U.S. patent application number 10/852642 was filed with the patent office on 2005-06-16 for variable valve apparatus and methods.
This patent application is currently assigned to 3M Innovative Properties Company. Invention is credited to Bedingham, William, Ericson, Katya, Parthasarathy, Ranjani V., Robole, Barry W..
Application Number | 20050130177 10/852642 |
Document ID | / |
Family ID | 34714388 |
Filed Date | 2005-06-16 |
United States Patent
Application |
20050130177 |
Kind Code |
A1 |
Bedingham, William ; et
al. |
June 16, 2005 |
Variable valve apparatus and methods
Abstract
Sample processing devices with variable valve structures and
methods of using the same are disclosed. The valve structures allow
for removal of selected portions of the sample material located
within the process chamber. Removal of the selected portions is
achieved by forming an opening in a valve septum at a desired
location. The valve septums may be large enough to allow for
adjustment of the location of the opening based on the
characteristics of the sample material in the process chamber. If
the sample processing device is rotated after the opening is
formed, the selected portion of the material located closer to the
axis of rotation exits the process chamber through the opening. The
remainder of the sample material cannot exit through the opening
because it is located farther from the axis of rotation than the
opening.
Inventors: |
Bedingham, William;
(Woodbury, MN) ; Robole, Barry W.; (Woodville,
WI) ; Parthasarathy, Ranjani V.; (Woodbury, MN)
; Ericson, Katya; (Fairburn, GA) |
Correspondence
Address: |
3M INNOVATIVE PROPERTIES COMPANY
PO BOX 33427
ST. PAUL
MN
55133-3427
US
|
Assignee: |
3M Innovative Properties
Company
|
Family ID: |
34714388 |
Appl. No.: |
10/852642 |
Filed: |
May 24, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10852642 |
May 24, 2004 |
|
|
|
10734717 |
Dec 12, 2003 |
|
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60532523 |
Dec 24, 2003 |
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Current U.S.
Class: |
435/6.16 ;
435/287.2; 73/863.01 |
Current CPC
Class: |
B01L 2400/0409 20130101;
B01L 3/502738 20130101; B01L 2400/0677 20130101; B01L 2300/0806
20130101 |
Class at
Publication: |
435/006 ;
073/863.01; 435/287.2 |
International
Class: |
C12Q 001/68; C12M
001/34 |
Claims
1. A valved process chamber on a sample processing device, the
valved process chamber comprising: a process chamber comprising a
process chamber volume located between opposing first and second
major sides of the sample processing device, wherein the process
chamber occupies a process chamber area on the sample processing
device, and wherein the process chamber area comprises a length and
a width transverse to the length, and further wherein the length is
greater than the width; a valve chamber located within the process
chamber area, the valve chamber located between the process chamber
volume and the second major side of the sample processing device,
wherein the valve chamber is isolated from the process chamber by a
valve septum separating the valve chamber and the process chamber,
and wherein a portion of the process chamber volume lies between
the valve septum and a first major side of the sample processing
device; and a detection window located within the process chamber
area, wherein the detection window is transmissive to selected
electromagnetic energy directed into and/or out of the process
chamber volume.
2. A valved process chamber according to claim 1, wherein the
detection window is coextensive along the length of the process
chamber with the valve septum.
3. A valved process chamber according to claim 1, wherein the
detection window is formed through the first major side of the
sample processing device.
4. A valved process chamber according to claim 1, wherein the
detection window is formed through the second major side of the
sample processing device.
5. A valved process chamber according to claim 1, wherein the valve
chamber and the detection window occupy mutually exclusive portions
of the process chamber area.
6. A valved process chamber according to claim 1, wherein the
detection window is formed through the second major side of the
sample processing device, and wherein the valve chamber and the
detection window occupy mutually exclusive portions of the process
chamber area.
7. A valved process chamber according to claim 1, wherein the valve
septum extends along the length of the process chamber area for 30%
or more of a maximum length of the process chamber area.
8. A valved process chamber according to claim 1, wherein the valve
septum extends for a length of 1 millimeter or more along the
length of the process chamber.
9. A valved process chamber according to claim 1, wherein the
sample processing device is opaque between the process chamber
volume and the first major side of the sample processing
device.
10. A valved process chamber according to claim 1, wherein at least
a portion of the valve chamber is located within a valve lip
extending into the process chamber area, and wherein the valve
septum is formed in the valve lip.
11. A valved process chamber according to claim 10, wherein the
valve lip occupies only a portion of the width of the process
chamber area.
12. A valved process chamber according to claim 11, wherein the
detection window occupies at least a portion of the width of the
process chamber area that is not occupied by the valve lip.
13. A valved process chamber on a sample processing device, the
valved process chamber comprising: a process chamber comprising a
process chamber volume located between opposing first and second
major sides of the sample processing device, wherein the process
chamber occupies a process chamber area on the sample processing
device, and wherein the process chamber area comprises a length and
a width transverse to the length, and further wherein the length is
greater than the width; a valve chamber located within the process
chamber area, the valve chamber located between the process chamber
volume and the second major side of the sample processing device,
wherein the valve chamber is isolated from the process chamber by a
valve septum separating the valve chamber and the process chamber,
and wherein a portion of the process chamber volume lies between
the valve septum and a first major side of the sample processing
device, and further wherein the valve chamber and the detection
window occupy mutually exclusive portions of the process chamber
area, and still further wherein at least a portion of the valve
chamber is located within a valve lip extending into the process
chamber area, and wherein the valve septum is formed in the valve
lip; and a detection window located within the process chamber
area, wherein the detection window is transmissive to selected
electromagnetic energy directed into and/or out of the process
chamber volume.
14. A method of selectively removing sample material from a process
chamber, the method comprising: providing a sample processing
device comprising: a process chamber comprising a process chamber
volume, wherein the process chamber occupies a process chamber area
on the sample processing device, and wherein the process chamber
area comprises a length and a width transverse to the length, and
further wherein the length is greater than the width; a valve
chamber located within the process chamber area, wherein the valve
chamber is isolated from the process chamber by a valve septum
located between the valve chamber and the process chamber; and a
detection window located within the process chamber area, wherein
the detection window is transmissive for selected electromagnetic
energy; providing sample material in the process chamber; detecting
a characteristic of the sample material in the process chamber
through the detection window; forming an opening in the valve
septum at a selected location along the length of the process
chamber, wherein the selected location is correlated to the
detected characteristic of the sample material; and moving only a
portion of the sample material from the process chamber into the
valve chamber through the opening formed in the valve septum.
15. A method according to claim 14, wherein moving only a portion
of the sample material from the process chamber into the valve
chamber comprises rotating the sample processing device.
16. A method according to claim 14, wherein the process chamber
area comprises a length and a width transverse to the length, and
further wherein the length is greater than the width.
17. A method according to claim 14, wherein the detected
characteristic comprises a free surface of the sample material, and
wherein the portion of the sample material moved from the process
chamber into the valve chamber comprises a selected volume of the
sample material.
18. A method according to claim 14, further comprising rotating the
sample processing device to separate components of the sample
material in the process chamber.
19. A method according to claim 18, wherein the detected
characteristic of the sample material comprises a boundary between
the separated components of the sample material, and wherein the
portion of the sample material moved from the process chamber into
the valve chamber comprises a portion of a selected component of
the sample material.
20. A method according to claim 14, wherein moving only a portion
of the sample material from the process chamber into the valve
chamber comprises moving a selected volume of the sample material
from the process chamber into the valve chamber.
21. A method according to claim 14, wherein the sample material
comprises blood.
22. A method of selectively removing sample material from a process
chamber, the method comprising: providing a sample processing
device comprising: a process chamber comprising a process chamber
volume, wherein the process chamber occupies a process chamber area
on the sample processing device, and wherein the process chamber
area comprises a length and a width transverse to the length, and
further wherein the length is greater than the width; a valve
chamber located within the process chamber area, wherein the valve
chamber is isolated from the process chamber by a valve septum
located between the valve chamber and the process chamber; and a
detection window located within the process chamber area, wherein
the detection window is transmissive for selected electromagnetic
energy; providing sample material in the process chamber; detecting
a characteristic of the sample material in the process chamber
through the detection window; forming an opening in the valve
septum at a selected location within the process chamber area,
wherein the selected location is correlated to the detected
characteristic of the sample material; and moving only a portion of
the sample material from the process chamber into the valve chamber
through the opening formed in the valve septum by rotating the
sample processing device.
23. A method of isolating nucleic acid from whole blood, the method
comprising: providing a device comprising a loading chamber and a
variable valved process chamber; placing whole blood in the loading
chamber; transferring the whole blood to a valved process chamber;
centrifuging the whole blood in the valved process chamber to form
a plasma layer, a red blood cell layer, and an interfacial layer
comprising white blood cells; removing at least a portion of the
interfacial layer comprising white blood cells; and lysing the
white blood cells in the separated interfacial layer and optionally
lysing the nuclei therein to release inhibitors and/or nucleic
acid.
24. The method of claim 23 wherein prior to lysing the white blood
cells, the method includes diluting the separated interfacial layer
of the sample with water or buffer, optionally further
concentrating the diluted layer to increase the concentration of
nucleic acid material, optionally separating the further
concentrated region, and optionally repeating this process of
dilution followed by concentration and separation to reduce the
inhibitor concentration to that which would not interfere with an
amplification method.
25. The method of claim 23 wherein the device further comprises a
separation chamber comprising a solid phase material.
26. The method of claim 25 wherein the solid phase material
comprises capture sites, a coating reagent coated on the solid
phase material, or both; wherein the coating reagent is selected
from the group consisting of a surfactant, a strong base, a
polyelectrolyte, a selectively permeable polymeric barrier, and
combinations thereof.
27. The method of claim 25 further comprising contacting the lysed
sample with the solid phase material in the separation chamber to
preferentially adhere at least a portion of the inhibitors to the
solid phase material; wherein lysing can occur before, simultaneous
with, or after contacting the solid phase material.
28. The method of claim 25 further comprising separating at least a
portion of the nuclei and/or nucleic acid from the solid phase
material having at least a portion of the inhibitors adhered
thereto.
29. The method of claim 23 wherein lysing comprises subjecting the
white blood cells to a strong base with optional heating to release
nucleic acid.
30. The method of claim 29 further comprising adjusting the pH of
the sample comprising the released nucleic acid to be within a
range of 7.5 to 9.
31. The method of claim 23 further comprising diluting the lysed
sample with water to reduce the inhibitor concentration to that
which would not interfere with an amplification method; optionally
further lysing the nuclei to release nucleic acid; and optionally
adjusting the pH of the sample comprising released nucleic
acid.
32. The method of claim 31 wherein diluting the lysed sample
comprises diluting with water to reduce the concentration of heme
to less than 2 micromolar.
33. The method of claim 31 wherein diluting the lysed sample
comprises diluting sufficiently with water to form a
20.times.-1000.times. dilution of the lysed sample.
34. The method of claim 31 wherein the water is RNAse-free sterile
water.
35. A method of isolating nucleic acid from whole blood, the method
comprising: providing a device comprising a loading chamber and a
variable valved process chamber; placing whole blood in the loading
chamber; transferring the whole blood to a valved process chamber;
contacting the whole blood with a density gradient material;
centrifuging the whole blood and density gradient material in the
valved process chamber to form layers, at least one of which
contains cells of interest; removing at least a portion of the
layer comprising the cells of interest; and lysing the separated
cells of interest to release nucleic acid.
36. The method of claim 35 wherein prior to lysing the separated
cells of interest, the method includes diluting the separated cells
of interest with water or buffer, optionally further concentrating
the diluted layer to increase the concentration of cells of
interest, optionally separating the further concentrated region,
and optionally repeating this process of dilution followed by
concentration and separation.
37. The method of claim 35 wherein prior to lysing the separated
cells of interest, the method includes diluting the separated cells
of interest with water.
38. The method of claim 37 wherein diluting the separated cells of
interest comprises diluting sufficiently with water to form a
20.times.-1000.times. dilution.
39. A method of isolating nucleic acid from whole blood comprising
one or more pathogens, the method comprising: providing a device
comprising a loading chamber, a variable valved process chamber,
and a separation chamber comprising pathogen capture material;
placing whole blood in the loading chamber; transferring the whole
blood to a valved process chamber; centrifuging the whole blood in
the valved process chamber to form a plasma layer comprising one or
more pathogens, a red blood cell layer, and an interfacial layer
comprising white blood cells; transferring at least a portion of
the plasma layer comprising one or more pathogens to the separation
chamber comprising pathogen capture material; separating at least a
portion of the one or more pathogens from the pathogen capture
material; and lysing the one or more pathogens to release nucleic
acid.
40. A kit for isolating nucleic acid from a sample, the kit
comprising: a device comprising a loading chamber and a variable
valved process chamber; and instructions for centrifuging whole
blood and removing a portion of the whole blood according to the
method of claim 23.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a Continuation-In-Part of U.S.
patent application Ser. No. 10/734,717, filed on Dec. 12, 2003, and
claims priority to U.S. Provisional Patent Application Ser. No.
60/532,523, filed on Dec. 24, 2003, both of which are incorporated
herein by reference in their entireties.
BACKGROUND
[0002] Sample processing devices including process chambers in
which various chemical or biological processes are performed play
an increasing role in scientific and/or diagnostic investigations.
The process chambers provided in such devices are preferably small
in volume to reduce the amount of sample material required to
perform the processes.
[0003] One persistent issue associated with sample processing
devices including process chambers is in the transfer of fluids
between different features in the devices. Conventional approaches
to separate and transfer fluidic contents of process chambers have
often required human intervention (e.g., manual pipetting) and/or
robotic manipulation. Such transfer processes suffer from a number
of disadvantages including, but not limited to, the potential for
errors, complexity and associated high costs, etc.
[0004] Attempts to address the fluid transfer issues have focused
on transferring the entire fluid contents of the process chambers
through, e.g., valves, tortuous paths, etc.
SUMMARY OF THE INVENTION
[0005] The present invention provides sample processing devices
with valve structures. The valve structures allow for removal of
selected portions of the sample material located within the process
chamber. Removal of the selected portions is achieved by forming an
opening in a valve septum at a desired location.
[0006] The valve septums are preferably large enough to allow for
adjustment of the location of the opening based on the
characteristics of the sample material in the process chamber. If
the sample processing device is rotated after the opening is
formed, the selected portion of the material located closer to the
axis of rotation exits the process chamber through the opening. The
remainder of the sample material cannot exit through the opening
because it is located farther from the axis of rotation than the
opening.
