U.S. patent application number 13/816648 was filed with the patent office on 2013-06-13 for microdroplet-manipulation systems and methods for automated execution of molecular biological protocols.
This patent application is currently assigned to the Arizona Board of Regents on Behalf of the University of Arizona. The applicant listed for this patent is Jeong-Yeol Yoon, David J. You. Invention is credited to Jeong-Yeol Yoon, David J. You.
Application Number | 20130149710 13/816648 |
Document ID | / |
Family ID | 45810942 |
Filed Date | 2013-06-13 |
United States Patent
Application |
20130149710 |
Kind Code |
A1 |
Yoon; Jeong-Yeol ; et
al. |
June 13, 2013 |
MICRODROPLET-MANIPULATION SYSTEMS AND METHODS FOR AUTOMATED
EXECUTION OF MOLECULAR BIOLOGICAL PROTOCOLS
Abstract
Disclosed herein are automated systems for performing various
biochemical and molecular biological procedures, including
processor-controlled execution of protocols involving multiple
steps performed in, on, or with liquid microdroplets. Example
protocols are the various Polymerase Chain Reaction (PCR)
protocols, but the subject systems are not limited to performing
PCR protocols. Formation of a microdroplet of the sample for use in
the described systems is achieved by bringing an amount of the
sample into contact with a hydrophobic milieu, such as a
superhydrophobic surface or hydrophobic liquid.
Inventors: |
Yoon; Jeong-Yeol; (Tucson,
AZ) ; You; David J.; (Tucson, AZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Yoon; Jeong-Yeol
You; David J. |
Tucson
Tucson |
AZ
AZ |
US
US |
|
|
Assignee: |
the Arizona Board of Regents on
Behalf of the University of Arizona
Tucson
AZ
|
Family ID: |
45810942 |
Appl. No.: |
13/816648 |
Filed: |
September 6, 2011 |
PCT Filed: |
September 6, 2011 |
PCT NO: |
PCT/US2011/050553 |
371 Date: |
February 12, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61402901 |
Sep 7, 2010 |
|
|
|
Current U.S.
Class: |
435/6.12 ;
435/287.2; 435/289.1; 435/303.1; 435/91.2 |
Current CPC
Class: |
B01L 7/52 20130101; G01N
2035/1037 20130101; G01N 35/1009 20130101; C12Q 1/686 20130101 |
Class at
Publication: |
435/6.12 ;
435/289.1; 435/303.1; 435/287.2; 435/91.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. An apparatus, comprising: a movement and placement device having
a range of movement in at least two dimensions; a
microdroplet-manipulating device coupled to the movement and
placement device and placeable by the movement and placement device
within the range of movement; a hydrophobic milieu located within
the range of movement; and a controller operably connected to the
movement and placement device and to the microdroplet-manipulating
device, the controller being programmable with a protocol in which
the movement and placement device is commanded to place the
microdroplet-manipulating device relative to the hydrophobic
milieu, and the microdroplet-manipulating device is commanded to
perform automatically at least two of placing an amount of a
hydrophilic liquid in contact with the hydrophobic milieu
sufficiently to form at least one microdroplet of the hydrophilic
liquid on or in the hydrophobic milieu, manipulating the
microdroplet while the microdroplet is in contact with the
hydrophobic milieu, and removing at least a portion of a
microdroplet from contact with the hydrophobic milieu.
2. The apparatus of claim 1, wherein the controller is further
configured to: receive a command to stop execution of the protocol;
accept from the user a change, in the protocol, of steps as
programmed in the controller; and resume execution of the protocol
with the change.
3. The apparatus of claim 1, wherein the controller is further
configured, as requested by a user: to stop execution of a first
protocol, to accept from the user a new protocol, and to commence
the second protocol in place of the first protocol.
4. The apparatus of claim 1 wherein the controller is further
configured, as requested by a user: to except from the user a
change in a preset protocol of steps as programmed in the
controller; and execute the protocol with the change without the
user having to change a component of the apparatus.
5. The apparatus of claim 1, wherein: the hydrophobic milieu
comprises a superhydrophobic surface and a hydrophobic liquid
having different respective locations in the range of movement; and
the controller is further programmed to command the
microdroplet-manipulating device to place an amount of a first
hydrophilic liquid on the superhydrophobic surface to form a first
microdroplet on the superhydrophobic surface and to place an amount
of a second hydrophilic liquid in the hydrophobic liquid to form a
second microdroplet in the hydrophobic liquid.
6. The apparatus of claim 5, wherein the controller is further
programmed to command the microdroplet-manipulating device to
manipulate the first microdroplet on the superhydrophobic surface
and to command the microdroplet-manipulating device to manipulate
the second microdroplet in the hydrophobic liquid.
7. The apparatus of claim 6, wherein: the command to manipulate the
first microdroplet on the superhydrophobic surface is part of a
first protocol; and the command to manipulate the second
microdroplet in the hydrophobic liquid is part of a second
protocol.
8. The apparatus of claim 7, wherein: the first protocol comprises
extracting genetic material from a sample; and the second protocol
comprises amplifying a predetermined portion of the extracted
genetic material.
9. The apparatus of claim 1, wherein the hydrophobic milieu
comprises a superhydrophobic surface.
10. The apparatus of claim 9, wherein manipulating the microdroplet
on the superhydrophobic surface comprises at least one of: moving
the microdroplet, adding a substance to the microdroplet, removing
a substance from the microdroplet, mixing contents of the
microdroplet, concentrating the microdroplet, agitating the
microdroplet, changing a volume of the microdroplet, changing a
shape of the microdroplet, changing a density of the microdroplet,
changing a composition of the microdroplet, changing a position of
the microdroplet, holding a substance relative to the microdroplet,
merging the microdroplet with another microdroplet, splitting the
microdroplet, and rotating the microdroplet.
11. The apparatus of claim 9, wherein manipulating the microdroplet
on the superhydrophobic surface comprises rotating the microdroplet
relative to the surface sufficiently to centrifuge the
microdroplet.
12. The apparatus of claim 9, wherein manipulating the microdroplet
on the superhydrophobic surface comprises extracting genetic
material from the microdroplet.
13. The apparatus of claim 9, wherein manipulating the microdroplet
on the superhydrophobic surface comprises serially diluting the
microdroplet.
14. The apparatus of claim 9, wherein the controller is further
configured to: receive a program comprising a first series of steps
in the protocol in which at least one step involves automatically
manipulating a microdroplet on the superhydrophobic surface; and
execute the first series of steps automatically upon command.
15. The apparatus of claim 14, wherein manipulating a microdroplet
on the superhydrophobic surface comprises at least one of: moving
the microdroplet, adding a substance to the microdroplet, removing
a substance from the microdroplet, mixing contents of the
microdroplet, concentrating the microdroplet, agitating the
microdroplet, changing a volume of the microdroplet, changing a
shape of the microdroplet, changing a density of the microdroplet,
changing a composition of the microdroplet, changing a position of
the microdroplet, holding a substance relative to the microdroplet,
merging the microdroplet with another microdroplet, splitting the
microdroplet, and rotating the microdroplet.
16. The apparatus of claim 9, wherein manipulating the microdroplet
further comprises: removing the microdroplet from the
superhydrophobic surface; and amplifying a nucleotide sequence in
the microdroplet.
17. The apparatus of claim 1, wherein the hydrophobic milieu
comprises a hydrophobic liquid.
18. The apparatus of claim 17, wherein manipulating a microdroplet
in the hydrophobic liquid comprises at least one of: moving the
microdroplet, adding heat to the microdroplet, removing heat from
the microdroplet, placing the microdroplet in a pendant condition
relative to the hydrophobic liquid, and retracting the
microdroplet.
19. The apparatus of claim 17, wherein the controller is further
configured to: receive a program comprising a first series of steps
in the protocol in which at least one step involves automatically
manipulating a microdroplet in the hydrophobic liquid; and execute
the first series of steps automatically upon command.
20. The apparatus of claim 17, further comprising a vessel
comprising at least one temperature-regulated chamber located
within the range of movement, the vessel containing the hydrophobic
liquid at a preset temperature.
21. The apparatus of claim 20, wherein the hydrophobic liquid
comprises a silicone oil.
22. The apparatus of claim 20, wherein the protocol comprises:
commanding the microdroplet-manipulating device to move a
microdroplet to the hydrophobic liquid in the temperature-regulated
chamber; commanding the microdroplet-manipulating device to
manipulate the microdroplet while the microdroplet is submerged in
the hydrophobic liquid in the temperature-regulated chamber; and
commanding the microdroplet-manipulating device to remove the
microdroplet from the temperature-regulated chamber.
23. The apparatus of claim 20, wherein the vessel comprises at
least three temperature-regulated chambers each containing a
respective volume of the hydrophobic liquid at a respective
temperature.
24. The apparatus of claim 23, wherein: the vessel further
comprises respective channels connecting the chambers together in a
cyclical manner; each channel also contains a respective volume of
the hydrophobic liquid; and the channels open into the respective
chambers.
25. The apparatus of claim 20, wherein the vessel comprises: a
first temperature-regulated chamber in which the hydrophobic liquid
is at a nucleic acid-denaturation temperature; a second
temperature-regulated chamber in which the hydrophobic liquid is at
a nucleic acid annealing temperature; and a third
temperature-regulated chamber in which the hydrophobic liquid is at
a nucleic acid extension temperature.
26. The apparatus of claim 25, wherein: the denaturation
temperature is in a range of about 94-96.degree. C.; the annealing
temperature is in the range of about 50-65.degree. C.; and the
extension temperature is in a range of about 70-74.degree. C.
27. The apparatus of claim 26, wherein the controller is further
configured to command the movement and positioning device to move
the microdroplet-manipulating device in a manner such that the
microdroplet, while being kept submerged in the hydrophobic liquid,
is moved from one chamber of the vessel to the next according to a
preset thermal cycle.
28. The apparatus of claim 27, wherein the controller is further
configured to command the microdroplet-manipulating device to hold
the microdroplet in each chamber for a preset respective length of
time.
29. The apparatus of claim 28, wherein the
microdroplet-manipulating device is configured to hold the
microdroplet in each chamber while the microdroplet is pendant from
the microdroplet-manipulating device and the
microdroplet-manipulating device moves the pendant droplet relative
to the hydrophobic liquid.
30. The apparatus of claim 29, wherein: the
microdroplet-manipulating device is further configured to retract
the microdroplet as the microdroplet-manipulating device is moving
the microdroplet through a respective channel from one chamber to
the next in the cycle; and the microdroplet-manipulating device is
further configured to de-retract the microdroplet as the
microdroplet is being held by the microdroplet-manipulating device
in a subsequent chamber of the cycle.
31. The apparatus of claim 30, wherein the cycle is a PCR
cycle.
32. The apparatus of claim 1, wherein the microdroplet-manipulating
device comprises a syringe fitted with a tip.
33. An apparatus, comprising: a movement and placement device
defining a preset motion range in at least two of x-, y-, and
z-dimensions; a microdroplet-manipulating device coupled to and
movable by the controlled movement and placement device in the
preset range; a superhydrophobic surface located in the preset
range; a temperature-controlled vessel located in the preset range
and containing a hydrophobic liquid, the vessel comprising multiple
interconnected chambers each held at a respective temperature; and
a controller operably connected to the movement and placement
device and to the microdroplet-manipulating device, the controller
being configured to command the movement and positioning device to
place the microdroplet-manipulating device relative to the
superhydrophobic surface and to command the movement and placement
device to perform automatically at least two of placing an aqueous
liquid microdroplet on the superhydrophobic surface, manipulating
the microdroplet on the superhydrophobic surface, removing at least
a portion of the microdroplet from the superhydrophobic surface,
placing the microdroplet in a chamber of the temperature-controlled
vessel, manipulating the microdroplet in the chamber, and removing
at least a portion of the microdroplet from the chamber.
34. The apparatus of claim 33, wherein the
microdroplet-manipulating device comprises a syringe fitted with a
tip.
35. The apparatus of claim 34, wherein the controller and syringe
are further configured, whenever the microdroplet is in a chamber
of the vessel and submerged in the hydrophobic liquid, to hold the
microdroplet in a pendant manner from the tip and to move the
microdroplet relative to the hydrophobic liquid.
36. The apparatus of claim 34, wherein the
microdroplet-manipulating device further comprises a
vibration-producing device disposed in contact with the syringe so
as to, when actuated, vibrate the syringe in an orbital manner
sufficiently to impart a corresponding stirring motion of the
microdroplet in contact with the tip.
37. The apparatus of claim 33, wherein the vessel comprises a first
chamber containing a hydrophobic liquid held at a nucleic
acid-denaturation temperature, a second chamber containing the
hydrophobic liquid held at a nucleic acid-annealing temperature,
and a third chamber containing the hydrophobic liquid held at a
nucleic acid-extending temperature.
38. The apparatus of claim 37, wherein the vessel comprises
respective channels connecting the chambers together, each channel
containing a respective volume of the hydrophobic liquid.
39. The apparatus of claim 38, wherein: the
microdroplet-manipulating device comprises a syringe fitted with a
tip; and the controller and syringe are further configured to
retract the microdroplet into the tip whenever the microdroplet is
being moved, by respective motion of the syringe imparted by the
movement and placement device, through a channel from one chamber
to the next.
40. The apparatus of claim 33, wherein the controller is programmed
to execute at least two protocols on a microdroplet, the protocols
including a first protocol comprising at least one step performed
with the microdroplet on the superhydrophobic surface and a second
protocol comprising at least one step performed with the
microdroplet in the vessel submerged in the hydrophobic liquid.
41. The apparatus of claim 40, wherein the first protocol is
directed to at least one of releasing genetic material from cells
in the microdroplet, diluting the microdroplet, centrifuging the
microdroplet, lysing the microdroplet, precipitating genetic
material in the microdroplet, collecting genetic material from the
microdroplet, washing and drying collected genetic material from
the microdroplet, and rehydrating collected genetic material from
the microdroplet.
42. The apparatus of claim 41, wherein: the
microdroplet-manipulating device comprises a syringe fitted with a
tip; and collecting genetic material from the microdroplet
comprises adhering the genetic material to the tip.
43. The apparatus of claim 42, wherein collecting genetic material
further comprises rotating the tip as the genetic material adheres
to the tip.
44. An apparatus for amplifying genetic material, obtained from a
sample, by a thermocycling protocol, the apparatus comprising: a
movement and placement device; a microdroplet-manipulating device
coupled to the movement and placement device and movable by the
movement and placement device in a region of space defined by the
movement and placement device; a multi-chamber array comprising at
least three chambers hydraulically connected together and
containing a hydrophobic liquid, each channel being held at a
respective temperature for a respective stage in the thermocycling
protocol; and a controller operably connected to the movement and
placement device and to the microdroplet-manipulating device, the
controller being configured to command the movement and placement
device to place the microdroplet-manipulating device relative to
the chambers of the array in a cyclic manner at respective times,
each cycle comprising placing the microdroplet-manipulating device
relative to a first chamber of the array at which the
microdroplet-manipulating device submerges a microdroplet being
carried by the microdroplet-manipulating device in the hydrophobic
liquid in the first chamber for a first defined length of time, to
transport the microdroplet through the respective channel to a
second chamber while keeping the droplet submerged in the
hydrophobic liquid for a defined second length of time, to
transport the microdroplet through the respective channel to a
third chamber while keeping the droplet submerged in the
hydrophobic liquid for a defined third length of time, and to
return the microdroplet through the respective channel to the first
chamber while keeping the droplet submerged in the hydrophobic
liquid; and repeating the cycle to produce a desired amplification
of the genetic material.
45. The apparatus of claim 44, further comprising a
superhydrophobic surface situated within the region of space and
reachable by the microdroplet-manipulating device being
controllably moved by the movement and placement device.
