U.S. patent application number 14/177194 was filed with the patent office on 2017-02-23 for multivolume devices, kits and related methods for quantification and detection of nucleic acids and other analytes.
The applicant listed for this patent is California Institute of Technology, University of Chicago. Invention is credited to Wenbin Du, Rustem F. Ismagilov, Jason E. Kreutz, Feng Shen, Bing Sun.
Application Number | 20170051365 14/177194 |
Document ID | / |
Family ID | 53774433 |
Filed Date | 2017-02-23 |
United States Patent
Application |
20170051365 |
Kind Code |
A9 |
Ismagilov; Rustem F. ; et
al. |
February 23, 2017 |
MULTIVOLUME DEVICES, KITS AND RELATED METHODS FOR QUANTIFICATION
AND DETECTION OF NUCLEIC ACIDS AND OTHER ANALYTES
Abstract
Provided are devices comprising multivolume analysis regions,
the devices being capable of supporting amplification, detection,
and other processes. Also provided are related methods of detecting
or estimating the presence nucleic acids, viral levels, and other
biological markers of interest.
Inventors: |
Ismagilov; Rustem F.;
(Altadena, CA) ; Shen; Feng; (Pasadena, CA)
; Kreutz; Jason E.; (Marysville, WA) ; Du;
Wenbin; (Wenzhou, CN) ; Sun; Bing; (Pasadena,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
California Institute of Technology
University of Chicago |
Pasadena
Chicago |
CA
IL |
US
US |
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Prior
Publication: |
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Document Identifier |
Publication Date |
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US 20150225803 A1 |
August 13, 2015 |
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Family ID: |
53774433 |
Appl. No.: |
14/177194 |
Filed: |
February 10, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13467482 |
May 9, 2012 |
9464319 |
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14177194 |
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13440371 |
Apr 5, 2012 |
9447461 |
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13467482 |
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13257811 |
Sep 20, 2011 |
9415392 |
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PCT/US2010/028316 |
Mar 23, 2010 |
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13440371 |
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61262375 |
Nov 18, 2009 |
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61162922 |
Mar 24, 2009 |
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61340872 |
Mar 22, 2010 |
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61518601 |
May 9, 2011 |
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61516628 |
Apr 5, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6851 20130101;
C12Q 1/707 20130101; C12Q 2561/113 20130101; C12Q 1/6851 20130101;
C12Q 2600/158 20130101; C12Q 1/703 20130101 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70 |
Goverment Interests
STATEMENT OF GOVERNMENT RIGHTS
[0004] The United States Government has certain rights in this
invention pursuant to Grant Nos. 1 R01 EB012946, GM074961, and
DP1OD003584, awarded by the National Institutes of Health; and
Grant No. CHE-0526693, awarded by the National Science Foundation.
Claims
1-36. (canceled)
37. A method, comprising: a) introducing an amount of a target
molecule from an original sample into a device; b) effecting
distribution of said amount of said target molecule into at least
two isolated areas of the device, said at least two isolated areas
defining volumes that differ from one another; c) effecting a
reaction on said target molecule so as to give rise to a reaction
product in the said at least two isolated areas; d) detecting said
reaction product optically in said at least two isolated areas; and
e) estimating, from said reaction product, the level of said target
molecule in said original sample.
38. The method of claim 37, wherein said target molecule comprises
a nucleic acid.
39. The method of claim 38, further comprising contacting an
amplification reagent with said nucleic acid.
40. The method of claim 37, wherein at least one of said at least
two isolated areas is estimated to contain only one nucleic acid
molecule.
41. The method of claim 37, wherein said reaction comprises nucleic
acid amplification.
42. The method of claim 41, wherein said nucleic acid amplification
comprises polymerase chain reaction, room-temperature polymerase
chain reaction, nested polymerase chain reaction, multiplex
polymerase chain reaction, arbitrarily primed polymerase chain
reaction, nucleic acid sequence-based amplification, transcription
mediated amplification, strand displacement amplification, branched
DNA probe target amplification, ligase chain reaction, cleavase
invader amplification, anti DNA-RNA hybrid antibody amplification,
or any combination thereof.
43. The method of claim 41, wherein said nucleic acid amplification
is essentially isothermal.
44. The method of claim 37, further comprising estimating the level
of nucleic acid in said original sample.
45. The method of claim 38, wherein at least one of said at least
two isolated areas is estimated to comprise about one molecule of
nucleic acid.
46. The method of claim 37, wherein at least one of said at least
two isolated areas defines a volume in the range of from about 1
picoliter to about 1 microliter.
47. The method of claim 37, wherein distribution of said amount of
said target molecule is effected by effecting relative motion
between a first and second component so as to distribute said
amount of said target molecule into said at least two isolated
areas.
48. The method of claim 47, wherein said relative motion gives rise
to said amount of said target molecule being divided among at least
10 isolated areas.
49. The method of claim 48, wherein said relative motion gives rise
to said amount of said target molecule being divided among at least
50 isolated areas.
50. The method of claim 37, wherein said reaction is effected at
two or more areas essentially simultaneously.
51-60. (canceled)
Description
RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Application
61/518,601, "Quantification of Nucleic Acids With Large Dynamic
Range Using Multivolume Digital Reverse Transcription PCR (RT-PCR)
On A Rotational Slip Chip Tested With Viral Load," filed on May 9,
2011.
[0002] The present application is also a continuation-in-part of
U.S. application Ser. No. 13/440,371, "Analysis Devices, Kits, And
Related Methods For Digital Quantification Of Nucleic Acids And
Other Analytes," filed on Apr. 5, 2012. U.S. application Ser. No.
13/440,371 claimed priority to U.S. Application 61/516,628,
"Digital Isothermal Quantification of Nucleic Acids Via
Simultaneous Chemical Initiation of Recombinase Polymerase
Amplification (RPA) Reactions on Slip Chip," filed on Apr. 5, 2011,
and also to U.S. Application 61/518,601, "Quantification of Nucleic
Acids With Large Dynamic Range Using Multivolume Digital Reverse
Transcription PCR (RT-PCR) On A Rotational Slip Chip Tested With
Viral Load," filed on May 9, 2011.
[0003] U.S. application Ser. No. 13/440,371 is also a continuation
in part of U.S. application Ser. No. 13/257,811, "Slip Chip Device
and Methods," filed on Sep. 20, 2011. That United States
application is a national stage entry of international application
PCT/US2010/028361, "Slip Chip Device and Methods," filed on Mar.
23, 2010. That international application claimed priority to U.S.
Application 61/262,375, "Slip Chip Device and Methods," filed on
Nov. 18, 2009, to U.S. Application 61/162,922, "Sip Chip Device and
Methods," filed on Mar. 24, 2009, and to U.S. Application
61/340,872, "Slip Chip Device and Methods," filed on Mar. 22, 2010.
All of the foregoing applications are incorporated herein by
reference in their entireties for any and all purposes.
TECHNICAL FIELD
[0005] The present application relates to the field of
microfluidics and to the fields of detection and amplification of
biological entities.
BACKGROUND
[0006] Real-time quantitative RT-PCR is an existing technique for
monitoring viral load for HIV, HCV, and other viral infections.
However, this test is cost-prohibitive in some resource-limited
settings and can require multiple instruments, skilled technicians,
and isolated rooms to prevent contamination. The test can thus be
inaccessible to patients in some resource-limited settings.
Moreover, the efficiency of RT-PCR, the quality of sample and
selection of targets, and the methods for interpretation of the
data may in some cases present concerns for the accuracy of
quantifying RNA using RT-PCR.
[0007] Although dipstick-type devices may provide semiquantitative
measurements of viral load after amplification in resource limited
settings, no quantitative test exists to resolve a 3-fold (i.e.,
appx. 0.5 log.sup.10) change in HIV RNA viral load, which change is
considered clinically significant. Accordingly, there is a
long-felt need in the art for devices and methods for quantitative
measurement, estimates, and/or even detection of viral load or
other parameters.
SUMMARY
[0008] In meeting the described challenges, the present disclosure
first provides devices. These devices comprise a first component
comprising a population of first areas; a second component
comprising a population of second areas; the first and second
components being engageable with one another such that relative
motion between the first and second components exposes at least
some of the first population of areas to at least some of the
second population of areas so as to form a plurality of analysis
regions. At least some of the analysis regions suitably differ in
volume from others of the analysis regions.
[0009] Also disclosed are devices. The devices suitably include a
first component comprising a population of first areas; a second
component comprising a population of second areas; the first and
second components being engageable with one another such that when
the first and second components are in a first position relative to
one another a fluidic path is formed between at least some of the
first areas and at least some of the second areas, and when the
first and second components are in a second position relative to
one another, the fluidic path is interrupted so as to isolate at
least some of the first areas from at least some of the second
areas.
[0010] Additionally provided are methods. These methods include
distributing one or more target molecules from an original sample
into a plurality of analysis regions, the distribution being
effected such that at least some of the analysis regions are
statistically estimated to each contain a single target molecule,
at least two of the analysis regions defining different volumes;
and effecting, in parallel, a reaction on at least some of the
single target molecules.
[0011] Other methods presented in this disclosure include
introducing an amount of a target molecule from an original sample
into a device; effecting distribution of the amount of the target
molecule into at least two isolated areas of the device, the at
least two isolated areas defining volumes that differ from one
another; effecting a reaction on the target molecule so as to give
rise to a reaction product in the at least two isolated areas; and
estimating, from the reaction product, the level of a target in the
original sample.
[0012] Also provided are methods, comprising distributing a
plurality of target molecules--suitably nucleic acids--from an
original sample into a plurality of analysis regions, the
distribution being effected such that at least some of the analysis
regions are estimated to each contain a single target molecule, at
least two of the analysis regions defining different volumes; and
effecting, in parallel, a nucleic acid amplification reaction on at
least some of the single target molecules.