[0007] The openings in the valve septum may be formed at locations
based on one or more characteristics of the sample material
detected within the process chamber. It may be preferred that the
process chambers include detection windows that transmit light into
and/or out of the process chamber. Detected characteristics of the
sample material may include, e.g., the free surface of the sample
material (indicative of the volume of sample material in the
process chamber). Forming an opening in the valve septum at a
selected distance radially outward of the free surface can provide
the ability to remove a selected volume of the sample material from
the process chamber.
[0008] For sample materials that can be separated into various
components, e.g., whole blood, rotation of the sample processing
device may result in separation of the plasma and red blood cell
components, thus allowing for selective removal of the components
to, e.g., different process chambers.
[0009] In some embodiments, it may be possible to remove selected
aliquots of the sample material by forming openings at selected
locations in one or more valve septums. The selected aliquot volume
can be determined based on the radial distance between the openings
(measured relative to the axis of rotation) and the cross-sectional
area of the process chamber between the opening.
[0010] The openings in the valve septums are preferably formed in
the absence of physical contact, e.g., through laser ablation,
focused optical heating, etc. As a result, the openings can
preferably be formed without piercing the outermost layers of the
sample processing device, thus limiting the possibility of leakage
of the sample material from the sample processing device.
[0011] In one aspect, the present invention provides a valved
process chamber on a sample processing device, the valved process
chamber including a process chamber having a process chamber volume
located between opposing first and second major sides of the sample
processing device, wherein the process chamber occupies a process
chamber area on the sample processing device, and wherein the
process chamber area has a length and a width transverse to the
length, and further wherein the length is greater than the width.
The valved process chamber also includes a valve chamber located
within the process chamber area, the valve chamber located between
the process chamber volume and the second major side of the sample
processing device, wherein the valve chamber is isolated from the
process chamber by a valve septum separating the valve chamber and
the process chamber, and wherein a portion of the process chamber
volume lies between the valve septum and a first major side of the
sample processing device. A detection window is located within the
process chamber area, wherein the detection window is transmissive
to selected electromagnetic energy directed into and/or out of the
process chamber volume.
[0012] In another aspect, the present invention provides a valved
process chamber on a sample processing device, the valved process
chamber including a process chamber having a process chamber volume
located between opposing first and second major sides of the sample
processing device, wherein the process chamber occupies a process
chamber area on the sample processing device, and wherein the
process chamber area has a length and a width transverse to the
length, and further wherein the length is greater than the width.
The valved process chamber also includes a valve chamber located
within the process chamber area, the valve chamber located between
the process chamber volume and the second major side of the sample
processing device, wherein the valve chamber is isolated from the
process chamber by a valve septum separating the valve chamber and
the process chamber, and wherein a portion of the process chamber
volume lies between the valve septum and a first major side of the
sample processing device, and further wherein the valve chamber and
the detection window occupy mutually exclusive portions of the
process chamber area, and still further wherein at least a portion
of the valve chamber is located within a valve lip extending into
the process chamber area, and wherein the valve septum is formed in
the valve lip. A detection window is located within the process
chamber area, wherein the detection window is transmissive to
selected electromagnetic energy directed into and/or out of the
process chamber volume.
[0013] In another aspect, the present invention includes a method
of selectively removing sample material from a process chamber. The
method includes providing a sample processing device that includes
a process chamber having a process chamber volume, wherein the
process chamber occupies a process chamber area on the sample
processing device; a valve chamber located within the process
chamber area, wherein the valve chamber is isolated from the
process chamber by a valve septum located between the valve chamber
and the process chamber; and a detection window located within the
process chamber area, wherein the detection window is transmissive
for selected electromagnetic energy. The method further includes
providing sample material in the process chamber; detecting a
characteristic of the sample material in the process chamber
through the detection window; and forming an opening in the valve
septum at a selected location along the length of the process
chamber, wherein the selected location is correlated to the
detected characteristic of the sample material. The method also
includes moving only a portion of the sample material from the
process chamber into the valve chamber through the opening formed
in the valve septum.
[0014] In another aspect, the present invention provides a method
of selectively removing sample material from a process chamber. The
method includes providing a sample processing device having a
process chamber with a process chamber volume, wherein the process
chamber occupies a process chamber area on the sample processing
device, and wherein the process chamber area includes a length and
a width transverse to the length, and further wherein the length is
greater than the width. The sample processing device also includes
a valve chamber located within the process chamber area, wherein
the valve chamber is isolated from the process chamber by a valve
septum located between the valve chamber and the process chamber;
and a detection window located within the process chamber area,
wherein the detection window is transmissive for selected
electromagnetic energy. The method also includes providing sample
material in the process chamber; detecting a characteristic of the
sample material in the process chamber through the detection
window; forming an opening in the valve septum at a selected
location within the process chamber area, wherein the selected
location is correlated to the detected characteristic of the sample
material; and moving only a portion of the sample material from the
process chamber into the valve chamber through the opening formed
in the valve septum by rotating the sample processing device.
[0015] In another embodiment, the present invention provides a
method of isolating nucleic acid from whole blood, the method
including: providing a device that includes a loading chamber and a
variable valved process chamber; placing whole blood in the loading
chamber; transferring the whole blood to a valved process chamber;
centrifuging the whole blood in the valved process chamber to form
a plasma layer (often the upper layer), a red blood cell layer
(often the lower layer), and an interfacial layer that includes
white blood cells; removing at least a portion of the interfacial
layer; and lysing the white blood cells in the separated
interfacial layer and optionally lysing the nuclei therein to
release inhibitors and/or nucleic acid.
[0016] If desired, prior to lysing the white blood cells, the
method can include diluting the separated interfacial layer of the
sample with water (preferably, RNAse-free sterile water) or buffer,
optionally further concentrating the diluted layer to increase the
concentration of nucleic acid material, optionally separating the
further concentrated region, and optionally repeating this process
of dilution followed by concentration and separation to reduce the
inhibitor concentration to that which would not interfere with an
amplification method.
[0017] Alternatively, before, simultaneously with, or after lysing
the white blood cells, if desired, the method can include
transferring the separated interfacial layer to a separation
chamber for contact with solid phase material to preferentially
adhere at least a portion of the inhibitors to the solid phase
material; wherein the solid phase material includes capture sites
(e.g., chelating functional groups), a coating reagent coated on
the solid phase material, or both; wherein the coating reagent is
selected from the group consisting of a surfactant, a strong base,
a polyelectrolyte, a selectively permeable polymeric barrier, and
combinations thereof.
[0018] Another embodiment of the present invention involves a
method of isolating nucleic acid from whole blood using a density
gradient material. In this embodiment, the method includes:
providing a device that includes a loading chamber and a variable
valved process chamber; placing whole blood in the loading chamber;
transferring the whole blood to a valved process chamber;
contacting the whole blood with a density gradient material;
centrifuging the whole blood and density gradient material in the
valved process chamber to form layers, at least one of which
contains cells of interest; removing at least a portion of the
layer that includes the cells of interest; and lysing the separated
cells of interest to release nucleic acid.
[0019] In another embodiment, the present invention provides a
method of isolating nucleic acid from whole blood that includes a
pathogen, the method includes: providing a device that includes a
loading chamber, a variable valved process chamber, and a
separation chamber with pathogen capture material therein; placing
whole blood in the loading chamber; transferring the whole blood to
a valved process chamber; centrifuging the whole blood in the
valved process chamber to form a plasma layer that includes a
pathogen, a red blood cell layer, and an interfacial layer that
includes white blood cells; transferring at least a portion of the
plasma layer with the pathogen to the separation chamber including
pathogen capture material; separating at least a portion of the
pathogen from the pathogen capture material; and lysing the
pathogen to release nucleic acid.
[0020] The present invention also provides kits for carrying out
the various methods of the present invention.
[0021] These and other features and advantages of the present
invention are described below in connection with various
illustrative embodiments of the devices and methods of the present
invention.
[0022] Definitions
[0023] "Nucleic acid" shall have the meaning known in the art and
refers to DNA (e.g., genomic DNA, cDNA, or plasmid DNA), RNA (e.g.,
mRNA, tRNA, or rRNA), and PNA. It can be in a wide variety of
forms, including, without limitation, double-stranded or
single-stranded configurations, circular form, plasmids, relatively
short oligonucleotides, peptide nucleic acids also called PNA's (as
described in Nielsen et al., Chem. Soc. Rev., 26, 73-78 (1997)),
and the like. The nucleic acid can be genomic DNA, which can
include an entire chromosome or a portion of a chromosome. The DNA
can include coding (e.g., for coding mRNA, tRNA, and/or rRNA)
and/or noncoding sequences (e.g., centromeres, telomeres,
intergenic regions, introns, transposons, and/or microsatellite
sequences). The nucleic acid can include any of the naturally
occurring nucleotides as well as artificial or chemically modified
nucleotides, mutated nucleotides, etc. The nucleic acid can include
a non-nucleic acid component, e.g., peptides (as in PNA's), labels
(radioactive isotopes or fluorescent markers), and the like.
[0024] "Nucleic acid-containing material" refers to a source of
nucleic acid such as a cell (e.g., white blood cell, enucleated red
blood cell), a nuclei, or a virus, or any other composition that
houses a structure that includes nucleic acid (e.g., plasmid,
cosmid, or viroid, archeobacteriae). The cells can be prokaryotic
(e.g., gram positive or gram negative bacteria) or eukaryotic
(e.g., blood cell or tissue cell). If the nucleic acid-containing
material is a virus, it can include an RNA or a DNA genome; it can
be virulent, attenuated, or noninfectious; and it can infect
prokaryotic or eukaryotic cells. The nucleic acid-containing
material can be naturally occurring, artificially modified, or
artificially created.
[0025] "Isolated" refers to nucleic acid (or nucleic
acid-containing material) that has been separated from at least a
portion of the inhibitors (i.e., at least a portion of at least one
type of inhibitor) in a sample. This includes separating desired
nucleic acid from other materials, e.g., cellular components such
as proteins, lipids, salts, and other inhibitors. More preferably,
the isolated nucleic acid is substantially purified. "Substantially
purified" refers to isolating nucleic acid of at least 3 picogram
per microliter (pg/.mu.L), preferably at least 2
nanogram/microliter (ng/.mu.L), and more preferably at least 15
ng/.mu.L, while reducing the inhibitor amount from the original
sample by at least 20%, preferably by at least 80% and more
preferably by at least 99%. The contaminants are typically cellular
components and nuclear components such as heme and related products
(hemin, hematin) and metal ions, proteins, lipids, salts, etc.,
other than the solvent in the sample. Thus, the term "substantially
purified" generally refers to separation of a majority of
inhibitors (e.g., heme and it degradation products) from the
sample, so that compounds capable of interfering with the
subsequent use of the isolated nucleic acid are at least partially
removed.
[0026] "Adheres to" or "adherence" or "binding" refer to reversible
retention of inhibitors to an optional solid phase material via a
wide variety of mechanisms, including weak forces such as Van der
Waals interactions, electrostatic interactions, affinity binding,
or physical trapping. The use of this term does not imply a
mechanism of action, and includes adsorptive and absorptive
mechanisms.
[0027] "Solid phase material" (which can optionally be included
within a device in methods of the present invention) refers to an
inorganic and/or organic material, preferably a polymer made of
repeating units, which may be the same or different, of organic
and/or inorganic compounds of natural and/or synthetic origin. This
includes homopolymers and heteropolymers (e.g., copolymers,
terpolymers, tetrapolymers, etc., which may be random or block, for
example). This term includes fibrous or particulate forms of a
polymer, which can be readily prepared by methods well-known in the
art. Such materials typically form a porous matrix, although for
certain embodiments, the solid phase also refers to a solid
surface, such as a nonporous sheet of polymeric material.
[0028] The optional solid phase material may include capture sites.
"Capture sites" refer to sites on the solid phase material to which
a material adheres. Typically, the capture sites include functional
groups or molecules that are either covalently attached or
otherwise attached (e.g., hydrophobically attached) to the solid
phase material.
[0029] The phrase "coating reagent coated on the solid phase
material" refers to a material coated on at least a portion of the
solid phase material, e.g., on at least a portion of the fibril
matrix and/or sorptive particles.
[0030] "Surfactant" refers to a substance that lowers the surface
or interfacial tension of the medium in which it is dissolved.
[0031] "Strong base" refers to a base that is completely
dissociated in water, e.g., NaOH.
[0032] "Polyelectrolyte" refers to an electrolyte that is a charged
polymer, typically of relatively high molecular weight, e.g.,
polystyrene sulfonic acid.
[0033] "Selectively permeable polymeric barrier" refers to a
polymeric barrier that allows for selective transport of a fluid
based on size and charge.
[0034] "Concentrated region" refers to a region of a sample that
has a higher concentration of nucleic acid-containing material,
nuclei, and/or nucleic acid, which can be in a pellet form,
relative to the less concentrated region.
[0035] "Substantially separating" as used herein, particularly in
the context of separating a concentrated region of a sample from a
less concentrated region of a sample, means removing at least 40%
of the total amount of nucleic acid (whether it be free, within
nuclei, or within other nucleic acid-containing material) in less
than 25% of the total volume of the sample. Preferably, at least
75% of the total amount of nucleic acid in less than 10% of the
total volume of sample is separated from the remainder of the
sample. More preferably, at least 95% of the total amount of
nucleic acid in less than 5% of the total volume of sample is
separated from the remainder of the sample.
[0036] "Inhibitors" refer to inhibitors of enzymes used in
amplification reactions, for example. Examples of such inhibitors
typically include iron ions or salts thereof (e.g., Fe.sup.2+ or
salts thereof) and other metal salts (e.g., alkali metal ions,
transition metal ions). Other inhibitors can include proteins,
peptides, lipids, carbohydrates, heme and its degradation products,
urea, bile acids, humic acids, polysaccharides, cell membranes, and
cytosolic components. The major inhibitors in human blood for PCR
are hemoglobin, lactoferrin, and IgG, which are present in
erythrocytes, leukocytes, and plasma, respectively. The methods of
the present invention separate at least a portion of the inhibitors
(i.e., at least a portion of at least one type of inhibitor) from
nucleic acid-containing material. As discussed herein, cells
containing inhibitors can be the same as the cells containing
nuclei or other nucleic acid-containing material. Inhibitors can be
contained in cells or be extracellular. Extracellular inhibitors
include all inhibitors not contained within cells, which includes
those inhibitors present in serum or viruses, for example.
[0037] "Preferentially adhere at least a portion of the inhibitors
to the solid phase material" means that one or more types of
inhibitors will adhere to the optional solid phase material to a
greater extent than nucleic acid-containing material (e.g., nuclei)
and/or nucleic acid, and typically without adhering a substantial
portion of the nucleic acid-containing material and/or nuclei to
the solid phase material.