46. The apparatus of claim 45, wherein the controller is further
configured to command the movement and placement device before,
during, or after performing the thermocyclic protocol, to place the
microdroplet-manipulating device relative to the superhydrophobic
surface and to command the microdroplet-manipulating device to
perform automatically at least two of placing a liquid microdroplet
on the superhydrophobic surface, manipulating the microdroplet on
the superhydrophobic surface, and removing at least a portion of
the microdroplet from the superhydrophobic surface.
47. The apparatus of claim 44, further comprising a real-time
cycle-detection device associated with the multi-chamber array.
48. The apparatus of claim 47, wherein the real-time
cycle-detection device comprises, in association with the channel
between the third and first chamber, a light source directing light
across the chamber so as to be incident on liquid passing through
said channel, and a light detector situated to detect light from
the source that has passed through said channel.
49. An apparatus, comprising: surface means for forming a
microdroplet whenever a corresponding volume of a hydrophilic
liquid is situated on the surface means; means for placing the
microdroplet on the surface means and removing the microdroplet
from the surface means; means for manipulating the microdroplet
including while the microdroplet is situated on the surface means;
and control means for actuating said placing means and said
manipulating means at preset moments in at least one protocol
including at least one manipulation of the microdroplet on said
surface means.
50. The apparatus of claim 49, wherein said manipulating means
comprises one or more of: means for moving a microdroplet, means
for adding a substance to a microdroplet, means for removing a
substance from a microdroplet, means for mixing contents of a
microdroplet, means for concentrating a microdroplet, means for
agitating a microdroplet, means for removing a portion of a
microdroplet, means for changing a volume of a microdroplet, means
for changing a shape of a microdroplet, means for changing a
density of a microdroplet, means for changing a composition of a
microdroplet, means for changing a position of a microdroplet,
means for holding a substance relative to a microdroplet, means for
merging the microdroplet with another microdroplet, means for
splitting the microdroplet, and means for rotating the
microdroplet.
51. The apparatus of claim 49, further comprising thermocycling
means, wherein said control means further actuates said placing
means and said manipulating means to place a microdroplet at preset
temperatures in said thermocycling means.
52. The apparatuss of claim 51, further comprising real-time
cycle-quantification means.
53. A method for performing a protocol on a hydrophilic liquid
sample, comprising: placing an amount of the sample in contact with
a first hydrophobic milieu to form at least one microdroplet of the
sample in or on the first hydrophobic milieu; and performing at
least one step of a first protocol on, in, or with the microdroplet
in or on the first hydrophobic milieu.
54. The method of claim 53, wherein: the first hydrophobic milieu
comprises a superhydrophobic surface; and the at least one step is
selected from the group consisting of: moving the microdroplet,
adding a substance to the microdroplet, removing a substance from
the microdroplet, mixing contents of the microdroplet,
concentrating the microdroplet, agitating the microdroplet,
changing a volume of the microdroplet, changing a shape of the
microdroplet, changing a density of the microdroplet, changing a
composition of the microdroplet, changing a position of the
microdroplet, holding a substance relative to the microdroplet,
merging the microdroplet with another microdroplet, splitting the
microdroplet, and rotating the microdroplet.
55. The method of claim 53, further comprising: placing an amount
of a second sample in contact with a second hydrophobic milieu to
form at least one microdroplet of the second sample in or on the
second hydrophobic milieu; and performing at least one step of a
second protocol on, in, or with the microdroplet of the second
sample in or on the second hydrophobic milieu.
56. The method of claim 55, wherein: the second protocol comprises
thermocycling the second sample; and the second hydrophobic milieu
is a hydrophobic liquid; and the at least one step of the second
protocol is produced with the microdroplet of the second sample
immersed in the hydrophobic liquid.
57. The method of claim 56, wherein the thermocycling is a
respective portion of a PCR protocol.
58. The method of claim 57, wherein the PCR protocol comprises
quantitative monitoring, in real time, products of the PCR
protocol.
59. The method of claim 58, wherein thermocycling the second sample
comprises: while keeping the microdroplet of the second sample
immersed in the hydrophobic liquid, placing the microdroplet in a
first chamber containing the hydrophobic liquid heated to a first
temperature; while keeping the microdroplet of the second sample
immersed in the hydrophobic liquid, placing the microdroplet in a
second chamber containing the hydrophobic liquid heated to a second
temperature; while keeping the microdroplet of the second sample
immersed in the hydrophobic liquid, placing the microdroplet in a
third chamber containing the hydrophobic liquid heated to a third
temperature; and repeating at least once the placements in the
first, second, and third chambers.
60. The method of claim 59, wherein: at least one of the first,
second, and third placements comprises a respective incubation time
of the microdroplet in the heated hydrophobic liquid in the
respective chamber; and the respective incubation is conducted
while the microdroplet is in a pendant condition in the hydrophobic
liquid in the respective chamber.
61. The method of claim 60, wherein the respective incubation is
conducted while the microdroplet is also being moved relative to
the hydrophobic liquid to achieve convective heating of the
microdroplet in the hydrophobic liquid in the respective
chamber.
62. The method of claim 55, wherein the second sample is a product
of the first protocol performed on the first sample.
63. The method of claim 53, wherein: the first hydrophobic milieu
comprises a hydrophobic liquid; and the at least one step comprises
exposing the first microdroplet to a first temperature while the
first microdroplet is immersed in the hydrophobic liquid.
64. The method of claim 63, wherein the at least one step further
comprises exposing the first microdroplet to a second temperature
while the first microdroplet is immersed in the hydrophobic
oil.
65. The method of claim 64, wherein the first and second
temperatures are respective portions of a PCR thermocycle.
66. The method of claim 53, further comprising: placing an amount
of the sample in contact with a second hydrophobic milieu to form
at least one microdroplet of the sample in or on the second
hydrophobic milieu; and performing at least one step of a second
protocol on, in, or with the microdroplet in or on the second
hydrophobic milieu, the second protocol comprising performing PCR
on a genetic material in the microdroplet.
67. The method of claim 66, wherein: the first hydrophobic milieu
comprises a superhydrophobic surface; the microdroplet is formed on
the superhydrophobic surface; and performing PCR comprises removing
the microdroplet from the superhydrophobic surface to a PCR cycler
comprising a first chamber, a second chamber, and a third chamber,
each containing an inert hydrophobic liquid heated to a respective
temperature, wherein the microdroplet is held in each chamber for a
specified time according to the respective step in PCR; and moving
the microdroplet from chamber to chamber in a cyclic manner to
achieve thermocycling of the microdroplet according to PCR.
68. The method of claim 67, wherein: the PCR cycler further
comprises respective channels connecting the chambers together,
each channel also containing the inert hydrophobic liquid; and
movement of the microdroplet from chamber to chamber is conducted
through the respective channels while the microdroplet remains
submerged in the inert hydrophobic liquid.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to, and the benefit of,
U.S. Provisional Patent Application No. 61/402,901, filed Sep. 7,
2010, which is incorporated herein by reference in its
entirety.
FIELD
[0002] This disclosure pertains to, inter alia, automated systems
for performing various biochemical and molecular biological
procedures, including processor-controlled execution of protocols
involving multiple steps performed in, on, or with liquid
microdroplets. Example protocols are the various Polymerase Chain
Reaction (PCR) protocols, but the subject systems are not limited
to performing PCR protocols.
BACKGROUND
[0003] Most protocols used in biochemistry and molecular biology
comprise multiple steps that must be executed properly and in the
proper order. Exemplary steps include mixing, diluting,
centrifuging, separating, extracting, suspending, heating, cooling,
reacting, dispensing, and the like. Many of these steps are
similar, at least in part, in various protocols. A principal
difference of one protocol versus another is often the particular
order in which the steps are executed. Another difference is a
different value of a parameter (e.g., temperature or reaction time)
in one protocol versus another. Yet another difference is the
inherent variability of different people performing nominally
similar steps.
[0004] An example of a molecular biological protocol involving a
defined series of steps, in which most of the steps are repeated
multiple times, is the Polymerase Chain Reaction (PCR) protocol.
Since its inception, PCR has been modified and tailored for use in
multiple specific situations. Consequently, although there are
currently multiple PCR protocols, many of the steps in them are
substantially the same because, for example, substantially all
existing protocols involve performing multiple temperature
"cycles." This has generated interest in automating PCR protocols.
In addition, there have been attempts to derive PCR protocols that
use progressively smaller sample and reagent volumes and that can
perform a PCR cycle in less time.
[0005] For example, there have been various attempts to incorporate
the complete process of PCR assays into microchannels, using
microfluidics, to reduce sample volumes. Since a microchannel is a
fluid passageway formed with a specific configuration, it cannot be
changed easily to accommodate departures from the particular
protocol for which the microchannel was configured. Consequently, a
key problem with this approach is that the user cannot change the
assay protocol easily. Also, the small size of the microchannel
device complicates isolating the different locations at which
respective thermal steps in the PCR cycle are conducted, and
prevent the removal and characterization of sample aliquots at any
stage in the PCR cycle.
[0006] Decreasing the sample size inevitably involves the
production and manipulation of small droplets (e.g.,
"microdroplets") of liquid. In the various efforts at automating
laboratory procedures, manipulating small droplets has been the
focus of much attention in recent years. Berthier and Silberzan,
Microfluidics for Biotechnology, Artech House, Norwood, 2006. Use
of small droplets allows significantly smaller reaction volumes and
decreased assay times. The two primary modes of conventional
droplet manipulations are: (1) manipulating discrete liquid plugs
in pre-defined microchannels (Belder, Angew. Chem. Int. Ed.
44:3521-3522, 2005; Joanicot and Ajdari, Science 309:887-888,
2005), and (2) manipulating liquid droplets resting on an open,
flat surface (Mugele and Baret, J. Phys: Condens. Matter
17:R705-R774, 2005; Su et al., ACM Trans. Design Autom. Electron
Syst. 11:442-464, 2006). The first mode (1) incorporates "liquid
plug" techniques and the second mode (2) incorporates
"open-surface" techniques. Changing an automated liquid-plug
technique is difficult essentially because changing the technique
involves changing the microfluidic plumbing. Open-surface
techniques, involving the manipulation of liquid droplets on an
open, flat surface, has been demonstrated most notably in
association with magnetofluidics, in which the droplets containing
paramagnetic particles move over a hydrophobic surface under the
influence of an external magnetic field. Egatz-Gomez et al., Appl.
Phys. Lett. 89:034106, 2006. A difficulty with this technique is
that paramagnetic particles have not yet been designed that do not
interfere with biological reactions. Another technique in this
general category is called electrowetting on dielectrics ("EWOD").
Cho et al., J. Microelectromech. Syst. 12:70-80, 2003. Although
this technique allows precise droplet movement, splitting, and
merging, an apparatus performing this method is comparatively
difficult to fabricate and operate, and has problems with
diffusional mixing and contamination from increased wetting on the
surface. Thus, a continuing need exists for improved systems and
methods of manipulating biological liquids in a microfluidic
environment.
SUMMARY
[0007] Disclosed herein are methods and apparatus for performing,
in a controlled and programmed automatic manner, various protocols
on microdroplet-sized samples of hydrophilic liquids. The protocols
are particularly directed to respective processes performed in
biochemical and/or molecular biological investigations. The
protocols are executed by respective steps involving respective
manipulations of microdroplets.
[0008] Formation of a microdroplet of the sample is achieved by
bringing an amount of the sample into contact with a "hydrophobic
milieu." As the amount of the sample interacts with the hydrophobic
milieu, a microdroplet of the sample is formed. The microdroplet
form is retained as long as it remains in contact with the
hydrophobic milieu. One form of the hydrophobic milieu is a
superhydrophobic surface on which the sample forms microdroplets
having a contact angle of at least 150.degree. but desirably close
to, but no greater than, 180.degree.. Another form of the
hydrophobic milieu is a hydrophobic liquid in which the sample is
immersed to form the microdroplet. With respect to either milieu,
the microdroplet retains its microdroplet form so long as it
remains in contact with the respective milieu. The volume of a
particular microdroplet is substantially unlimited in terms of
minimal volume. In other words, the microdroplets can be as small
as desired, so long as they can be manipulated. Maximum volume of a
microdroplet is determined by the particular volume limit past
which the microdroplet is no longer substantially spherical.
Depending upon the particular sample and particular hydrophobic
milieu, the maximum volume can range from about 20 .mu.L, to about
100 .mu.L, more typically in the range of 20-50 .mu.L.
[0009] The protocols can be any of various "wet" processes that can
be performed on a chemical or biological sample, such as a sample
collected in the field or a sample produced in a laboratory. In an
advantageous embodiment the processes are of generally two types:
thermocycling protocols and pre-thermocycling protocols. Not
intending to be limiting, an important thermocycling protocol is
one of the Polymerase Chain Reaction (PCR) protocols. Important
pre-thermocycling protocols include, but are not limited to,
dilutions of the sample, extraction of genetic material from the
sample, centrifuging the sample, and extracting genetic material
from the sample.
[0010] The systems are of a size permitting them to be placed on a
benchtop, such as in a laboratory. Some embodiments are
miniaturized to an extent that they can be taken into the field for
on-site processing and identification of samples collected
there.
[0011] PCR protocols performable using the subject apparatus are
rapid compared to most conventional apparatus. A key reason for
this speed is the inclusion, in many embodiments, of a
thermocycling vessel or "PCR vessel" having multiple chambers each
containing the hydrophobic liquid. The chambers have respective
heaters and temperature sensors to provide respective volumes of
the hydrophobic liquid at respective temperatures. In many
embodiments three chambers are provided so as to provide a
respective chamber for each of the temperatures normally required
in a PCR cycle. The chambers are connected together by channels
that also contain the hydrophobic liquid. The channels allow
microdroplets to be transferred from chamber to chamber without
being removed from the hydrophobic milieu.
[0012] The subject apparatus also include a "movement and placement
device" to which a "microdroplet manipulation device" is coupled.
The movement and placement device in many embodiments comprises a
processor-controlled manipulator such as an x-y or x-y-z
manipulator to which a block or member (called herein a "carriage")
is mounted. In this disclosure the processor-controlled manipulator
is also called a "wire-guide system" for reasons discussed
elsewhere herein.
[0013] Formed microdroplets tend to remain as microdroplets so long
as they remain in contact with their respective hydrophobic milieu.
Substantially all the protocols performable using the system
involve manipulations of one or more microdroplets. Example
protocols include, but are not limited to, dilution of
microdroplets (e.g., serial dilution), centrifugation of
microdroplets in situ, extraction of DNA or other genetic material,
and rapid PCR thermocycling. Depending upon the specific protocol,
it is performed either on a superhydrophobic surface or in a
hydrophobic liquid. Some protocols can be performed on either
hydrophobic milieu. The system has substantially no limitations on
the complexity and configuration of protocols that it can perform,
making it extremely versatile and far-reaching in its
applications.
[0014] The system can perform substantially any
microdroplet-manipulation-based protocol pre-programmed into the
system (more specifically into the controller). Modifications
required for adding a protocol or changing a protocol are simply
made by changing the programming, which can be easily accomplished
by the operator. Because of this flexibility, the user has the
ability to start and stop execution of a protocol at substantially
any time, manually control the droplet-manipulation system, make
adjustments to the protocol on-the-fly, and resume a suspended
protocol where it left off. The system can be used to execute
pre-programmed droplet movements and manipulations for the rapid
detection of a particular microorganism by PCR. In such an
application, the microdroplet movements and manipulations can
include serial dilutions, centrifugation, extraction of genetic
material from a droplet, and amplification of the genetic material
by PCR thermocycling. As confirmed by post-PCR gel electrophoresis,
the processes performed by the system produced greater positive
band intensity over a non-centrifuged sample. Thus, the system has
the adaptability to replace many common laboratory tasks on a
single platform (through re-programmability), and rapid succession
(using droplets) and with a high level of accuracy and
automation.