[0013] Also disclosed are devices, comprising a first component
comprising a population of first wells formed in a first surface of
the first component, the population of wells being arranged in a
radial pattern; a second component comprising a population of
second wells formed in a first surface of the second component, the
population of wells being arranged in a radial pattern; the first
and second components being engageable with one another such that
relative rotational motion between the first and second components
exposes at least some of the first population of wells to at least
some of the second population of wells so as to form a plurality of
analysis regions, an analysis region comprising a first well and a
second well in pairwise exposure with one another.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The summary, as well as the following detailed description,
is further understood when read in conjunction with the appended
drawings. For the purpose of illustrating the invention, there are
shown in the drawings exemplary embodiments of the invention;
however, the invention is not limited to the specific methods,
compositions, and devices disclosed. In addition, the drawings are
not necessarily drawn to scale. In the drawings:
[0015] FIG. 1 illustrates a rotationally-configured multivolume
device according to the present disclosure;
[0016] FIG. 2 illustrates end-point fluorescence images of
multivolume digital RT-PCR performed on a rotational device
according to the present disclosure;
[0017] FIG. 3 illustrates performance of digital RT-PCR with
synthetic RNA template on an exemplary multivolume device over a 4
log.sub.10 dynamic range;
[0018] FIG. 4 illustrates performance of an exemplary device;
[0019] FIG. 5 illustrates an exemplary device for multiplexed,
multivolume digital RT-PCR with high dynamic range;
[0020] FIG. 6 illustrates representative multivolume digital RT-PCR
for quantification of HIV viral load in two patients' samples;
[0021] FIG. 7 illustrates a representative experiment performing
RT-PCR of HIV viral RNA at an expected concentration of 51
molecules/mL in a RT-PCR mix;
[0022] FIG. 8 illustrates a representative negative control for HIV
viral load;
[0023] FIG. 9 illustrates in tabular form detection and
quantification data;
[0024] FIG. 10 presents a tabular summary of HIV quantification
performance;
[0025] FIG. 11 presents a tabular summary of detection range
data;
[0026] FIG. 12 shows an image, obtained with a iPhone 4S.TM.
camera, of an exemplary multivolume device filled with LAMP
reaction mix;
[0027] FIG. 13 shows a close-up of the center of the image in FIG.
12;
[0028] FIG. 14 presents a schematic view of a radially-arranged
device for performing MV digital PCR, the device design consisting
of 160 wells each at 125, 25, 5, and 1 nL. A sample is loaded from
the center and after filling the device components are relatively
rotated so as to isolate wells--after reaction, wells containing
template have enhanced signal and are counted;
[0029] FIG. 15 presents, in tabular form, a summary of the
specifications of an exemplary device according to the present
disclosure;
[0030] FIG. 16 presents experimental results for MV digital PCR on
an exemplary device using control DNA. Representative false color
(shaded) images (lighter shading represents positive wells that
showed at least a 3-fold increase in intensity compared to negative
wells) for solutions with input concentrations of (a) 1500
molecules/mL and (b) 600,000 molecules/mL (zoomed in on smaller
wells). (c, d) Graphical summary of all experiments comparing the
input concentration, based on UV-vis measurements (black curve),
and observed concentrations using MV digital PCR (.times. and +)
over the entire dynamic range. Represented as (c) the actual
concentration and (d) as a ratio to better show distribution of
results. Stock samples were approximately 500, 1500, 8000, 20,000,
30,000, 100,000, 600,000, and 3,000,000 molecules/mL. The
confidence intervals (CI) for the combined system (solid curves)
indicate where 95% of the experiments should fall. CI curves for
the individual volumes (dashed curves) are also provided to
indicate over what range of concentration each volume contributes;
and
[0031] FIG. 17 illustrates a separate analysis of 10 experimental
results for different well volumes with an input concentration of
30,000 molecules/mL, showing distribution of measured
concentrations for each volume and the overall agreement of
results.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0032] The present invention may be understood more readily by
reference to the following detailed description taken in connection
with the accompanying figures and examples, which form a part of
this disclosure. It is to be understood that this invention is not
limited to the specific devices, methods, applications, conditions
or parameters described and/or shown herein, and that the
terminology used herein is for the purpose of describing particular
embodiments by way of example only and is not intended to be
limiting of the claimed invention. Also, as used in the
specification including the appended claims, the singular forms
"a," "an," and "the" include the plural, and reference to a
particular numerical value includes at least that particular value,
unless the context clearly dictates otherwise.
[0033] The term "plurality" as used herein, means more than one.
When a range of values is expressed, another embodiment includes
from the one particular value and/or to the other particular value.
Similarly, when values are expressed as approximations, by use of
the antecedent about, it will be understood that the particular
value forms another embodiment. All ranges are inclusive and
combinable. All documents cited herein are incorporated herein by
reference in their entireties for any and all purposes.
[0034] In one embodiment, the present disclosure provides devices.
These devices suitably include a first component comprising a
population of first areas and a second component comprising a
population of second areas. The first and second components are
suitably engageable with one another such that relative motion
between the first and second components exposes at least some of
the first population of areas to at least some of the second
population of areas so as to form a plurality of analysis regions.
As described herein, at least some of the analysis regions may
suitably differ in volume from other analysis regions.
[0035] In some embodiments, the first and second components are
engaged so as to permit rotational motion of one component relative
to the other component. This may be in the form of two plates that
are rotatably engaged with one another, as shown in exemplary FIG.
1. As shown in panels A-D of that figure, the two plates may be
rotatably engaged such that relative rotation between the plates
gives rise to wells formed in the plates aligning with one another
(i.e., being placed into at least partial register) or de-aligning
from one another.
[0036] Also as shown in that exemplary figure (FIG. 1, panel G), a
device may provide analysis regions that differ from one another in
volume. For example, the analysis regions (formed between FIG. 1
panels F and G by relative rotational motion between two plates
that gives rise to pairwise exposure of wells formed in the plates
to one another) shown in FIG. 1 have volumes of about 1, 5, 25, and
125 nL.
[0037] Although the analysis regions shown in FIG. 1 increase in
volume with increasing radial distance outward from axis of
rotation between the plates, there is no requirement that analysis
regions vary such a size and/or spatial manners. Other embodiments
of the disclosed devices feature first and second components
engaged so as to permit linear movement of one component relative
to the other component.
[0038] It should be understood that although exemplary FIG. 1 shows
first and second components engaged with one another, the present
disclosure is not limited to devices that have only two components.
For example, a user may construct a device that has first, second,
and third components that are engageable with one another. As but
one example, the device shown in FIG. 1 may include a first
well-bearing component that is engaged with one face of a second
well-bearing component. The second face of the second well-bearing
component may, in turn, be engageable with a third well-bearing
component, such that the wells of the third component may be placed
into overlap with the wells of the second face of the second
well-bearing component. Such a structure may be in a layer-cake or
sandwich form. These configurations enable increased information
density, as such configurations allow creation of additional areas
on a device where reactions and analysis may take place.
[0039] In an alternative embodiment, one surface of a component may
be engaged with two other components. For example, a base component
having formed thereon first and second circular banks of wells that
are separate from one another may be engageable with [1] a first
component that features wells that may be placed into overlap with
the first bank of wells of the base component and [2] a second
component that features wells that may be placed into overlap with
the second bank of wells of the base component. In this way, a
single device may feature multiple components so as to increase the
number and diversity of reaction and/or analysis locations on the
device.
[0040] The first and second components may be of a range of sizes.
In some embodiments, at least one of the first or second components
has a thickness in the range of from about 10 micrometers to about
5000 micrometers, or in the range of from about 50 micrometers to
about 1000 micrometers, or even from about 200 micrometers to about
500 micrometers. These dimensions are particularly useful in
applications where a user may desire a reduced-size device or a
device having a relatively compact form factor. Components may be
formed of a glass, a polymer, and the like.
[0041] Soda-lime glass is considered especially suitable; other
component materials may also be used. Exemplary fabrication methods
for such devices are set forth in Du et al., Lab Chip 2009, 9,
2286-2292. In some embodiments, components of the device are
fabricated from a glass substrate by way of wet etching. In some
other embodiments, the device can be fabricated from plastic
materials, such as polycarbonate, Poly(methyl methacrylate) (PMMA),
polypropylene, polyethylene, cyclo olefin copolymer (COC), cyclo
olefin polymer (COP), and fluorinated polymers including but not
limited to fluorinated ethylene-propylene FEP (fluorinated
ethylene-propylene), perfluoroakoxy (PFA) and
polytetrafluoroethylene (PTFE). Surfaces may be treated with
methods such as silanization and physical deposition, such as vapor
deposition techniques. As one example, a surface of the device may
be treated with dichlorodimethylsilane by vapor silanization. The
surface of the device can be coated with silicone or fluorinated
polymers. Reaction fluids used in the devices may also include
ingredients related to the surfaces of the devices. As one such
example, bovine serum albumin is added to a PCR mixture to prevent
adsorption and denaturation of molecules on a surface of the
device.
[0042] In some embodiments (e.g., exemplary FIG. 5A), a component
comprises a conduit that places at least some of the first
population of wells into fluidic communication with the environment
exterior to the component. In some embodiments, a population of
wells is characterized as being radially disposed relative to a
location on the first component, as shown in exemplary FIG. 5.
Populations of wells may also be present in a circular pattern, a
grid pattern, or virtually any other conformation. In some
embodiments, a device (e.g., exemplary FIG. 5) may include several
populations of wells that are each in fluid communication with
their own conduits, which in turn enables a user to introduce
different materials to different populations of wells. Exemplary
FIG. 5 shows this by reference to a device having five separate
banks of wells, each separate bank of wells containing a different
sample (samples I-V). As shown in this exemplary figure, the banks
of wells may be configured such that a given bank of wells
maintains a sample (e.g., sample I) in isolation from other samples
(e.g., sample II). In the exemplary FIG. 5a, each bank of separate
wells in a form that is roughly a wedge or a pie-slice in shape;
banks of wells may have other layouts (grids, lines, and the like),
depending on the device and on the user's needs. The device may be
configured such that each bank of wells has an inlet configured to
supply material (e.g., sample) to that bank of wells only. In this
way, a device may have five banks of wells, each bank of wells
being supplied by one or more separate inlet. A bank of wells may,
of course, be supplied by one, two, three, or more inlets,
depending on the user's needs. A device may also be configured such
that a given inlet supplies material to at least some wells in two
or more separate banks of wells.
