reservoir may also be a locus on a substrate surface
within which a fluid is constrained.
[0050] The term "substrate" as used herein refers to any item
having a surface onto which one or more fluids may be deposited.
The substrate may be constructed in any of a number of forms
including, for example, wafers, slides, well plates, or membranes.
In addition, the substrate may be porous or nonporous as required
for deposition of a particular fluid. Suitable substrate materials
include, but are not limited to, supports that are typically used
for solid phase chemical synthesis, such as polymeric materials
(e.g., polystyrene, polyvinyl acetate, polyvinyl chloride,
polyvinyl pyrrolidone, polyacrylonitrile, polyacrylamide,
polymethyl methacrylate, polytetrafluoroethylene, polyethylene,
polypropylene, polyvinylidene fluoride, polycarbonate, and
divinylbenzene styrene-based polymers), agarose (e.g.,
Sepharose.RTM.), dextran (e.g., Sephadex.RTM.), cellulosic polymers
and other polysaccharides, silica and silica-based materials, glass
(particularly controlled pore glass, or "CPG"), functionalized
glasses, and ceramics, as well as such substrates treated with
coatings that cover the entirety or a portion of a surface , e.g.,
treated with microporous polymers (particularly cellulosic polymers
such as nitrocellulose), microporous metallic compounds
(particularly microporous aluminum), antibody-binding proteins
(available from Pierce Chemical Co., Rockford, Ill.), bisphenol A
polycarbonate, poly-L-lysine and the like. Such coatings may be
deposited via acoustic ejection or other means, to form arrays or
other patterns on the substrate surface. Additional information
relating to the term "substrate" can be found in U.S. Patent
Application Publication No. 20020037579 to Ellson et al.
[0051] The invention thus generally relates to methods that employ
focused radiation to eject droplets of a fluid from a reservoir and
to handle pathogenic materials. Typically, focused acoustic
radiation is employed to eject droplets of a pathogenic fluid from
a reservoir. In addition, the inventive method may be used in
conjunction with a pathogen-impermeable enclosure. For example, the
method may involve positioning a substrate so that a designated
site on a substrate surface is placed in droplet-receiving
relationship with respect to the reservoir. After focused radiation
is applied to the fluid in the reservoir such that a droplet of the
fluid is deposited at the designated site, the droplet may be
sealed in the pathogen-impermeable enclosure.
[0052] The invention also provides a method of monitoring for a
change in the amount and/or concentration of a pathogen in a
pathogenic fluid. Instead of using focused acoustic radiation to
eject droplets from a reservoir, a pathogen-impermeable enclosure
is provided enclosing a pathogenic fluid that is comprised of a
pathogen and a carrier fluid. Acoustic radiation is then generated
to monitor for a change in the amount and/or concentration of the
pathogen enclosed in the pathogen-impermeable enclosure. Generally,
the invention is suited for use with biological pathogens such as
viral and bacterial matter.
[0053] FIG. 1 illustrates in simplified cross-sectional view a
system for studying the interaction between a candidate compound
and a bacterial pathogen. As with all figures referenced herein, in
which like parts are referenced by like numerals, FIG. 1 is not to
scale, and certain dimensions may be exaggerated for clarity of
presentation. The system 1 includes an enclosure 11 that serves to
enclose the other components of the system. A reservoir 13 is
provided containing a pathogen-containing fluid 15 having a fluid
surface 17. The pathogen-containing fluid is comprised of at least
one discrete bacterial pathogenic particle 19 suspended in a
carrier fluid 21. A plurality of pathogenic particles 19 is
depicted in FIG. 1A, though the pathogen-containing fluid may be
comprised of a single pathogenic particle as well. As shown,
reservoir 13 may be axially symmetric, having a vertical wall 23
extending upward from a circular reservoir base 25 and terminating
at an opening 27. The material and thickness of the reservoir base
should be such that acoustic radiation may be transmitted
therethrough and into the pathogen-containing fluid 15 within the
reservoir 13.