[0015] It will be understood that the microdroplet-manipulating
device may have a first portion that remains stationary and a
second portion that is moved by the movement and placement
device.
[0016] One aspect as disclosed herein is directed to apparatus as
summarized above. An embodiment of such an apparatus comprises a
movement and placement device having a range of movement in at
least two dimensions (e.g., x and y, or x, y, and z). A
microdroplet-manipulating device is coupled to the movement and
placement device so as to be placeable by the movement and
placement device within the range of movement. A hydrophobic milieu
is located within the range of movement. A controller is operably
connected to the movement and placement device and to the
microdroplet-manipulating device. The controller is programmable
with a protocol in which the movement and placement device is
commanded to place the microdroplet-manipulating device relative to
the hydrophobic milieu. The microdroplet-manipulating device is
commanded to perform automatically at least two of the following:
(a) placing an amount of a hydrophilic liquid in contact with the
hydrophobic milieu sufficiently to form at least one microdroplet
of the hydrophilic liquid on or in the hydrophobic milieu, (b)
manipulating the microdroplet while the microdroplet is in contact
with the hydrophobic milieu, and (c) removing at least a portion of
a microdroplet from contact with the hydrophobic milieu.
[0017] In this embodiment the hydrophobic milieu can comprise: (1)
a superhydrophobic surface, and (2) a hydrophobic liquid. These two
milieu have different respective locations in the range of
movement. In such an embodiment the controller is further
programmed to command the microdroplet-manipulating device to place
an amount of a first hydrophilic liquid on the superhydrophobic
surface to form a first microdroplet on the superhydrophobic
surface and to place an amount of a second hydrophilic liquid in
the hydrophobic liquid to form a second microdroplet in the
hydrophobic liquid. The controller can be further programmed to
command the microdroplet-manipulating device to manipulate the
first microdroplet on the superhydrophobic surface and to command
the microdroplet-manipulating device to manipulate the second
microdroplet in the hydrophobic liquid. The command to manipulate
the first microdroplet on the superhydrophobic surface can be part
of a first protocol, while the command to manipulate the second
microdroplet in the hydrophobic liquid can be part of a second
protocol. The first protocol can comprise extracting genetic
material from a sample, and the second protocol can comprise
amplifying a predetermined portion of the extracted genetic
material, particularly by thermocycling.
[0018] By way of example, any of the following can be within the
scope of manipulating the microdroplet on the superhydrophobic
surface: (a) moving the microdroplet, (b) adding a substance to the
microdroplet, (c) removing a substance from the microdroplet, (d)
mixing contents of the microdroplet, (d) concentrating the
microdroplet, (e) changing a composition of the microdroplet, (f)
changing a position of the microdroplet, (g) holding a substance
relative to the microdroplet, (h) merging the microdroplet with
another microdroplet, (i) splitting the microdroplet, and (j)
rotating the microdroplet.
[0019] By way of example, any of the following can be within the
scope of manipulating the microdroplet in the hydrophobic liquid:
(a) moving the microdroplet, (b) adding heat to the microdroplet,
(c) removing heat from the microdroplet, (d) placing the
microdroplet in a pendant condition relative to the hydrophobic
liquid, and (e) retracting the microdroplet.
[0020] For performing thermocycling, the apparatus desirably
includes a vessel that comprises at least three
temperature-regulated chambers each containing a respective volume
of the hydrophobic liquid at a respective temperature. In addition,
the vessel desirably also comprises respective channels connecting
the chambers together in a cyclical manner, wherein each channel
also contains a respective volume of the hydrophobic liquid and the
channels open into the respective chambers. Of these chambers, a
first temperature-regulated chamber is one in which the hydrophobic
liquid is at a nucleic acid-denaturation temperature. The second
temperature-regulated chamber is one in which the hydrophobic
liquid is at a nucleic acid annealing temperature, and the third
temperature-regulated chamber is one in which the hydrophobic
liquid is at a nucleic acid extension temperature. By way of
example the denaturation temperature is in a range of about
94-96.degree. C., the annealing temperature is in the range of
about 50-65.degree. C., and the extension temperature is in a range
of about 70-74.degree. C.
[0021] Desirably, the controller is further configured to command
the movement and positioning device to move the
microdroplet-manipulating device in a manner such that the
microdroplet, while being kept submerged in the hydrophobic liquid,
is moved from one chamber of the vessel to the next according to a
preset thermal cycle. The controller desirably is further
configured to command the microdroplet-manipulating device to hold
the microdroplet in each chamber for a preset respective length of
time. Further desirably, the microdroplet-manipulating device is
configured to hold the microdroplet in each chamber while the
microdroplet is pendant from the microdroplet-manipulating device
and while the microdroplet-manipulating device moves the pendant
droplet relative to the hydrophobic liquid to ensure good
convective heating of the microdroplet. If required or as desired,
the microdroplet-manipulating device is further configured to
retract the microdroplet as the microdroplet-manipulating device is
moving the microdroplet through a respective channel from one
chamber to the next in the cycle. Also, the
microdroplet-manipulating device is further configured to
de-retract the microdroplet as the microdroplet is being held by
the microdroplet-manipulating device in a subsequent chamber of the
cycle.
[0022] In many of the manipulations summarized above, the
microdroplet manipulating device is simply a small syringe (e.g.,
1-mL capacity) fitted with a blunt hypodermic needle or pipetter
tip. The pipetter tips are advantageous for many of the
manipulations and offer the benefit of being inexpensive and
commercially available in large quantities. The can also be arrayed
at a rack or the like in the range of movement of the placement and
movement device for automatic re-tipping as required.
[0023] Whereas most of the manipulations (e.g., performing
dilutions) are readily understood, certain manipulations, such as
"centrifuging" and "mixing" the contents of a microdroplet
(particularly on the superhydrophobic surface) are surprising
result obtained by appropriately "vibrating" the syringe and/or tip
thereof. In many embodiments a "vibration motor" is placed adjacent
the tip of the syringe and commanded to actuate (e.g., by a
pulse-width-modulated drive signal) in a manner resulting in a
stirring motion of the syringe tip, which appropriately agitates
the microdroplet without breaking it up.
[0024] The foregoing and additional features and advantages of the
subject of this disclosure will be more apparent from the following
detailed description, which proceeds with reference to the
accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 is a schematic diagram of the first representative
embodiment of a microdroplet-protocol system.
[0026] FIG. 2 is a perspective view of the x-y wire-guide system of
the first representative embodiment, the system being an exemplary
controlled movement and placement device having a range of movement
in at least two dimensions.
[0027] FIG. 3A is a plan view of a first example PCR vessel
comprising three respective chambers arranged substantially
linearly.
[0028] FIG. 3B is a plan view of a second example of PCR vessel
comprising three respective chambers configured in a
two-dimensional arrangement.
[0029] FIG. 3C is a perspective view of the PCR vessel shown in
FIG. 3B.
[0030] FIG. 4 is a plot of temperature versus time as obtained in a
CFD simulation showing that a single PCR cycle performed with a 10
.mu.L microdroplet can be completed in 13 seconds with the
microdroplet remaining for two seconds in each chamber of the PCR
vessel (typical extension rate=80 nucleotides per second, with a
2-second holding time required per step for a 160 bp
amplification), indicating that a 30-cycle PCR can be completed in
six minutes 30 seconds.
[0031] FIGS. 5A-5H are a series of images of a serial-dilution
protocol performed using the first representative embodiment. In
the protocol a guided pipetter tip manipulates, splits, and mixes
the contents of discrete microdroplets situated on a
superhydrophobic surface as an example of a hydrophobic milieu.
FIG. 5A depicts placing multiple 20-.mu.L microdroplets of diluent
(phosphate-buffered saline (PBS)) on the superhydrophobic surface;
FIG. 5B shows 2 .mu.L of E. coli suspension being added to the
first microdroplet for dilution; FIG. 5C shows the first
microdroplet being mixed (mixing is performed by vibrating the
syringe to which the pipetter tip is attached, which causes
corresponding vibration of the syringe tip); FIG. 5D shows the
syringe being withdrawn momentarily for retipping; FIG. 5E shows 2
.mu.L being withdrawn from the first microdroplet for further
dilution; FIG. 5F shows the withdrawn liquid from the first
microdroplet being added to and mixed with the second microdroplet;
FIG. 5G shows the syringe being withdrawn momentarily for
retipping; and FIG. 5H shows the last microdroplet receiving a
2-.mu.L aliquot from the third microdroplet, thereby producing a
10.sup.-4 dilution of the original E. coli suspension.
[0032] FIGS. 6A-6E are a series of images of a protocol for
concentrating a sample by droplet centrifugation performed on the
superhydrophobic surface. FIG. 6A shows a needle tip immersed in
the microdroplet as a blunt, hollow needle is being induced to
undergo circular motion relative to the superhydrophobic surface
(motion is imparted by a vibration motor contacting the syringe and
induced to vibrate in a desired manner by pulse-width modulation);
FIG. 6B shows spinning of the microdroplet being induced by the
vibrating needle (microdroplet spinning at 2264 rpm, which produces
22.times.g centrifugal force); FIG. 6C depicts liquid from the
center of the microdroplet being drawn up by the needle; and FIG.
6D depicts the remaining microdroplet of concentrated sample
remaining on the superhydrophobic surface after withdrawal of the
needle.
[0033] FIGS. 7A-7H are a series of images of a protocol in which
DNA is extracted from a microdroplet of a concentrated sample on
the superhydrophobic surface. FIG. 7A shows a droplet (foremost
microdroplet) of concentrated sample as placed on the
superhydrophobic surface; FIG. 7B shows a pipetter tip adding
cell-lysis solution to the microdroplet; FIG. 7C shows the
microdroplet being heated (while submerged in silicone oil in a
lysis chamber at 80.degree. C., the silicone oil being another
example of a hydrophobic milieu); FIG. 7D shows a microdroplet of
the lysed solution being placed on the superhydrophobic surface;
FIG. 7E shows isopropyl alcohol being added to the microdroplet of
lysed cells to precipitate the DNA, wherein the precipitated DNA
adheres to the pipetter tip; FIG. 7F shows the pipetter tip (with
adhered DNA) being washed in a microdroplet of ethanol; FIG. 7G
depicts the pipetter tip (with adhered DNA) being air-dried; and
FIG. 7H shows the pipetter tip immersed in a hydration solution in
which the DNA is resuspended. FIGS. 8A-8F are schematic elevational
views of respective steps in the protocol for DNA extraction shown
in FIGS. 7A-7H. FIG. 8A shows the pipetter tip placing a
microdroplet of concentrated E. coli produced in the centrifugation
protocol (FIGS. 6A-6E) on the superhydrophobic surface; FIG. 8B
shows cell-lysis solution being added to the microdroplet and mixed
by vibrating the pipetter tip; FIG. 8C shows isopropanol being
added to the microdroplet to precipitate to the DNA, and also shows
the pipetter tip being slowly rotated to gather the precipitated
DNA; FIG. 8D shows the precipitated DNA on the tip being dried by
immersion of the tip in 70% ethanol (tip is then withdrawn for air
drying); FIG. 8E shows the DNA on the tip being resuspended in a
hydration solution (tip being vibrated to hasten resuspension and
thus detachment of the DNA from the tip); and FIG. 8F shows
primers, Taq polymerase, and water being added to prepare the DNA
in the microdroplet for PCR or other downstream protocol.
[0034] FIG. 9 is a perspective view showing a general layout of
components of a microdroplet manipulation system according to the
second representative embodiment.
[0035] FIG. 10 is an image of a gel in which RT-PCR results as
described in Example 6 are shown.
[0036] FIG. 11 is an image of a gel in which PCR results as
described in Example 4 are shown.
SEQUENCE LISTING
[0037] The nucleic and/or amino acid sequences provided herewith
are shown using standard letter abbreviations for nucleotide bases,
and three letter code for amino acids, as defined in 37 C.F.R.
1.822. Only one strand of each nucleic acid sequence is shown, but
the complementary strand is understood as included by any reference
to the displayed strand. The Sequence Listing is submitted as an
ASCII text file named SeqList-87746-01.txt, created Aug. 29, 2011,
about 1.2 KB, which is incorporated by reference herein. In the
accompanying sequence listing:
[0038] SEQ ID NOs: 1 and 2 are forward and reverse primers for
amplification of a nucleic acid sequence of the Escherichia coli
K12 aminoacyl-histidine dipeptidase (pepD) gene.
[0039] SEQ ID NOs: 3 and 4 are forward and reverse primers for
amplification of a nucleic acid sequence of the H1N1 influenza A
RNA polymerase subunit PA gene.
DETAILED DESCRIPTION
[0040] The disclosure is set forth below in the context of multiple
representative embodiments, which are not intended to be limiting
in any way.
[0041] The drawings are intended to illustrate the general manner
of construction and are not necessarily to scale. In the detailed
description and in the drawings themselves, specific illustrative
examples are shown and described herein in detail. It will be
understood, however, that the drawings and the detailed description
are not intended to limit the invention to the particular forms
disclosed, but are merely illustrative and intended to teach one of
ordinary skill how to make and/or use the invention claimed
herein.
[0042] As used in this application and in the claims, the singular
forms "a," "an," and "the" include the plural forms unless the
context clearly dictates otherwise. Additionally, the term
"includes" means "comprises." Further, the term "coupled"
encompasses mechanical as well as other practical ways of coupling
or linking items together, and does not exclude the presence of
intermediate elements between the coupled items.
[0043] The described things and methods described herein should not
be construed as being limiting in any way. Instead, this disclosure
is directed toward all novel and non-obvious features and aspects
of the various disclosed embodiments, alone and in various
combinations and sub-combinations with one another. The disclosed
things and methods are not limited to any specific aspect or
feature or combinations thereof, nor do the disclosed things and
methods require that any one or more specific advantages be present
or problems be solved.
[0044] Although the operations of some of the disclosed methods are
described in a particular, sequential order for convenient
presentation, it should be understood that this manner of
description encompasses rearrangement, unless a particular ordering
is required by specific language set forth below. For example,
operations described sequentially may in some cases be rearranged
or performed concurrently. Moreover, for the sake of simplicity,
the attached figures may not show the various ways in which the
disclosed things and methods can be used in conjunction with other
things and method. Additionally, the description sometimes uses
terms like "produce" and "provide" to describe the disclosed
methods. These terms are high-level abstractions of the actual
operations that are performed. The actual operations that
correspond to these terms will vary depending on the particular
implementation and are readily discernible by one of ordinary skill
in the art.
[0045] In the following description, certain terms may be used such
as "up," "down,", "upper," "lower," "horizontal," "vertical,"
"left," "right," and the like. These terms are used, where
applicable, to provide some clarity of description when dealing
with relative relationships. But, these terms are not intended to
imply absolute relationships, positions, and/or orientations. For
example, with respect to an object, an "upper" surface can become a
"lower" surface simply by turning the object over. Nevertheless, it
is still the same object.
[0046] The use of controlled mechanisms for manipulating liquid
microdroplets on an open, superhydrophobic surface was previously
investigated (You et al., Faraday Discuss. 149: 159-170, 2011,
incorporated herein by reference in its entirety). Briefly, a clean
metal wire was used for guiding motion of a hydrophilic-liquid
microdroplet that had formed on a superhydrophobic surface. This is
the source of the term "wire-guide," Yoon and You, J. Biol. Eng.
2:15, 2008 (incorporated herein by reference). In addition to using
wires for manipulating microdroplets, also evaluated was the use of
analogous structures for microdroplet manipulation (e.g., blunt
hypodermic needles and pipetter tips) having any of a variety of
materials, sizes, and configurations for applying different forces
to liquid microdroplets. Thus, as used herein, a "wire-guide"
system includes use of any of the microdroplet-manipulating devices
described herein. The wire-guide system proved to be adaptable to a
wide range of microdroplet volumes and properties. It was also
discovered that a blunt, hollow needle or disposable pipetter tip
attached to a mechanized syringe or analogous device can be
vibrated or otherwise moved in a particular manner for use in
precisely dividing and splitting liquid microdroplets, for mixing
the contents of a microdroplet, and for rotating the microdroplet.