[0038] "Microfluidic" (where used herein) refers to a device with
one or more fluid passages, chambers, or conduits that have at
least one internal cross-sectional dimension, e.g., depth, width,
length, diameter, etc., that is less than 500 .mu.m, and typically
between 0.1 .mu.m and 500 .mu.m. In the devices used in the present
invention, the microscale channels or chambers may preferably have
at least one cross-sectional dimension between 0.1 .mu.m and 200
.mu.m, more preferably between 0.1 .mu.m and 100 .mu.m, and often
between 1 .mu.m and 20 .mu.m. Typically, a microfluidic device
includes a plurality of chambers (process chambers, separation
chambers, mixing chambers, waste chambers, diluting reagent
chambers, amplification reaction chambers, loading chambers, and
the like), each of the chambers defining a volume for containing a
sample; and at least one distribution channel connecting the
plurality of chambers of the array; wherein at least one of the
chambers within the array can include a solid phase material
(thereby often being referred to as a separation chamber) and/or at
least one of the process chambers within the array can include a
lysing reagent (thereby often being referred to as a mixing
chamber), for example.
[0039] The terms "comprises" and variations thereof do not have a
limiting meaning where these terms appear in the description and
claims.
[0040] Also herein, the recitations of numerical ranges by
endpoints include all numbers subsumed within that range (e.g., 1
to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
[0041] The above summary of the present invention is not intended
to describe each disclosed embodiment or every implementation of
the present invention. The description that follows more
particularly exemplifies illustrative embodiments. In several
places throughout the application, guidance is provided through
lists of examples, which examples can be used in various
combinations. In each instance, the recited list serves only as a
representative group and should not be interpreted as an exclusive
list. Furthermore, various embodiments are described in which the
various elements of each embodiment could be used in other
embodiments, even though not specifically described.
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] FIG. 1 is a plan view of one exemplary sample processing
device according to the present invention.
[0043] FIG. 2 is an enlarged cross-sectional view of a portion of
the sample processing device of FIG. 1, taken along line 2-2 in
FIG. 1.
[0044] FIGS. 3A-3D depict one exemplary method of moving fluid
through a process array including a process chamber and a valve
chamber.
[0045] FIG. 4 is a plan view of an alternative process chamber and
multiple valve chambers in accordance with the present
invention.
[0046] FIG. 5 is a cross-sectional view of another alternative
process chamber and valve chamber construction according to the
present invention, including optional detection apparatus facing
both major sides of the sample processing device.
[0047] FIG. 6 is a representation of a device used in certain
methods of the present invention.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS OF THE
INVENTION
[0048] In the following detailed description of illustrative
embodiments of the invention, reference is made to the accompanying
figures of the drawing which form a part hereof, and in which are
shown, by way of illustration, specific embodiments in which the
invention may be practiced. It is to be understood that other
embodiments may be utilized and structural changes may be made
without departing from the scope of the present invention.
[0049] The present invention provides a sample processing device
that can be used in the processing of liquid sample materials (or
sample materials entrained in a liquid) in multiple process
chambers to obtain desired reactions, e.g., PCR amplification,
ligase chain reaction (LCR), self-sustaining sequence replication,
enzyme kinetic studies, homogeneous ligand binding assays, and
other chemical, biochemical, or other reactions that may, e.g.,
require precise and/or rapid thermal variations. More particularly,
the present invention provides sample processing devices that
include one or more process arrays, each of which may preferably
include a loading chamber, at least one process chamber, a valve
chamber, and conduits for moving fluids between various components
of the process arrays. The devices of the present invention may or
may not include microfluidic features.
[0050] Although various constructions of illustrative embodiments
are described below, sample processing devices of the present
invention may be similar to those described in, e.g., U.S. Patent
Application Publication Nos. US2002/0064885 (Bedingham et al.);
US2002/0048533 (Bedingham et al.); US2002/0047003 (Bedingham et
al.), and US2003/138779 (Parthasarathy et al.); as well as U.S.
Pat. No. 6,627,159 B1 (Bedingham et al.). The documents identified
above all disclose a variety of different constructions of sample
processing devices that could be used to manufacture sample
processing devices according to the principles of the present
invention.
[0051] One illustrative sample processing device manufactured
according to the principles of the present invention is illustrated
in FIGS. 1 & 2, where FIG. 1 is a plan view of one sample
processing device 10 and FIG. 2 is an enlarged cross-sectional view
of a portion of the sample processing device 10 (taken along line
2-2 in FIG. 1). The sample processing device 10 may preferably be
in the shape of a circular disc as illustrated in FIG. 1, although
any other shape that can be rotated could be used in place of a
circular disc.
[0052] The sample processing device 10 includes at least one, and
preferably multiple process arrays 20. If the sample processing
device 10 is circular as depicted, it may be preferred that each of
the depicted process arrays 20 extends from proximate a center 12
of the sample processing device 10 towards the periphery of the
sample processing device 10. The process arrays 20 are depicted as
being substantially aligned radially with respect to the center 12
of the sample processing device 10. Although this arrangement may
be preferred, it will be understood that any arrangement of process
arrays 20 may alternatively be used. Also, although the illustrated
sample processing device 10 includes four process arrays 20, the
exact number of process arrays provided in connection with a sample
processing device manufactured according to the present invention
may be greater than or less than four.
[0053] Each of the process arrays 20 (in the embodiment depicted in
FIG. 1) includes a loading chamber 30 connected to a process
chamber 40 along a conduit 32. The process arrays 20 also include a
valve chamber 60 connected to a second process chamber 70 by a
conduit 62. The valve chamber 60 may preferably be located within a
valve lip 50 extending into the area occupied by the process
chamber 40 on the sample processing device 10.
[0054] It should be understood that a number of the features
associated with one or more of the process arrays 20 may be
optional. For example, the loading chambers 30 and associated
conduits 32 may be optional where sample material can be introduced
directly into the process chambers 40 through a different loading
structure. At the same time, additional features may be provided
with one or more of the process arrays 20. For example, two or more
valve chambers 60 may be associated with one or more of the process
arrays 20. Additional valve chambers may be associated with
additional process chambers or other features.
[0055] Any loading structure provided in connection with the
process arrays 20 may be designed to mate with an external
apparatus (e.g., a pipette, hollow syringe, or other fluid delivery
apparatus) to receive the sample material. The loading structure
itself may define a volume (as, e.g., does loading chamber 30 of
FIG. 1) or the loading structure may define no specific volume,
but, instead, be a location at which sample material is to be
introduced. For example, the loading structure may be provided in
the form of a port through which a pipette or needle is to be
inserted. In one embodiment, the loading structure may be, e.g., a
designated location along a conduit that is adapted to receive a
pipette, syringe needle, etc. The loading may be performed manually
or by an automated system (e.g., robotic, etc.). Further, the
sample processing device 10 may be loaded directly from another
device (using an automated system or manually).
[0056] FIG. 2 is an enlarged cross-sectional view of the processing
device 10 taken along line 2-2 in FIG. 1. Although sample
processing devices of the present invention may be manufactured
using any number of suitable construction techniques, one
illustrative construction can be seen in the cross-sectional view
of FIG. 2. The sample processing device 10 includes a base layer 14
attached to a valve layer 16. A cover layer 18 is attached to the
valve layer 16 over the side of the valve layer 16 that faces away
from the base layer 14.
[0057] The layers of sample processing device 10 may be
manufactured of any suitable material or combination of materials.
Examples of some suitable materials for the base layer 14 and/or
valve layer 16 include, but are not limited to, polymeric material,
glass, silicon, quartz, ceramics, etc. For those sample processing
devices 10 in which the layers will be in direct contact with the
sample materials, it may be preferred that the material or
materials used for the layers be non-reactive with the sample
materials. Examples of some suitable polymeric materials that could
be used for the substrate in many different bioanalytical
applications may include, but are not limited to, polycarbonate,
polypropylene (e.g., isotactic polypropylene), polyethylene,
polyester, etc.
[0058] The layers making up sample processing device 10 may be
attached to each other by any suitable technique or combination of
techniques. Suitable attachment techniques preferably have
sufficient integrity such that the attachment can withstand the
forces experienced during processing of sample materials in the
process chambers. Examples of some of the suitable attachment
techniques may include, e.g., adhesive attachment (using pressure
sensitive adhesives, curable adhesives, hot melt adhesives, etc.),
heat sealing, thermal welding, ultrasonic welding, chemical
welding, solvent bonding, coextrusion, extrusion casting, etc. and
combinations thereof. Furthermore, the techniques used to attach
the different layers may be the same or different. For example, the
technique or techniques used to attach the base layer 14 and the
valve layer 16 may be the same or different as the technique or
techniques used to attach the cover layer 18 and the valve layer
16.
[0059] FIG. 2 depicts a process chamber 40 in its cross-sectional
view. Also seen in FIG. 2 is the valve lip 50 that, in the depicted
embodiment is located within the area occupied by the process
chamber, i.e., the process chamber area. The process chamber are
may preferably be defined by projecting the process chamber
boundaries onto either of the major sides of the sample processing
device 10. In the embodiment depicted in FIG. 2, a first major side
15 of the sample processing device 10 is defined by the lowermost
surface of base layer 14 (i.e., the surface facing away from valve
layer 16) and a second major side 19 is defined by the uppermost
surface of cover layer 18 (i.e., the surface facing away from the
valve layer 16). It should be understood that "upper" and "lower"
as used herein are with reference to FIG. 2 only and are not to be
construed as limiting the orientation of the sample processing
device 10 in use.
[0060] The valve lip 50 is depicted as extending into the process
chamber area as defined by the outermost boundaries of process
chamber 40. Because the valve lip 50 is located within the process
chamber area, the valve lip 50 may be described as overhanging a
portion of the process chamber 40 or being cantilevered over a
portion of the process chamber 40.
[0061] Preferred process chambers of the present invention may
include a detection window that allows the detection of one or more
characteristics of any sample material in the process chamber 40.
It may be preferred that the detection be achieved using selected
light, where the term "light" refers to electromagnetic energy,
whether visible to the human eye or not. It may be preferred that
the light fall within a range of ultraviolet to infrared
electromagnetic energy, and, in some instances, it may be preferred
that light include electromagnetic energy in the spectrum visible
to the human eye. Furthermore, the selected light may be, e.g.,
light of one or more particular wavelengths, one or more ranges of
wavelengths, one or more polarization states, or combinations
thereof.
[0062] In the embodiment depicted in FIG. 2, the detection window
may be provided in the cover layer 18 or in the base layer 14 (or
both). Regardless of which component is used as the detection
window, the materials used preferably transmit significant portions
of selected light. For the purposes of the present invention,
significant portions may be, e.g., 50% or more of normal incident
selected light, more preferably 75% or more of normal incident
selected light. Examples of some suitable materials for the
detection window include, but are not limited to, e.g.,
polypropylenes, polyesters, polycarbonates, polyethylenes,
polypropylene-polyethylene copolymers, cyclo-olefin polymers (e.g.,
polydicyclopentadiene), etc.
[0063] In some instances, it may be preferred that the base layer
14 and/or the cover layer 18 of the sample processing device 10 be
opaque such that the sample processing device 10 is opaque between
the volume of the process chamber volume 14 and at least one side
of the sample processing device 10. By opaque, it is meant that
transmission of the selected light as described above is
substantially prevented (e.g., 5% or less of such normally incident
light is transmitted).
[0064] Valve chamber 60 is depicted in FIG. 2 and may preferably be
at least partially located within the valve lip 50 as seen in FIG.
2. At least a portion of the valve chamber 60 may preferably be
located between the second major side 19 of the sample processing
device 10 and at least a portion of the process chamber 40. The
valve chamber 60 is also preferably isolated from the process
chamber 40 by a valve septum 64 separating the valve chamber 64 and
the process chamber 40, such that a portion of the volume of the
process chamber 40 lies between the valve septum 64 and the first
major side 15 of the sample processing device 10. In the depicted
embodiment, the cover layer 18 is preferably sealed to the valve
lip 50 along surface 52 to isolate the valve chamber 60 from the
process chamber 50.
[0065] The valve septum 64 is preferably formed of material in
which openings can be formed by non-contact methods, e.g., laser
ablation, etc. As such the material or materials used in the septum
64 may include materials that preferentially absorb the energy used
to open the septum 64. For example, the septum 64 may include
materials such as, e.g., carbon black, UV/IR absorbers. etc.
[0066] The energy used to form openings in the valve septum 64 can
be directed onto the valve septum 64 either through the cover layer
18 or through the base layer 14 (or through both). It may be
preferred, however, that the energy be directed at the valve septum
64 through the cover layer 18 to avoid issues that may be
associated with directing the energy through the sample material in
the process chamber 40 before it reaches the valve septum 64.
[0067] One illustrative method of using a process array 120 will
now be described with respect to FIGS. 3A-3D, each of which is a
plan view of the process array in various stages of one
illustrative method according to the present invention. The process
array 120 depicted in each of the figures includes a loading
chamber 130 connected to a process chamber 140 through conduit 132.
The process array also includes a valve lip 150 and a valve chamber
160 located within a portion of the valve lip 150. The valve lip
150 and the valve chamber 160 define a valve septum 164 separating
and isolating the valve chamber 160 from the process chamber 140
before any openings are formed through the valve septum 164. The
valve septum 164 boundary is depicted as a broken line in the
figures because it may not be visible to the naked eye.
[0068] Another feature of the process array 120 is a detection
window 142 through selected light can be transmitted into and/or
out of the process chamber 140. The detection window 142 may be
formed through either major side of the device in which process
array 120 is located (or through both major sides if so desired).
In the depicted embodiment, the detection window 142 may preferably
be defined by that portion of the area occupied by the process
chamber 140 that is not also occupied by the valve lip 150. In
another manner of characterizing the detection window 142, the
detection window 142 and the valve lip 150 (and/or valve chamber
160 contained therein) may be described as occupying mutually
exclusive portions of the area of the process chamber 140.
[0069] The process array 120 also includes an output process
chamber 170 connected to the valve chamber 160 through conduit 162.
The output process chamber 170 may include, e.g., one or more
reagents 172 located therein. The reagent 172 may be fixed within
the process chamber 170 or it may be loose within the process
chamber. Although depicted in process chamber 170, one or more
reagents may be provided at any suitable location or locations
within the process array 120, e.g., the loading chamber 130,
conduits 132 & 162, process chamber 140, valve chamber 160,
etc.
[0070] The use of reagents is optional, i.e., sample processing
devices of the present invention may or may not include any
reagents in the process chambers. In another variation, some of the
process chambers in different process arrays may include a reagent,
while others do not. In yet another variation, different process
chambers may contain different reagents. Further, the interior of
the process chamber structures may be coated or otherwise processed
to control the adhesion of reagents.