[0054] The system 1 also includes an acoustic device 29 comprised
of an acoustic generator 31 for generating acoustic radiation and a
focusing means 33 for focusing the acoustic radiation at a focal
point within the fluid from which a droplet is to be ejected near
the fluid surface. The acoustic device may be used as an ejector
and/or as an analyzer. The acoustic generator contains a
transducer, e.g., a piezoelectric element 35, commonly shared by an
analyzer. As shown, a combination unit 37 is provided that both
serves as a controller and a component of an analyzer. Operating as
a controller, the combination unit 37 provides the piezoelectric
element 35 with electrical energy that is converted into mechanical
and acoustic energy. Operating as a component of an analyzer, the
combination unit receives and analyzes electrical signals from the
transducer. The electrical signals are produced as a result of the
absorption and conversion of mechanical and acoustic energy by the
transducer 35.
[0055] As shown in FIG. 1, the focusing means 33 may comprise a
single solid piece having a concave surface 39 for focusing
acoustic radiation, but the focusing means may be constructed in
other ways as discussed below. The acoustic device 29 is thus
adapted to generate and focus acoustic radiation near a fluid
surface 17 when acoustically coupled to a reservoir 13. The
acoustic generator 31 and the focusing means 33 may function as a
single unit controlled by a single controller, or they may be
independently controlled, depending on the desired performance of
the device.
[0056] There are a number of ways to acoustically couple the
acoustic device 29 to the reservoir 13 and thus to the fluid
therein. One such approach is through direct contact as is
described, for example, in U.S. Pat. No. 4,308,547 to Lovelady et
al., wherein a focusing means constructed from a hemispherical
crystal having segmented electrodes is submerged in a liquid to be
ejected. However, a preferred approach is to acoustically couple
the acoustic device to the reservoir 13 and fluid 15 without
contacting any portion of the acoustic device, e.g., the focusing
means, with the fluid 15. To this end, as illustrated in FIG. 1,
the acoustic device is positioned in controlled and repeatable
acoustic coupling with fluid 15 in reservoir 13. This typically
involves direct or indirect contact between the acoustic device and
the external surface of the reservoir 13. When direct contact is
used in order to acoustically couple the acoustic device to the
reservoir, it is preferred that the direct contact be wholly
conformal to ensure efficient acoustic energy transfer. That is,
the acoustic device and the reservoir should have corresponding
surfaces adapted for mating contact. Thus, if acoustic coupling is
achieved between the acoustic device and reservoir through the
focusing means, it is desirable for the reservoir to have an
outside surface that corresponds to the surface profile of the
focusing means. Without conformal contact, efficiency and accuracy
of acoustic energy transfer may be compromised. In addition, since
many focusing means have a curved surface, the direct contact
approach may necessitate the use of a reservoir having a specially
formed inverse surface.
[0057] Optimally, acoustic coupling is achieved between the
acoustic device and the reservoir through indirect contact, as
illustrated in FIG. 1A. In this figure, an acoustic coupling medium
41 is placed between the focusing means 33 and the base 25 of
reservoir 13, with the acoustic device and reservoir located at a
predetermined distance from each other. The acoustic coupling
medium may be an acoustic coupling fluid, preferably an
acoustically homogeneous material, in conformal contact with both
the acoustic focusing means 33 and each reservoir. In addition, it
is important to ensure that the acoustic coupling medium is
substantially free of material having different acoustic properties
than the fluid medium itself. Furthermore, it is preferred that the
acoustic coupling medium be comprised of a material having acoustic
properties that facilitate the transmission of acoustic radiation
without significant attenuation of acoustic pressure and intensity.
Also, the acoustic impedance of the coupling medium should
facilitate the transfer of energy from the coupling medium into the
container. As shown, the first reservoir 13 is acoustically coupled
to the acoustic focusing means 33, such that an acoustic wave is
generated by the acoustic generator and directed by the focusing
means 37 into the acoustic coupling medium 41, which then transmits
the acoustic radiation into the reservoir 13.
[0058] Also provided is a substrate 51 having designated site 53 on
a surface 55 thereof. As shown, the substrate 51 may be a well that
together with a cover 57 form an enclosure adapted to contain a
volume of fluid, and the designated site 53 may be located within
the interior of the enclosure. A culturing fluid 59 for sustaining
and growing bacterial pathogens and, optionally, for containing a
candidate compound, may be provided in the interior of the
enclosure.