It was also found that wire-guiding can allow protocols to be
performed on, in, or with microdroplets depending from the ends of
hollow needles or pipetter tips. These protocols include any of
various dilution protocols and various PCR protocols, in which
movement of a pendant microdroplet while submerged in an inert
hydrophobic oil provides good and rapid convective transfer of heat
to and from the microdroplet as required.
Polymerase Chain Reaction as an Exemplary Protocol
[0047] The Polymerase Chain Reaction (PCR) is a well-established
method for detecting and amplifying DNA and RNA. PCR has unlimited
sensitivity and unparalleled specificity. It is an essential tool
and medical research and clinical medicine. It is used extensively
for detecting infectious disease organisms and detecting gene
mutations. Reverse transcription PCR (RT-PCR) is a variant of PCR
in which an RNA strand is first reverse transcribed to
complementary DNA (cDNA) using the enzyme reverse transcriptase.
The cDNA is then amplified using traditional PCR.
[0048] PCR is an in vitro amplification technique that increases
the number of copies of a nucleic acid molecule (for example, a
nucleic acid molecule in a sample or specimen). In particular
examples, amplification of a nucleic acid molecule of defined
length is achieved by multiple cycles of a three-step procedure
involving denaturing a DNA template, annealing oligonucleotide
primer pairs to opposite strands of the template, and extending the
primers with a thermally stable DNA polymerase to copy each strand
of the template. Each step of a PCR cycle is carried out at a
specific temperature. Target DNA is denatured at high temperature
(such as at 95-98.degree. C., such as about 95, 96, 97, or
98.degree. C.). The temperature for annealing primers to
complementary target DNA strands by nucleic acid hybridization is
typically sequence-specific. Common primer-template annealing
temperatures are between about 50-56.degree. C., such as about 50,
51, 52, 53, 54, 55, or 56.degree. C. Primer extension is carried
out at a polymerase-specific temperature. Repeated polymerase
exposure to high temperature in the denaturing step necessitates
use of a thermal-stable polymerase, many of which are known in the
art. In particular examples, the Taq DNA polymerase is used, and
extension is carried out at about 72.degree. C.
[0049] Exemplary embodiments of systems disclosed herein are used
to amplify DNA by PCR. However, one of skill in the art will
recognize that the described systems can be used in other exemplary
methods of DNA amplification such as isothermal amplification
methods, which can use fewer than three temperature-controlled
controlled chambers of the described PCR vessel. Representative and
non-limiting examples of isothermal in vitro amplification
techniques include strand-displacement amplification (see U.S. Pat.
No. 5,744,311); transcription-free isothermal amplification (see
U.S. Pat. No. 6,033,881); repair chain-reaction amplification (see
WO 90/01069); ligase chain-reaction amplification (see EP-A-320
308); gap-filling ligase chain-reaction amplification (see U.S.
Pat. No. 5,427,930); coupled ligase detection and PCR (see U.S.
Pat. No. 6,027,889); and NASBA.TM. RNA transcription-free
amplification (see U.S. Pat. No. 6,025,134).
[0050] The product of a PCR or other amplification technique can be
characterized by various standard techniques known in the art, such
as electrophoresis, restriction endonuclease cleavage patterns,
oligonucleotide hybridization or ligation, and/or nucleic acid
sequencing.
[0051] PCR and other amplification methods and techniques for
characterizing amplification products are well-known in the art,
and are described, for example, in Ausubel et al. (eds.), Current
Protocols in Molecular Biology, published by Wiley InterScience,
2011 (ISSN 1934-3639). Additionally, methods for preparing and
using nucleic acid primers are described, for example, in Sambrook
et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York,
1989), and Innis et al. (PCR Protocols, A Guide to Methods and
Applications, Academic Press, Inc., San Diego, Calif., 1990).
[0052] In particular embodiments of PCR, known as real-time or
quantitative PCR, methods and devices are used for detecting and
measuring products generated during each PCR cycle, which are
proportionate to the amount of template nucleic acid prior to the
start of PCR. The information obtained, such as an amplification
curve, can in some examples be used to quantify the initial amounts
of template nucleic acid sequence. In particular embodiments,
real-time PCR is accomplished using nucleic acid probes that can be
included in an amplification reaction, for example to permit
detection of formed amplicons (such as in real time). In one
example, the detectable label associated with the probe is a
fluorophore. The fluorescence signal intensity can be related to
the amount of PCR product (amplicon) by a product-dependent
decrease of the quench of a reporter fluorophore, or by an increase
of the Forster resonance energy transfer (FRET) from a donor to an
acceptor fluorophore. FRET is the radiationless transfer of
excitation energy by dipole-dipole interaction between fluorophores
with overlapping emission and excitation spectra. Because the FRET
and the quench efficiency are strongly dependent on the distance
between the fluorophores, the PCR-product-dependent change in the
distance between the fluorophore can be used to generate the
sequence-specific signals.
[0053] Several different probes can be used in real-time PCR
methods. All can function by a decrease of quench or an increase of
FRET. In one example, 5' nuclease fluorogenic target-specific
oligonucleotide probes are utilized. One particular example of such
a probe is a TaqMan probe (Applied Biosystems, Foster City,
Calif.), which includes a reporter fluorophore at the 5' end, and a
quencher internally or at the 3' end. An exemplary 5' reporter
fluorophore is FAM (5-carboxyfluorescein), and an exemplary 3'
quencher fluorophore is TAMARA (6-carboxy-tetramethylrhodamine).
Intact probes do not produce a fluorescent signal because they are
quenched. In one example, during extension of the primers, the
TaqMan probe, which is complementary to the amplicon sequence, is
bound to the single-stranded PCR product like the primers. Upon
reaching the probe, Taq DNA polymerase (due to its intrinsic
nuclease activity) cuts the probe, releasing the quencher from the
reporter fluorophore, which now fluoresces after excitation with
the appropriate wavelength of light. The signal generated by the
reporter fluorophore is detected, and quantitation of the amplicons
can be made by analysis of the resulting amplification curve.
[0054] Commercial PCR systems generally utilize a single Peltier
module for heating and cooling a sample-containing vial to the
various temperatures required in a PCR cycle. This is a major
disadvantage of conventional automated PCR systems. Among the three
principal modes of heat transfer (conduction, convection, and
radiation), conduction is the least efficient for heating and
cooling liquid samples. Also, since most commercial PCR systems
utilize only a single heating block instead of separate blocks (one
for each temperature in the PCR cycle) the temperature of the
single block must be changed repeatedly for use in the next thermal
step. Repeatedly changing the temperature of a single heating block
consumes valuable time (usually a significant portion of the total
PCR cycling time) as well as power. In methods as disclosed herein,
a separate respective heater is used for each temperature in the
cycle, the heaters are held, as required, at constant respective
temperatures, and the sample is moved from one heater to the next
as it progresses through a PCR cycle. By eliminating single-block
warm-up and cool-off, the PCR cycle times are substantially
reduced.
[0055] An approach to reducing PCR cycling times in conventional
PCR protocols is focused on reducing sample volumes. Much of this
work has been directed to using of microfluidics, in which small
volumes of liquid travel through microchannels. Unfortunately,
these microchannel devices rely upon conductive heat transfer for
heat cycling of the sample. Since conductive heat transfer is
relatively inefficient and slow, several minutes are usually
required to complete each cycle. This results in over an hour being
required to perform typically 25-30 cycles of PCR. See, e.g., Kopp
et al., Science 280:1046-1048, 1998.
[0056] In conventional PCR devices employing microchannels, liquid
flows continuously through the microchannel, or flows within a
microchannel as discrete liquid plugs. Although microchannel PCR
can be performed more rapidly than certain other conventional PCR
assays, a serious limitation is the lack of adaptability of the
microchannel PCR device. Namely, microchannels formed in a unit of
material are difficult if not impossible to reconfigure for a
different protocol or change in the usual protocol. Usually,
replacement of the existing microchannel piece with a new one
specifically configured for the new protocol is required. Changing
the protocol even slightly typically requires a redesign of the
microchannel piece. Also, sample-dependent pre-processing steps
such as cell lysis, DNA/RNA extraction, and/or reverse
transcription (RT) are difficult to implement in a microchannel
piece not specifically configured for performing these additional
steps. Additionally, these conventional systems are closed systems,
meaning that once a sample has been introduced as a droplet into a
microchannel, the process cannot be interrupted or augmented until
the droplet has completed its course or the sample has been
expelled from the system.
[0057] Conventional techniques for manipulating droplets on open,
flat surfaces include electrowetting-based microfluidics (Chang et
al., Biomed. Microdevices 8:215-225, 2006) and magnetofluidics
(Ohashi et al., Biomed. Microdevices 9:695-702, 2007). Apparatus
and methods focused primarily on electrowetting on dielectrics are
difficult to fabricate and to operate, and the technique has
problems with diffusional mixing and contamination due to increased
wetting on the surface. Magnetofluidics includes adding magnetic
particles to the droplets to facilitate moving the droplets over a
superhydrophobic surface using a magnet. This raises concerns of
adverse effects being introduced to the samples as a result of
adding magnetic particles to them, e.g., particle interference with
biological or biochemical reactions.
[0058] To resolve the issues with conventional techniques as
summarized above, Applicants arrived at wire-guided microdroplet
microfluidics (using a microdroplet-manipulating device) as being
the simplest and most efficient technique for performing various
protocols with the desired speed and efficiency. No magnetic
particles are required, and wire-guided microdroplet microfluidics
can be implemented easily in a variety of microdroplet environments
and situations. Samples having microdroplet-sized volumes (1-50
.mu.L, such as any of 1-10, 10-20, 20-30, 30-40, and 40-50 .mu.L,
e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 .mu.L) allow for lower
reaction volumes and decreased assay times for many common
laboratory procedures. Using a microdroplet-manipulating device,
such as a wire, syringe, pipetter tip, or the like, multiple-step
protocols can be performed upon, in, and with microdroplets on or
in a hydrophobic milieu, such as on open, flat, superhydrophobic
surfaces or within biologically inert, hydrophobic oil or other
suitable hydrophobic liquid. These protocols are easily
reconfigurable at any time before, during, or after performing a
particular protocol. For example, an extra step(s) can be added on
the fly to an ongoing protocol, as required or desired, without
having to change the system. In addition, the system can be
programmed to perform multiple microdroplet-manipulation protocols,
or portions thereof, in combination. A combination can include, for
example, all of one protocol plus all of another, all of one and
part of another, part of a first protocol and all or part of
another, or multiple protocols or parts thereof. In addition, any
of the microdroplet-manipulation protocols described herein can be
combined with one or more thermal-cycling protocols such as PCR or
a portion thereof, performed using the system as discussed later
below.
[0059] Another advantage of wire-guided microfluidics is its ready
utility with microdroplet volumes that are not excessively small.
There are conventional "nanodrop" and related schemes in which the
PCR assay is performed in very small, stationary droplets (smaller
than most microdroplets). The droplet volumes are extremely small
(nanoliter volumes), mostly in an attempt to speed up
conduction-based heat transfer. Even though this "stationary
droplet PCR" approach has allowed significant reduction in PCR
thermal cycling time compared to other conventional methods, the
extremely tiny volume (typically in the nanoliter range) makes it
very difficult to confirm the products of the PCR thus performed
(e.g., by gel electrophoresis, subsequent imaging, and/or gene
sequencing). Nanodrop PCR also raises concerns of assay
reproducibility and reliability. For example, dividing a
low-concentration sample into nanoliter volumes may result in one
or more droplets not receiving any of the target molecules. In
wire-guided microfluidics, in contrast, the volume of a
microdroplet can be kept sufficiently large (e.g., 1-10 .mu.L) to
allow confirmation of its products and ensuring the reliability of
the assay.
[0060] Moreover, the systems described herein can be additionally
configured to perform real-time PCR protocols. Such systems include
means for exciting and detecting of a fluorescent probe, which are
well known in the art.
First Representative Embodiment
[0061] This embodiment is directed to a microdroplet-protocol
system comprising a movement and placement device. The movement and
placement device in this embodiment is a
computer-numerical-controlled (CNC) system ("wire-guide system")
that provides controlled motion in at least two dimensions (e.g., x
and y) and more desirably in three dimensions (x, y, and z).
[0062] The system of this embodiment is configured for performing
various biochemical and molecular biological protocols, such as
(but not limited to) microdroplet serial dilution, microdroplet
centrifugation, DNA extraction from a microdroplet, and PCR
(including thermocycling) of the extracted (or other) DNA. DNA
extractions and microdroplet centrifugations can be performed in
air (or inert gas) on a superhydrophobic surface as an exemplary
hydrophobic milieu. PCR thermocycling desirably is performed under
an inert hydrophobic liquid in a multi-chambered PCR vessel
including multiple heaters. The system has essentially no
limitations on the complexity and/or configuration of procedures
that it can perform. Le., the range of possible protocols
represents any of various combinations of microdroplet-manipulation
protocols and cyclic-heating protocols. Also, a user can start
and/or stop at any time a protocol being performed by the system
and insert a change to the protocol (including execution of one or
more manual operations, if desired), followed by automatic
resumption of the original protocol or another protocol.
[0063] As used herein, a "hydrophobic milieu" is an either
surficial or liquid environment that favors formation of
substantially spherical microdroplets of a hydrophilic liquid
placed in contact with the milieu. Substantially spherical
microdroplets can be manipulated in the various ways described
herein. Significant deviation from sphericity tends to make a
microdroplet more difficult to manipulate. A first example of a
hydrophobic milieu is a superhydrophobic surface (i.e., a surface
on which a microdroplet of hydrophilic liquid tends to assume a
substantially spherical shape (contact angle usually 150.degree. or
more, but desirably less than)180.degree.. Another example of a
hydrophilic milieu for a microdroplet is immersion in a hydrophobic
liquid, such as an inert hydrophobic oil (e.g., silicone oil).
Either type of hydrophobic milieu shapes a hydrophilic droplet into
a substantially spherical shape, which is important for
manipulating the microdroplet. Note that "super" hydrophobicity is
important for the surface but not for the liquid.
[0064] A general system configuration of this embodiment is shown
in FIG. 1. The depicted system 10 resembles, in part, a
computer-controlled or robotic system configure to perform various
programmed tasks. In this embodiment, the system 10 is configured
to perform various actions on, in, and with microdroplets. The
system 10 of this embodiment comprises a PCR vessel 12, a
temperature controller 14, a power supply 16, a controller or
computer 18, a user interface 20, a motor driver 22, an x-y
wire-guide system 24, an array 27 of pipetter tips, vials (some
possibly containing reagents and/or samples as required), and a
planar superhydrophobic surface 25. FIG. 1 depicts the following
example electrical interconnections: The PCR vessel 12 includes
multiple heated zones and temperature sensors that are connected to
the temperature controller 14. The temperature controller 14 is
connected to the power supply 16 and the controller 18. The user
interface 20 is connected to the controller 18. The controller 18
is connected to the power supply 16. The power supply 16 is
connected to the motor driver 22. The motor driver 22 is connected
to stepper motors 26, 28, 30 of the x-y wire-guide system 24, to
the power supply 16, and to the controller 18. The entire system 10
is mounted to a base 32 that can be placed, for example, on a
laboratory bench-top. Details on these individual components are
provided later below.
[0065] Referring further to FIG. 1, the x-y wire-guide system 24 of
this embodiment includes a carriage 34 that rides on and is guided
by guide rods while moving in the x- and y-directions. Further
details of the wire-guide system 24 are provided later below in the
discussion of FIG. 2. Motion of the carriage 34 in the x- and
y-directions is actuated by respective stepper motors 26, 28, 30.