[0071] The process chamber 140 (and its associated process chamber
area) may preferably have a length (measured along, e.g., axis 121
in FIG. 3A) that is greater than the width of the process chamber
140, where the process chamber width is measured perpendicular to
the process chamber length. As such, the process chamber 140 may be
described as "elongated." It may be preferred that the axis 121
along which the process chamber 140 is elongated be aligned with a
radial direction extending from an axis of rotation about which the
sample processing device containing process array is rotated (if
rotation is the driving force used to effect fluid transfer).
[0072] In other aspects, it may be preferred that the detection
window 142 be at least coextensive along the length of the process
chamber 140 with the valve septum 164. Although the depicted
detection window 142 is a single unitary feature, it will be
understood that more two or more detection windows could be
provided for each process chamber 140. For example, a plurality of
independent detection windows could be distributed along the length
of the process chamber 140 (e.g., alongside the valve septum
164.
[0073] Another manner of characterizing the relative sizes of the
various features may be, e.g., that the valve septum 164 extends
along the length of the process chamber area for 30% or more (or,
alternatively, for 50% or more) of a maximum length of the process
chamber 140 (along its elongation axis 121). Such a
characterization of the dimensions of valve septum 164 may be
expressed in actual measurements for many sample processing
devices, e.g., the valve septum 164 may be described as extending
for a length of 1 millimeter or more along the length of the
process chamber 140.
[0074] The first stage of the depicted method is seen in FIG. 3A,
where the loading chamber 130 includes sample material 180 located
therein. For the purposes of the illustrated method, the sample
material 180 is whole blood. After loading, the blood 180 is
preferably transferred to the process chamber 140 through conduit
132. The transfer may preferably be effected by rotating the
process array 120 about an axis of rotation 111. The rotation may
preferably occur, for example, in the plane of the paper on which
FIG. 3A is located, although any rotation about point 111 in which
process chamber 140 is moved in an arc about a point located on the
opposite side of the loading chamber 130 from the process chamber
140 may be acceptable. A further description of a preferred process
for processing whole blood to remove the nucleic acid is provided
below.
[0075] The process arrays used in sample processing devices of the
present invention may preferably be "unvented." As used in
connection with the present invention, an "unvented process array"
is a process array (i.e., at least two connected chambers) in which
the only openings leading into the process array are located in the
loading structure, e.g., the loading chamber. In other words, to
reach the process chamber within an unvented process array, sample
materials must be delivered to the loading chamber. Similarly, any
air or other fluid located within the process array before loading
of the sample material must also escape from the process array
through the loading chamber. In contrast, a vented process array
would include at least one opening outside of the loading chamber.
That opening would allow for the escape of any air or other fluid
located within the process array before loading.
[0076] Moving sample material through sample processing devices
that include unvented process arrays may be facilitated by
alternately accelerating and decelerating the device during
rotation, essentially burping the sample materials through the
conduits and process chambers. The rotating may be performed using
at least two acceleration/deceleratio- n cycles, i.e., an initial
acceleration, followed by deceleration, second round of
acceleration, and second round of deceleration. It may further be
helpful if the acceleration and/or deceleration are rapid. The
rotation may also preferably only be in one direction, i.e., it may
not be necessary to reverse the direction of rotation during the
loading process. Such a loading process allows sample materials to
displace the air in those portions of the process arrays that are
located farther from the center of rotation of the device. The
actual acceleration and deceleration rates may vary based on a
variety of factors such as temperature, size of the device,
distance of the sample material from the axis of rotation,
materials used to manufacture the devices, properties of the sample
materials (e.g., viscosity), etc.
[0077] FIG. 3B depicts the process array after movement of the
blood 180 into the process chamber 140. The blood 180 remains in
the process chamber 140, i.e., does not travel into the valve
chamber 160, because the valve chamber 160 is isolated from the
process chamber 140 by the valve septum 164.
[0078] Additional rotation of the process array 120 may preferably
result in separation of the components of the blood 180 into, as
seen in FIG. 3C, red blood cells 182, a buffy coat layer 184, and
plasma 186. The separation is typically a result of centrifugal
forces and the relative densities of the materials.
[0079] If the precise volume of the different components in each
sample of blood 180 (or if the volume of the blood sample 180
itself) is not known, the location of the boundaries between the
different separated layers may not be known. In connection with the
present invention, however, it may preferably be possible to detect
the locations of the boundaries between the different separated
components.
[0080] Such detection may preferably occur through the detection
window using any suitable selected light. The light may be
transmitted through or reflected from the blood components 182, 184
& 186 to obtain an image of the sample material in the process
chamber 140. In another alternative, absorbance of light may be
used to detect the boundaries or locations of one or more selected
components. For example, after spinning blood, it may be possible
to detect the interfaces between the packed red blood cell layer,
the buffy layer (white blood cells), and plasma. After spinning
beads, it may be possible to detect the interface between the
packed bead layer and a supernatant layer.
[0081] It may be preferable to determine the location of all
features or characteristics of the sample material, i.e., the
location of all boundaries, including the free surface 187 of the
plasma 186. In other instances, it may be sufficient to determine
the location of only one feature, e.g., the boundary between the
buffy coat layer 184 and the plasma 186, where the detected
characteristic provides sufficient information to perform the next
step in the method.
[0082] After the suitable characteristic or characteristics of the
materials in the process chamber 140 have been detected, an opening
168 is preferably formed in the valve septum 164 at the desired
location. In the depicted method, the desired location for opening
168 is chosen to remove a portion of the plasma 186 from the
process chamber 140. It may be desirable that substantially all of
the plasma 186 be removed, leaving only a small amount (see 186r in
FIG. 3D) in the process chamber 140. It may be necessary to leave a
small amount of plasma in the process chamber 140 to limit or
prevent the transfer of red blood cells 182 out of the process
chamber 140.
[0083] The opening 168 can be formed by any suitable non-contact
technique. One such technique may be, e.g., laser ablation of the
valve septum 168. Other techniques may include, but are not limited
to, e.g., focused optical heating, etc.
[0084] After the opening 168 is formed, additional rotation of the
process array 120 preferably moves the plasma 186 from the process
chamber 140 into the valve chamber 160 through opening 168,
followed by transfer into the output process chamber 170 through
conduit 162. As a result, the plasma 186 is located in the process
chamber 170, with a small remainder of plasma 186r in the process
chamber 140 along with the buffy coat layer 184 and red blood cells
182.
[0085] A portion of another embodiment of a process array 220
including a process chamber 240 and valve structures according to
the present invention is depicted in FIG. 4. In the depicted
embodiment, the process chamber 240 is elongated along axis 221 and
the process array 220 is designed for rotation to provide the force
to move fluids. The rotation may be about point 211 which, in the
depicted embodiment, lies on axis 221. It should, however, be
understood that the point about which the process array is rotated
is not required to lie on axis 221.
[0086] The process chamber 240 is shown in broken lines where the
valve lips 250a, 250b and 250c extend into the process chamber area
and in solid lines where the valve lips 250a, 250b and 250c do not
extend into the process chamber area. It may be preferred that in
those portions of the process chamber area that are not occupied by
the valve lips 250a, 250b and 250c, the process chamber 240 include
a detection window 242 that allows for the transmission of selected
light into and/or out of the process chamber 240 to allow for
detection of sample material 280 in the process chamber 240.
[0087] The process array 220 also includes valve chambers 260a,
260b, and 260c isolated and separated from the process chamber 240.
The valve chambers 260a, 260b, and 260c are each in communication
with a chamber 270a, 270b, and 270c (respectively). The valve
chambers 260a, 260b, and 260c may be connected to their respective
chambers 270a, 27ob, and 270c by a conduit as shown in FIG. 4.
[0088] Each of the valve chambers 260a, 260b, and 260c may
preferably be located, at least in part, on a valve lip 250a, 250b
and 250c (respectively). Each of the valve chambers 260a, 260b, and
260c may also preferably be isolated and separated from the process
chamber 240 by a valve septum 264a, 264b, and 264c located within
each of the valve chambers 260a, 260b, and 260c. Each of the valve
septums 264a, 264b, and 264c is defined, in part, by the broken
lines of process chamber 240.
[0089] The multiple valve chambers 260a, 260b, and 260c provided in
connection with the process chamber 240 may provide the ability to
selectively remove different portions of any sample material in the
process chamber and to move that sample material to different
chambers 270a, 270b, and 270c. For example, a first portion of
sample material 280 in the process chamber 240 may be moved into
chamber 270a by forming an opening 268a in valve septum 264a of
valve chamber 260a.
[0090] After moving the first portion of sample material 280 into
chamber 270a through opening 268a in valve chamber 260a, another
opening 268b may be provided in valve septum 264b of valve chamber
260b to move a second portion of the sample material 280 into
chamber 270b. The second portion will typically include the sample
material 280 located between openings 268a and 268b. The distance
separating those two openings along the length of the process
chamber 240 is indicated by x in FIG. 4. As a result, the volume of
the second portion of sample material 280 can be determined if the
cross-sectional area of the process chamber 240 (taken in a plane
perpendicular to the axis 221) is known. As a result, it may be
possible to move a known or selected volume of sample material into
chamber 270b by forming openings 268a and 268b a selected distance
apart from each other.
[0091] The process chamber 240 also includes a third valve chamber
260c located in a valve lip 250c at the end of the process chamber
240 farthest from the point 211 about which the process array 220
may be rotated. The valve lip 250c extends over the entire width of
the process chamber 240 (in contrast to the valve lips 250a and
250b that extend over only a portion of the width of the process
chamber 240).
[0092] FIG. 5 depicts another process chamber 340 in connection
with the present invention in cross-section. The process chamber
340 is formed in a sample processing device 310 that includes a
base layer 313, intermediate layer 314, valve layer 316 and cover
layer 318. The various layers may be attached to each other by any
suitable combination of techniques.
[0093] Although the layers are depicted as single, homogeneous
constructions, it will be understood that one or more of the layers
could be formed of multiple materials and/or layers. Furthermore,
it may be possible to combine some of the layers. For example,
layers 313 and 314 may be combined (as an example, see layer 14 in
the cross-sectional view of FIG. 2). Alternatively, it may be
possible to combine layers 314 and 316 into a single structure that
could be formed by, e.g., molding, extrusion, etc.
[0094] The construction seen in FIG. 5 includes a valve chamber 360
separated from the process chamber 340 by a valve septum 364. The
valve chamber 360 is further defined by the cover layer 318. A
device 390 is also depicted in FIG. 5 that can be used to, e.g.,
form an opening in the valve septum 364. The device 390 may be,
e.g., a laser, etc. that can preferably deliver the energy
necessary to form an opening in the valve septum 364 without
forming an opening in the cover layer 318.
[0095] If the energy required to form openings in the valve septum
364 can be directed through the cover layer 318, then the base
layer 313 may be formed of any material that may block such energy.
For example, the base layer 313 may be made of, e.g., a metallic
foil or other material. If the valve layer 316 and/or valve septum
364 allow for the passage of sufficient amounts of selected
wavelengths of light, it may be possible to detect sample material
in the process chamber 340 through the valve layer 316 and/or valve
septum 364.
[0096] If, alternatively, the valve layer 316 and valve septum 364
block the passage of light such that detection of sample material
in the process chamber 340 cannot be performed, then it may be
desirable to detect sample material in the process chamber 340
through the base layer 313. Such detection may be accomplished
using detection device 392 as seen in FIG. 5 that can detect sample
material in the process chamber 340 through the layer 313. In some
instances, it may be possible to form openings in the valve septum
364 using device 392 directing energy through layer 313 (if the
passage of such energy through sample material in the process
chamber 340 is acceptable).
[0097] Illustrative Method using Whole Blood
[0098] The present invention also provides methods and kits for
isolating nucleic acid from a whole blood that includes nucleic
acid (e.g., DNA, RNA, PNA), which is included within
nuclei-containing cells (e.g., white blood cells).
[0099] It should be understood that although the methods are
directed to isolating nucleic acid from a sample, the methods do
not necessarily remove the nucleic acid from the nucleic
acid-containing material (e.g., nuclei). That is, further steps may
be required to further separate the nucleic acid from the nuclei,
for example.
[0100] Certain methods of the present invention may involve
ultimately separating nucleic acid from inhibitors, such as heme
and degradation products thereof (e.g., iron salts), which are
undesirable because they can inhibit amplification reactions (e.g.,
as are used in PCR reactions). More specifically, certain methods
of the present invention may involve separating at least a portion
of the nucleic acid in a sample from at least a portion of at least
one type of inhibitor. Preferred methods may involve removing
substantially all the inhibitors in a sample containing nucleic
acid such that the nucleic acid is substantially pure. For example,
the final concentration of iron-containing inhibitors may
preferably be no greater than about 0.8 micromolar (.mu.M), which
is the current level tolerated in conventional PCR systems.
[0101] In order to get clean DNA from whole blood, removal of
hemoglobin as well as plasma proteins is typically desired. When
red blood cells are lysed, heme and related compounds are released
that inhibit Taq Polymerase. The normal hemoglobin concentration in
whole blood is 15 grams (g) per 100 milliliters (mL) based on which
the concentration of heme in hemolysed whole blood is around 10
millimolar (mM). For PCR to work out satisfactorily, the
concentration of heme should be reduced to the micromolar (.mu.M)
level. This can be achieved, for example, by dilution or by removal
of inhibitors using a material that binds inhibitors.
[0102] In one embodiment, the present invention provides a method
of isolating nucleic acid from whole blood, the method includes:
providing a device that includes a loading chamber and a variable
valved process chamber; placing whole blood in the loading chamber;
transferring the whole blood to a valved process chamber;
centrifuging the whole blood in the valved process chamber to form
a plasma layer (often the upper layer), a red blood cell layer
(often the lower layer), and an interfacial layer (located between
the plasma layer and the red blood cell layer) that includes white
blood cells; removing at least a portion of the interfacial layer;
and lysing the white blood cells in the separated interfacial layer
and optionally lysing the nuclei therein to release inhibitors
and/or nucleic acid. In certain embodiments, the lysing involves
subjecting the white blood cells to a strong base with optional
heating to release nucleic acid. If desired, the method can further
include adjusting the pH of the sample that includes the released
nucleic acid to be within a range of 7.5 to 9. Alternatively, the
lysing can involve subjecting the white blood cells to a
surfactant.
[0103] If desired, before, simultaneously with, or after lysing the
white blood cells, the method can include transferring the
separated interfacial layer to a separation chamber for contact
with solid phase material to preferentially adhere at least a
portion of the inhibitors to the solid phase material. More
specifically, in certain embodiments of this method, the device
further includes a separation chamber having a solid phase material
therein. The solid phase material preferably includes capture sites
(e.g., chelating functional groups), a coating reagent coated on
the solid phase material, or both; wherein the coating reagent is
selected from the group consisting of a surfactant, a strong base,
a polyelectrolyte, a selectively permeable polymeric barrier, and
combinations thereof.