[0059] In operation as an analyzer, the acoustic device 33 may
first be positioned below reservoir 13, so as to be acoustically
coupled to the reservoir through acoustic coupling medium 41. Once
the acoustic device and the reservoir are in proper alignment, the
transducer 35 of the acoustic generator 31, as depicted in FIG. 1A,
is activated to produce acoustic radiation that is directed toward
surface 17 of the reservoir, with the amount of energy being
insufficient to eject fluid. This is typically accomplished by
using an extremely short pulse (on the order of tens of
nanoseconds) relative to that required for droplet ejection (on the
order of microseconds). The acoustic radiation will then travel in
a generally upward direction toward the free fluid surface 17. The
acoustic radiation will be reflected under different circumstances.
Typically, reflection will occur when there is a change in an
acoustic property of the medium through which the acoustic
radiation is transmitted. Thus, this first emission of focused
acoustic energy permits sonic detection of the presence of a
bacterial pathogenic particle sufficiently close to the surface for
ejection by virtue of reflection of acoustic energy by the
particle. Methods for determining the position of the particles by
sonic detection are readily apprehended by those of ordinary skill
in the art of acoustic microscopy and related arts. After a
pathogenic particle is detected and located, other properties
and/or characteristics of the carrier fluid and the pathogenic
particle may be measured before the decision to eject is made as
described in U.S. patent application Ser. No. 10/010,972,
Publication No. 20030101819, entitled "Acoustic Assessment of
Fluids in a Plurality of Reservoirs," filed Dec. 4, 2001, by
inventors Mutz, Ellson, and Foote.
[0060] It will be appreciated by those of ordinary skill in the art
that conventional or modified sonar techniques may be employed to
locate a pathogenic particle. For example, the acoustic radiation
may be reflected back at the piezoelectric element 35, where the
acoustic energy will be converted into electrical energy for
analysis. Once the analysis has been performed, a decision may be
made as to whether and/or how to dispense fluid from the reservoir.
If no particle is sufficiently close to the surface for ejection,
the acoustic energy may be focused at progressively greater
distances from the fluid surface until a pathogenic particle is
located and driven closer to the surface by focused acoustic energy
or other means. Similarly, the optimum intensity and directionality
of the ejection acoustic wave may be determined from similar types
of acoustic analysis, optionally in combination with additional
data. For example, the desired intensity and directionality of the
ejection acoustic wave may be determined by using the data from the
above-described assessment relating to reservoir volume or fluid
property data, as well as geometric data associated with the
reservoir. In addition, the data may show the need to reposition
the acoustic device so as to reposition the acoustic generator
and/or focusing means with respect to the fluid surface, in order
to ensure that the focal point of the ejection acoustic wave is
near the fluid surface where desired. Thus, positioning means 36
may be used to ensure that the acoustic device 29 and reservoir 13
are appropriately positioned to carry out acoustic
ejection/detection with proper focus.
[0061] Thus, one advantage of the invention is the ability to
selectively dispense components of the pathogenic fluid. For
example, one could select a droplet size too small to entrain a
host cell and enable separation of non-cell containing liquid from
the pathogenic fluid. There are other methods to accomplish this
type of separation including detecting the presence or absence of
cells in the ejection zone before opting to dispense the droplet.
Such sorting/selective dispensation functionalities are known in
the art See, e.g., U.S. Patent Application Publication No.
20020064808 to Mutz et al.; U.S. patent application Ser. No.
09/999,166, Publication No. 20020142286, filed Nov. 29, 2001, for
"Focused Acoustic Energy for Ejection Cells from a Fluid,"
inventors Mutz and Ellson, assigned to Picoliter Inc. (Mountain
View, Calif.); U.S. Patent Application Publication No. 20020064809
to Mutz et al.; and U.S. patent application Ser. No. 10/040,926,
Publication No. 20020090720, filed Dec. 28, 2001, for "Focused
Acoustic Ejection Cell Sorting System and Method," inventors Mutz,
Ellson, and Lee, assigned to Picoliter, Inc. (Mountain View,
Calif.).