Mounted to the carriage 34 are a syringe holder 36 or analogous
device and drive motors 38 for moving the syringe holder (and thus
the syringe) up and down (z-direction movement) and for moving the
syringe plunger up and down (z-direction movement) relative to the
syringe holder 36. Operably coupled to the syringe (particularly at
or near the "tip end" of the syringe) is a vibration motor 40 used
for imparting desired rapid periodic motions of the syringe end
(and thus of a needle, wire, pipettor tip, or the like mounted to
the syringe end). For these various motions, the controller 16 is
electrically connected to the syringe holder 36, the motors 38, the
vibration motor 40, and to the carriage 34.
[0066] This system 10 is configured to position a controllably
actuatable syringe 36 and needle (or pipetter tip or other
implement), used for manipulating microdroplets, relative to the
hydrophobic milieu, such as the superhydrophobic surface 25 (on
which serial dilutions and DNA extractions, for example, can be
performed). The system also controls operation of the syringe and
needle (or pipetter tip or other implement) for manipulating
microdroplets in the PCR vessel 12 (in which PCR thermocycling, for
example, can be performed).
[0067] The x-y wire-guide system 24 of this embodiment is shown in
FIG. 2. The x-y wire-guide system 24 is an example of a "movement
and placement device" because it moves and places the carriage 24,
and hence the syringe, and hence a needle, tip or other implement
mounted to the tip end of the syringe, at a desired location and
placement to execute a respective action on a microdroplet as set
forth in a protocol. The wire-guide system 24 includes support
members 50a-50b and 52a-52b mounted vertically (z-direction) to the
base 32. Extending in the x-direction between the support members
50a and 52a are parallel guide bars 54a, 56a, and extending in the
x-direction between the support members 50b, 52b are parallel guide
bars 54b, 56b. Journaled in both support members 50a, 52a parallel
to the guide bars 54a, 56a is a threaded rod 58a. Similarly,
journaled in both support members 50b, 52b parallel to the guide
bars 54b, 56b is a threaded rod 58b. The guide bars 54a, 56a also
extend through respective slide bushings (not shown) in a movable
member 62a, and the guide bars 54b, 56b also extend through
respective slide bushings (not shown) in a movable member 62b. The
bushings (and thus the movable members 62a, 62b) move with
extremely low friction along the guide bars 54a, 54b, 54b, 56b.
Meanwhile, the threaded rod 58a is threaded through a ball screw
(not shown) mounted in the movable member 62a so that axial
rotation of the threaded rod 58a causes linear movement of the
movable member 62a in the x-direction. Similarly, the threaded rod
58b is threaded through a ball screw (not shown) in the movable
member 62b so that axial rotation of the threaded rod 58b causes
linear movement of the movable member 62b in the x-direction.
Simultaneous rotation of the threaded rods 58a, 58b is achieved by
respective stepper motors 26, 28 simultaneously rotating at
identical angular velocities as controlled by the motor driver 22.
Spanning between the movable members 62a, 62b and the y-direction
are parallel guide bars 64, 66. The guide bars 64, 66 extend
through respective slide bushings (not shown) in the carriage 34.
The bushings (and thus the carriage 34) move with extremely low
friction along the guide bars 64, 66. Meanwhile, a threaded rod is
journaled in the movable members 62a, 62b parallel to the guide
bars 64, 66. The threaded rod 67 is threaded through a ball screw
(not shown) in the carriage 34 so that axial rotation of the
threaded rod 67 causes movement of the carriage 34 in the
y-direction. Rotation of the threaded rod 67 is caused by a
respective stepper motor 30 controlled by the motor driver 22. The
particular configuration of the wire-guide system 24 described
above is one example of a mechanical structure capable of providing
controlled movement and placement of the syringe or other implement
relative to microdroplets on or within a hydrophobic milieu, such
as on a superhydrophobic surface or in an inert hydrophobic oil.
Any of various other x-y-z manipulator structures can alternatively
be employed to provide the same motions as the wire-guide system
24. It will also be understood that the guide bars, threaded rods,
and ball screws can be replaced with analogous mechanisms such as,
but not limited to, linear motors and analogous devices, or
belt-driven mechanisms.
[0068] As noted, mounted to the carriage 34 is a syringe holder 36
operably coupled to the motor 38 for raising and lowering the
syringe 68 in the z-direction. The motor 38 is connected to and
controlled by the motor driver 22. Also, a vibration motor 40 is
mounted to the carriage 34 and coupled to the syringe 68 (or at
least to the tip-end of the syringe) to impart, as needed, a
circular or otherwise suitable vibratory motion to the syringe tip
as the syringe is being held by the syringe holder 36. Yet another
actuator (not shown) mounted to the carriage 34 is a linear
actuator that controllably moves the syringe plunger (not detailed)
in the z-direction to enable the syringe to aspirate or release a
microdroplet of liquid, for example.
[0069] The combinations of x-, y-, and z-motions of the syringe 68
produced by the wire-guided system 24 allows a needle, pin, pipette
tip, or other suitable tool 70 attached to the syringe to be placed
anywhere in three-dimensional space defined by the wire-guide
system 24. Motions of the motors 26, 28, 30, 38, 40 are achieved
using respective motor-control circuits provided in the motor
driver 22 as controlled by the controller 18 and powered by the
power supply 16. Since a stepper motor operates by a train of
electrical pulses that can be counted by the respective
motor-control circuit, there is usually no need for a device, such
as a linear or rotary encoder, that monitors position of either the
movable members 62a, 62b or the carriage 34.
[0070] The syringe 68 can be of a disposable nature for
convenience. It will be understood that the term "syringe" is not
limited to conventional clinical syringes but rather encompasses
various implements having the ability to aspirate, release, and/or
manipulate liquid microdroplets in the conditions prevailing in the
system 10. These implements include pipetters and the like that
typically use disposable tips, as used widely used in biochemistry
and molecular biology. The term "syringe" also encompasses various
devices that are analogous to syringes and pipetters. Use of a
clinical syringe 68 as the implement is not limited only to
syringes to which conventional hollow needles are attached. Certain
types of syringes may utilize, for example, disposable pipetter
tips instead of needles. Thus, for microdroplet insertion and
extraction, a syringe 68 mounted to the syringe holder 36 can be
fitted with a disposable pipetter tip or a modified conventional
(e.g., Luer-Lok.TM.) blunt-ended needle or other suitable tool. As
noted, the syringe holder 36 includes at least one linear actuator
38 that drives the syringe plunger in the z-direction as required
and moves the syringe 68 up and down, as required, relative to the
carriage 34. Also, as noted previously, a vibration motor 40 causes
rapid periodic motion of the syringe and/or its needle as described
later below. In addition, the syringe can be provided with yet
another actuator that provides controlled motion of the syringe (or
at least a tip fitted thereto) around the z-axis (as used in, e.g.,
collecting precipitated DNA on the syringe needle or pipetter tip
during execution of the DNA extraction protocol.
[0071] Therefore, the syringe 68 as an exemplary tool is not
intended to be limiting in any way. Substantially any implement or
tool useful for microdroplet manipulations can be mounted to the
carriage 34. No matter which implement is used, the system 10
provides x-y-z motion of the implement to any location reachable by
the carriage 34 riding on the guide rods and lead screws. It will
also be understood that more than one implement can be mounted to
the carriage 34; additional drive mechanisms can be added to the
carriage 34 to provide independent manipulation (including motion
relative to the carriage 34) of multiple implements mounted to the
carriage. Alternatively, each of multiple carriages can include
respective implements that are independently movable relative to
their respective carriages.
[0072] Furthermore, although a clean, metal wire was initially used
to guide an aqueous microdroplet on the superhydrophobic surface,
the wire can be replaced with any of a variety of
microdroplet-manipulating devices of various materials and sizes to
modulate the force of the microdroplet to the wire (W.sub.a),
making the system 10 highly adaptable to a wide range of
microdroplet volumes and properties. Syringe needles or disposable
pipetter tips have ready utility for use in, for example, precise
splitting and mixing of microdroplets as aided by actuation of the
vibration motor 34 and the motor moving the syringe plunger
relative to the syringe itself.
[0073] This embodiment is particularly well suited for performing
PCR thermocycling and for performing auxiliary protocols such as,
but not limited to, serial dilution of a sample, concentration of a
sample by centrifugation, and DNA extraction, all being preparatory
to performing PCR. For example, an exemplary process performed by
the system 10 can begin with serial dilutions of a sample (e.g.,
10-fold serial dilutions). Following dilution, the system 10 can
concentrate a sample microdroplet by "centrifugation" achieved by
spinning the microdroplet in situ at a high angular velocity using
the syringe needle actuated by the vibration motor 40. During
centrifugation, the more dilute portions of the microdroplet at the
axis of rotation can be is drawn into the syringe, leaving the more
concentrated portion of the sample for further analysis. The sample
can then be subjected to DNA extraction, in which a wire-guided
needle performs extraction, separation, and removal of the
precipitated genetic material. Afterward, the system 10 can amplify
a particular genetic sequence(s) in the sample by PCR thermocycling
or analogous technique. The results of the amplification can be
confirmed by gel electrophoresis or other suitable method.
[0074] Most tasks other than PCR cycling can be performed on the
open, flat superhydrophobic surface 25. PCR cycling is not suitable
for performance on the superhydrophobic surface 25 because the high
temperatures involved in PCR thermocycling can evaporate very
quickly a sample exposed to air. Consequently, PCR thermocycling
desirably is performed in the PCR vessel 12. The hydrophobic milieu
is provided by submersion in a suitable (i.e., hydrophobic and
chemically inert) oil that maintains the microdroplets as spherical
entities amenable to manipulation, that prevents sample
evaporation, and speeds up thermocycling.
[0075] Thus, microdroplet manipulations take place on or in a
hydrophobic milieu. As discussed previously, a hydrophobic milieu
can be a surface or substance (e.g., liquid) that, when contacted
by a hydrophilic (e.g., aqueous) liquid, tends to form the
hydrophilic liquid into substantially spherical microdroplet(s).
Spherical microdroplets of the hydrophilic liquid are important for
performing any of various manipulations on, in, or with the
microdroplets. In particular examples, the hydrophobic milieu is a
superhydrophobic surface. In other examples the hydrophobic milieu
is a hydrophobic, inert liquid, such as silicone oil. The
superhydrophobic surface 25 is essentially an open, flat (planar)
substrate (e.g., a glass substrate) having a surficial treatment or
inherent property in which the upper surface thereof is provided
with a superhydrophobic property. In this embodiment, the
superhydrophobic surface used a commercial product having planar
dimensions of 25.times.55 mm, although this is not limiting in any
way. In general, a "hydrophobic" surface exhibits contact angles,
with aqueous microdroplets (e.g., microdroplets of water, buffered
solution, and the like) on the surface, of greater than 90.degree..
For example, a smooth, glass surface coated with a film of
alkylsilane or Teflon.TM. exhibits a contact angle to aqueous
microdroplets of approximately 110.degree., and hence is a
hydrophobic surface. But, a simply hydrophobic surface does not
provide sufficiently high contact angles with which to perform
certain mcrodroplet manipulations, such as centrifugation, on the
superhydrophobic surface. A "superhydrophobic" surface is one on
which the contact angle of an aqueous microdroplet is in the range
of approximately 150.degree. to 180.degree.. In this range, contact
angles of approximately 150-155.degree. are optimal for
microdroplet manipulations on the superhydrophobic surface. A
droplet having a contact angle greater than approximately
155.degree. may be manipulatable with the systems described herein,
but may also exhibit difficulty in remaining on the
superhydrophobic surface. Achieving superhydrophobicity is not
limited to application of a superhydrophobic substance to the
surface. Other ways of achieving super hydrophobicity include, but
are not limited to, surface roughening to increase the effective
unit area of the surface (Wenzel, Ind. Eng. Chem. 28:988, 1936; and
Wenzel, J. Phys. Colloid Chem. 53:1466, 1949), or treating the
surface to form an array of micron or submicron sized asperities
with hollows between them to provide two different contact angles,
according to the Cassie model (Cassie and Baxter, Trans. Faraday
Soc. 40:546-551, 1944; Cassie, Discuss. Faraday Soc. 3:11-15,
1948).
[0076] The PCR vessel 12 provides an array of multiple respective
temperature zones ("chambers") in which the respective temperature
steps in the PCR protocol are performed on a microdroplet. The PCR
vessel 12 is also configured to contain hydrophobic oil in which
the aqueous microdroplet can be immersed to prevent evaporation of
the microdroplet and to accelerate heating of the microdroplet. The
microdroplet typically remains in the oil throughout the entire
time the microdroplet is in the PCR vessel 12, including during
transport from one chamber thereof to the next. For most PCR
protocols, the PCR vessel comprises three chambers. However, this
is not limiting, and the PCR vessel can have, for example, greater
or fewer than three chambers for greater flexibility and/or as
required. One of skill will appreciate that the "PCR vessel" is not
limited solely for the purpose of performing PCR reactions. Rather
the "PCR vessel" can be used in any protocol requiring one or more
multiple temperature stages and which could result in microdroplet
evaporation if otherwise performed in air. Moreover, any one of the
chambers can be used independently in any application or procedure
that requires a temperature-controlled environment produced by a
heat block, water bath, and the like.
[0077] Whereas PCR reactions are performed using the system 10,
several different configurations of multi-chamber PCR vessels were
considered. The first configuration 100 had three temperature zones
("chambers") 102, 104, 106 connected in line, as shown in FIG. 3A.
Each chamber 102, 104, 106 included its own heater 102a, 104a, 106a
and its own temperature sensor 102b, 104b, 106b. The chambers 102
and 104 were connected by a channel 108, and the chambers 104 and
106 were connected by a channel 110. The chambers 106 and 102 were
also connected by a channel 112. The chambers 102, 104, and 106 and
channels 108, 110, and 112 were all open on top. A sample
microdroplet being subjected to PCR thermal cycling was passed,
using a needle or pipetter tip attached to the syringe,
sequentially through the first chamber 102, the channel 108, the
second chamber 104, the channel 110, the channel 106, and the
channel 112 to return to the chamber 102 to repeat the cycle. The
in-line PCR vessel 100 worked fine for PCR thermocycling, and each
chamber 102, 104, and 106 maintained its respective temperature
even though the chambers were connected together by the channels
108, 110, and 112. However, the non-cyclic arrangement of the
chambers required an unnecessarily long travel of the microdroplet
(via the channel 112) at the end of each cycle as the microdroplet
returned from the third chamber 106 to the first chamber 102.
Therefore, a more cyclic arrangement 120 of chambers 122, 124, and
126 was considered a more desirable arrangement (e.g., the
arrangement shown in FIG. 3B), in which the channel 132 is
substantially shorter than the channel 112 in FIG. 3A. In FIG. 3B,
the chambers 122, 124, and 126 correspond to the chambers 102, 104,
and 106, respectively, in FIG. 3A. Similarly, the channels 128,
130, and 132 correspond to the channels 108, 110, and 112 in FIG.
3A. (See also FIG. 3C.) At the bottom of each chamber 122, 124, and
126 is a respective heater 122a, 124a, and 126a that is
independently controlled to maintain a respective temperature in
the respective chamber. To such end, each chamber includes a
respective temperature sensor 122b, 124b, and 126b. The heaters and
sensors are connected to the temperature controller 14 (FIG. 1).