[0043] Certain embodiments of the disclosed devices feature
components where the first areas are wells formed on (or in) a
component. In some embodiments, a well suitably has a volume in the
range of from about 0.1 picoliter to about 10 microliters. In some
variations, the second areas may be wells. Such wells suitably have
volumes in the range of from about 0.1 picoliter to about 10
microliters.
[0044] When areas are placed into exposure with one another so as
to give rise to analysis regions, an analysis region may, in some
embodiments, have a volume in the range of from about 0.1 picoliter
to about 20 microliters. The volumes of two analysis regions may
differ from one another. As one example, the ratio of the volumes
defined by two analysis regions is in the range of from about 1:1
to about 1:1,000,000, or from about 1:50 to about 1:1,000, or from
about 1:100 to about 1:500. In the example shown in FIG. 1, the
ratio between some of the analysis region volumes is 1 n1:125
nL=1:125.
[0045] The devices may also include an imager configured to capture
at least one image of an analysis region. The imager may be a
camera, CCD, PMT, or other imaging device. Portable imagers, such
as digital cameras and cameras on mobile devices such as
smartphones/mobile phones are considered suitable imagers. The
device may be configured such that the imager is positioned such
that it may capture an image of some of the analysis regions of a
device or even an image of all of the analysis regions.
[0046] The devices may also be configured to display an image of an
analysis region for capture of at least one image by an imager. As
one example, the device may be configured, as shown in exemplary
FIG. 2, to present or otherwise display the analysis regions in
such a manner that the analysis regions (including their contents)
may be imaged. Exemplary images are shown in FIGS. 12 and 13, which
images were obtained using the camera of an iPhone 4S.TM. mobile
device.
[0047] The devices may also include a processor configured to
estimate a concentration of an analyte residing in one or more
analysis regions. The processor may be present in the device
itself; in such cases, the device may include the imager and a
processor that is configured to estimate a concentration of an
analyte residing in one or more analysis regions. This may be
effected by, for example, a computer imaging routine configured to
take as an input an image of the analysis regions of a device and
then operate on that input (as described elsewhere herein) to
estimate the concentration of an analyte in the one or more
analysis regions. In one exemplary embodiment, the processor
operates on an image of the analysis regions of the device and,
from that, estimates the presence of an analyte in a sample.
[0048] As one example, a user may extract a blood sample from a
subject to determine whether a particular virus is present in the
subject. The user may then process the blood sample (e.g., cell
isolation, cell lysis, and the like) and assay the sample (e.g.,
via PCR) for the presence of a particular nucleic acid that is a
marker for the virus of interest. The processor may then, by
analyzing the image of the analysis regions in which the nucleic
acid may be present, statistically estimate the presence (if any)
of the virus in the subject. The processor may, alternatively, be
configured to detect the presence of the analyte in a yes/no
fashion; this may be useful in situations where the user is
interested only in knowing whether the subject has a virus and is
less interested in knowing the level of that virus in the subject.
Further information regarding exemplary processing methods is found
in Kreutz et al., JACS 2011 133: 17705-17712; Kreutz et al., Anal.
Chem. 2011 83: 8158-8168; and Shen et al., Anal. Chem. 2011 83:
3533-3540.
[0049] The disclosed devices are suitably configured to permit
formation of 5, 10, 100, 500 or more analysis regions. In some
embodiments, the devices are adapted to place at least about 10
first areas into pairwise exposure with at least 10 second areas.
This pairwise exposure in turn effects formation of 10 analysis
regions. The devices may also be adapted so as to be capable of
placing at least about 100 first areas into pairwise exposure with
at least 100 second areas, or even placing at least about 200 first
areas into pairwise exposure with at least 200 second areas.
Exemplary FIG. 1 shows (by way of the "slip" rotational motion
shown between FIG. 1 panels C and D) placement of filled wells
(darkened circles) into exposure with unfilled wells (unfilled
circles).
[0050] Devices according to the present disclosure may also include
a quantity of a reagent disposed within the device. The reagent may
be a salt, a buffer, an enzyme, and the like. Reagents that are
useful in an amplification reaction are considered especially
suitable. Such reagents may be disposed within an area of the
device in dried or liquid form. Reagents may also be disposed
within the device within a well; fluid reagents may be preloaded
into wells where the reagents remain until the device is used.
[0051] In one such embodiment, a sample is loaded into a first
population of wells on a first component of a device. The device
may also include a second population of wells on a second component
of the device, the second population of wells being pre-filled with
a reagent selected to react with the sample. Relative motion
between the first and second components exposes at least some of
the first population of wells to at least some of the second
population of wells (e.g., FIG. 1), and the pre-stored reagent may
then react with the sample in individual analysis regions formed
from first and second wells exposed to one another.
[0052] The present disclosure provides other devices. These devices
suitably include a first component comprising a population of first
areas and a second component comprising a population of second
areas, with the first and second components suitably being
engageable with one another such that when the first and second
components are in a first position relative to one another a
fluidic path is formed between at least some of the first areas and
at least some of the second areas. The devices are also suitably
configured such that when the first and second components are in a
second position relative to one another, the fluidic path is
interrupted so as to isolate at least some of the first areas from
at least some of the second areas.
[0053] One such exemplary embodiment is shown by FIG. 1. As shown
in that figure, first and second components (well-bearing plates,
in this figure) are engaged with one another (right side of panel
A). In a first position (panels B and C), wells formed in the upper
component (shown with dotted lines) and wells formed in the lower
component (shown by solid lines) form a fluidic path, which path is
shown by the fluid filling illustrated in panel C. The filling may
be effected by an inlet (not shown in FIG. 1) that is formed in (or
through) a component so as to connect a well of the component to
the exterior of the device.
[0054] In a second position (shown in panel D), the fluidic path is
interrupted by way of relative motion of the well-bearing
components so as to isolate some of the wells that formerly defined
the fluidic path from one another. In this way, the device allows a
user to [1] introduce a material into multiple areas (e.g., wells)
and then [2] isolate those areas from one another so as to allow
processing of that material in individualized quantities. Suitable
components and the characteristics of these components (e.g.,
wells, well volumes) are described elsewhere herein. It should be
understood that the devices may include embodiments where two or
more first areas may differ from one another in terms of volume.
For example, a first component may include wells of 1 nL, 10 nL,
and 100 nL formed therein. Likewise, the devices may include
embodiments where two or more second areas may differ from one
another in terms of volume. For example, a second component may
include wells of 1 nL, 10 nL, and 100 nL formed therein. As shown
by exemplary panel C of FIG. 1, the fluidic path may comprise at
least one first area at least partially exposed to (e.g.,
overlapping with) at least one second area. The overlap between
first and second areas may give rise to analysis regions, which
analysis regions may (as described elsewhere herein) have different
volumes from one another.
[0055] In some particularly suitable embodiments, the fluidic path
is configured to permit the passage of aqueous media. This may be
accomplished, for example, by placing a layer of material (e.g.,
lubricating fluid or oil) between the first and second components.
As explained in the other documents cited herein, the layer of
lubricating oil may act to isolate a well formed in the first
component from other wells formed in the first component and also
from wells formed in the second component, except when those wells
are exposed (e.g., placed into at least partial register) to one
another. The lubricating oil may be chosen such that it does not
permit the passage of aqueous media. In some embodiments, the
lubricating fluid may be mineral oil, tetradecane, long chain
hydrocarbon, silicone oil, fluorocarbon, and the like, as well as
combinations of the foregoing.
[0056] It should be understood that in some embodiments, an oil or
other non-aqueous material may also be disposed within a well. This
may be shown by reference to exemplary FIG. 1. In one embodiment,
certain first and second wells may be filled with an aqueous
material (panel C in FIG. 1). Other wells on the device that are
not filled with the aqueous material may be filled with an oil (not
shown). When the fluidic path between the aqueous-filled wells is
broken (panels C and D), the aqueous-filled wells are exposed
pairwise to wells that are filled with oil.
[0057] In some of devices, an analysis region may include an
isolated first area or an isolated second area. In one such
embodiment, the first component comprises wells formed therein and
the second compartment comprises wells formed therein. The
components may be positioned such that (e.g., FIG. 1, panel C) at
least some of the first and second wells form a fluidic path. The
components may then be positioned such that the fluidic path is
interrupted and, further, that some of the first wells are (not
shown) positioned opposite to a flat (i.e., non-well bearing)
portion of the second component, and some of the second wells are
positioned opposite to a flat (i.e., non-well bearing) portion of
the first component. In other embodiments, an analysis region may
comprise an isolated first area and an isolated second area that
are exposed only to one another. This is shown by panel D of
non-limiting FIG. 1.
[0058] Also provided are methods. These methods suitably include
distributing one or more target molecules from an original sample
into a plurality of analysis regions, the distribution being
effected such that at least some of the analysis regions are
statistically estimated to each contain a single target molecule.
Embodiments where at least two of the analysis regions have
different volumes from another are considered especially suitable.
In some embodiments, the methods include effecting a reaction on at
least some of the single target molecules. The reaction may take
place in parallel, i.e., the reaction occurs on two or more target
molecules at the same time. The reaction may be also performed in
multiple analysis regions at the same time. It should be understood
that different types reactions (e.g., amplification, lysing) may
take place at the same time at different analysis regions.