[0104] When a solid phase material is present, the method includes
contacting the lysed sample with the solid phase material in the
separation chamber to preferentially adhere at least a portion of
the inhibitors to the solid phase material; wherein lysing can
occur before, simultaneous with, or after contacting the solid
phase material. The method typically then includes separating at
least a portion of the nuclei and/or nucleic acid from the solid
phase material having at least a portion of the inhibitors adhered
thereto.
[0105] In certain embodiments wherein no solid phase material is
used, this method can involve diluting the lysed sample with water
(preferably, RNAse-free sterile water) or buffer to reduce the
inhibitor concentration to that which would not interfere with an
amplification method; optionally further lysing the nuclei to
release nucleic acid; optionally heating the sample to denature
proteins and optionally adjusting the pH of the sample that
includes released nucleic acid and optionally carrying out PCR.
Diluting can be accomplished with sufficient water to reduce the
concentration of heme to less than 2 micromolar. Alternatively,
diluting can be accomplished with sufficient water to form a
2.times. to 1000.times. dilution of the lysed sample.
[0106] Alternatively, if desired, prior to lysing the white blood
cells, the method can include diluting the separated interfacial
layer of the sample with water or buffer, optionally further
concentrating the diluted layer to increase the concentration of
nucleic acid material, optionally separating the further
concentrated region, and optionally repeating this process of
dilution followed by concentration and separation to reduce the
inhibitor concentration to that which would not interfere with an
amplification method.
[0107] Referring to FIG. 6, an example of one potentially preferred
embodiment of the device suitable for use with these embodiments
includes a loading chamber 670, a variable valved process chamber
672, an optional separation chamber 676, an eluting reagent chamber
678, a waste chamber 680, and an optional amplification chamber
682. These chambers are in fluid communication with each other such
that a whole blood sample can be loaded into the loading chamber
670, which can then be transferred to the variable valved process
chamber 672. Upon centrifuging the whole blood in the valved
process chamber 672 to form a plasma layer (often the upper layer),
a red blood cell layer (often the lower layer), and an interfacial
layer that includes white blood cells, at least a portion (and
preferably a substantial portion) of the interfacial layer is
transferred to the optional separation chamber 676 to separate the
white blood cells (buffy coat) from at least the red blood cell
layer and preferably from both of the other two (the plasma layer
and the red blood cell layer) layers of the whole blood, which can
be transferred to the optional waste chamber 680. Therein the white
blood cells in the buffy coat can be lysed to release inhibitors
and nuclei and/or nucleic acid. If the separation chamber 676
includes a solid phase material, the process can include
preferentially adhering at least a portion of the inhibitors to the
solid phase material. The eluting reagent in the eluting reagent
chamber 678 is then transferred to the separation chamber 676 to
remove at least a portion of the target nucleic acid-containing
material and/or nucleic acid. In certain embodiments, this material
can be directly transferred to an amplification reaction chamber
682 for carrying out a PCR process, for example. The amplification
reaction chamber 682 can optionally include pre-deposited reactants
for the amplification reaction (e.g., PCR).
[0108] Lysing Reagents and Conditions
[0109] For certain embodiments of the invention, at some point
during the process, cells within the sample, particularly nucleic
acid-containing cells (e.g., white blood cells, bacterial cells,
viral cells) are lysed to release the contents of the cells and
form a sample (i.e., a lysate). Lysis, as used herein, is the
physical disruption of the membranes of the cells, referring to the
outer cell membrane and, when present, the nuclear membrane. This
can be done using standard techniques, such as by hydrolyzing with
proteinases followed by heat inactivation of proteinases, treating
with surfactants (e.g., nonionic surfactants or sodium dodecyl
sulfate), guanidinium salts, or strong bases (e.g., NaOH),
disrupting physically (e.g., with ultrasonic waves), boiling, or
heating/cooling (e.g., heating to at least 55.degree. C. (typically
to 95.degree. C.) and cooling to room temperature or below
(typically to 8.degree. C.)), which can include a freezing/thawing
process. Typically, if a lysing reagent is used, it is in aqueous
media, although organic solvents can be used, if desired.
[0110] Lysing of red blood cells (RBC's) without the destruction of
white blood cells (WBC's) in whole blood can occur to release
inhibitors through the use of water (i.e., aqueous dilution) as the
lysing agent (i.e., lysing reagent). Alternatively, ammonium
chloride or quaternary ammonium salts can also be used to break
RBC's. The RBC's can also be lysed by hypotonic shock with the use
of a hypotonic buffer. The intact WBC's or their nuclei can be
recovered by centrifugation, for example.
[0111] Typically, a stronger lysing reagent, such as a surfactant,
can be used to lyse RBC's as well as nucleic acid-containing cells
(e.g., white blood cells (WBC's), bacterial cells, viral cells) to
release inhibitors, nuclei, and/or nucleic acid. For example, a
nonionic surfactant can be used to lyse RBC's as well as WBC's
while leaving the nuclei intact. Nonionic surfactants, cationic
surfactants, anionic surfactants, and zwitterionic surfactants can
be used to lyse cells. Particularly useful are nonionic
surfactants. Combinations of surfactants can be used if desired. A
nonionic surfactant such as TRITON X-100 can be added to a TRIS
buffer containing sucrose and magnesium salts for isolation of
nuclei.
[0112] The amount of surfactant used for lysing is sufficiently
high to effectively lyse the sample, yet sufficiently low to avoid
precipitation, for example. The concentration of surfactant used in
lysing procedures is typically at least 0.1 wt-%, based on the
total weight of the sample. The concentration of surfactant used in
lysing procedures is typically no greater than 4.0 wt-%, and
preferably, no greater than 1.0 wt-%, based on the total weight of
the sample. The concentration is usually optimized in order to
obtain complete lysis in the shortest possible time with the
resulting mixture being PCR compatible. In fact, the nucleic acid
in the formulation added to the PCR cocktail should allow for no
inhibition of real-time PCR.
[0113] If desired, a buffer can be used in admixture with the
surfactant. Typically, such buffers provide the sample with a pH of
at least 7, and typically no more than 9.
[0114] Typically, an even stronger lysing reagent, such as a strong
base, can be used to lyse any nuclei contained in the nucleic
acid-containing cells (as in white blood cells) to release nucleic
acid. For example, the method described in U.S. Pat. No. 5,620,852
(Lin et al.), which involves extraction of DNA from whole blood
with alkaline treatment (e.g., NaOH) at room temperature in a time
frame as short as 1 minute, can be adapted to certain methods of
the present invention. Generally, a wide variety of strong bases
can be used to create an effective pH (e.g., 8-13, preferably 13)
in an alkaline lysis procedure. The strong base is typically a
hydroxide such as NaOH, LiOH, KOH; hydroxides with quaternary
nitrogen-containing cations (e.g., quaternary ammonium) as well as
bases such as tertiary, secondary or primary amines. Typically, the
concentration of the strong base is at least 0.01 Normal (N), and
typically, no more than 1 N. Typically, the mixture can then be
neutralized, particularly if the nucleic acid is to subjected to
PCR. In another procedure, heating can be used subsequent to lysing
with base to further denature proteins followed by neutralizing the
sample.
[0115] One can also use Proteinase K with heat followed by heat
inactivation of proteinase K at higher temperatures for isolation
of nucleic acids from the nuclei or WBC.
[0116] One can also use a commercially available lysing agent and
neutralization agent such as in Sigma's Extract-N-Amp Blood PCR kit
scaled down to, e.g., microfluidic dimensions if desired. Stonger
lysing solutions such as POWERLYSE from GenPoint (Oslo, Norway) for
lysing difficult bacteria such as Staphylococcus, Streptococcus,
etc. can be used to advantage in certain methods of the present
invention.
[0117] In another procedure, a boiling method can be used to lyse
cells and nuclei, release DNA, and precipitate hemoglobin
simultaneously. The DNA in the supernatant can be used directly for
PCR without a concentration step, making this procedure useful for
low copy number samples.
[0118] For infectious diseases, it may be necessary to analyze
bacterial or viruses from whole blood. For example, in the case of
bacteria, white blood cells may be present in conjunction with
bacterial cells. In a device, it would be possible to lyse red
blood cells to release inhibitors, and then separate out bacterial
cells and white blood cells by centrifugation, for example, prior
to further lysing. This concentrated slug of nucleic
acid-containing cells (bacterial and white blood cells/nuclei) can
be moved further into a chamber for removal of inhibitors. Then,
the bacterial cells, for example, can be lysed.
[0119] Bacterial cell lysis, depending on the type, may be
accomplished using heat. Alternatively, bacterial cell lysis can
occur using enzymatic methods (e.g., lysozyme, mutanolysin) or
chemical methods. The bacterial cells are preferably lysed by
alkaline lysis.
[0120] The use of bacteria for propagation of plasmids is common in
the study of genomics, analytic molecular biology, preparatory
molecular biology, etc. In the case of the bacterium containing
plasmid, genetic material from both the bacterium and the plasmid
are present. A clean-up procedure to separate cellular proteins and
cellular fragments from genomic DNA can be carried out using a
method of the present invention. The supernatant thus obtained,
which contains the plasmid DNA, is called the "cleared lysate." The
cleared lysate can be further purified using a variety of means,
such as anion-exchange chromatography, gel filtration, or
precipitation with alcohol.
[0121] In a specific example of a protocol for bacterial cultures,
which can be incorporated into a device, an E. Coli cell culture is
centrifuged and resuspended in TE buffer (10 mM TRIS, 1 mM EDTA, pH
7.5) and lysed by the addition of 0.1 M NaOH/1% SDS (sodium dodecyl
sulfate). The cell lysis is stopped by the addition of 1 volume of
3 M (three molar) potassium acetate (pH 4.8) and the supernatant
centrifuged. The cell lysate is further purified to get clean
plasmid DNA.
[0122] Plasma and serum represent the majority of specimens
submitted for molecular testing that include viruses. After
fractionation of whole blood, plasma or serum samples can be used
for the extraction of viruses (i.e., viral particles). For example,
to isolate DNA from viruses, it may be possible to first separate
out the serum by spinning blood. By the use of the variable valve,
the serum alone can be emptied into another chamber. The serum can
then be centrifuged to concentrate the virus or can be used
directly in subsequent lysis steps after removal of the inhibitors
using a solid phase material, for example, as described herein. The
solid phase material could absorb the solution such that the virus
particles do not go through the material. The virus particles can
then be eluted out in a small elution volume. The virus can be
lysed by heat or by enzymatic or chemical means, for example, by
the use of surfactants, and used for downstream applications, such
as PCR or real-time PCR. In cases where viral RNA is required, it
may be necessary to have an RNAse inhibitor added to the solution
to prevent degradation of RNA.
[0123] Optional Solid Phase Material
[0124] For certain embodiments of the invention, it has been found
that inhibitors will adhere to solid phase materials that include a
solid matrix in any form (e.g., particles, fibrils, a membrane),
preferably with capture sites (e.g., chelating functional groups)
attached thereto, a coating reagent (preferably, surfactant) coated
on the solid phase material, or both. The coating reagent can be a
cationic, anionic, nonionic, or zwitterionic surfactant.
Alternatively, the coating reagent can be a polyelectrolyte or a
strong base. Various combinations of coating reagents can be used
if desired.
[0125] The solid phase material useful in the methods of the
present invention may include a wide variety of organic and/or
inorganic materials that retain inhibitors such as heme and heme
degradation products, particularly iron ions, for example. Such
materials are functionalized with capture sites (preferably,
chelating groups), coated with one or more coating reagents (e.g.,
surfactants, polyelectrolytes, or strong bases), or both.
Typically, the solid phase material includes an organic polymeric
matrix.
[0126] Generally suitable materials are chemically inert,
physically and chemically stable, and compatible with a variety of
biological samples. Examples of solid phase materials include
silica, zirconia, alumina beads, metal colloids such as gold,
gold-coated sheets that have been functionalized through mercapto
chemistry, for example, to generate capture sites. Examples of
suitable polymers include for example, polyolefins and fluorinated
polymers. The solid phase material is typically washed to remove
salts and other contaminants prior to use. It can either be stored
dry or in aqueous suspension ready for use. The solid phase
material is preferably used in a flow-through receptacle, for
example, such as a pipet, syringe, or larger column, microtiter
plate, or other device, although suspension methods that do not
involve such receptacles could also be used.
[0127] The solid phase material useful in the methods of the
present invention can include a wide variety of materials in a wide
variety of forms. For example, it can be in the form of particles
or beads, which may be loose or immobilized, fibers, foams, frits,
microporous film, membrane, or a substrate with microreplicated
surface(s). If the solid phase material includes particles, they
are preferably uniform, spherical, and rigid to ensure good fluid
flow characteristics.
[0128] For flow-through applications of the present invention, such
materials are typically in the form of a loose, porous network to
allow uniform and unimpaired entry and exit of large molecules and
to provide a large surface area. Preferably, for such applications,
the solid phase material has a relatively high surface area, such
as, for example, more than one meter squared per gram (m.sup.2/g).
For applications that do not involve the use of a flow-through
device, the solid phase material may or may not be in a porous
matrix. Thus, membranes can also be useful in certain methods of
the present invention.
[0129] For applications that use particles or beads, they may be
introduced to the sample or the sample introduced into a bed of
particles/beads and removed therefrom by centrifuging, for example.
Alternatively, particles/beads can be coated (e.g., pattern coated)
onto an inert substrate (e.g., polycarbonate or polyethylene),
optionally coated with an adhesive, by a variety of methods (e.g.,
spray drying). If desired, the substrate can be microreplicated for
increased surface area and enhanced clean-up. It can also be
pretreated with oxygen plasma, e-beam or ultraviolet radiation,
heat, or a corona treatment process. This substrate can be used,
for example, as a cover film, or laminated to a cover film, on a
reservoir in a device.
[0130] In one embodiment, the solid phase material includes a
fibril matrix, which may or may not have particles enmeshed
therein. The fibril matrix can include any of a wide variety of
fibers. Typically, the fibers are insoluble in an aqueous
environment. Examples include glass fibers, polyolefin fibers,
particularly polypropylene and polyethylene microfibers, aramid
fibers, a fluorinated polymer, particularly,
polytetrafluoroethylene fibers, and natural cellulosic fibers.
Mixtures of fibers can be used, which may be active or inactive
toward binding of nucleic acid. Preferably, the fibril matrix forms
a web that is at least about 15 microns, and no greater than about
1 millimeter, and more preferably, no greater than about 500
microns thick.