The PCR vessel 120 is made of ABS polymer, for example, although
any of a large variety of inert, rigid, heat-tolerant, and
oil-tolerant materials could alternatively be used, including many
plastics and metals, and certain glasses and ceramics. The
rectilinear (square) profile of the chambers as shown is not
limiting. Square chambers correlate to the shape of the particular
heaters selected for use in this embodiment. By changing the shape
of the heaters, the chambers could be made in any shape. For
example, the chambers could be hexagonally shaped or circularly
shaped to shorten the intervening channels or to eliminate them
entirely. (However, the channels may be useful for thermal
isolation of the chambers from each other.) In the particular
embodiment shown in FIGS. 3B and 3C, the respective heater of each
chamber was configured as exposed copper traces on a single-cited
printed circuit board, and the temperature sensor was a
thermocouple. Other types of heaters and temperature sensors can
alternatively be used.
[0078] In particular examples, the PCR vessel 120 can include a
real-time monitoring circuit that is connected to means for
exciting and detecting an excited fluorophore in the PCR reaction
microdroplet. Exemplary means includes a photodiode that could be
situated adjacent a channel connecting together two chambers in the
PCR vessel 132 (see second representative embodiment). The
photodiode is situated to receive light from a laser diode or the
like disposed on the opposite side of the channel, and the
photodiode and laser diode together can be used to monitor the
progress of a PCR reaction as the microdroplet moves between the
diodes in the channel. (If the microdroplet passes through the
channel while retracted in the needle or pipetter tip, the needle
or pipetter tip should be transmissive to the wavelength(s) of
light produced by the laser diode.) The monitoring circuit monitors
amplified products produced by real-time PCR and other protocols
performed in the PCR vessel. The monitoring circuit is connected to
a user interface to display the results of the monitoring of the
PCR reaction.
[0079] With the PCR vessel 120 shown in FIGS. 3B and 3C,
steady-states for the three necessary PCR cycling temperatures (for
example, about 95.degree. C., about 54.degree. C., and about
72.degree. C. in the chambers 122, 124, and 126, respectively) were
achieved in a compact, connected configuration. In this exemplary
embodiment, actual oil temperatures were set to 105.degree. C.,
55.degree. C. and 80.degree. C., respectively, to ensure rapid
heating of a microdroplet in a very short time and to compensate
for thermal losses from the chambers and channels.
[0080] Temperature variations parallel to the heater 122a, 124a,
and 126a in any chamber 122, 124, and 126 did not vary more than
.+-.1.degree. C. except near the respective walls of the chamber.
The depth of the temperature sensor (thermocouple) resulted in a
more significant temperature variation of .+-.2.degree. C. Thus,
the depth of the sensor 122b, 124b, and 126b relative to the depth
of the microdroplet in each chamber was considered to ensure proper
heating of the microdroplet in the chamber. During normal use, the
PCR vessel 120 contains the inert hydrophobic oil in which the
microdroplet(s) is submerged during execution of PCR thermocycling
(or any other incubation procedure). The oil desirably is
chemically inert, of relatively low viscosity, stable to the
temperatures encountered in the PCR vessel, and does not contribute
any substance to the microdroplet. For this purpose, silicone oil
is particularly desirable, but any other oil or hydrophobic liquid
meeting these criteria will suffice.
[0081] CFD modeling was performed on a microdroplet as it traveled
in silicone oil through the three chambers of the PCR vessel. The
parameters of the simulation were; a 10 .mu.L microdroplet
traveling at a rate of 2 cm/s through uniformly heated silicone
oil. The simulation revealed the effects of convective heat
transfer on the microdroplet at its surface and throughout its
volume. Data obtained from the simulation of convective heat
transfer to the microdroplet were data-logged and used to plot a
graph of temperature versus time (FIG. 4). The plot shows the
temperature of the microdroplet at its center, on its surface, and
throughout its overall volume. Heating of the microdroplet from
72.degree. C. to 95.degree. C. occurred in approximately 2 seconds
in the 95.degree. chamber 122 (actual temperature 105.degree. C.),
cooling from 95.degree. C. to 62.degree. C. occurred in
approximately 4 seconds in the 54.degree. C. chamber 124 (actual
temperature 55.degree. C.), and heating from 62.degree. C. to
72.degree. C. occurred in approximately 1 second in the 72.degree.
C. chamber 126 (actual temperature 80.degree. C.).
[0082] Therefore, the overall time consumed in heating and cooling
a 10-.mu.L microdroplet for one PCR cycle is theoretically seven
seconds. However, because particles will accumulate primarily at
the oil-to-water interface of the microdroplet, these times may be
significantly reduced. If the microdroplet is additionally
maintained at the correct temperature for two seconds per chamber,
then the overall PCR cycle takes approximately 13 seconds. With
these times, performing 30 cycles of PCR of a 10-.mu.L microdroplet
would only require six minutes 30 seconds. Use of a smaller
microdroplet size may further shorten the PCR time.
[0083] Microdroplets of 10 .mu.L size were selected for use because
such volumes closely match the volumes used in typical PCR assays.
Also, 10 .mu.L provides sufficient volume for further analysis to
be conducted on the sample, such as gel electrophoresis and/or DNA
sequencing. (Without performing these post-analysis procedures, the
product of a PCR protocol may not be definitively determined or may
lead to unreliable conclusions.) The range of microdroplet size
usable with the system 10 is governed principally by the range of
microdroplet size that can be manipulated, particularly on the
superhydrophobic surface. Microdroplets that are too large collapse
on the surface and hence lose the roughly spherical shape desired
for manipulation. Microdroplets that are too small evaporate too
quickly, even on the superhydrophobic surface. Hence, a practical
range is about 1 .mu.L to 50-100 .mu.L, such as about 1-10 .mu.L,
about 10-20 .mu.L, about 20-30 .mu.L, about 30-40 .mu.L, about
40-50 .mu.L, about 50-60 .mu.L, about 60-70 .mu.L, about 70-80
.mu.L, about 80-90 .mu.L or about 90-100 .mu.L.
[0084] Microdroplet manipulation in the PCR vessel 120 was
performed using a hollow blunt needle fitted to the syringe 68,
with the needle pointing downward. While in a particular chamber of
the vessel 120, the microdroplet can remain hanging (i.e., pendant)
from the blunt tip of the needle or pipetter tip to facilitate
convective heat transfer from the oil to the microdroplet or vice
versa. Keeping the microdroplet pendant is readily achieved because
the microdroplet only needs to stay in a given chamber for four to
five seconds. The microdroplet can also remain pendant when
traveling from one chamber to the next if the velocity of this
travel is sufficiently low. Within each chamber 122, 124, and 126
of the PCR vessel 120, the velocity of the controlled movements of
the syringe 68 was set to 2 cm/s, and this same velocity can be
used when transporting the microdroplet from one chamber to the
next. If the velocity of inter-chamber transfer is increased (above
2 cm/s), then the resultant force of oil on the pendant
microdroplet may cause the microdroplet to detach and fall from the
needle. To achieve faster PCR cycle times while preventing
detachment of the micridroplet from the needle, the microdroplet
can be retracted up into the needle by backing the syringe plunger
slightly, prior to commencing inter-chamber movement of the
microdroplet. Then, when the needle arrives in the next chamber,
the syringe plunger can be moved to push the microdroplet back out
of the needle (while keeping the microdroplet pendant from the
needle). This action, including microdroplet retraction during
inter-chamber movement, significantly reduces the time needed for
PCR thermocycling.
[0085] The motor (e.g., a linear stepping motor) used for moving
the syringe plunger relative to the syringe 68 was calibrated to
insert and retract exactly a 10 .mu.L aqueous microdroplet into
each chamber of the PCR vessel. The microdroplet was pushed
completely out of the syringe needle (while remaining pendant) in
each chamber. Conductive heat loss or gain between the needle and
microdroplet was considered; but, since the microdroplet was
pendant, the contact area of the microdroplet with the needle tip,
relative to the contact area of the microdroplet to the surrounding
oil, was quite small. Also, since the microdroplet and syringe
needle move together and remain in direct contact with each other,
the method of heat transfer between the two is conduction. Heat
transfer between the microdroplet and the oil, on the other hand,
is primarily convection, which provides a much higher rate of heat
transfer. Therefore, Applicants concluded that the effects of the
needle on the thermocycling temperatures in the chambers of the PCR
vessel were minimal.
Microdroplet Insertion and Extraction
[0086] For insertions and extractions of microdroplets, a
disposable syringe can be used that is fitted with a disposable
pipetter tip or a blunt-ended hypodermic needle, for example. An
exemplary syringe size is a disposable 1-mL plastic syringe, but a
syringe of any volume capacity that can fit into the syringe holder
36 can be used. For such a syringe, the syringe holder 36 can be a
quick-release type that holds the syringe parallel to the z-axis of
the system with the needle pointing downward. The syringe plunger
is actuated (by a motor, piezoelectric stack, or the like) to move
the plunger in either z-axis direction relative to the syringe.
Serial Dilution
[0087] The apparatus 10 can readily perform serial dilutions of a
sample containing any of various analytes of interest. The analyte
can be living (e.g., bacterial cells or plant cells or animal
cells) or inanimate (e.g., DNA or RNA). Serial dilutions desirably
and conveniently are performed in microdroplets on the
superhydrophobic surface 25, although they alternatively can be
performed in microdroplets under oil in the PCR vessel. Serial
dilutions are performed using the syringe 68, the movements of
which are largely produced and guided by the x-y wire-guide system
24 or other controlled movement and positioning device.
[0088] The sequence of events of sample dilution, including serial
dilution, is shown in FIGS. 5A-5H; see also Example 1. Using the
syringe 68 fitted with a disposable pipetter tip or needle, an
array of microdroplets of diluent is placed on the superhydrophobic
surface (FIG. 5A). An exemplary microdroplet volume is 20 .mu.L. A
predetermined volume of sample (e.g., 2 .mu.L) is added to the
first microdroplet (FIG. 5B). The first microdroplet is then mixed
(FIG. 5C) by keeping the distal end of the needle or pipetter tip
immersed in the microdroplet and agitating the syringe using the
vibration motor 40. The syringe is retipped (FIG. 5D) by moving the
syringe 68 to the rack 27 where the used needle or pipette tip is
discarded and a fresh one attached to the syringe (automatically by
executing appropriate motions of the syringe). During this moment,
the system may automatically re-calibrate itself to insure
precision of subsequent actions. The syringe is moved back to the
superhydrophobic surface to remove an aliquot (e.g., 2 .mu.L) from
the first microdroplet and add the aliquot into the second
microdroplet (FIG. 5E). The second microdroplet is then mixed (FIG.
5F) in the same manner as the first microdroplet, followed by
retipping the syringe (FIG. 5G) as previously. These steps are
repeated (FIG. 5H) for each remaining microdroplet of diluent in
the array on the superhydrophobic surface.
[0089] Clearing the superhydrophobic surface for its next use is as
simple as either aspirating the microdroplet(s) using the syringe
as a pipette and then discarding the pipette tip, or simply guiding
the microdroplet off the surface into a tissue in which the
microdroplet is absorbed and later discarded. Further
alternatively, the superhydrophobic surface can simply be replaced
(e.g., with a sterile one).
Microdroplet Centrifugation
[0090] The system disclosed herein performs concentration of a
diluted sample by microdroplet "centrifugation," as controlled by a
controlled movement and placement device. For such action, the
syringe is fitted with a blunt-ended tip or needle, and the tip is
inserted into a microdroplet (resting on the superhydrophobic
surface) containing a substance to be separated from the carrier
liquid of the microdroplet. Hence, this is an in situ
centrifugation technique. The needle tip is inserted into the
microdroplet, and the needle is actuated by the vibration motor to
vibrate in two dimensions while immersed in the microdroplet. The
resulting spinning of the microdroplet tends to concentrate the
substance in outer regions of the microdroplet relative to the
center of the microdroplet. In this embodiment the vibration motor
40 is operated by a pulse-width-modulated (PWM) algorithm to
generate the most favorable 2-dimensional motion path (e.g., oval
or circular), which can be verified by high-speed imaging. To
maximize the number of rotations per minute (RPM), the PWM
algorithm was executed to isolate a resonant frequency that induced
the most stable and rapid 2-D path (e.g., elliptical or
substantially circular) of the needle in the microdroplet. RPMs of
over 2000 have resulted in useful analyte concentration. RPMs of
10,000 or more are readily producible.
[0091] One of ordinary skill in the art will appreciate that the
frequency of motion of the needle may change between samples due to
variations from one sample to the next of needle height from the
superhydrophobic surface, changes in contact angle of the
microdroplet to the superhydrophobic surface due to variations in
microdroplet content, and/or changes in the connection between the
needle and the syringe. In such event, requisite small changes can
be made to the PWM frequency using the user interface 20. Example
frequencies, not intending to be limiting, range from approximately
1000 rpm to 10,000 rpm or more.
[0092] Centrifugation of a microdroplet is illustrated in FIGS.
6A-6E, involving a microdroplet containing bacterial cells. FIG. 6A
depicts the syringe needle undergoing PWM vibration. Also shown is
the microdroplet resting on the superhydrophobic surface. FIG. 6B
shows the needle tip inserted in the microdroplet and causing the
microdroplet to spin as a result of the vibration imparted to the
syringe needle. In an example, the microdroplet is caused to spin
at approximately 2000 RPM, which produces a centrifugation force of
22.times.g. FIG. 6C shows the needle aspirating sample-poor liquid
from the center of the microdroplet after concluding spinning. FIG.
6D shows the needle being withdrawn from the remaining microdroplet
of concentrated sample. FIG. 6E is a bar graph showing the
concentration of bacteria in a 10-.mu.L sample before and after
microdroplet centrifugation, wherein a three-minute centrifugation
produced a 3.06-fold increase in mean concentration of bacterial
cells.
[0093] In FIG. 6C, after a specific period of centrifugation, the
syringe extracts liquid from the center of the microdroplet. This
extraction can be done at a slower rate (e.g., .about.0.5 .mu.L/s)
compared to sample dispensation, to reduce potential turbulence
induced by the aspiration and subsequent decrease in microdroplet
diameter.
Extraction and Amplification of Genetic Material
[0094] The system of this embodiment also performs extraction and
amplification of genetic material from living cells using the same
apparatus configuration and on the same superhydrophobic surface as
the procedures described above. The only difference is the
execution of a different preprogrammed algorithm for extraction and
amplification of genetic material, compared to the other
procedures.
[0095] Rapid DNA extraction from a concentrated sample is
illustrated in FIGS. 7A-7H and GA-GF. In FIG. 7A a microdroplet of
concentrated sample (containing living cells) is placed on the
superhydrophobic surface. In FIG. 7B a cell-lysis solution is added
to the microdroplet using the syringe fitted with a pipetter tip.
In FIG. 7C, the syringe aspirates the microdroplet from the
superhydrophobic surface and moves the drop to a lysis chamber
heated to 80.degree. C. The lysis chamber can be either one of the
chambers of the PCR vessel, notably the nominally 72.degree. C.
chamber (actual temperature is 80.degree. C.) 126 or a separate
chamber. If the lysis chamber is a separate chamber, the lysis
chamber can be constructed similarly to one of the chambers of the
PCR vessel in that it includes a heater and a temperature sensor.
The lysis chamber also includes a volume of an inert hydrophobic
oil, such as silicone oil, in which the microdroplet can be
submerged during this step. In FIG. 7D the microdroplet now
containing lysed cells is returned, using the syringe, from the
lysis chamber to the superhydrophobic surface. In FIG. 7E,
isopropyl alcohol solution is added to the microdroplet to
precipitate the DNA in the microdroplet. Particularly if the
pipetter tip is plastic, the DNA adheres to the tip. This
collection can be facilitated by slowly rotating the tip by slow
"centrifugation" using the vibration motor 40. In particular
examples, wherein the precipitating DNA does not adhere to the
pipette or other "wire guide" tip, precipitating DNA can be
concentrated and recovered by the microdroplet centrifuging method
described above. In FIG. 7F the pipetter tip (with attached DNA) is
washed in 70% ethanol. In FIG. 7G the syringe is retracted and the
DNA on the surface of the pipetter tip is allowed to air dry. In
FIG. 7H the syringe tip is lowered into a microdroplet of hydration
solution on the superhydrophobic surface to resuspend the DNA in
the solution. To hasten resuspension of the DNA, the syringe needle
may be vibrated using the vibration motor 40.