[0059] The distribution may be effected by dividing an area within
which one or more target molecules resides into at least two
analysis regions. Panels C and D of FIG. 1 are illustrative of this
aspect of the method. In panel C, a fluid containing one or more
target molecules is introduced into a fluidic path that comprises,
as described elsewhere herein, wells formed in first and second
components. When the fluidic path is interrupted (panel D of FIG.
1), the fluid is subdivided into various analysis regions. The
volumes of the analysis regions and the target-molecule containing
fluid itself may be configured such that at least some of the
analysis regions each contain (or at estimated to each contain) a
single target molecule.
[0060] The user may also estimate the concentration of a target
compound in the original sample. This estimation may be effected by
application of most probable number theory, as described in Shen et
al., JACS 2011 133: 17705-17712, Kreutz et al., Analytical
Chemistry 2011 83: 8158-8168, and Shen et al., "Digital Isothermal
Quantification of Nucleic Acids via Simultaneous Chemical
Initiation of Recombinase Polymerase Amplification Reactions on
SlipChip", Analytical Chemistry 2011 83:3533-3540. The estimation
may be performed such that the estimation has a lower detection
limit, at a 95% confidence value, of more than about 0.1
molecules/mL, and an upper level of quantification of less than
about 10.sup.12 molecules/mL.
[0061] In some embodiments, the target molecule comprises a target
nucleic acid, and the method is capable of estimating the
concentration of the target nucleic acid in the original sample
with at least about 3-fold resolution for original samples with
concentrations of about 500 molecules or more of target nucleic
acid per milliliter.
[0062] Nucleic acids and proteins are considered especially
suitable target molecules. In some embodiments, the reaction is an
amplification, such as a nucleic acid amplification. The
amplification may be performed within 5, 10, 20, 50, 100, 500, or
even 1000 analysis regions. The amplification may be performed in
such a way that amplification occurs in at least two analysis
regions at the same time, although it is not necessary that
amplification begin or end at the same time in the different
analysis regions. The amplification may be performed in an
essentially isothermal manner such that the process takes place
within a temperature range of plus or minus about 10 degrees C. For
example, the amplification may take place at within 10 degrees C.
of ambient conditions.
[0063] A variety of amplification techniques may be used, as
described elsewhere herein. Some such suitably techniques include a
polymerase chain reaction, a room-temperature polymerase chain
reaction, a nested polymerase chain reaction, a multiplex
polymerase chain reaction, an arbitrarily primed polymerase chain
reaction, a nucleic acid sequence-based amplification, a
transcription mediated amplification, a strand displacement
amplification, a branched DNA probe target amplification, a ligase
chain reaction, a cleavase invader amplification, an anti DNA-RNA
hybrid antibody amplification, and the like.
[0064] An analysis region may, as described elsewhere herein,
comprise first and second areas in pairwise exposure with one
another. The user may effect relative motion between a first
component comprising a plurality of first areas and a second
component comprising a plurality of second areas, the relative
motion placing at least one first area and at least one second area
into pairwise exposure with one another to define at least one
analysis region. This is illustrated in FIG. 1, panels C and D,
where first and second areas are placed into pairwise exposure with
one another so as to define analysis regions. The relative motion
may place at least about 10 first areas into pairwise exposure with
at least about 10 second areas, or may even place at least about
100 first areas into pairwise exposure with at least about 100
second areas.
[0065] The instant disclosure also provides methods. These methods
include introducing an amount of a target molecule from an original
sample into a device; effecting distribution of the amount of the
target molecule into at least two isolated areas of the device, the
at least two isolated areas defining volumes that differ from one
another; effecting a reaction on the target molecule so as to give
rise to a reaction product in the at least two isolated areas; and
estimating, from the reaction product, the level of target molecule
in the original sample.
[0066] The target molecule may be, for example, a nucleic acid. The
methods may also include contacting an amplification reagent--such
as a reagent useful in PCR--with the nucleic acid. In some
embodiments, at least one isolated area is estimated to contain one
nucleic acid molecule, as described elsewhere herein. One
particularly suitable reaction to perform within the disclosed
methods is nucleic acid amplification; suitable amplification
techniques are described elsewhere herein. The nucleic acid
amplification may be essentially isothermal, as described elsewhere
herein.
[0067] The methods may also include estimating the level of a
nucleic acid in the original sample. In some embodiments, at least
one of the isolated areas is estimated to comprise about one
molecule of nucleic acid. This facilitates application of the
estimation methods described in Kreutz et al., JACS 2011 133:
17705-17712; Kreutz et al., Anal. Chem. 2011 83: 8158-8168; and
Shen et al., Anal. Chem. 2011 83: 3533-3540. The disclosed methods
may be capable of estimating the concentration of target nucleic
acid in the original sample with at least about 3-fold resolution
for original samples with concentrations of about 500 molecules or
more of target nucleic acid per milliliter.
[0068] An isolated area, e.g., a well, may suitably have a volume
in the range of from about 1 picoliter to about 10 microliters, as
described elsewhere herein. Volumes in the range of from about 1 nL
to about 500 nL, or even from about 5 nL to about 100 nL are
considered suitable.
[0069] Distribution of some amount of target molecules may be
effected by effecting relative motion between a first and second
component so as to distribute the amount of the target molecule
into at least two isolated areas, as described elsewhere herein and
as shown by exemplary FIG. 1 panels B-D. The relative motion may
give rise to the amount of the target molecule being divided among
at least 10 isolated areas, or even among at least 50 isolated
areas.
[0070] According to the disclosed methods, a reaction may be
effected at two or more areas essentially simultaneously. The
reactions need not necessarily (but can be) the same in two or more
areas. For example, a user may effect an amplification reaction at
three areas while effecting a different reaction (e.g., denaturing)
at three other areas.
[0071] Other disclosed methods include distributing a plurality of
target molecules from an original sample into a plurality of
analysis regions, the distribution being effected such that at
least some of the analysis regions are estimated to each contain a
single target molecule, and at least two of the analysis regions
defining different volumes; effecting, in parallel, a nucleic acid
amplification reaction on at least some of the single target
molecules. Suitable amplification techniques are described
elsewhere herein. The amplification may, in some embodiments, be
effected essentially isothermally.
[0072] In some embodiments, the methods further include removing a
product of the nucleic acid amplification reaction. Such recovery
may be carried out, in some embodiments, by accessing individual
wells of a device. In some embodiments, recovery is achieved by
combining material from multiple wells, for example by placing a
device into the loading position and using a carrier fluid
(including a gas) to expel the material from the device. Recovery
may also be accomplished by pipetting material out of a device.
Such recovery may be used for additional analysis of nucleic acid
products, such as sequencing, genotyping, analysis of methylation
patterns, and identification of epigenetic markers.
[0073] Recovered material may be removed from the device. In some
embodiments, recovered material may be transferred to another
device, or another region of the same device. Amplification may be
carried out by the methods described herein or by other methods
known in the art or by their combinations. As one non-limiting
example, a user may detect the presence of a target nucleic acid,
e.g., by PCR. Once the presence of the target is confirmed, the
user may remove the product from the device. This may be
accomplished by pipetting the product out of an individual well and
transferring that product to another device or container. The
recovered product may be further processed, e.g., sequenced. A
variety of sequencing methods are known to those of ordinary skill
in the art, including Maxam-Gilbert sequencing, chain-termination
sequencing, polony sequencing, 454 Sequencing.TM., SOLiD
Sequencing.TM., ion semiconductor sequencing, nanoball sequencing,
Helioscope.TM. sequencing, nanopore sequencing, single-molecule
SMRT.TM. sequencing, single molecule real time sequencing (RNAP),
and the like.
[0074] The present disclosure also provides devices. These devices
include a first component comprising a population of first wells
formed in a first surface of the first component, the population of
wells being arranged in a radial pattern; a second component
comprising a population of second wells formed in a first surface
of the second component, the plurality of wells being arranged in a
radial pattern; the first and second components being engageable
with one another such that relative rotational motion between the
first and second components exposes at least some of the first
population of wells to at least some of the second population of
wells so as to form a plurality of analysis regions, an analysis
region comprising a first well and a second well in pairwise
exposure with one another.
[0075] In some embodiments, at least two analysis regions have
volumes that differ from one another, as described elsewhere
herein. The first component may include a channel having an inlet,
the channel configured so as to place at least some of the first
wells into fluid communication with the environment exterior to the
channel. The inlet may reside in a surface of the first component
other than the surface of the first component in which the first
wells are formed. In this way, when the two components are
assembled together such that the wells of the first component face
the wells of the second component, the user may fill the wells of
the first component without dissembling the device.
[0076] It should be understood that the second component may also
include a channel and inlet, configured such that the channel inlet
is formed in a surface of the second component other than the
surface of that component in which the wells reside.
[0077] The devices may include, e.g., from about 10 to about 10,000
first wells. The devices may also include, e.g., from about 10 to
about 10,000 second wells.
[0078] The disclosed methods may further include estimating the
level, presence, or both of the one or more nucleic acids in the
biological sample. Estimating may comprise (a) estimating the
presence or absence of the one or more nucleic acids in two or more
wells of different volumes and (b) correlating the estimated
presence or absence of the one or more nucleic acids in the two or
more wells of different volumes to a level of the one or more
nucleic acids in the biological sample, or, alternatively, to the
level of some other target in a biological sample or even in a
subject. Exemplary estimation methods are described elsewhere
herein.
[0079] The present disclosure provides estimating the level of a
target present in a sample (e.g., estimating viral load in a
subject by determining the presence or concentration of a nucleic
acid marker in a sample). The present disclosure, however, also
provides detecting the presence of a target in a sample so as to
provide the user with a yes/no determination concerning whether a
particular analyte is present in a subject. In these embodiments,
the user may perform a reaction (e.g., amplification, labeling) on
a sample and merely assay for the presence of a "positive" result
of the reaction.
[0080] One estimation method is provided in Kreutz et al., Anal.
Chem. 2011 83: 8158-8168. As explained in that publication,
theoretical methods may be used--in conjunction with software
analysis tools--to design and analyze multivolume analysis devices.