[0131] If used, the particles are typically insoluble in an aqueous
environment. They can be made of one material or a combination of
materials, such as in a coated particle. They can be swellable or
nonswellable, although they are preferably nonswellable in water
and organic liquids. Preferably, if the particle is doing the
adhering, it is made of nonswelling, hydrophobic material. They can
be chosen for their affinity for the nucleic acid. Examples of some
water swellable particles are described in U.S. Pat. Nos. 4,565,663
(Errede et al.), 4,460,642 (Errede et al.), and 4,373,519 (Errede
et al.). Particles that are nonswellable in water are described in
U.S. Pat. Nos. 4,810,381 (Hagen et al.), 4,906,378 (Hagen et al.),
4,971,736 (Hagen et al.); and 5,279,742 (Markell et al.). Preferred
particles are polyolefin particles, such as polypropylene particles
(e.g., powder). Mixtures of particles can be used, which may be
active or inactive toward binding of nucleic acid.
[0132] If coated particles are used, the coating is preferably an
aqueous- or organic-insoluble, nonswellable material. The coating
may or may not be one to which nucleic acid will adhere. Thus, the
base particle that is coated can be inorganic or organic. The base
particles can include inorganic oxides such as silica, alumina,
titania, zirconia, etc., to which are covalently bonded organic
groups. For example, covalently bonded organic groups such as
aliphatic groups of varying chain length (C2, C4, C8, or C18
groups) can be used.
[0133] Examples of suitable solid phase materials that include a
fibril matrix are described in U.S. Pat. Nos. 5,279,742 (Markell et
al.), 4,906,378 (Hagen et al.), 4,153,661 (Ree et al.), 5,071,610
(Hagen et al.), 5,147,539 (Hagen et al.), 5,207,915 (Hagen et al.),
and 5,238,621 (Hagen et al.). Such materials are commercially
available from 3M Company (St. Paul, Minn.) under the trade
designations SDB-RPS (Styrene-Divinyl Benzene Reverse Phase
Sulfonate, 3M Part No. 2241), cation-SR membrane (3M Part No.
2251), C-8 membrane (3M Part No. 2214), and anion-SR membrane (3M
Part No. 2252).
[0134] Those that include a polytetrafluoroethylene matrix (PTFE)
are particularly preferred. For example, U.S. Pat. No. 4,810,381
(Hagen et al.) discloses a solid phase material that includes: a
polytetrafluoroethylene fibril matrix, and nonswellable sorptive
particles enmeshed in the matrix, wherein the ratio of nonswellable
sorptive particles to polytetrafluoroethylene being in the range of
19:1 to 4:1 by weight, and further wherein the composite solid
phase material has a net surface energy in the range of 20 to 300
milliNewtons per meter. U.S. Pat. No. RE 36,811 (Markell et al.)
discloses a solid phase extraction medium that includes: a PTFE
fibril matrix, and sorptive particles enmeshed in the matrix,
wherein the particles include more than 30 and up to 100 weight
percent of porous organic particles, and less than 70 to 0 weight
percent of porous (organic-coated or uncoated) inorganic particles,
the ratio of sorptive particles to PTFE being in the range of 40:1
to 1:4 by weight.
[0135] Particularly preferred solid phase materials are available
under the trade designation EMPORE from the 3M Company, St. Paul,
Minn. The fundamental basis of the EMPORE technology is the ability
to create a particle-loaded membrane, or disk, using any sorbent
particle. The particles are tightly held together within an inert
matrix of polytetrafluoroethylene (90% sorbent: 10% PTFE, by
weight). The PTFE fibrils do not interfere with the activity of the
particles in any way. The EMPORE membrane fabrication process
results in a denser, more uniform extraction medium than can be
achieved in a traditional Solid Phase Extraction (SPE) column or
cartridge prepared with the same size particles.
[0136] In another preferred embodiment, the solid phase (e.g., a
microporous thermoplastic polymeric support) has a microporous
structure characterized by a multiplicity of spaced, randomly
dispersed, nonuniform shaped, equiaxed particles of thermoplastic
polymer connected by fibrils. Particles are spaced from one another
to provide a network of micropores therebetween. Particles are
connected to each other by fibrils, which radiate from each
particle to the adjacent particles. Either, or both, the particles
or fibrils may be hydrophobic. Examples of preferred such materials
have a high surface area, often as high as 40 meters.sup.2/gram as
measured by Hg surface area techniques and pore sizes up to about 5
microns.
[0137] This type of fibrous material can be made by a preferred
technique that involves the use of induced phase separation. This
involves melt blending a thermoplastic polymer with an immiscible
liquid at a temperature sufficient to form a homogeneous mixture,
forming an article from the solution into the desired shape,
cooling the shaped article so as to induce phase separation of the
liquid and the polymer, and to ultimately solidify the polymer and
remove a substantial portion of the liquid leaving a microporous
polymer matrix. This method and the preferred materials are
described in detail in U.S. Pat. Nos. 4,726,989 (Mrozinski),
4,957,943 (McAllister et al.), and 4,539,256 (Shipman). Such
materials are referred to as thermally induced phase separation
membranes (TIPS membranes) and are particularly preferred.
[0138] Other suitable solid phase materials include nonwoven
materials as disclosed in U.S. Pat. No. 5,328,758 (Markell et al.).
This material includes a compressed or fused particulate-containing
nonwoven web (preferably blown microfibrous) that includes high
sorptive-efficiency chromatographic grade particles.
[0139] Other suitable solid phase materials include those known as
HIPE Foams, which are described, for example, in U.S. Pat.
Publication No. 2003/0011092 (Tan et al.). "HIPE" or "high internal
phase emulsion" means an emulsion that includes a continuous
reactive phase, typically an oil phase, and a discontinuous or
co-continuous phase immiscible with the oil phase, typically a
water phase, wherein the immiscible phase includes at least 74
volume percent of the emulsion. Many polymeric foams made from
HIPE's are typically relatively open-celled. This means that most
or all of the cells are in unobstructed communication with
adjoining cells. The cells in such substantially open-celled foam
structures have intercellular windows that are typically large
enough to permit fluid transfer from one cell to another within the
foam structure.
[0140] The solid phase material can include capture sites for
inhibitors. Herein, "capture sites" refer to groups that are either
covalently attached (e.g., functional groups) or molecules that are
noncovalently (e.g., hydrophobically) attached to the solid phase
material.
[0141] Preferably, the solid phase material includes functional
groups that capture the inhibitors. For example, the solid phase
material may include chelating groups. In this context, "chelating
groups" are those that are polydentate and capable of forming a
chelation complex with a metal atom or ion (although the inhibitors
may or may not be retained on the solid phase material through a
chelation mechanism). The incorporation of chelating groups can be
accomplished through a variety of techniques. For example, a
nonwoven material can hold beads functionalized with chelating
groups. Alternatively, the fibers of the nonwoven material can be
directly functionalized with chelating groups.
[0142] Examples of chelating groups include, for example,
--(CH.sub.2--C(O)OH).sub.2, tris(2-aminoethyl)amine groups,
iminodiacetic acid groups, nitrilotriacetic acid groups. The
chelating groups can be incorporated into a solid phase material
through a variety of techniques. They can be incorporated in by
chemically synthesizing the material. Alternatively, a polymer
containing the desired chelating groups can be coated (e.g.,
pattern coated) on an inert substrate (e.g., polycarbonate or
polyethylene). If desired, the substrate can be microreplicated for
increased surface area and enhanced clean-up. It can also be
pretreated with oxygen plasma, e-beam or ultraviolet radiation,
heat, or a corona treatment process. This substrate can be used,
for example, as a cover film, or laminated to a cover film, on a
reservoir in a device.
[0143] Chelating solid phase materials are commercially available
and could be used as the solid phase material in the present
invention. For example, for certain embodiments of the present
invention, EMPORE membranes that include chelating groups such as
iminodiacetic acid (in the form of the sodium salt) are preferred.
Examples of such membranes are disclosed in U.S. Pat. No. 5,147,539
(Hagen et al.) and commercially available as EMPORE Extraction
Disks (47 mm, No. 2271 or 90 mm, No. 2371) from the 3M Company. For
certain embodiments of the present invention, ammonium-derivatized
EMPORE membranes that include chelating groups are preferred. To
put the disk in the ammonium form, it can be washed with 50 mL of
0.1M ammonium acetate buffer at pH 5.3 followed with several
reagent water washes.
[0144] Examples of other chelating materials include, but are not
limited to, crosslinked polystyrene beads available under the trade
designation CHELEX from Bio-Rad Laboratories, Inc. (Hercules,
Calif.), crosslinked agarose beads with tris(2-aminoethyl)amine,
iminodiacetic acid, nitrilotriacetic acid, polyamines and
polyimines as well as the chelating ion exchange resins
commercially available under the trade designation DUOLITE C-467
and DUOLITE GT73 from Rohm and Haas (Philadelphia, Pa.), AMBERLITE
IRC-748, DIAION CR11, DUOLITE C647.
[0145] Typically, a desired concentration density of chelating
groups on the solid phase material is about 0.02 nanomole per
millimeter squared, although it is believed that a wider range of
concentration densities is possible.
[0146] Other types of capture materials include anion exchange
materials, cation exchange materials, activated carbon, reverse
phase, normal phase, styrene-divinyl benzene, alumina, silica,
zirconia, and metal colloids. Examples of suitable anion exchange
materials include strong anion exchangers such as quaternary
ammonium, dimethylethanolamine, quaternary alkylamine,
trimethylbenzyl ammonium, and dimethylethanolbenzyl ammonium
usually in the chloride form, and weak anion exchangers such as
polyamine. Examples of suitable cation exchange materials include
strong cation exchangers such as sulfonic acid typically in the
sodium form, and weak cation exchangers such as carboxylic acid
typically in the hydrogen form. Examples of suitable carbon-based
materials include EMPORE carbon materials, carbon beads, Examples
of suitable reverse phase C8 and C18 materials include silica beads
that are end-capped with octadecyl groups or octyl groups and
EMPORE materials that have C8 and C18 silica beads (EMPORE
materials are available from 3M Co., St. Paul, Minn.). Examples of
normal phase materials include hydroxy groups and dihydroxy
groups.
[0147] Commercially available materials can also be modified or
directly used in methods of the present invention. For example,
solid phase materials available under the trade designation LYSE
AND GO (Pierce, Rockford, Ill.), RELEASE-IT (CPG, NJ), GENE FIZZ
(Eurobio, France), GENE RELEASER (Bioventures Inc., Murfreesboro,
Tenn.), and BUGS N BEADS (GenPoint, Oslo, Norway), as well as
Zymo's beads (Zymo Research, Orange, Calif.) and Dynal's beads
(Dynal, Oslo, Norway) can be incorporated into the methods of the
present invention, particularly into a device as the solid phase
capture material.
[0148] In certain embodiments of such methods, the solid phase
material includes a coating reagent. The coating reagent is
preferably selected from the group consisting of a surfactant, a
strong base, a polyelectrolyte, a selectively permeable polymeric
barrier, and combinations thereof. In certain embodiments of such
methods, the solid phase material includes a
polytetrafluoroethylene fibril matrix, sorptive particles enmeshed
in the matrix, and a coating reagent coated on the solid phase
material, wherein the coating reagent is selected from the group
consisting of a surfactant, a strong base, a polyelectrolyte, a
selectively permeable polymeric barrier, and combinations thereof.
Herein, the phrase "coating reagent coated on the solid phase
material" refers to a material coated on at least a portion of the
solid phase material, e.g., on at least a portion of the fibril
matrix and/or sorptive particles.
[0149] Examples of suitable surfactants are listed below.
[0150] Examples of suitable strong bases include NaOH, KOH, LiOH,
NH.sub.4OH, as well as primary, secondary, or tertiary amines.
[0151] Examples of suitable polyelectrolytes include, polystyrene
sulfonic acid (e.g., poly(sodium 4-styrenesulfonate) or PSSA),
polyvinyl phosphonic acid, polyvinyl boric acid, polyvinyl sulfonic
acid, polyvinyl sulfuric acid, polystyrene phosphonic acid,
polyacrylic acid, polymethacrylic acid, lignosulfonate,
carrageenan, heparin, chondritin sulfate, and salts or other
derivatives thereof.
[0152] Examples of suitable selectively permeable polymeric
barriers include polymers such as acrylates, acryl amides,
azlactones, polyvinyl alcohol, polyethylene imine, polysaccharides.
Such polymers can be in a variety of forms. They can be
water-soluble, water-swellable, water-insoluble, hydrogels, etc.
For example, a polymeric barrier can be prepared such that it acts
as a filter for larger particles such as white blood cells, nuclei,
viruses, bacteria, as well as nucleic acids such as human genomic
DNA and proteins. These surfaces could be tailored by one of skill
in the art to separate on the basis of size and/or charge by
appropriate selection of functional groups, by cross-linking, and
the like. Such materials would be readily available or prepared by
one of skill in the art.
[0153] Preferably, the solid phase material is coated with a
surfactant without washing any surfactant excess away, although the
other coating reagents can be rinsed away if desired. Typically,
the coating can be carried out using a variety of methods such as
dipping, rolling, spraying, etc. The coating reagent-loaded solid
phase material is then typically dried, for example, in air, prior
to use.
[0154] Particularly desirable are solid phase materials that are
coated with a surfactant, preferably a nonionic surfactant. This
can be accomplished according to the procedure set forth in the
Examples Section. Although not intending to be limited by theory,
the addition of the surfactant is believed to increase the
wettability of the solid phase material, which allows the
inhibitors to soak into the solid phase material and bind
thereto.
[0155] The coating reagent for the solid phase materials are
preferably aqueous-based solutions, although organic solvents
(alcohols, etc.) can be used, if desired. The coating reagent
loading should be sufficiently high such that the sample is able to
wet out the solid phase material. It should not be so high,
however, that there is significant elution of the coating reagent
itself. Preferably, if the coating reagent is eluted with the
nucleic acid, there is no more than about 2 wt-% coating reagent in
the eluted sample. Typically, the coating solution concentrations
can be as low as 0.1 wt-% coating reagent in the solution and as
high as 10 wt-% coating reagent in the solution.