[0062] In order to deposit droplets of fluid from the reservoir
into the well 51, a positioning means 52 is employed to align well
51 with reservoir 13. The cover 57, as depicted in FIG. 1B, is
removed from the well 51, and the well 51 is positioned above the
reservoir 13 by the positioning means 52 such that the designated
site 53 located on the interior surface of the substrate faces the
surface 17 of the fluid 15 in the reservoir. Also as shown in FIG.
1B, culturing fluid 59 may be constrained within the well 51
through surface forces. Once a particle that is sufficiently close
to the fluid surface 17 of reservoir 13 is located and is
determined to meet any other criteria for ejection, the acoustic
device 29 serves as an ejector. The acoustic generator 31 is
activated to produce an acoustic wave that is focused by the
focusing means 33 to eject a volume of fluid that forms droplet 61,
which contains a pathogenic particle 19. Generally, an ejected
droplet may not contain more than one particle when the droplet to
particle volume ratio is less than about 2:1. In some instances,
however, a droplet may contain a plurality of pathogenic particles.
For example, a single viral particle may have a cross-sectional
dimension of about 10 nm. A 1 pL droplet may contain a plurality of
viral particles of this size. One way in which the precise amount
of energy required to eject only the required volume and no more
can be determined by slowly increasing the energy applied, from an
amount insufficient to eject a particle desired for ejection, until
there is just enough energy applied to eject the droplet the
desired distance to the targeted substrate locale. After this
initial determination, approximately the same energy, with
adjustment for any change in fluid level, may be applied to eject
particles of substantially the same volume as the initial
calibration particle. As a result, droplet 61 may be ejected from
fluid surface 17 into the interior of well 51. As depicted in FIG.
1B, the droplet 61 may contain a single pathogenic particle 19.
However, a plurality of pathogenic may be ejected under some
instances. In either case, the particle or particles are thus
exposed to the candidate compound in culturing fluid 59.
[0063] Then, as shown in FIG. IC, the cover 57 is placed over the
well 51 to form an enclosure that contains the ejected pathogenic
particle 19 suspended in the culturing fluid 59 with the candidate
compound. Typically, the enclosure is sealed such that no matter
enters or exits from the enclosure while sealed. The enclosure may
then be acoustically coupled to the acoustic device so as to allow
acoustic monitoring for changes in the amount and/or concentration
of the pathogen enclosed in the enclosure. As depicted, the
acoustic device 29 may be positioned below the well 51, in order to
achieve acoustic coupling between the acoustic device and the well
through the acoustic coupling medium 41. Once the acoustic device
and the well are in proper alignment, the transducer 35 of the
acoustic generator 31 may be activated to produce acoustic
radiation in order to perform an initial assessment of the contents
of the well. The assessment may be carried out in a similar manner
as set forth herein and in U.S. patent application Ser. No.
10/010,972, Publication No. 20030101819, entitled "Acoustic
Assessment of Fluids in a Plurality of Reservoirs," filed Dec. 4,
2001, by inventors Mutz, Ellson, and Foote, and may establish a
baseline to which later assessments may be compared. As depicted in
FIG. 1D, heating elements 63 within the system enclosure 11 are
activated to bring the contents within the well to culturing
conditions and the number of pathogenic particles 19 within well 51
are increased. The acoustic assessment may then be carried out at
predetermined intervals to determine whether there are any changes
in the amount and/or concentration of the pathogen as a result of
interaction with the candidate compound. Other forms of assessment,
such as optical density, may be used.
[0064] It should be apparent that in some embodiments, the
invention relates to the use of a pathogen-impermeable enclosure as
well as to the ejection of a droplet of pathogenic fluid from a
reservoir to a substrate. Accordingly, the invention also provides
a device for dispensing one or more droplets of fluid. The device
includes a reservoir adapted to contain a fluid, an ejector for
applying focused radiation to the reservoir in a manner effective
to eject a droplet of fluid from the reservoir, and a means for
positioning a substrate such that a substrate is positioned to
receive a droplet of fluid from the reservoir. A
pathogen-impermeable enclosure is also provided for isolating the
reservoir and substrate therein.