[0096] The steps described above are also shown in FIGS. 8A-8F. The
protocol shown in FIGS. 8A-8F is particularly directed to preparing
a sample for PCR. In FIG. 8A a microdroplet of concentrated sample
of E. coli (from a microdroplet-centrifugation procedure) is placed
on the superhydrophobic surface. In FIG. 8B cell-lysis solution is
added to the microdroplet and mixed there with by vibrating the
syringe needle. The microdroplet is then transferred to and heated
in the lysis chamber at 80.degree. C. for three minutes. In FIG.
8C, after completion of cell lysis, the microdroplet is removed
from the lysis chamber, and isopropanol is added to precipitate the
DNA. While slowly rotating the needle tip, the precipitated DNA
adheres to the tip. In FIG. 8D, the precipitated DNA is washed in
70% ethanol, and the syringe tip is removed from the microdroplet
for air drying. In FIG. 8E, after air-drying the DNA is resuspended
in hydration solution, as aided by vibration of the needle as
required. In FIG. 8F, the microdroplet is prepared for PCR.
[0097] An innovative step in the DNA-extraction procedures
discussed above is the one in which the precipitated DNA is
conveniently extracted from the microdroplet using the syringe tip
as a substrate. Since the microdroplet is resting on the
superhydrophobic surface, the predominant force is the
electrostatic interaction of the genetic material toward the
pipetter tip during removal, making the extraction process fast and
easy with minimal residual fluid needing to be evaporated.
Polymerase Chain Reaction
[0098] Using the described system 10, any PCR protocol can be
conducted on a sample immediately following extraction of DNA
therefrom or at any other time when a DNA- or RNA-containing sample
is available for analysis.
[0099] For performing PCR, a PCR vessel, such as the PCR vessel
described in FIGS. 3B and 3C is utilized. The PCR vessel contains a
volume of silicone or other inert hydrophobic oil in which the
microdroplets are manipulated and heated at each step of the PCR
cycle. The PCR process is essentially a thermocycling process that
occurs over three different temperatures: denaturation at
94-96.degree. C., annealing at 50-65.degree. C., and extension at
72.degree. C. Each stage in the cycle occurs in a separate chamber
of the PCR vessel, beginning with denaturation. A syringe needle
tip or pipette tip is used to control the movement of the
microdroplet while the microdroplet is submerged in oil during each
stage, and is used to transfer the microdroplet from one chamber to
the next. During the time the microdroplet is in a chamber, the
microdroplet is pendant from the tip of the needle, and the needle
continuously moves the microdroplet relative to the oil in the
chamber to ensure good convective heat transfer from the oil to the
microdroplet (or vice versa). During transfer from one chamber to
the next, the microdroplet desirably is retracted into the tip and
pushed back out after it has entered the next chamber. An exemplary
PCR procedure involves 30 cycles. After PCR, the sample can be
analyzed using a technique such as gel electrophoresis.
[0100] In addition to PCR, the system can also perform related
techniques that require multiple temperature incubation steps, such
as reverse transcriptase PCR (RT-PCR).
[0101] During PCR, the Taq DNA polymerase, or any other thermally
stable DNA polymerase, can be used to amplify the DNA sequence of
interest. Typical extension rates of the polymerase utilized thus
far are about 80 nucleotides per second. Thus, for a template 160
nucleotides or less, about two seconds of holding time at each
temperature of the cycle is usually needed for each step of the
cycle, for a total of six seconds per cycle. In addition, about two
seconds are required at each step for heating up or cooling down
the microdroplet, with the exception of the initial denaturation
step that takes about three seconds, yielding a total of about
seven seconds. Therefore, a single cycle of PCR can be completed
within 13 seconds for a 10-.mu.L microdroplet. If a smaller
microdroplet is used, e.g., 1 .mu.L, the heating/cooling time can
be reduced to less than one second each, allowing a single cycle of
PCR to be completed in as few as six seconds. Therefore, using any
of the embodiments disclosed herein, a 20-cycle PCR amplification
of a 160-bp gene can be completed in just two minutes. One of
ordinary skill will appreciate that, for nucleic acid templates
that are longer or shorter than 160 base pairs, incubation time can
be adjusted accordingly to enable complete copying of the nucleic
acid sequence of interest. Similarly, incubation times can also be
adjusted to account for variations in polymerization rates of
different thermal stable DNA polymerases.
Second Representative Embodiment
[0102] This embodiment of a microdroplet-protocol system is
illustrated in FIG. 9. The system is directed to a hand-held system
200 similar in many ways to the first representative embodiment 10,
but substantially reduced in size. Similar to the first
representative embodiment 10, the second representative embodiment
200 performs the entire PCR process, as well other thermal
processes performable under oil, and various processes performable
on the superhydrophobic surface. These processes include, but are
not limited to, serial dilution, centrifugation, DNA extraction,
sample incubation (such as for reverse transcription), and
thermocycling for PCR, all being performed on, in, or with
microdroplets.
[0103] A schematic diagram of this embodiment 200 is shown in FIG.
9, and includes a three-axis CNC manipulator (an exemplary
controlled movement and placement device) 202, a superhydrophobic
surface 204, a rack 206 of vials, a rack 208 of pipetter tips
and/or needles, a PCR vessel 210 comprising at least three chambers
210a, 210b, 210c, a user interface 212, a heating circuit 214, a
microcontroller 216, a real-time monitoring circuit 218, and a
rechargeable battery 220. At least the racks 206, 208, PCR vessel
210, controlled movement and placement device 202, and
superhydrophobic surface 204 are sequestered from the external
environment, but are visible to a user, through an overlying window
224. The system 200 is hand-held or usable on a work surface.
[0104] The user may choose a pre-programmed protocol that is stored
within the microcontroller 216, or may create his/her own protocol
using the user interface 212. To such end, the user interface 212
comprises requisite buttons, icons, user-prompts, joystick, or
other data-entry means. Also if desired, the user can interrupt an
ongoing process at substantially any time during the process, enter
a modification to the process, and then resume the process or
restart the process.
[0105] Since the system is battery-operated and has its own user
interface 212 and its own microcontroller, there is no need for an
external computer to enable use of the system except for upgrading
the software in the microcontroller. Pipetter tips (or syringe
needles or other tools) are used to withdraw microdroplets of
sample/reagent from a vial in the rack 206, to transfer the
microdroplet(s) to the superhydrophobic surface 204, and to
manipulate individual microdroplets on the superhydrophobic surface
(for serial dilution/centrifuging/DNA extraction, for example).
Pipetter tips and/or needles are also used for delivering
microdroplets to and moving the microdroplets in the PCR vessel
210. By observing through the window 224, the user can monitor
microdroplet manipulations and interrupt and reprogram as needed.
Pipette tips are typically used only once and discarded. The rack
206 of vials, rack 208 of pipetter tips, the superhydrophobic
surface 204, and the PCR vessel 210 collectively can be made as a
single disposable cartridge for use with only a single set of
samples.
[0106] In this and other embodiments, the controlled movement and
positioning device provides a simple manner in which microdroplets
are manipulated, compared to a conventional system. Manipulations
of the microdroplet can be performed on an open surface or under
oil using a wire or needle, wherein the needle is either solid or
hollow, or any of various pipetter tips and the like. These tools
can be selected with respect to materials, sizes, and
configurations thereof relative to the size, shape, volume, and
density of the microdroplets, thereby making the system highly
adaptable to a wide range of microdroplet volumes and properties.
Furthermore, the use of a syringe needle or disposable pipetter tip
can be used to perform precise microdroplet splitting, mixing,
diluting, as well as microdroplet centrifugation, by appropriately
moving and/or vibrating the tool.
[0107] The controlled movement and positioning device 202 in this
embodiment may include a first carriage 226a and a second carriage
226b, both being positionable as commanded by the microcontroller
216 in the three-dimensional space encompassed by the controlled
movement and positioning device. Mounted to the first carriage 226a
is a first syringe 228a, and mounted to the second carriage 226b is
a second syringe 228b. Providing multiple syringes allows the
system 200 to perform correspondingly multiple protocols
simultaneously or correspondingly different steps of a particular
protocol simultaneously, as commanded by the microcontroller
216.
[0108] This embodiment includes a real-time monitoring circuit 220
that is connected to a photodiode 232 situated adjacent a channel
connecting together two chambers in the PCR vessel 210. The
photodiode 232 is situated to receive light from a laser diode 234
or the like disposed on the opposite side of the channel. The
monitoring circuit monitors amplified products produced by PCR and
other protocols performed in the PCR vessel 210. The photodiode 232
desirably is an avalanche photodiode. The voltage signal from the
photodiode is conditioned and amplified using an analog op-amp
circuit, converted to digital signal using an open source
microcontroller board such as Arduino, and the integrated signal is
displayed on a small liquid crystal display that is connected to a
microcontroller board. This feature provides real-time
quantification of the PCR process. Depending upon the outcome of
the real-time quantification, the user can interrupt the PCR
protocol at any time and restart the protocol using a different or
modified protocol.
[0109] This and other embodiments, such as the first representative
embodiment, can be used in a wide variety of applications,
including but not limited to, detecting and quantifying infectious
agents in various settings, medical diagnostics, veterinary
diagnostics, environmental monitoring, and general food safety. A
list of potential users is as follows: (1) research laboratories
(microbiology, food science, veterinary medicine, medicine,
pharmacy, public health, biological/biomedical engineering,
environmental science/engineering, etc.); (2) food industry (fresh
produce, dairy products, meats, etc.); (3) hospital laboratories
(disease diagnostics); (4) veterinary diagnostic laboratories; (5)
environmental monitoring firms (e.g., air quality and water
quality); (6) defense industry (e.g., biological warfare); and
government agencies (e.g., NIH, EPA, CDC, USDA, DOD, etc.).
EXAMPLE 1
Serial Dilution of a Bacterial Culture in a Microdroplet
[0110] This example was directed to controlled serial dilution of a
concentrated sample of E. coli culture (in LB broth) in respective
microdroplets on the superhydrophobic surface.
[0111] The serial dilution was performed in a respective array of
microdroplets of diluent (e.g., phosphate-buffered saline or "PBS")
placed on the superhydrophobic surface, as described above (see
FIGS. 5A-5H). The volume of each microdroplet was 20 .mu.L, and the
volume transferred from one microdroplet to the next was 2 .mu.L.
Four dilutions were performed, yielding a final 10.sup.-4 dilution
of the original suspension. Before performing each dilution, the
syringe was moved to a location at which the syringe tip could (and
was) replaced. The accuracy and precision by which the system
performed the serial dilutions were evaluated using standard
plate-counting methods for E. coli.
[0112] The standard plate-count revealed low variance in the final
E. coli concentrations obtained after performing multiple serial
dilutions.
EXAMPLE 2
Microdroplet Centrifugation on a Superhydrophobic Surface
[0113] In this example, selected diluted samples of suspended E.
coli cells were concentrated by microdroplet centrifugation.
Microdroplet centrifugations were performed using a 22-gauge
blunt-ended syringe needle. The vibration motor 40 executed a
useful vibration of the needle by pulse-width-modulation (PWM).
Each microdroplet was centrifuged in this manner for five minutes,
at which point the liquid in the center of the microdroplet was
aspirated therefrom using the syringe, leaving the concentrated
(outer) portion of the microdroplet. Standard colony plate-counting
methods were used to determine the concentrations of E. coli in the
respective samples before and after centrifugation.
[0114] A high-speed video camera was used to obtain video images of
microdroplet rotation about the syringe needle at 480 frames per
second. The microdroplet rotated at 2,300 round per minute (rpm),
which can further be increased by optimizing the needle size, type
of vibration motor and the pulse-width-modulation (PWM).
[0115] The effects of evaporation on sample concentrations (not yet
microdroplet-centrifuged) and on extracted DNA samples were also
evaluated. Three case studies were compared by measuring the DNA
concentration: (1) stock concentration, (2) concentration after
leaving the microdroplet stationary on the superhydrophobic surface
for five minutes, and (3) concentration after stirring the
microdroplet for five minutes by wire-guided microdroplet
centrifugation.
[0116] FIGS. 6A-6E depict the sequence of events of controlled
centrifugation. The microdroplet to be centrifuged was from a
liquid culture of E. coli. The respective concentrations of E. coli
in the microdroplet before and after centrifugation were analyzed
using standard plate-counting methods. As shown in FIG. 6E,
microdroplet centrifugation produced a 3.06-fold increase in mean
E. coli concentration after only three minutes.
[0117] Computational fluid dynamic (CFD) simulations allowed
analysis of the movements of E. coli cells (as particles) in the
microdroplets using cells of varying sizes (1, 2, 5, 7, 10 .mu.m
diameter) and their locations (as measured from the bottom of the
microdroplet) versus time. When cell diameter was 5, 7, or 10
.mu.m, 120, 60, and 30 seconds, respectively, were required for the
cells to settle to the bottom of the respective microdroplet.
Particles having diameters of 1 and 2 .mu.m never reached the
bottom of the microdroplet even after 300 seconds; however, they
did move by .about.100 .mu.m and .about.300 .mu.m,
respectively.
[0118] These results show that the system can centrifuge E. coli
cells in a microdroplet in a short period of time. The simulation
results also indicated that longer periods of centrifugation for
single colonies may be sufficient for complete centrifugation.
EXAMPLE 3
DNA Extraction from E. coli in a Microdroplet
[0119] This example was directed to controlled DNA extraction from
cells suspended in microdroplets. DNA was extracted from E. coli
cells that were cultured in Luria-Bertani (LB) broth overnight at
room temperature to prevent cells from reaching death phase. Cells
were collected either by conventional centrifugation or by
controlled in situ centrifugation of a microdroplet containing
suspended cells, as described in Example 2.
[0120] Following concentration of the E. coli samples, DNA
extractions were performed in the microdroplets. Nuclei-lysis
solution (e.g., Promega's Cell Lysis Solution Part #A793A included
in Wizard.RTM. Genomic DNA Purification Kit) was added to the
microdroplets. After mixing, the droplets were heated in the
80.degree. C. PCR chamber for 5 min. The resulting lysed samples
were redeposited as microdroplets onto the superhydrophobic surface
and allowed to cool. The DNA in each microdroplet on the surface
was precipitated by addition of 70% isopropanol solution. After an
initial mixing, the pipetter tip was rotated slowly to allow
precipitated DNA to adhere to the tip. The syringe was then moved
to allow air-drying of the tip for 1 min. The tip was then washed
in 70% ethanol and air dried again for 1 min. Finally, the tip was
immersed in DNA rehydration solution for 3 min.
EXAMPLE 4
PCR Thermocycling in a Microdroplet
[0121] This example was directed to controlled PCR thermocycling of
DNA obtained from bacterial cells (see, e.g., Example 3) and
suspended in a microdroplet. Control PCR was performed using a
conventional PCR thermocycler to ensure proper design of primers
and to serve as a positive control. PCR was run according to
manufacturer's instructions from the GoTaq Green Master Mix
(Promega Bio-Tek, Madison, Wis.). Once positive results were
verified, the controlled movement and placement device was used to
detect the presence of E. coli DNA in the microdroplet.