Multivolume digital PCR ("MV digital PCR") is a reaction that is
especially amenable to these methods. MV digital PCR minimizes the
total number of wells required for "digital" (single molecule)
measurements while also maintaining high dynamic range and high
resolution.
[0081] As one illustrative example, a multivolume device having
fewer than 200 total wells is predicted to provide dynamic range
with a 5-fold resolution. Without being bound to any particular
theory, this resolution is similar to that of single-volume designs
that use approximately 12,000 wells.
[0082] Mathematical techniques, such as application of the Poisson
distribution and binomial statistics, may be used to process
information obtained from an experiment and to quantify performance
of devices. These techniques were experimentally validated using
the disclosed devices.
[0083] MV digital PCR has been demonstrated to perform reliably,
and results from wells of different volumes agreed with one
another. In using the devices, no artifacts due to different
surface-to-volume ratios were observed, and single molecule
amplification in volumes ranging from 1 to 125 nL was
self-consistent.
[0084] An exemplary device according to the present disclosure was
constructed to meet the testing requirements for measuring
clinically relevant levels of HIV viral load at the point-of-care
(in plasma, <500 molecules/mL to >1,000,000 molecules/mL).
The predicted resolution and dynamic range was experimentally
validated using a control sequence of DNA, as described in Kreutz
et al., Anal. Chem. 2011 83: 8158-8168.
[0085] The estimation theory applied in the above publication may
be summarized as follows. First, there are two assumptions that are
maintained: (1) having at least one target molecule in a well is
necessary and sufficient for a positive signal, and (2) target
molecules do not interact with one another or device surfaces, to
avoid biasing their distribution. At the simplest level of
analysis, when molecules are at low enough densities that there is
either 0 or 1 molecule within a well, concentrations can be
estimated simply by counting wells displaying a "positive" signal.
Under the above assumptions, Poisson and binomial statistics may be
used to obtain quantitative results from experiments resulting in
one positive well to experiments resulting in one negative well.
The Poisson distribution (eq. 1), in the context of digital PCR,
gives the probability, p, that there are k target molecules in a
given well based on an average concentration per well, v.lamda.
where v is the well volume (mL) and .lamda. is the bulk
concentration (molecules/mL). In digital PCR, the same readout
occurs for all k>0, so if k=0, then eq. 1 simplifies to give the
probability, p, that a given well will not contain target molecules
(the well is "negative").
p=((v.lamda.).sup.k.sup.e-(v.lamda.))/k,
and for k=0(empty well), p=e.sup.-(v.lamda.) (1)
[0086] In single-volume systems, the number of negative wells, b,
out of total wells, n, can serve as an estimate for p, so expected
results can be estimated from known concentrations, or observed
results can be used to calculate expected concentrations (eq.
2).
b=ne.sup.-(v.lamda.)
or
.lamda.=-ln(b/n)/v (2)
[0087] The binomial equation is used to determine the probability,
P, that a specific experimental result (with a specific number of
negatives, b, and positives, n-b, out of the total number of wells,
n, at each volume) will be observed, on the basis of .lamda., (eq.
3),
where ( n b ) = n ! b ! ( n - b ) ! P = ( n b ) p b ( 1 - p ) n - k
or P = ( n b ) ( e - v .lamda. ) b ( 1 - e - v .lamda. ) n - b ( 3
) ##EQU00001##
[0088] An incomplete analysis of multivolume systems may be
performed by simply selecting a single volume and analyzing it as
described above; this is the approach that has typically been taken
in serial dilution systems. The single volume that minimizes the
standard error is generally chosen; this typically occurs when
10-40% of wells are negative. This method, however, does not
utilize the information from the other "dilutions" (or volumes),
and would require using different dilutions for different sample
concentrations. Combining the results from wells of different
volumes fully minimizes the standard error and provides
high-quality analysis across a very large dynamic range. This is
achieved by properly combining the results of multiple binomial
distributions (one for each volume); specifically, the probability
of a specific experimental result P (defined above) is the product
of the binomials for each volume (eq. 4), where P is defined as a
function of the bulk concentration .lamda., P=f(.lamda.),
f ( .lamda. ) = P = ( n i b i ) ( e - v i .lamda. ) ( 1 - e - v i
.lamda. ) n i - b i ( 4 ) ##EQU00002##
[0089] For a given set of results, the MPN is found by solving for
the value of that maximizes P. In general, taking the derivative of
an equation and solving for zero provides the maximum and/or
minimum values of that equation; as a binomial distribution (and
subsequently the product of binomials) has only a single maximum,
solving the derivative of eq. 4 for zero provides the "most
probable" concentration. The standard deviation, .sigma., is more
appropriately applied to ln (.lamda.) than to .lamda., because the
distribution of P based on ln (.lamda.) is more symmetrical than
that for .lamda.. In addition, this approach provides better
accuracy for low concentrations by enforcing the constraint that
concentrations must be positive. Thus, a change of variables is
needed during the derivations so .sigma. can be found for ln
(.lamda.). Therefore, f(.lamda.) (eq. 4) is converted to
F(.LAMBDA.) (eq. 5), where .theta.=e.sup.-v and
.LAMBDA.=ln(.lamda.).
F ( .LAMBDA. ) = P ( n i b i ) ( .theta. i e .LAMBDA. ) b i ( 1 -
.theta. i e .LAMBDA. ) n i - b i ( 5 ) ##EQU00003##
[0090] The derivative is easier to handle if the natural log is
applied to eq. 5, as the individual components are separated, but
the overall result is unchanged (eq. 6).
L ( .LAMBDA. ) = ln F ( .LAMBDA. ) = i = 1 m ( ln ( n i b i ) + b i
e .LAMBDA. ln ( .theta. 1 ) + ( n i - b i ) ln ( 1 - .theta. i e
.LAMBDA. ) ) ( 6 ) ##EQU00004##
[0091] The first derivative is then
ln ( .theta. i ) can be replaced with - v i : e .LAMBDA. i = 1 m (
- b i v i + ( n i - b i ) v i .theta. i e .LAMBDA. ( 1 - .theta. i
i .LAMBDA. ) ) ##EQU00005##
[0092] ln(.theta.i) can be replaced with vi:
= e .LAMBDA. i = 1 m ( - b i v i + ( n i - b i ) v i .theta. i e
.LAMBDA. ( 1 - .theta. i e .LAMBDA. ) ) ##EQU00006##
substituting (ni ti) for bi (where ti is the number of positive
wells):
= e .LAMBDA. i = 1 m ( - n i v i + t i n i + t i v i .theta. i e
.LAMBDA. ( 1 - .theta. i e .LAMBDA. ) ) ##EQU00007##
rearranging to put all t.sub.i's over the denominator
= e .LAMBDA. i = 1 m ( - n i v i + t i v i ( 1 - .theta. i e
.LAMBDA. ) - t i v i .theta. i e .LAMBDA. ( 1 - .theta. i e
.LAMBDA. ) + t i v i .theta. i e .LAMBDA. ( 1 - .theta. i e
.LAMBDA. ) ) ##EQU00008##
and simplifying and rearranging in terms of b.sub.i
.differential. L ( .LAMBDA. ) .differential. .LAMBDA. = e .LAMBDA.
i = 1 m ( - n i v i + ( n i - b i ) v i ( 1 - .theta. i e .LAMBDA.
) ) ( 7 ) ##EQU00009##
[0093] Setting eq. 7 equal to 0, re-substituting .lamda., and
rearranging then gives eq. 8. By solving eq. 8 for .lamda., the
expected concentration can be determined from the number of empty
wells. This can be done using any solver function; the code
MVdPCR_MLE.m (described in Kreutz et al., Analytical Chemistry 2011
83: 8158-8168) performs this step using a globalized Newton
method.
i = 1 m n i v i = i = 1 m ( n i - b i ) v i ( 1 - e - v i .lamda. )
( 8 ) ##EQU00010##
[0094] The standard error, .sigma., for a result can be found using
the Fisher information, I(X), for ln(.lamda.), 44 requiring the
change of variable to .LAMBDA.. The Fisher information is defined
in eq. 9, where E[ ] represents the expected value of the given
variable.
1 variance = 1 .sigma. 2 = I ( .LAMBDA. ) = - .intg. .differential.
2 I ( .LAMBDA. ) .differential. .LAMBDA. 2 f ( x ; .theta. ) x = E
[ - .differential. 2 L ( .LAMBDA. ) .differential. .LAMBDA. 2 ] ( 9
) ##EQU00011##
[0095] In eq. 10, the second derivative of eq. 6 is found.
.differential. 2 L ( .LAMBDA. ) .differential. .LAMBDA. 2 = e
.LAMBDA. i = 1 m ( - n i v i + ( n i - b i ) v i ( 1 - .theta. i e
.LAMBDA. ) ) + e .LAMBDA. i = 1 m ( e .LAMBDA. ( n i - b i )
.theta. i e .LAMBDA. v i ( ln .theta. ) ( 1 - .theta. i e .LAMBDA.