[0065] Any of a number of different ejectors that apply focused
radiation may be used. For instance, the ejector may apply focused
electromagnetic radiation of appropriate wavelengths to eject
droplets from the reservoir. Typically, the ejector is an acoustic
ejector. Although ordinary inkjet technologies involving
piezoelectric elements may be employed, it is preferred that the
ejector be a nozzleless acoustic device that employs an acoustic
generator and a focusing means for focusing the acoustic radiation
generated thereby. Exemplary ejectors are described above and in
U.S. Patent Application Publication No. 20020037579 to Ellson et
al.
[0066] The device typically includes a detachable reservoir to
provide modularity and interchangeability of components. In
addition, the reservoir may be adapted for single use. However,
integrated or permanently attached reservoirs may be employed as
well. For any of these reservoirs, a pathogen-impermeable cover may
be provided for establishing sealing contact therewith.
[0067] As alluded to above, the invention may be used for pathogen
formatting or reformatting purposes. Thus, a plurality of
reservoirs and/or substrates may be provided for use with the
present invention. The reservoirs and/or substrates are preferably
substantially acoustically indistinguishable from each other. In
addition, the reservoirs may be provided in a single-piece unit,
such as when the reservoirs represent wells of a well plate. Many
well plates suitable for use with the device are commercially
available and may contain, for example, 96, 384, 1536, or 3456
wells per well plate. Manufacturers of suitable well plates for use
in the employed device include Corning, Inc. (Corning, N.Y.) and
Greiner America, Inc. (Lake Mary, Fla.). However, the availability
of such commercially produced well plates does not preclude the
manufacture and use of custom-made well plates containing at least
about 10,000 wells, or as many as 100,000 to 500,000 wells, or
more. To facilitate handling of multiple reservoirs, it is
preferred that the reservoirs be substantially acoustically
indistinguishable from one another. In addition, the reservoirs
should be generally arranged in a pattern or an array to provide
each reservoir with individual systematic addressability.
[0068] The reservoirs or substrates may come into contact with
pathogenic fluids, nonpathogenic fluids, or a combination of
pathogenic and nonpathogenic fluids. Furthermore, the material used
in the construction of reservoirs and substrates should be
compatible with the fluids in contact therewith. Thus, if it is
intended that the reservoirs or substrates contact an organic
solvent such as acetonitrile, polymers that dissolve or swell in
acetonitrile would be unsuitable for use in forming the reservoirs
or substrates. Similarly, reservoirs or substrates intended to
contact DMSO should be compatible with DMSO. Additional information
relating to materials selection and construction of reservoirs and
substrates, and to fluids with which the reservoirs and substrate
may come into contact, are described, for example, in U.S. Patent
Application Publication Nos. 20020037579 and 20020037375, each to
Ellson et al., and in U.S. Pat. Nos. 5,520,715 and 5,722,479 to
Oeftering.
[0069] In some instances, a microfluidic device may be used with
the invention in place of the substrate and/or reservoir.
Microfluidic devices typically have fluid-transporting features of
micrometer or submicrometer dimensions in which any number of
processes and or analytical techniques involving very small amounts
of fluid may be carried out. Microfluidic devices are available
from ACLARA BioSciences, Inc. (Mountain View, Calif.), Caliper
Technologies Corp. (Mountain View, Calif.), and Fluidigm Corp.
(South San Francisco, Calif.). The combined employment of focused
acoustic ejection and microfluidic devices is discussed in greater
detail in U.S. patent application Ser. No. 10/066,546, Publication
No. 20020125424, entitled "Acoustic Sample Introduction for
Analysis and/or Processing," filed Jan. 30, 2002, by inventors
Ellson and Mutz.
[0070] Similarly, the device may further include a means for
positioning the acoustic ejector as well as a means for positioning
the substrate, such as described, for example, in U.S. Patent
Application Publication Nos. 20020037579 and 20020037375, each to
Ellson et al. As described in these publications, either or both
positioning means may be constructed from, for example, motors,
levers, pulleys, gears, a combination thereof, or other
electromechanical or mechanical means known to one of ordinary
skill in the art. Furthermore, it will be appreciated that various
components of the device may require individual control or
synchronization. Such control or synchronization may allow for the
ejection droplets to prepare an array on a substrate surface.