[0156] Surfactants
[0157] Nonionic Surfactants. A wide variety of suitable nonionic
surfactants are known that can be used as a lysing reagent
(discussed above), an eluting reagent (discussed below), and/or as
a coating on the solid phase material. They include, for example,
polyoxyethylene surfactants, carboxylic ester surfactants,
carboxylic amide surfactants, etc. Commercially available nonionic
surfactants include, n-dodecanoylsucrose,
n-dodecyl-.beta.-D-glucopyranoside,
n-octyl-.beta.-D-maltopyranoside,
n-octyl-.beta.-D-thioglucopyranoside, n-decanoylsucrose,
n-decyl-.beta.-D-maltopyranoside, n-decyl-.beta.-D-thiomaltoside,
n-heptyl-.beta.-D-glucopyranoside,
n-heptyl-.beta.-D-thioglucopyranoside,
n-hexyl-.beta.-D-glucopyranoside, n-nonyl-.beta.-D-glucopyranoside,
n-octanoylsucrose, n-octyl-.beta.-D-glucopyranoside,
cyclohexyl-n-hexyl-.beta.-D-maltoside,
cyclohexyl-n-methyl-.beta.-D-maltoside, digitonin, and those
available under the trade designations PLURONIC, TRITON, TWEEN, as
well as numerous others commercially available and listed in the
Kirk Othmer Technical Encyclopedia. Examples are listed in Table 1
below. Preferred surfactants are the polyoxyethylene surfactants.
More preferred surfactants include octyl phenoxy
polyethoxyethanol.
1TABLE 1 SURFACTANT TRADE NAME NONIONIC SURFACTANT SUPPLIER
PLURONIC F127 Modified oxyethylated alcohol and/or Sigma
oxypropylated straight chain alcohols St. Louis, MO TWEEN 20
Polyoxyethylene (20) sorbitan Sigma monolaurate St. Louis, MO
TRITON X-100 t-Octyl phenoxy polyethoxyethanol Sigma St. Louis, MO
BRIJ 97 Polyoxyethylene (10) oleyl ether Sigma St. Louis, MO IGEPAL
CA-630 Octyl phenoxy poly (ethyleneoxy) Sigma ethanol St. Louis, MO
TOMADOL 1-7 Ethoxylated alcohol Tomah Products Milton, WI Vitamin E
TPGS d-Alpha tocopheryl polyethylene glycol Eastman 1000 Kingsport,
TN
[0158] Also suitable are fluorinated nonionic surfactants of the
type disclosed in U.S. Pat. Publication Nos. 2003/0139550 (Savu et
al.) and 2003/0139549 (Savu et al.). Other nonionic fluorinated
surfactants include those available under the trade designation
ZONYL from DuPont (Wilmington, Del.).
[0159] Zwitterionic Surfactants. A wide variety of suitable
zwitterionic surfactants are known that can be used as a coating on
the solid phase material, as a lysing reagent, and/or as an eluting
reagent. They include, for example, alkylamido betaines and amine
oxides thereof, alkyl betaines and amine oxides thereof, sulfo
betaines, hydroxy sulfo betaines, amphoglycinates,
amphopropionates, balanced amphopolycarboxyglycinates, and alkyl
polyaminoglycinates. Proteins have the ability of being charged or
uncharged depending on the pH; thus, at the right pH, a protein,
preferably with a pI of about 8 to 9, such as modified Bovine Serum
Albumin or chymotrypsinogen, could function as a zwitterionic
surfactant. A specific example of a zwitterionic surfactant is
cholamido propyl dimethyl ammonium propanesulfonate available under
the trade designation CHAPS from Sigma. More preferred surfactants
include N-dodecyl-N,N dimethyl-3-ammonia-1-propane sulfonate.
[0160] Cationic Surfactants. A wide variety of suitable cationic
surfactants are known that can be used as a lysing reagent, an
eluting reagent, and/or as a coating on the solid phase material.
They include, for example, quaternary ammonium salts,
polyoxyethylene alkylamines, and alkylamine oxides. Typically,
suitable quaternary ammonium salts include at least one higher
molecular weight group and two or three lower molecular weight
groups are linked to a common nitrogen atom to produce a cation,
and wherein the electrically-balancing anion is selected from the
group consisting of a halide (bromide, chloride, etc.), acetate,
nitrite, and lower alkosulfate (methosulfate etc.). The higher
molecular weight substituent(s) on the nitrogen is/are often (a)
higher alkyl group(s), containing about 10 to about 20 carbon
atoms, and the lower molecular weight substituents may be lower
alkyl of about 1 to about 4 carbon atoms, such as methyl or ethyl,
which may be substituted, as with hydroxy, in some instances. One
or more of the substituents may include an aryl moiety or may be
replaced by an aryl, such as benzyl or phenyl. Among the possible
lower molecular weight substituents are also lower alkyls of about
1 to about 4 carbon atoms, such as methyl and ethyl, substituted by
lower polyalkoxy moieties such as polyoxyethylene moieties, bearing
a hydroxyl end group, and falling within the general formula:
R(CH.sub.2CH.sub.2O).sub.(n-1)CH.sub.2CH.sub.2OH
[0161] where R is a (C1-C4)divalent alkyl group bonded to the
nitrogen, and n represents an integer of about 1 to about 15.
Alternatively, one or two of such lower polyalkoxy moieties having
terminal hydroxyls may be directly bonded to the quaternary
nitrogen instead of being bonded to it through the previously
mentioned lower alkyl. Examples of useful quaternary ammonium
halide surfactants for use in the present invention include but are
not limited to methyl-bis(2-hydroxyethyl)coco-ammonium chloride or
oleyl-ammonium chloride, (ETHOQUAD C/12 and O/12, respectively) and
methyl polyoxyethylene (15) octadecyl ammonium chloride (ETHOQUAD
18/25) from Akzo Chemical Inc.
[0162] Anionic Surfactants. A wide variety of suitable anionic
surfactants are known that can be used as a lysing reagent, an
eluting reagent, and/or as a coating on the solid phase material.
Surfactants of the anionic type that are useful include sulfonates
and sulfates, such as alkyl sulfates, alkylether sulfates, alkyl
sulfonates, alkylether sulfonates, alkylbenzene sufonates,
alkylbenzene ether sulfates, alkylsulfoacetates, secondary alkane
sulfonates, secondary alkylsulfates and the like. Many of these can
include polyalkoxylate groups (e.g., ethylene oxide groups and/or
propylene oxide groups, which can be in a random, sequential, or
block arrangement) and/or cationic counterions such as Na, K, Li,
ammonium, a protonated tertiary amine such as triethanolamine or a
quaternary ammonium group. Examples include: alkyl ether sulfonates
such as lauryl ether sulfates available under the trade designation
POLYSTEP B12 and B22 from Stepan Company, Northfield, Ill., and
sodium methyl taurate available under the trade designation NIKKOL
CMT30 from Nikko Chemicals Co., Tokyo, Japan); secondary alkane
sulfonates available under the trade designation HOSTAPUR SAS,
which is a sodium (C14-C17)secondary alkane sulfonates
(alpha-olefin sulfonates), from Clariant Corp., Charlotte, N.C.;
methyl-2-sulfoalkyl esters such as sodium
methyl-2-sulfo(C12-C16)ester and disodium 2-sulfo(C12-C16)fatty
acid available from Stepan Company under the trade designation
ALPHASTE PC-48; alkylsulfoacetates and alkylsulfosuccinates
available as sodium laurylsulfoacetate (trade designation LANTHANOL
LAL) and disodiumlaurethsulfosuccinate (trade designation
STEPANMILD SL3), both from Stepan Co.; and alkylsulfates such as
ammoniumlauryl sulfate commercially available under the trade
designation STEPANOL AM from Stepan Co.
[0163] Another class of useful anionic surfactants include
phosphates such as alkyl phosphates, alkylether phosphates,
aralkylphosphates, and aralkylether phosphates. Many of these can
include polyalkoxylate groups (e.g., ethylene oxide groups and/or
propylene oxide groups, which can be in a random, sequential, or
block arrangement). Examples include a mixture of mono-, di- and
tri-(alkyltetraglycolether)-o-phosphoric acid esters generally
referred to as trilaureth-4-phosphate commercially available under
the trade designation HOSTAPHAT 340KL from Clariant Corp., and
PPG-5 ceteth 10 phosphate available under the trade designation
CRODAPHOS SG from Croda Inc., Parsipanny, N.J., as well as alkyl
and alkylamidoalkyldialkylamine oxides. Examples of amine oxide
surfactants include those commercially available under the trade
designations AMMONYX LO, LMDO, and CO, which are
lauryldimethylamine oxide, laurylamidopropyldimethylamine oxide,
and cetyl amine oxide, all from Stepan Co.
[0164] Elution Techniques
[0165] For embodiments that use a solid phase material for
retaining inhibitors, the more concentrated region of the sample
that includes nucleic acid-containing material (e.g., nuclei)
and/or released nucleic acid can be eluted using a variety of
eluting reagents. Such eluting reagents can include water
(preferably RNAse free water), a buffer, a surfactant, which can be
cationic, anionic, nonionic, or zwitterionic, or a strong base.
[0166] Preferably the eluting reagent is basic (i.e., greater than
7). For certain embodiments, the pH of the eluting reagent is at
least 8. For certain embodiments, the pH of the eluting reagent is
up to 10. For certain embodiments, the pH of the eluting reagent is
up to 13. If the eluted nucleic acid is used directly in an
amplification process such as PCR, the eluting reagent should be
formulated so that the concentration of the ingredients will not
inhibit the enzymes (e.g., Taq Polymerase) or otherwise prevent the
amplification reaction.
[0167] Examples of suitable surfactants include those listed above,
particularly, those known as SDS, TRITON X-100, TWEEN, fluorinated
surfactants, and PLURONICS. The surfactants are typically provided
in aqueous-based solutions, although organic solvents (alcohols,
etc.) can be used, if desired. The concentration of a surfactant in
an eluting reagent is preferably at least 0.1 weight/volume percent
(w/v-%), based on the total weight of the eluting reagent. The
concentration of a surfactant in an eluting reagent is preferably
no greater than 1 w/v-%, based on the total weight of the eluting
reagent. A stabilizer, such as polyethylene glycol, can optionally
be used with a surfactant.
[0168] Examples of suitable elution buffers include TRIS-HCl,
N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES),
3-[N-morpholino]propanesulfonic acid (MOPS),
piperazine-N,N'-bis[2-ethane- sulfonic acid] (PIPES),
2-[N-morpholino]ethansulfonic acid (MES), TRIS-EDTA (TE) buffer,
sodium citrate, ammonium acetate, carbonate salts, and bicarbonates
etc.
[0169] The concentration of an elution buffer in an eluting reagent
is preferably at least 10 millimolar (mM). The concentration of a
surfactant in an eluting reagent is preferably no greater than 2
weight percent (wt-%).
[0170] Typically, elution of the nucleic acid-containing material
and/or released nucleic acid is preferably accomplished using an
alkaline solution. Although not intending to be bound by theory, it
is believed that an alkaline solution allows for improved binding
of inhibitors, as compared to elution with water. The alkaline
solution also facilitates lysis of nucleic acid-containing
material. Preferably, the alkaline solution has a pH of 8 to 13,
and more preferably 13. Examples of sources of high pH include
aqueous solutions of NaOH, KOH, LiOH, quaternary nitrogen base
hydroxide, tertiary, secondary or primary amines, etc. If an
alkaline solution is used for elution, it is typically neutralized
in a subsequent step, for example, with TRIS buffer, to form a
PCR-ready sample.
[0171] The use of an alkaline solution can selectively destroy RNA,
to allow for the analysis of DNA. Otherwise, RNAse can be added to
the formulation to inactivate RNA, followed by heat inactivation of
the RNAse. Similarly, DNAse can be added to selectively destroy DNA
and allow for the analysis of RNA; however, other lysis buffers
(e.g., TE) that do not destroy RNA would be used in such methods.
The addition of RNAse inhibitor such as RNAsin can also be used in
a formulation for an RNA preparation that is subjected to real-time
PCR.
[0172] Elution is typically carried out at room temperature,
although higher temperatures may produce higher yields. For
example, the temperature of the eluting reagent can be up to
95.degree. C. if desired. Elution is typically carried out within
10 minutes, although 1-3 minute elution times are preferred.
[0173] Additional Embodiments
[0174] In other cases, it may be desirable to isolate various cell
types selectively using known density gradient materials. These
density gradient materials include sucrose and other commercially
available under the trade designations FICOLL (Amersham
Biosciences, Piscataway, N.J.), PERCOLL (Amersham Biosciences,
Piscataway, N.J.), HISTOPAQUE (Sigma, St. Louis, Mo.), ISOPREP
(Robbins Scientific Corporation, Sunnyvale, Calif.), HISTODENZ
(Sigma, St. Louis, Mo.), and OPTIPREP (Sigma, St. Louis, Mo.). The
specific cells of interest, for example, peripheral blood
mononuclear cells (PBMC's) can be selectively removed by the use of
a variable valve device. After extraction of the specific cells of
interest, PCR can be directly carried out after lysis as long as
the gradient material is PCR compatible. In cases where the
gradient material is not PCR compatible, care must be taken to
ensure adequate dilution of the sample (e.g., with water or buffer)
followed by concentration of cells and repeating this process a few
times to produce a PCR ready sample. Alternatively, simply diluting
significantly may be sufficient to produce a PCR ready sample
[0175] For example, in one embodiment of the present invention, a
method includes: providing a device including a loading chamber and
a variable valved process chamber; placing whole blood in the
loading chamber; transferring the whole blood to a valved process
chamber; contacting the whole blood with a density gradient
material; centrifuging the whole blood and density gradient
material in the valved process chamber to form layers, at least one
of which contains cells of interest; removing at least a portion of
the layer containing the cells of interest; and lysing the
separated cells of interest to release nucleic acid. In one aspect
of this method, prior to lysing the separated cells of interest,
the method includes diluting the separated cells of interest with
water or buffer, optionally further concentrating the diluted layer
to increase the concentration of cells of interest, optionally
separating the further concentrated region, and optionally
repeating this process of dilution followed by concentration and
separation. In another aspect of this method, prior to lysing the
separated cells of interest, the method includes diluting the
separated cells of interest with water, preferably sufficiently to
form a 20.times.-1000.times. dilution.
[0176] The inhibitors can be removed using solid phase materials,
as described herein (as well as described in U.S. patent
application Ser. No. ______, filed on ______, entitled METHODS FOR
NUCLEIC ACID ISOLATION AND KITS USING SOLID PHASE MATERIAL
(Attorney Docket No. 59073US003)), prior to or after capture of
viral particles onto the beads (for example, as discussed below).
Such solid phase materials can be used in various methods and with
various samples described herein.
[0177] In addition to this, the level of inhibitors can be reduced
using concentration/separation/optional dilution steps, for
example, as disclosed in U.S. patent application Ser. No. ______,
filed on ______, entitled METHODS FOR NUCLEIC ACID ISOLATION AND
KITS USING A MICROFLUIDIC DEVICE AND CONCENTRATION STEP (Attorney
Docket No. 59801US002).
[0178] In other embodiments, it may be necessary to capture viral
DNA/RNA in the white blood cell. In these cases, the white blood
cells can be isolated using a variable valve and beads can be used
to capture the viral DNA/RNA.