) 2 ) = e .LAMBDA. i = 1 m ( n i v i - ( n i - b i ) v i ( 1 -
.theta. i e .LAMBDA. ) ) - ( e .LAMBDA. ) 2 i = 1 m ( ( n i - b i )
v i 2 .theta. i e .LAMBDA. ( 1 - .theta. i e .LAMBDA. ) 2 ) ( 10 )
##EQU00012##
[0096] Using this expression in eq. 9 to then find the inverse
variance gives eq. 11
1 .sigma. 2 = - E [ e .LAMBDA. i = 1 m ( n i v i - ( n i - b i ) v
i ( 1 - .theta. i e .LAMBDA. ) ) - ( e .LAMBDA. ) 2 i = 1 m ( ( n i
- b i ) v i 2 .theta. i e .LAMBDA. ( 1 - .theta. i e .LAMBDA. ) 2 )
] = - e .LAMBDA. i = 1 m ( n i v i - ( n i - E [ b i ] ) v i ( 1 -
.theta. i e .LAMBDA. ) ) + ( e .LAMBDA. ) 2 i = 1 m ( ( n i - E [ b
1 ] ) v i 2 .theta. i e .LAMBDA. ( 1 - .theta. i e .LAMBDA. ) 2 )
With E [ b i ] coming from eq 2 = - e .LAMBDA. i = 1 m ( n i v i -
( n i - n i .theta. i e .LAMBDA. ) v i ( 1 - .theta. i e .LAMBDA. )
) + ( e .LAMBDA. ) 2 i = 1 m ( ( n i - n i .theta. i e .LAMBDA. ) v
i 2 .theta. i e .LAMBDA. ( 1 - .theta. i e .LAMBDA. ) 2 ) = - e
.LAMBDA. i = 1 m ( n i v i - n i v i ( 1 - .theta. i e .LAMBDA. ) (
1 - .theta. i e .LAMBDA. ) ) + ( e .LAMBDA. ) 2 i = 1 m ( n i ( 1 -
.theta. i e .LAMBDA. ) v i 2 .theta. i e .LAMBDA. ( 1 - .theta. i e
.LAMBDA. ) 2 ) = ( e .LAMBDA. ) 2 i = 1 m ( n i v i 2 .theta. i e
.LAMBDA. ( 1 - .theta. i e .LAMBDA. ) ) = .lamda. 2 i = 1 m ( n i v
i 2 e - v i .lamda. ( 1 - e - v i .lamda. ) ) = .lamda. 2 i = 1 m (
n i v i 2 ( e v i .lamda. - 1 ) ) ( 11 ) ##EQU00013##
[0097] This ultimately gives the standard error (eq. 12), from
which confidence intervals can be generated (eq. 13), where Z is
the upper critical value for the standard normal distribution.
.sigma. = 1 .lamda. 2 v i 2 n i e v i .lamda. - 1 ( 12 ) CI = ln (
.lamda. ) .+-. Z .sigma. ( 13 ) ##EQU00014##
[0098] One aspect of the disclosed devices achieves a certain
resolution (that is, to distinguish a certain difference in
concentration) at certain concentrations. As mentioned above for
HIV viral load monitoring, a system suitably achieves a 3-fold
resolution for as low as 500 molecules/mL. To correctly resolve two
different concentrations, the potential for false positives (Type I
error) and false negatives (Type II error) may be considered.
Samples suitably give results at the desired confidence level
(1-.alpha., measure of Type I error) and demonstrate this
confidence level again and again (Power: 1-.beta., measure of Type
II error).
[0099] When comparing two results, the null hypothesis is that the
results come from samples that have statistically the same
concentration. .alpha. is the probability that two results that are
determined to be statistically different are in fact from the same
sample, thus resulting in a false positive. A 95% confidence level
would correspond to .alpha.=0.05 and an accepted false positive
rate of 5%. The power level measures the probability, .beta., that
samples that are statistically different at the desired confidence
level give results that fall below this confidence level. A 95%
power level would correspond to .beta.=0.05 and thus an accepted
false negative rate of 5%. For the exemplary analysis described
herein, the 3-fold resolution is defined such that samples with a
3-fold difference in concentration (e.g., 500 and 1500
molecules/mL) gives results that are statistically different with
at least 95% confidence (.alpha.<0.05, less than 5% false
positives) at least 95% of the time (power level of 95%,
.beta.<0.05, no more than 5% false negatives). The Z-test (eq.
14) was chosen to measure the confidence level, where .lamda. and
.sigma. are calculated using eqs. 8 and 12, respectively, for a set
of two results (the specific number of negatives, b.sub.i, out of
the total number of wells, n.sub.i, at each volume i of wells). The
Z-test measures the probability that results are statistically
different, by assuming that the test statistics (left side of eq.
14) can be approximated by a standard normal distribution, so Z
corresponds to a known probability. Power level is measured by
simulating results from two different samples and determining the
probability that they will give results that at least meet the
desired confidence level.
.lamda. 1 - .lamda. 2 .sigma. 1 2 + .sigma. 2 2 = Z 1 for 95 %
confidence .lamda. 1 - .lamda. 2 .sigma. 1 2 + .sigma. 2 2 >
1.96 ( 14 ) ##EQU00015##
[0100] A multivolume device was designed with 160 wells each at
volumes of 125, 25, 5, and 1 nL (FIG. 15). A radial layout of wells
(FIG. 14) provides an efficient use of space when wells of
significantly different volumes are used. In the initial
orientation of the radial multivolume device, the wells are aligned
to create a continuous fluidic path that allows all of the sample
wells to be filled in one step using dead end filling. The
components of the device can then be rotationally slipped or
translated (by) .about.8.degree. to simultaneously isolate each
well and also overlap the well with an optional corresponding
thermal expansion well (FIG. 14). This device has a LDL of 120
molecules/mL and a dynamic range where at least 3-fold resolution
is achieved from 520 to 3,980,000 molecules/mL (FIG. 15). A control
631 bp sequence of DNA was used to validate the MV digital PCR
approach. The initial concentration of this stock solution was
determined by UV-vis, and the stock was then diluted to levels
required for testing of the chip. Concentrations were tested across
the entire dynamic range of the device: approximately 500, 1500,
8000, 20,000, 30,000, 100,000, 600,000, and 3,000,000 molecules/mL.
A total of 80 experiments and 29 additional controls were
performed, and the observed concentrations showed excellent
agreement with the expected concentrations and demonstrated the
accuracy of the device performance over the entire dynamic range
(FIG. 16). The experimental results consist of a "digital" pattern
of positive and negative wells. At an input concentration of 1500
molecules/mL (FIG. 3a), the larger 125 and 25 nL wells provide the
majority of the information to determine the concentration. As
expected, at a higher concentration of 600,000 molecules/mL,
positives were found in the smaller 5 and 1 nL wells also (FIG.
16b), and these smaller wells provide the majority of the
information used to determine the concentration. Excellent
agreement was found between the input concentration and the
measured concentration over 4 orders of magnitude (FIG. 16c, d). In
this multivolume design, the 95% confidence interval is narrow at a
consistent level over a very large range of concentrations: the CI
is within 13.8-15% of the expected value from 9500 to 680,000
molecules/mL and within 13.8-17.5% from 5400 to 1,700,000
molecules/mL. The experimental data closely tracked the
theoretically predicted CI (FIG. 16d).
[0101] As expected, the largest wells (125 nL) provided the largest
contribution to the overall confidence interval for samples in the
10.sup.2-10.sup.4 molecules/mL range while the use of smaller and
smaller wells down to 1 nL in volume extended the dynamic range
with a 95% confidence interval above 10.sup.6 molecules/mL (FIG.
16c, d). For each concentration, there was excellent agreement
among the individual results obtained from the wells of different
volumes, consistent with the accuracy of the overall device. This
agreement is illustrated for an input concentration of 30,000
molecules/mL (FIG. 17). At this concentration, the wells of all
volumes provided a reasonable number of positives and negatives for
quantification, and we found that the concentration calculated from
the results fell within the 95% confidence intervals for individual
volumes of wells (38 of 40 results), and also, the averages from
wells of each volumes were internally consistent (FIG. 17).
[0102] The estimation may, in some embodiments, have a lower
detection limit, at a 95% confidence value, in the range of between
about 40 molecules/mL sample to about 120 molecules/mL of sample.
The methods may suitably be capable of resolving a three-fold
difference in viral load of the biological sample based on an
estimate of the level of the one or more nucleic acids.
[0103] In other embodiments, the methods include estimating the
level, presence, or both of a protein in the biological sample.
This estimation may be effected by (a) contacting the sample with a
detection moiety capable of binding to the protein so as to give
rise to a population of labeled proteins, the detection moiety
comprising the one or more nucleic acids, (b) disposing the labeled
proteins into two or more wells of different volumes, (c)
amplifying the one or more nucleic acids of the detection moiety in
the two or more wells of different volumes, and (d) correlating an
estimated presence or absence of the one or more nucleic acids in
the two or more wells of different volumes to a level of the
protein in the biological sample.
[0104] As one example, anti-PSA capture antibody coated fluorescent
magnetic beads are used to capture the target PSA molecule. The
concentration of PSA may be controlled so there was less than one
molecule on one bead. A dsDNA tag is attached to an anti-PSA
detection antibody and used as signal probe. After incubation
between antibodies and antigen, magnetic beads with
captured/labeled PSA are loaded into pL wells with PCR supermix.
Each well contains either one or no bead. After amplification, only
wells containing beads are counted. The ratio between "on" wells
and the total number of wells is used to determine the
concentration of target.
[0105] As explained above, relative motion between first and second
components effects distribution of any contents of the population
of first wells between the first population of wells and an
additional population of wells. The relative motion may effect
distribution of any contents of the population of second wells
between the second population of wells and an additional population
of wells.
[0106] It should be understood that the methods may include one,
two, or more applications of relative motion between components.
For example, a first relative motion (e.g., rotation) may be
applied so as to place first and second sets of wells into fluid
communication with one another. After the contents of the first and
second wells contact one another, additional rotation may be
applied to place the wells with mixed contents into fluid
communication with another set of wells with different contents,
which in turns enables the user to effect processes that require
separate and/or sequential mixing steps of two, three, or more
sample volumes. This may be done, for example, to (1) mix materials
in well A and well B in well A; and then (2) to contact the mixed
materials in well A with a buffer in well C so as to dilute the
contents of well A. Alternatively, the mixed contents of well A may
be contacted (via relative motion of components) with well C such
that the contents of well C may react with the contents of well A
(which well included the contents of well A and well B).
[0107] Also provided are kits. The kits suitably include device as
set forth elsewhere herein, and also a supply of a reagent selected
to participate in nucleic acid amplification. The reagent may be
disposed in a container adapted to engage with a the conduit of the
first component, the conduit of the second component, or both. Such
a container may be a pipette, a syringe, and the like.