[0071] Furthermore, the device may also include certain
performance-enhancing features. For example, the device may include
a cooling means for lowering the temperature of the substrate to
ensure, for example, that the ejected droplets adhere to the
substrate. The cooling means may be adapted to maintain the
substrate surface at a temperature that allows fluid to partially,
or preferably substantially, solidify after the fluid comes into
contact therewith. The device may also include a means for
maintaining fluid in the reservoirs at an appropriate temperature,
since repeated application of acoustic energy to a fluid will
result in heating, which can in turn cause unwanted changes in
fluid properties such as viscosity, surface tension, and density.
Design and construction of such temperature-maintaining means are
known to one of ordinary skill in the art and will involve
incorporation of at least one heating element and/or at least one
cooling element.
[0072] In addition, the device may further include a means for
manually manipulating items within the enclosure without
compromising the pathogenic impermeability of the enclosure. In
addition, a locating means may be provided for locating a discrete
particle in the pathogenic fluid. Exemplary locating means are
described in U.S. patent application Ser. Nos. 09/727,391,
09/999,166, and 10/033,739 (Publication Nos. 20020064808,
20020142286, and 20020090720 respectively). When the device
includes an acoustic generator, the locating means may include an
analyzer for analyzing acoustic radiation generated by the acoustic
generator. Such an analyzer is typically positioned to receive
acoustic radiation generated by the acoustic generator and
transmitted through fluid contained in the reservoir. In some
instances, as described above, the analyzer is positioned to
receive acoustic radiation reflected by a free fluid surface
contained in the reservoir. In such a case, the analyzer may
include a component common to the acoustic generator, such as a
piezoelectric element. Exemplary analyzers are described in U.S.
patent application Ser. No. 10/010,972, Publication No.
20030101819, entitled "Acoustic Assessment of Fluids in a Plurality
of Reservoirs," filed Dec. 4, 2001, by inventors Mutz, Ellson, and
Foote.
[0073] It should be noted that acoustic assessment of the
pathogenic contents of a pathogen-impermeable enclosure may be
performed in transmissive mode rather than in reflective mode. As
depicted in FIG. 2, a device 22 may be provided that includes a
reservoir 13 adapted to contain a fluid 14. As depicted, the
reservoir 13 is in a sealed state. An acoustic radiation generator
35 is positioned below the reservoirs, and analyzer 38 is
positioned in opposing relationship with the acoustic radiation
generator 35 above the reservoirs. That is, the contents 14 of the
reservoir 13 may undergo acoustic assessment when the reservoir 13
is interposed between the acoustic radiation generator 35 and the
analyzer 38. The acoustic radiation generator 35 and the analyzer
38 are acoustically coupled to the reservoir via coupling media 41
and 42, respectively. Once the acoustic radiation generator 35, the
reservoir 13, and the analyzer 38 are in proper alignment, the
acoustic radiation generator 35 is activated to produce acoustic
radiation that is transmitted through the reservoir 13 and its
contents 14 toward the analyzer 38. The received acoustic radiation
is analyzed by analyzer 38.
[0074] Thus, the invention also provides a method of monitoring for
a change in the amount and/or concentration of a pathogen in a
pathogenic fluid. As before, a pathogen-impermeable enclosure is
provided that encloses a pathogenic fluid comprising a pathogen and
a carrier fluid. The method also involves acoustically monitoring
for a change in the amount and/or concentration of the pathogen
enclosed in the pathogen-impermeable enclosure.
[0075] It should be apparent, then, that the invention permits
previously unrealized opportunities in pathogenic studies through
the use of focused acoustic radiation. For example, acoustic drop
ejection may be used to array and/or rearray infectious pathogenic
agents. This may be accomplished, for instance, by using ordinary
culturing techniques to grow a stock of bacterial pathogen in a
single reservoir that contains culturing media. In some instances,
the culturing medium may be provided as a coating on a solid
surface. For example, the bacterial pathogen may be immobilized on
a substrate surface selected for facile immobilization of cells.
Such surfaces include, for example, a collagen-derivatized surface,
dextran, polyacrylamide, nylon, polystyrene, and combinations
thereof. In some instances, the surfaces are inherently cytophilic.