[0179] For example, beads can be functionalized with the
appropriate groups to isolate specific cells, viruses, bacteria,
proteins, nucleic acids, etc. The beads can be segregated from the
sample by centrifugation and subsequent separation. The beads could
be designed to have the appropriate density and sizes (nanometers
to microns) for segregation. For example, in the case of viral
capture, beads that recognize the protein coat of a virus can be
used to capture and concentrate the virus prior to or after removal
of small amounts of residual inhibitors from a serum sample.
[0180] Nucleic acids can be extracted out of the segregated viral
particles by lysis. Thus, the beads could provide a way of
concentrating relevant material in a specific region within a
device, also allowing for washing of irrelevant materials and
elution of relevant material from the captured particle.
[0181] Examples of such beads include, but are not limited to,
crosslinked polystyrene beads available under the trade designation
CHELEX from Bio-Rad Laboratories, Inc. (Hercules, Calif.),
crosslinked agarose beads with tris(2-aminoethyl)amine,
iminodiacetic acid, nitrilotriacetic acid, polyamines and
polyimines as well as the chelating ion exchange resins
commercially available under the trade designation DUOLITE C-467
and DUOLITE GT73 from Rohm and Haas (Philadelphia, Pa.), AMBERLITE
IRC-748, DIAION CR11, DUOLITE C647. These beads are also suitable
for use as the solid phase material as discussed above.
[0182] Other examples of beads include those available under the
trade designations GENE FIZZ (Eurobio, France), GENE RELEASER
(Bioventures Inc., Murfreesboro, Tenn.), and BUGS N BEADS
(GenPoint, Oslo, Norway), as well as Zymo's beads (Zymo Research,
Orange, Calif.) and DYNAL beads (Dynal, Oslo, Norway).
[0183] Other materials are also available for pathogen capture. For
example, polymer coatings can also be used to isolate specific
cells, viruses, bacteria, proteins, nucleic acids, etc. in certain
embodiments of the invention. These polymer coatings could directly
be spray-jetted, for example, onto the cover film of a device.
[0184] Viral particles can be captured onto beads by covalently
attaching antibodies onto bead surfaces. The antibodies can be
raised against the viral coat proteins. For example, DYNAL beads
can be used to covalently link antibodies. Alternatively, synthetic
polymers, for example, anion-exchange polymers, can be used to
concentrate viral particles. Commercially available resins such as
viraffinity (Biotech Support Group, East Brunswick, N.J.) can be
used to coat beads or applied as polymer coatings onto select
locations in a device to concentrate viral particles. BUGS N BEADS
(GenPoint, Oslo, Norway) can, for example, be used for extraction
of bacteria. Here, these beads can be used to capture bacteria such
as Staphylococcus, Streptococcus, E coli, Salmonella, and Clamydia
elementary bodies.
[0185] Thus, in one embodiment of the present invention when the
sample includes viral particles or other pathogens (e.g.,
bacteria), a device can include solid phase material in the form of
viral capture beads or other pathogen capture material. In this
method, the sample contacts the viral capture beads. More
specifically, in one case, the viral capture beads can be used only
for concentration of virus or bacteria, for example, followed by
segregation of beads to another chamber, ending with lysis of virus
or bacteria. In another case, the beads can be used for
concentration of virus or bacteria, followed by lysis and capture
of nucleic acids onto the same bead, dilution of beads,
concentration of beads, segregation of beads, and repeating the
process multiple times prior to elution of captured nucleic
acid.
[0186] In a specific embodiment, a method includes: providing a
device including a loading chamber, a variable valved process
chamber, and a separation chamber including pathogen capture
material; placing whole blood in the loading chamber; transferring
the whole blood to a valved process chamber; centrifuging the whole
blood in the valved process chamber to form a plasma layer
including one or more pathogens, a red blood cell layer, and an
interfacial layer (therebetween) including white blood cells;
transferring at least a portion of the plasma layer including the
one or more pathogens to the separation chamber having pathogen
capture material therein; separating at least a portion of the one
or more pathogens from the pathogen capture material; and lysing
the one or more pathogens to release nucleic acid.
[0187] Alternatively, if beads (or other pathogen capture material)
are not the method of choice for viral capture (or other pathogen
capture), then one may choose to pellet out viral particles from
serum or plasma using an ultracentrifuge. These concentrated viral
particles can be transferred to the device for lysing with a
surfactant with the addition of an RNAse inhibitor, for example, if
viral RNA needs to be isolated followed by an amplification
reaction (RT-PCR).
[0188] If the downstream application of the nucleic acid is
subjecting it to an amplification process such as PCR, then all
reagents used in the method are preferably compatible with such
process (e.g., PCR compatible). Furthermore, the addition of PCR
facilitators may be useful, especially for diagnostic purposes.
Also, heating of the material to be amplified prior to
amplification can be beneficial.
[0189] In embodiments in which the inhibitors are not completely
removed, the use of buffers, enzymes, and PCR facilitators can be
added that help in the amplification process in the presence of
inhibitors. For example, enzymes other than Taq Polymerase, such as
rTth, that are more resistant to inhibitors can be used, thereby
providing a huge benefit for PCR amplification. The addition of
Bovine Serum Albumin, betaine, proteinase inhibitors, bovine
transferrin, etc. can be used as they are known to help even
further in the amplification process. Alternatively, one can use a
commercially available product such as Novagen's Blood Direct PCR
Buffer kit (EMD Biosciences, Darmstadt, Germany) for direct
amplification from whole blood without the need for extensive
purification.
[0190] Objects and advantages of this invention may be further
illustrated by the following examples, but the particular materials
and amounts thereof recited in these examples, as well as other
conditions and details, should not be construed to unduly limit
this invention.
EXAMPLES
Example 1
Preparation of Solid Phase Material: Ammonia Form with TRITON-X
100
[0191] A 3M No. 2271 EMPORE Extraction Chelating Disk was placed in
a glass filter holder. The extraction disk was converted into the
ammonia form, following the procedure printed on the package
insert. The disk placed in a vial and was submerged in a 1%
TRITON-X 100 (Sigma-Aldrich, St. Louis, Mo.) solution (0.1 gram (g)
of TRITON-X 100 in 10 mL of water), mixing for about 6-8 hours on a
Thermolyne Vari-Mix Model M48725 Rocker (Barnstead/Thermolyne,
Dubuque, Iowa). The disk was placed in glass filter holder, dried
by applying a vacuum for about 20 minutes (min), and then dried
overnight at room temperature (approximately 21.degree. C.), taking
care not to wash or rinse the disk.
Example 2A
Effect of Inhibitor/DNA on PCR: Varying Inhibitor Concentration
with Fixed DNA Concentration
[0192] A dilution series of inhibitors were made prior to spiking
with clean human genomic DNA in order to study the effect of
inhibitor on PCR. To 10 .mu.L of 15 nanograms per microliter
(ng/.mu.L) human genomic DNA, 1.mu.L of different Mix I (neat or
dilutions thereof) was added (Samples 2--no inhibitor added,
2D--neat, 2E--1:10, 2F--1:30, 2G--1:100, 2H--1:300) and vortexed.
Two (2) .mu.L aliquots of each sample were taken for 20 .mu.L PCR.
The results are shown in Table 2.
[0193] Mix I: one hundred (100) .mu.L of whole blood was added to 1
.mu.L of neat TRITON-X 100. The solution was incubated at room
temperature (approximately 21.degree. C.) for about 5 minutes,
vortexing the solution intermittently (for approximately 5 seconds
every 20 seconds). The solution was investigated to make sure that
it was transparent before proceeding to the next step. The solution
was spun in an Eppendorf Model 5415D centrifuge at 400 rcf for
about 10 minutes. Approximately 80 .mu.L from the top of the
microcentrifuge tube and designated Mix I.
Example 2B
Effect of Inhibitor/DNA on PCR: Varying DNA Concentration with
Fixed Inhibitor Concentration
[0194] To 10 .mu.L of human genomic DNA, 1 .mu.L of 1:3 diluted Mix
I (described above) was added. DNA concentrations that were
examined were the following: Samples 2J--15 ng/.mu.L, 2K--7.5
ng/.mu.L, 2L--3.75 ng/.mu.L, 2M--1.5 ng/.mu.L. Two (2) .mu.L
aliquots of each sample were taken for 20 .mu.L PCR. The results
are shown in Table 2.
Example 2C
Effect of Inhibitor/DNA on PCR: DNA with No Added Inhibitor
[0195] The following samples were prepared with 1 .mu.L of water
added to each DNA sample instead of inhibitor: Samples 2N--15
ng/.mu.L, 2P--7.5 ng/.mu.L, 2Q--3.75 ng/.mu.L, 2R--1.5 ng/.mu.L.
Two (2) .mu.L aliquots of each sample were taken for 20 .mu.L PCR.
The results are shown in Table 2.
2 TABLE 2 Ct (duplicate Sample No. samples) 2 19.10 19.06 2D 13.94
29.50 2E 27.39 26.22 2F 21.44 20.66 2G 19.90 19.30 2H 19.90 20.08
2J 28.45 28.61 2K 29.16 30.22 2L 30.47 29.96 2M 28.43 26.16 2N
20.05 19.80 2P 20.74 20.54 2Q 21.95 21.88 2R 22.67 23.10
Example 3
Procedure for Isolation of Genomic DNA from Whole Blood with the
Use of a Chelating Solid Phase Material
[0196] Six hundred (600) .mu.L of whole blood was spun at 2500 rpm
for 10 min. The supernatant was separated and discarded, and the
buffy coat was extracted from the interfacial layer. Five (5) .mu.L
of buffy coat was added to five (5) .mu.L of 2% TRITON-X. The
solution was mixed thoroughly, and placed onto a 3M No. 2271 EMPORE
Extraction Chelating Disk prepared as described in Example 1 using
10% TRITON-X 100 instead of 1% TRITON-X 100 as a loading solution.
After the solution had soaked into the disk, the sample was
extracted with a twenty (20) .mu.L aliquot of 0.1M NaOH. The
solution was briefly spun in an Eppendorf Model 5415D centrifuge at
400 rcf. An aliquot of eleven (11) .mu.L of sample was heated for 3
min at 95.degree. C., and then added to three (3) .mu.L of 1 M
TRIS-HCl (pH 7.4).
Example 4
Procedure for Isolation of Genomic DNA from Whole Blood
[0197] Six hundred (600) .mu.L of whole blood was spun at 2500 rpm
for 10 min. The supernatant was separated and discarded, and the
buffy coat was extracted from the interfacial layer. Five (5) .mu.L
of buffy coat was added to the ninety five (95) .mu.L of RNase-free
sterile water. The solution was mixed until the color became
uniform and spun in an Eppendorf Model 5415D centrifuge at 400 rcf
for about 2 minutes. An aliquot of ninety five (95) .mu.L of the
solution from the top was separated and discarded, leaving about
five (5) .mu.L of concentrated material at the bottom of the
centrifuge tube. To the last 5 .mu.L of concentrated material, 95
.mu.L of RNase-free sterile water was added. The sample was mixed
until the color became uniform. The solution was spun in an
Eppendorf Model 5415D centrifuge at 400 rcf for about 2 minutes. A
95 .mu.L of the solution on the top was separated and discarded,
leaving about ten (10) .mu.L of concentrated material at the bottom
of the centrifuge tube. To the last 10 .mu.L of concentrated
material, one (1) .mu.L of 1 M NaOH was added. After 1 min
incubation, the sample was heated for 3 min at 95.degree. C. A 3
.mu.L of 1 M TRIS-HCl (pH 7.4) was added to 11 .mu.L of sample.
[0198] Results
[0199] Table 3 reports results that were obtained on ABI 7700 QPCR
Machine (Applera, Foster City, Calif.) following the instructions
in QuantiTech SYBR Green PCR Handbook on p. 10-12 for preparation
of a 10 .mu.L PCR sample (2 .mu.L of sample in 10 .mu.L SYBR Green
Master Mix, 4 .mu.L .beta.-actin, 4 .mu.L of water) for Examples
1-2; Results for Examples 3-4 were obtained on LightCycler 2.0
(Roche Applied Science, Indianapolis, Ind.) following the
instructions in LightCycler Factor V Leiden Mutation Kit's package
insert on p. 2-3 for preparation of a 10 .mu.L PCR sample (2.5
.mu.L of sample in 5.5 .mu.L of RNase-free sterile water, 1 .mu.L
of 10.times. Factor V Leiden Reaction Mix and 1 .mu.L of 10.times.
Factor V Leiden Mutation Detection Mix). Spectra measurements were
run on a SpectraMax Plus.sup.384 spectrophotometer at 405 nm
(Molecular Devices Corporation, Sunnyvale, Calif.). Two, three or
four values for each sample represent duplicates, triplicates, or
quadruplicates.
3 TABLE 3 405 nm Samples Ct (avg) 1.5 ng/.mu.L human genomic 16.92
-- DNA in 0.1 M NaOH/40 mM 20.67 TRIS-HCl buffer 1.5 ng/.mu.L human
genomic 19.01 0 DNA in water 18.67 1.5 ng/.mu.L human genomic 16.18
-- DNA in water 16.28 Examples 2A and 2B Mix I -- 2.63 diluted 1:36
Examples 2A and 2B -- 0.38 Mix I diluted 1:360 Examples 2A and 2B
-- 0.036 Mix I diluted 1:3600 Examples 2A and 2B -- 0 Mix I diluted
1:36000 Example 3* 26.02, 24.93 -- Example 4* 22.73, 23.93 --
*Positive Control for Examples 3-4 was DNA extracted from two
hundred (200) .mu.L of whole blood following "Blood and Body Fluid
Spin Protocol" described in QIAamp DNA
[0200] Blood Mini Kit Handbook p. 27, eluting in 200 .mu.L of water
and had Ct value of 20-21. Negative Control (NTC or no template
control) did not amplify in these experiments.
[0201] As used herein and in the appended claims, the singular
forms "a," "and," and "the" include plural referents unless the
context clearly dictates otherwise. Thus, for example, reference to
"a valve lip" includes a plurality of valve lips and reference to
"the process chamber" includes reference to one or more process
chambers and equivalents thereof known to those skilled in the
art.
[0202] All references and publications cited herein are expressly
incorporated herein by reference in their entirety into this
disclosure. Illustrative embodiments of this invention are
discussed and reference has been made to possible variations within
the scope of this invention. These and other variations and
modifications in the invention will be apparent to those skilled in
the art without departing from the scope of the invention, and it
should be understood that this invention is not limited to the
illustrative embodiments set forth herein. Accordingly, the
invention is to be limited only by the claims provided below and
equivalents thereof.
* * * * *