[0108] The disclosed devices and kits may also include a device
capable of supplying or removing heat from the first and second
components. Such devices include heaters, refrigeration devices,
infrared or visible light lamps, and the like. The kits may also
include a device capable of collecting an image of at least some of
the first population of wells, the second population of wells, or
both.
[0109] Amplification Techniques
[0110] A non-exclusive listing of suitable isothermal amplification
techniques are provided below. These techniques are illustrative
only, and do not limit the present disclosure.
[0111] A first set of suitable isothermal amplification
technologies includes NASBA, and RT-RPA. These amplification
techniques can operate at 40 deg. C. (a lower temperature preferred
for certain POC devices): NASBA (product: RNA), RT-RPA (product:
DNA), RT-LAMP using one of LAMP HIV-RNA 6-primer sets,
transcription-mediated amplification (TMA, 41 deg. C), helicase
dependent amplification (HAD, 65 deg. C), and strand-displacement
amplification (SDA, 37 deg. C),
[0112] In addition to standard PCR techniques, the disclosed
methods and devices are also compatible with isothermal
amplification techniques such as loop-mediated amplification
(LAMP), Recombinase polymerase amplification (RPA), nucleic acid
sequence based amplification (NASBA), transcription-mediated
amplification (TMA), helicase-dependent amplification (HAD),
rolling circle amplification (RCA), and strand-displacement
amplification (SDA). The disclosed multivolume devices can be used
to digitize such platforms.
[0113] Other isothermal amplification methods are also suitable.
Isothermal exponential amplification reaction (EXPAR) may amplify a
10-20 bp trigger oligonucleotide generated from a genomic target
more than 106 times in less than 10 minutes at 55 deg. C. by
repeating cycles of polymerase and endonuclease activity, and has
been coupled with DNA-functionalized gold nanospheres for the
detection of herpes simplex virus. Isothermal and chimeric
primer-initiated amplification of nucleic acids (ICANs) amplify
target DNA at 55 deg. C using a pair of 50-DNA-RNA-30 primers and
the activity of RNase H and strand displacing polymerase.
[0114] Signal-mediated amplification of RNA technology (SMART)
produces copies of an RNA signal at 41 deg. C. in the presence of
an RNA or DNA target by way of the three-way junction formed
between the target and two probes, one of which contains the RNA
signal sequence and a T7 promoter sequence for T7 RNA polymerase.
The single stranded RNA product may be detected by
hybridization-based methods and because the signal is independent
of the target, SMART may be used for detection of different target
sequences. Cyclic enzymatic amplification method (CEAM) detects
nucleic acids in the picomolar range in less than 20 minutes at 37
deg. C. using a displacing probe and Exonuclease III (Exo III) to
generate amplification of fluorescent signal in the presence of a
target. Isothermal target and signaling probe amplification (iTPA)
combines the principle of ICAN and the inner-outer probe concept of
LAMP along with fluorescence resonance energy transfer cycling
probe technology (FRET CPT) for simultaneous target and signal
amplification in 90 minutes at 60 deg. C., and has been shown to
detect Chlamydia trachomatis at single copy level.
[0115] Other suitable amplification methods include ligase chain
reaction (LCR); amplification methods based on the use of Q-beta
replicase or template-dependent polymerase; helicase-dependent
isothermal amplification; strand displacement amplification (SDA);
thermophilic SDA nucleic acid sequence based amplification (3 SR or
NASBA) and transcription-associated amplification (TAA).
[0116] Non-limiting examples of PCR amplification methods include
standard PCR, AFLP-PCR, Allele-specific PCR, Alu-PCR, Asymmetric
PCR, Biased Allele-Specific (BAS) Amplification, Colony PCR, Hot
start PCR, Inverse PCR (IPCR), In situ PCR (ISH),
Intersequence-specific PCR (ISSR-PCR), Long PCR, Multiplex PCR,
Nested PCR, Quantitative PCR, Reverse Transcription PCR (RT-PCR),
Real Time PCR, Single cell PCR, Solid phase PCR, Universal
Size-Specific (USS-PCR), branched-DNA technology, and the like
[0117] Further amplification techniques are described below. Each
of these techniques is suitably performed by the disclosed devices
and methods. Allele-specific PCR is a diagnostic or cloning
technique based on single-nucleotide polymorphisms (SNPs)
(single-base differences in DNA). It requires some knowledge of a
DNA sequence, including differences between alleles, and uses
primers whose 3' ends encompass the SNP. PCR amplification may be
less efficient in the presence of a mismatch between template and
primer, so successful amplification with an SNP-specific primer
signals presence of the specific SNP in a sequence.
[0118] Assembly PCR or Polymerase Cycling Assembly (PCA) is an
artificial synthesis of long DNA sequences by performing PCR on a
pool of long oligonucleotides with short overlapping segments. The
oligonucleotides alternate between sense and antisense directions,
and the overlapping segments determine the order of the PCR
fragments, thereby selectively producing the final long DNA
product.
[0119] Asymmetric PCR preferentially amplifies one DNA strand in a
double-stranded DNA template. It is used in sequencing and
hybridization probing where amplification of only one of the two
complementary strands is required. PCR is carried out as usual, but
with a great excess of the primer for the strand targeted for
amplification. Because of the slow (arithmetic) amplification later
in the reaction after the limiting primer has been used up, extra
cycles of PCR are required. A recent modification on this process,
known as Linear-After-The-Exponential-PCR (LATE-PCR), uses a
limiting primer with a higher melting temperature (Tm) than the
excess primer to maintain reaction efficiency as the limiting
primer concentration decreases mid-reaction.
[0120] Helicase-dependent amplification is similar to traditional
PCR, but uses a constant temperature rather than cycling through
denaturation and annealing/extension cycles. DNA helicase, an
enzyme that unwinds DNA, is used in place of thermal
denaturation.
[0121] Hot start PCR is a technique that reduces non-specific
amplification during the initial set up stages of the PCR. It may
be performed manually by heating the reaction components to the
denaturation temperature (e.g., 95.degree. C.) before adding the
polymerase. Specialized enzyme systems have been developed that
inhibit the polymerase's activity at ambient temperature, either by
the binding of an antibody or by the presence of covalently bound
inhibitors that dissociate only after a high-temperature activation
step. Hot-start/cold-finish PCR is achieved with new hybrid
polymerases that are inactive at ambient temperature and are
activated at elongation temperature.
[0122] Inter-sequence-specific PCR (ISSR) is a PCR method for DNA
fingerprinting that amplifies regions between simple sequence
repeats to produce a unique fingerprint of amplified fragment
lengths.
[0123] Inverse PCR is commonly used to identify the flanking
sequences around genomic inserts. It involves a series of DNA
digestions and self-ligation, resulting in known sequences at
either end of the unknown sequence.
[0124] Ligation-mediated PCR: uses small DNA linkers ligated to the
DNA of interest and multiple primers annealing to the DNA linkers;
it has been used for DNA sequencing, genome walking, and DNA
footprinting.
[0125] Methylation-specific PCR (MSP) is used to detect methylation
of CpG islands in genomic DNA. DNA is first treated with sodium
bisulfite, which converts unmethylated cytosine bases to uracil,
which is in turn recognized by PCR primers as thymine. Two PCRs are
then carried out on the modified DNA, using primer sets identical
except at any CpG islands within the primer sequences. At these
points, one primer set recognizes DNA with cytosines to amplify
methylated DNA, and one set recognizes DNA with uracil or thymine
to amplify unmethylated DNA. MSP using qPCR can also be performed
to obtain quantitative rather than qualitative information about
methylation.
[0126] Miniprimer PCR uses a thermostable polymerase (S-Tbr) that
can extend from short primers ("smalligos") as short as 9 or 10
nucleotides. This method permits PCR targeting to smaller primer
binding regions, and is used to amplify conserved DNA sequences,
such as the 16S (or eukaryotic 18S) rRNA gene.
[0127] Multiplex Ligation-dependent Probe Amplification (MLPA)
permits multiple targets to be amplified with only a single primer
pair, as distinct from multiplex-PCR.
[0128] Multiplex-PCR: consists of multiple primer sets within a
single PCR mixture to produce amplicons of varying sizes that are
specific to different DNA sequences. By targeting multiple genes at
once, additional information may be gained from a single test-run
that otherwise would require several times the reagents and more
time to perform. Annealing temperatures for each of the primer sets
may be optimized to work correctly within a single reaction, and
amplicon sizes. That is, their base pair length may be different
enough to form distinct bands when visualized by gel
electrophoresis.
[0129] Nested PCR: increases the specificity of DNA amplification,
by reducing background due to non-specific amplification of DNA.
Two sets of primers are used in two successive PCRs. In the first
reaction, one pair of primers is used to generate DNA products,
which besides the intended target, may still consist of
non-specifically amplified DNA fragments. The product(s) are then
used in a second PCR with a set of primers whose binding sites are
completely or partially different from and located 3' of each of
the primers used in the first reaction.
[0130] Overlap-extension PCR or Splicing by overlap extension
(SOE): a genetic engineering technique that is used to splice
together two or more DNA fragments that contain complementary
sequences. The technique is used to join DNA pieces containing
genes, regulatory sequences, or mutations; the technique enables
creation of specific and long DNA constructs.
[0131] Quantitative PCR (Q-PCR): used to measure the quantity of a
PCR product (commonly in real-time). It quantitatively measures
starting amounts of DNA, cDNA, or RNA. Q-PCR is commonly used to
determine whether a DNA sequence is present in a sample and the
number of its copies in the sample. Quantitative real-time PCR can
have a high degree of precision. QRT-PCR (or QF-PCR) methods use
fluorescent dyes, such as Sybr Green, EvaGreen or
fluorophore-containing DNA probes, such as TaqMan, to measure the
amount of amplified product in real time. It is also sometimes
abbreviated to RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR
are more appropriate contractions, as RT-PCR commonly refers to
reverse transcription PCR (see below), often used in conjunction
with Q-PCR.