[0122] An AccessQuick.RTM. PCR system kit was used for PCR
performed using the controlled movement and placement device. A
mixture of 4 .mu.L GoTaq Green.RTM., PCR reaction mix, 1 .mu.L of
each 10 .mu.M forward and reverse primers, 1 .mu.L of DNA sample,
and 3 .mu.L of autoclaved water, for a total of 10 .mu.L per
microdroplet. The microdroplets were subjected to 30 PCR cycles
using the PCR vessel described in the First Representative
Embodiment. Following DNA extraction, the system automatically
attached a 14-gauge blunt-ended syringe needle (modified for
friction fit) onto the syringe (by moving the syringe to the rack
containing needles and lowering the syringe onto the needle). The
syringe was then used to extract the PCR-ready solution (DNA
solution+Taq solution+primers). The system was programmed to heat
the denaturing, annealing and extension chambers of the PCR vessel
to 105.degree. C., 55.degree. C., and 80.degree. C., respectively.
The microdroplet began the cycles in the 105.degree. C. chamber for
denaturing for 3 seconds, then the 55.degree. C. chamber for
annealing of primers for 3 seconds, and then the 80.degree. C.
chamber for extension of the products for 5 seconds. The very first
denaturation was conducted for 15 seconds. The final step consisted
of a final annealing for 20 seconds. As described elsewhere herein,
during each of these stages, the system moved the microdroplet in a
circular motion in the oil in the respective chamber to enhance
convective heat transfer from the oil to the droplet. The chambers
were connected together with narrow channels, which allowed the
syringe needle to stay submerged in the silicone oil while moving
from one chamber to the next, and preventing the possibility of
microdroplet loss or evaporation due to the effects of surface
tension that otherwise would arise from inserting and removing the
needle from the oil each time the microdroplet was transferred to
the next chamber.
[0123] To design the primers for PCR, nucleotide sequences of
aminoacyl-histidine dipeptidase (pepD) and 16S ribosomal RNA from
Escherichia coli K12 were pooled from GenBank. These sequences were
then subjected to multiple alignment analysis using ClustalX to
search for a conserved region so that consensus primers could be
designed. Selected primers were then analyzed for appropriate
melting temperature as well as any possible hairpin or
self-dimerization by using OligoAnalyzer 3.1 (IDT Corporation,
Coralville, Iowa, USA). Primers for pepD were 4F (5'-GGG AAT TCG
TCG ACG TGT CTG AAC TGT CTC AAT T-3'; SEQ ID NO: 1) and 4R (5'-GAG
CCG AAG CTT TTA CTT CGC CGG AAT TTC TT-3'; SEQ ID NO: 2) which
resulted in an amplicon of about 1500 base pairs.
[0124] Gel-electrophoresis results of the PCR are shown in FIG. 11.
Lane 1 shows positive control DNA extracted with culture media. The
first lane is the ladder and confirms the .about.1500-bp sequence
being amplified. Lane 2 shows positive-control DNA extracted
without culture media. Since culture media does not interfere with
DNA extraction, Lane 3 shows positive control DNA from a
centrifuged microdroplet. (Pelletizing the E. coli with
microdroplet certification was not possible; therefore the culture
media could not be completely removed). Therefore, the extent of
DNA amplification was qualitatively determined by the positive
controls and lanes 2 and 3, showing that there is a minimal
reduction in band intensity resulting from a lower concentration of
the amplified sequence. However, the strong positive band of lane 2
shows that microdroplet PCR amplification in the presence of
culture media remains a viable option. Lane 4 is the result of the
controlled microdroplet dilution, centrifugation, extraction, and
amplification of E. coli cultured in LB media. Lane 4 exhibits an
increase in signal intensity because of the PCR band achieved
through effective sample concentration by controlled in situ
microdroplet centrifugation. Lane 5 is the result using the
identical processes that produced lane 4, less the concentrating
step from microdroplet centrifugation. The band intensity of lane 4
was much stronger than that of lane 5, further indicating that
microdroplet centrifugation is a useful method for concentrating
samples in the interest of either increasing the positive signal of
PCR amplification or reducing the number of cycles required for a
positive result, thereby decreasing assay time.
[0125] Due to the long genetic sequence being amplified
(.about.1500 bp), and corresponding extension time of 10 minutes
overall (4 seconds for denaturation, 4 seconds for annealing and 8
seconds of extension, with an initial denaturation of 30 seconds
and a final annealing of 60 seconds) was required to perform 30
amplification cycles.
EXAMPLE 5
Modeling of Microdroplet Behavior During Controlled
Centrifugation
[0126] This example is directed to CFD modeling of events
associated with microdroplet centrifugation using the controlled
movement and placement device. In order to track E. coli particles
inside the microdroplet and to estimate the appropriate time for
microdroplet centrifugation prior to DNA extraction, the commercial
software (FLUENT 6.3 and GAMBIT 1.3; FLUENT Inc., Lebanon, N.H.,
USA) was used to build mathematical grids using the finite volume
method (FVM). A 2-D model was used in this study rather than a 3-D
model as the system was symmetric in shape. It was also assumed
that E. coli particles were generated and migratory at a face
parallel to the direction of gravity due to centrifugation and
perpendicular to the axis of rotation.
[0127] The grid size used in the simulation consisted of 70642
cells, 106277 faces and 35636 nodes. The shape of all meshes was
triangular. First-order schemes were used because they provided
better convergence of calculations than the second-order, although
they provided less accurate results due to the increased error in
numerical discretization. An unsteady-state solution was used in
order to track the movement of the E. coli cells by time. Since the
force of gravity from centrifugation was the dominant force of
interest inside the microdroplet, neither the energy equation nor
turbulent model options were chosen in FLUENT. Instead, a discrete
phase model was used to track the E. coli cellsparticles. It was
assumed that size of the microdroplet was 4 mm in diameter. The
boundary was divided evenly into four zones by distance from the
bottom of the microdroplet (zone 1; 2-3 mm from the bottom, zone 2;
1-2 mm from the bottom, zone 3; 0-1 mm from the bottom). Different
gravitational forces were applied to each zone (zone 1: 58.86
m/s.sup.2, zone 2: 107.91 m/s.sup.2, zone 3: 215.82 m/s.sup.2)
based on the distance of that zone from the axis of rotation. For
the migratory simulation, it was assumed that the E. coli particles
started from the top of zone 1 and migrated toward the bottom. Five
cases of simulations were conducted by changing the size of E. coli
cell (diameter of 1, 2, 5, 7, 10 .mu.m) to simulate colony
formation. Each E. coli cell was assumed to be a solid sphere with
density of 1.2 g/cm.sup.3. The time required for each E. coli
colony to migrate to the bottom of the microdroplet was estimated
for each case.
EXAMPLE 6
Detection of H1N1 Influenza A by PCR
[0128] In this example pendant microdroplets, being held on the
ends of blunt syringe needles were heated as required for rapid PCR
thermocycling, using the PCR vessel and automated aspects of the
controlled movement and placement device, as described elsewhere
herein. Droplet heating was by forced convective heat transfer
achieved by moving the microdroplets, while pendant and while
submerged in the oil in the respective chamber of the PCR vessel,
relative to the oil. In this example a 160-bp gene from influenza
A/H1N1/2009 was amplified by the system in 30 cycles of PCR in 6
minutes, 50 seconds in a 10 .mu.L microdroplet.
[0129] RNA polymerase subunit (PA) nucleotide sequences of H1N1
influenza A virus of human origin, swine origin, and avian origin
were pooled from GenBank. These RNA sequences were subjected to
multiple alignment analysis using ClustalX to search for conserved
regions among the three different species so that consensus primers
could be designed. Selected primers were analyzed for appropriate
melting temperature as well as any possible hairpin or
self-dimerization by using OligoAnalyzer 3.1 (IDT Corp.,
Coralville, Iowa). The primers designed to detect RNA polymerase
subunit PA were: PAhln1.sub.--583F (5'-CAGTCCGAAAGAGGCGAAG-3'; SEQ
ID NO: 3) and PAhln1.sub.--745R (5'-GAAAGCTTGCCCTCAATGCAG-3'; SEQ
ID NO: 4), resulting in a product of about 160 base pairs (bp).
[0130] For RNA extraction, NATtrol influenza A H1N1 virus external
round control from Zeptometrix Corporation (Buffalo N.Y.) in medium
was used as a target. This virus sample is 209 H1N1 flu virus of
human origin, as purified/modified by the manufacturer. Its RNA was
extracted using the E.Z.N.A. viral RNA kit (Promega Corp., Madison,
Wis.) through a spin-column protocol. Briefly, 500 .mu.L of QVL
lysis buffer was mixed with 150 .mu.L cell medium, then
precipitated with absolute ethanol. The mixture was passed through
a spin column and cleaned with RWB buffer provided with the kit.
Finally, RNA was eluted from the column using elution buffer. The
resulting RNA was then subjected to one-step RT-PCR.
[0131] Reverse transcription PCR (RT-PCR) was first performed using
a conventional PCR machine to ensure proper design of primers and
proper extraction of the RNA. RT-PCR was run according to
recommendations regarding the AccessQuick.TM. RT-PCR system
(Promega Corp., Madison Wis.). After positive results were
verified, the controlled movement and placement device was employed
to detect the presence of influenza A H1N1 virus in several
samples.
[0132] The AccessQuick.TM. RT-PCR system kit was used for the PCR
reactions performed by the controlled movement and placement
device. Ten-.mu.L microdroplets were subjected to 30 cycles of PCR.
After initial aspiration of the PCR solution into the syringe, the
system transferred the microdroplet to a 37.degree. C. chamber
containing silicone oil for four minutes for reverse transcription
to occur. After forming the cDNA template, the chamber was heated
to 54.degree. C. while the sample was undergoing an initial
30-second denaturation step performed in the 95.degree. C. chamber
of the PCR vessel. The microdroplet began each cycle in the
95.degree. C. chamber for denaturation for 3 seconds, then
transferred to the 54.degree. C. chamber for annealing of primers
for 3 seconds, and then moved to the 72.degree. C. chamber for
extension of the products for 5 seconds. The very first
denaturation was conducted for 15 seconds. The final step after 30
cycles was a final annealing for 20 seconds. During each of these
cycles and steps, the controlled movement and placement device
moved the pendant microdroplet in a circular motion in the oil in
the respective chamber to achieve good convective heat transfer to
or from the microdroplet as required. The chambers were connected
together with respective narrow channels to allow the syringe
needle to remain submerged in the silicone oil, thereby preventing
the possibility of microdroplet evaporation or loss due to the
effects of surface tension arising from insertion and removal of
the needle from the oil. During passage through a channel, the
microdroplet was retracted into the needle.
[0133] PCR products were determined by gel electrophoresis and
fluorescent imaging of the gel. The PCR products were applied to 2%
low-melting agarose gel. The gel was electrophoresed using an 80-V,
1-A power supply for 60 minutes in 1.times. Tris-EDTA (TE) buffer
solution. The gel was soaked in ethidium bromide (.about.1
.mu.g/mL) for approximately 6 minutes and imaged using a GelDoc
1000 imaging system (Bio-Rad Laboratories, Hercules, Calif.).
[0134] For DNA preparation and sequencing, PCR products from a
conventional PCR machine and from the controlled microdroplet
system were cleaned using a QIAquck PCR purification kit (Qiagen,
Valencia Calif.) for sequencing to insure proper detection of the
influenza A H1N1 virus. Amplified PCR products were sequenced using
the Applied Biosystems 3730 DNA analyzer (Carlsbad Calif.).
Sequences were viewed and corrected using a Chromos Lite V 2.01
(Technelysium Pty Ltd, Southport, Queensland, Australia) and
finally checked against the GenBank database.
[0135] The primers used for virus detection were specifically
designed to work across human, swine, and avian strains of
influenza A H1N1. FIG. 10 shows a gel image of about 160 bp
products of influenza A H1N1 (human origin), after 30 cycles of PCR
performed in 6 minutes 50 seconds (lane 3), with an additional four
minute reverse transcription step. This minimal reverse
transcription time was determined through a series of PCR
experiments. Additionally, the results of 30 cycles of PCR
performed in 8 minutes 10 seconds (lane 2) and in 10 minutes (lane
1) are also shown. For the results shown in lane 2 (8 minutes and
10 seconds total cycling time), the microdrop stayed in the
denaturation chamber for 3 seconds, annealing chamber for 4
seconds, and extension chamber for 7 seconds, with 15 seconds
initial denaturation and 30 seconds final annealing. For the
results shown in lane 1 (10 minutes total cycling time), the
microdrop stayed in the denaturation chamber for 4 seconds,
annealing chamber for 4 seconds, and extension chamber for 6
seconds, with 15 seconds initial denaturation and 30 seconds final
annealing. Lane 4 is the result of a negative control experiment in
which PCR cycling was performed under the same conditions but
without primers.
[0136] FIG. 10 showed that slightly less PCR amplification occurred
when the 8-minute, 10-second PCR was reduced to 6 minutes 50
seconds. However, the signal from the longer-cycle experiment was
still relatively strong and was considered a positive result
compared to the negative control. Longer thermocycling correlated
to brighter gel bands, suggesting that sufficient time is important
for the Taq polymerase to amplify the DNA. If there were more DNA
in the target sample or if the reverse transcription time were
increased, then 20 PCR cycles may have been sufficient, which would
require only (13 second).times.(20 cycles)=4 min, 20 seconds to
complete. Also, if a smaller microdroplet were used, e.g. 1 .mu.L,
then the time for each PCR cycle could be further reduced, making
possible a 20-cycle PCR regiment performed in less than three
minutes.
[0137] Although the PCR products are at the correct band size (FIG.
10), further verification was performed to validate the content of
the band. PCR products were cleaned and submitted for sequencing.
The genomic content of each band is then compared against the
nucleotide collection database on the National Center for
Biotechnology Information. The query sequence was 99% to 100%
homology to various samples of 2009 H1N1 influenza A virus with an
E-value of 3.times.10.sup.-50 (Table 1), suggesting that the
controlled movement and placement device worked properly and
successfully detected this virus (99 to 100% homology) in 10
minutes or less.
TABLE-US-00001 TABLE 1 Comparison of amplified viral sequences to
H1N1 genomic database Access Influenza A Max Total Query E- Max No.
Description Score Score Cover Value Ident CY056121.1A/ Dist.
Columbia/ 219 219 95% 3.00 .times. 10.sup.-54 100% 1NS31/2009(H1N1)
CY053282.1A/ Brownsville/ 219 219 95% 3.00 .times. 10.sup.-54 100%
39H/2009(H1N1) CY044906.1A/ New York/ 219 219 95% 3.00 .times.
10.sup.-54 100% 3611/2009 (H1N1) CY050026.1A/ Mexico City/ 215 215
96% 4.00 .times. 10.sup.-53 99% WR1307N/2009 (H1N1) CY061804.1A/
swine/Hong Kong/ 213 213 95% 2.00 .times. 10.sup.-52 99% 189/2101
(H1N1) CY062323.1A/ swine/Thailand/ 213 213 95% 2.00 .times.
10.sup.-52 99% CU-RA9/2009(H1N1) CY062255.1A/ California/ 213 213
95% 2.00 .times. 10.sup.-52 99% VRDL86/2009 (H1N1) CY062191.1A/ New
York/ 213 213 95% 2.00 .times. 10.sup.-52 99% 3251/2010 (H1N1)
CY061591.1A/ Texas/ 213 213 95% 2.00 .times. 10.sup.-52 99%
JMS403/2009(H1N1)
[0138] Whereas the invention has been described above in the
context of multiple representative embodiments, it is not limited
to those embodiments. On the contrary, it is intended to encompass
all modifications, alternatives, and equivalents as may be within
the spirit and scope thereof, as defined by the appended claims.
Sequence CWU 1
1
4134DNAArtificial sequenceSynthetic oligonucleotide 1gggaattcgt
cgacgtgtct gaactgtctc aatt 34232DNAArtificial sequenceSynthetic
oligonucleotide 2gagccgaagc ttttacttcg ccggaatttc tt
32319DNAArtificial sequenceSynthetic oligonucleotide 3cagtccgaaa
gaggcgaag 19421DNAArtificial sequenceSynthetic oligonucleotide
4gaaagcttgc cctcaatgca g 21
* * * * *