In other instances, a cytophilic substrate surface is provided as a
result of surface modification. In a simple embodiment, the
culturing medium may be provided as a collagen coating on an
interior surface of a well.
[0076] Focused radiation may then be used to transfer the culture
to the wells of a well plate, wherein each well contains a
different antibiotic candidate compound. Then, each well plate may
be sealed and put in an incubator for a bacterial growth assay.
Every 30 minutes, an acoustic transducer could pass under each well
to measure the reflected acoustic energy from a suitable acoustic
pulse sent into the well and detecting a change due to the increase
in the density of bacterial particles in the plate. In this way,
growth rate inhibition for each compound could be determined.
[0077] As another example, drug compounds could be arrayed in a
series of concentrations, allowing a dose response curve to be
constructed. Throughout the process of candidate compound arraying,
bacterial regrowth, and growth measurements, the assay plate would
remain sealed. As a result, problems such as compound
cross-contamination and the danger of exposure of lab personnel to
aerosolized pathogens are substantially reduced or eliminated.
Since the acoustic frequency content of pulses sent in the wells
may be varied, one could distinguish viral particles from
bacterial, or bacterial cells from mammalian cells, in situ. Thus,
acoustics could be used in the context of an assay where an
antibody library displayed on a phage would be used to assess the
ability of the antibody to protect mammalian cells from viral
infection. The growth of mammalian cells could be readily be
distinguished from the growth of bacteria or phage via acoustic
detection, since mammalian cells are 10-1000 fold larger than
bacterial or viral particles.
[0078] Furthermore, the assay system described herein can readily
be expanded to the analysis of patient blood samples. Antibody and
other tests can be run in the sealed plate format, reducing the
chance of exposure of laboratory workers to aerosolized toxins,
bacteria, or viruses, while providing a controlled environment for
the performance of high-precision diagnostics. In addition, health
workers could collect sample specimens and transfer them to
microfluidic chips through the use of focused radiation in sealed
systems, for use in a laboratory or in fieldwork.
[0079] As another example, the invention may be used with a well
plate comprised of a plurality of wells each containing a serum
sample and a cover or lid that has a substantially planar surface
coated with a fluorophore-conjugated antibody. The lid may be then
sealed against the well plate. After ejecting droplets from the
wells to the coated lid, a homogeneous fluorescence assay may be
performed to detect the pathogen which has affinity to the
antibody. Since the cover is sealed against the well plate, there
is a lowered risk of pathogenic contamination. As a result, the
invention provides an improved method for carrying out such assays
by reducing the risk of pathogenic exposure.
[0080] In some instance, the cover or lid may be patterned with a
plurality of different conjugated antibodies bound thereto. Each
region of the cover or lid may then be sealed over a well. The
antibodies provide multiple fluorescent indicators for droplets of
fluid ejected from the well. Based on the spatial position and
other aspects of the fluorescent signal, the antibody detection
event can be identified.
[0081] This flat cover approach provides added advantages when used
in conjunction with the invention. For example, it is a relatively
simple matter to coat antibodies onto a flat surface. In addition,
by using the flat cover, the depth of field for the optics in a
fluorescent scanner is reduced. Decoupling the fluorescent read
from the well plate is also advantageous. It eliminates the
background fluorescence of any materials in the well and issues
related to the opacity of the well plate itself to the fluorescent
wavelength used for detection. Also, if any well coating is
required to promote an environment for amplifying the pathogen, the
coating may be applied independently from the application of
antibody coating on the cover.
[0082] Another advantage of the present invention is that it may be
used in applications wherein nucleotidic materials associated with
pathogen are employed and/or analyzed. For example, in order to
determine whether an individual has been infected with a viral or
bacterial pathogen, bodily fluid from the individual may be
extracted to determine whether nucleotidic sequences associated
with the pathogen is present in the bodily fluid. As such sequences
may be present in a small amount, polymerase chain reaction (PCR)
may be used in conjunction with the invention.
[0083] PCR is a well known technique that makes it possible to
start with otherwise undetectable amounts of nucleotidic material
such as DNA and create ample amounts of the material for subsequent
analysis. In essence, PCR uses a repetitive series of steps to
create copies of polynucleotide sequences located between two
primer sequences. PCR first involves mixing a template (e.g.,
target DNA to be amplified), two primer sequences selected so as to
be complementary to a portion of the template, PCR buffer, free
deoxynucleotide triphosphates, such as dATP, dCTP, dGTP, and dTTP,
and thermostable DNA polymerase. When a duplex DNA molecule is used
as the template, the DNA is denatured, using heat, into two
complementary single strands. The primers then anneal to the
strands. A subsequent cooling step allows the primers to anneal to
complementary sequences on single-stranded DNA molecules containing
the sequence to be amplified. Replication of the target sequence is
then accomplished by the DNA polymerase which produces a strand of
DNA that is complementary to the template. That is, nucleotide
monophosphate residues are linked to the primers in the presence of
a thermostable DNA polymerase to create a primer extension product.
After primer extension, twice as many duplex DNA molecules exist.
Repetition of this process doubles the number of copies of the
sequence of interest, and multiple cycles increase the number of
copies exponentially.
[0084] Since PCR requires repeated cycling between higher and lower
temperatures, PCR devices must be fabricated from materials capable
of withstanding such temperature changes. In some instances,
thermocycling may involve a denaturing step at around 90.degree. C.
to around 95.degree. C. for 5 to 60 seconds, an annealing step at
around 50.degree. C. to around 65.degree. C. for 2 to 80 seconds,
and a polymerization step at around 72.degree. C. for 5 to 120
seconds. The sample may be subjected to 30 or more cycles to
produce the desired amplification. Thermocycling may be achieved by
any suitable and convenient method, e.g., using commercially
available thermocyclers, a heating block apparatus, and/or an
infrared radiation source in conjunction with cooling devices.
After thermocycling is complete, a PCR sample may be cooled to a
temperature of around 4.degree. C. for subsequent analysis,
processing, treatment or testing. Thus, the materials from which
the pathogen-impermeable enclosures of the present invention are
made, including for example, any wells, lids, covers, etc. that
serve as components of the pathogen-impermeable enclosures, should
be mechanically and chemically stable at high temperatures, and
capable of withstanding repeated temperature changes without
mechanical degradation. Furthermore, the materials should be
compatible with the PCR reaction itself, and not inhibit the
polymerase or bind DNA. Reactants for the PCR reaction may require
encapsulation in a water-impermeable substance such as mineral oil
to avoid drying in the thermocycling process.
[0085] It should be noted that other nucleic acid amplification
and/or reaction techniques are known in the art and that the term
"PCR" encompasses such additional techniques as well. That is, the
reference to the term "PCR" is intended to include ligase chain
reactions, rolling circle amplification, repair chain reactions and
other techniques involving reaction mixtures that undergo
denaturation, annealing and extension processes.
[0086] The assay system as described above may be adapted for use
with PCR techniques. For example, in certain PCR based techniques,
a sample may be mixed with PCR reagents as well as with a detector
for the presence of DNA to quantify the DNA generated by the
reaction and/or to determine the presence or identity of a specific
pathogen. Accordingly, an exemplary assay involves the ejection of
pathogen-containing sample droplets onto a lid, which may then be
placed in sealing contact with a well of a well plate to form a
pathogen-impermeable enclosure. The ejection may take place either
before or after the sample is combined with the PCR reagents. Then,
the enclosure is placed in a thermocycler for DNA amplification and
for subsequent pathogen identification. The results of the assay
can be determined by a variety of methods including the use of the
optional DNA quantification material added to the PCR reagent.
Methods for selection of primer pairs, both as positive and
negative controls for accurate pathogen identification and
determination of assay results, are known to those of skill in the
art.
[0087] It is to be understood that while the invention has been
described in conjunction with the preferred specific embodiments
thereof, the foregoing description is intended to illustrate and
not limit the scope of the invention. Other aspects, advantages,
and modifications will be apparent to those skilled in the art to
which the invention pertains.
[0088] All patents, patent applications, journal articles, and
other references cited herein are incorporated by reference in
their entireties.
* * * * *