[0132] Reverse Transcription PCR (RT-PCR): for amplifying DNA from
RNA. Reverse transcriptase reverse transcribes RNA into cDNA, which
is then amplified by PCR. RT-PCR is widely used in expression
profiling, to determine the expression of a gene or to identify the
sequence of an RNA transcript, including transcription start and
termination sites. If the genomic DNA sequence of a gene is known,
RT-PCR can be used to map the location of exons and introns in the
gene. The 5' end of a gene (corresponding to the transcription
start site) is typically identified by RACE-PCR (Rapid
Amplification of cDNA Ends).
[0133] Solid Phase PCR: encompasses multiple meanings, including
Polony Amplification (where PCR colonies are derived in a gel
matrix, for example), Bridge PCR (primers are covalently linked to
a solid-support surface), conventional Solid Phase PCR (where
Asymmetric PCR is applied in the presence of solid support bearing
primer with sequence matching one of the aqueous primers) and
Enhanced Solid Phase PCR (where conventional Solid Phase PCR can be
improved by employing high Tm and nested solid support primer with
optional application of a thermal step to favor solid support
priming).
[0134] Thermal asymmetric interlaced PCR (TAIL-PCR) may be useful
for isolation of an unknown sequence flanking a known sequence.
Within the known sequence, TAIL-PCR uses a nested pair of primers
with differing annealing temperatures; a degenerate primer is used
to amplify in the other direction from the unknown sequence.
[0135] Touchdown PCR (Step-down PCR) is a variant of PCR that aims
to reduce nonspecific background by gradually lowering the
annealing temperature as PCR cycling progresses. The annealing
temperature at the initial cycles is usually a few degrees
(3-5.degree. C.) above the Tm of the primers used, while at the
later cycles, it is a few degrees (3-5.degree. C.) below the primer
Tm. The higher temperatures give greater specificity for primer
binding, and the lower temperatures permit more efficient
amplification from the specific products formed during the initial
cycles.
[0136] PAN-AC uses isothermal conditions for amplification, and may
be used in living cells.
[0137] Universal Fast Walking is useful for genome walking and
genetic fingerprinting using a more specific two-sided PCR than
conventional one-sided approaches (using only one gene-specific
primer and one general primer) by virtue of a mechanism involving
lariat structure formation. Streamlined derivatives of UFW are LaNe
RAGE (lariat-dependent nested PCR for rapid amplification of
genomic DNA ends), 5' RACE LaNe, and 3' RACE LaNe.
[0138] COLD-PCR (co-amplification at lower denaturation
temperature-PCR) is a modified Polymerase Chain Reaction (PCR)
protocol that enriches variant alleles from a mixture of wildtype
and mutation-containing DNA.
[0139] Another alternative isothermal amplification and detection
method that is isothermal in nature is described at
http://www.invaderchemistry.com/(Invader Chemistry.TM.) This method
may be performed by the disclosed devices and methods. Another
alternative amplification technique (so-called qPCR) is disclosed
by MNAzyme (http://www.speedx.com.au/MNAzymeqPCR.html), which
technique is also suitable for the presently disclosed devices and
methods.
[0140] One may also effect amplification based on nucleic acid
circuits (which circuits may be enzyme-free). The following
references (all of which are incorporated herein by reference in
their entireties) describe exemplary circuits; all of the following
are suitable for use in the disclosed devices and methods: Li et
al., "Rational, modular adaptation of enzyme-free DNA circuits to
multiple detection methods," Nucl. Acids Res. (2011) doi:
10.1093/nar/gkr504; Seelig et al., "Enzyme-Free Nucleic Acid Logic
Circuits," Science (Dec. 8, 2006), 1585-1588; Genot et al, "Remote
Toehold: A Mechanism for Flexible Control of DNA Hybridization
Kinetics," JACS 2011, 133 (7), pp 2177-2182; Choi et al.,
"Programmable in situ amplification for multiplexed imaging of mRNA
expression," Nature Biotechnol, 28:1208-1212, 2010; Benner, Steven
A., and A. Michael Sismour. "Synthetic Biology." Nat Rev Genet 6,
no. 7 (2005): 533-543; Dirks, R. M., and N. A. Pierce. "Triggered
Amplification by Hybridization Chain Reaction." Proceedings of the
National Academy of Sciences of the United States of America 101,
no. 43 (2004): 15275; Graugnard, E., A. Cox, J. Lee, C. Jorcyk, B.
Yurke, and W. L. Hughes. "Kinetics of DNA and Rna Hybridization in
Serum and Serum-Sds." Nanotechnology, IEEE Transactions on 9, no. 5
(2010): 603-609; Li, Bingling, Andrew D. Ellington, and Xi Chen.
"Rational, Modular Adaptation of Enzyme-Free DNA Circuits to
Multiple Detection Methods." Nucleic Acids Research, (2011); Li,
Q., G. Luan, Q. Guo, and J. Liang. "A New Class of Homogeneous
Nucleic Acid Probes Based on Specific Displacement Hybridization."
Nucleic Acids Research 30, no. 2 (2002): e5-e5; Picuri, J. M., B.
M. Frezza, and M. R. Ghadiri. "Universal Translators for Nucleic
Acid Diagnosis." Journal of the American Chemical Society 131, no.
26 (2009): 9368-9377; Qian, Lulu, and Erik Winfree. "Scaling up
Digital Circuit Computation with DNA Strand Displacement Cascades."
Science 332, no. 6034 (2011): 1196-1201; Tsongalis, G. J. "Branched
DNA Technology in Molecular Diagnostics." American journal of
clinical pathology 126, no. 3 (2006): 448-453; Van Ness, Jeffrey,
Lori K. Van Ness, and David J. Galas. "Isothermal Reactions for the
Amplification of Oligonucleotides." Proceedings of the National
Academy of Sciences 100, no. 8 (2003): 4504-4509; Yin, Peng, Harry
M. T. Choi, Colby R. Calvert, and Niles A. Pierce. "Programming
Biomolecular Self-Assembly Pathways." Nature 451, no. 7176 (2008):
318-322; Zhang, D. Y., and E. Winfree. "Control of DNA Strand
Displacement Kinetics Using Toehold Exchange." Journal of the
American Chemical Society 131, no. 47 (2009): 17303-17314; Zhang,
David Yu, Andrew J. Turberfield, Bernard Yurke, and Erik Winfree.
"Engineering Entropy-Driven Reactions and Networks Catalyzed by
DNA." Science 318, no. 5853 (2007): 1121-1125; Zhang, Z., D. Zeng,
H. Ma, G. Feng, J. Hu, L. He, C. Li, and C. Fan. "A DNA-Origami
Chip Platform for Label-Free SNP Genotyping Using Toehold-Mediated
Strand Displacement." Small 6, no. 17 (2010): 1854-1858.
[0141] It should also be understood that the present disclosure is
not limited to application to molecules, as the disclosed devices
and methods may be applied to organisms (e.g., those described in
paragraph 0133 of priority application PCT/US2010/028316 and also
elsewhere in that application), single cells, single biological
particles (e.g., bacteria), single vesicles, single exosomes,
single viruses, single spores, lipoprotein particles, and the like,
and single non-biological particles. One exemplary analysis of
lipoprotein particles may be found at www.liposcience.com.
Furthermore, it should also be understood that the disclosed
devices and methods may be applied to stochastic confinement
(described in, for example, "Stochastic Confinement to Detect,
Manipulate, And Utilize Molecules and Organisms," patent
application PCT/US2008/071374), and reactions and manipulations of
stochastically confined objects. As one non-limiting example,
biological samples may be assessed for the presence or level of
certain bacteria, such as those organisms that serve as markers for
bacterial vaginosis. This assessment may be performed by amplifying
nucleic acids that may be present in the sample and correlating the
levels of those nucleic acids to the presence or absence of the
marker organisms. One exemplary analysis is found at
http://www.viromed.com/client/cats/BV%20LAB.pdf.
[0142] It should be understood that "nucleic acid" is not limited
to DNA. "Nucleic acid" should be understood as referring to RNA
and/or a DNA. Exemplary RNAs include, but are not limited to,
mRNAs, tRNAs, snRNAs, rRNAs, retroviruses, small non-coding RNA,
microRNAs, ploysomal RNAs, pre-mRNAs, intromic RNA, and viral RNA.
Exemplary DNAs include, but are not limited to, genomic DNA,
plasmid DNA, phage DNA, nucleolar DNA, mitochondrial DNA,
chloroplast DNA, cDNA, synthetic DNA, yeast artificial chromosomal
DNA, bacterial artificial chromosomal DNA, other extrachromosomal
DNA, and primer extension products.
[0143] In some embodiments, a nucleic acid comprises PNA and/or LNA
(locked nucleic acid). In still other embodiments, a nucleic acid
comprises one or more aptamers that can be in the form of single
stranded DNA, RNA, or modified nucleic acids. Nucleic acid aptamers
may be single stranded or double stranded. In some embodiments,
nucleic acid contains nucleotides with modified synthetic or
unnatural bases, including any modification to the base, sugar or
backbone. Further information is found in U.S. Pat. No. 7,790,385
and also in United States patent application publications
US2008/0032310, US2008/0050721, and US2005/0089864, all of which
are incorporated herein by reference in their entireties for any
and all purposes.
[0144] It should also be understood that in some embodiments, the
disclosed devices and methods provide for detection of target
molecules with or without quantification (or estimated
quantification) of the target molecules. Accordingly, it is not
necessary for a user to estimate the concentration or level of a
target in a sample; the disclosed devices and methods may be used
to detect the presence of a target in a yes/no fashion. This is
especially useful in applications where the user may desire only to
know whether a particular target (e.g., a virus) is present; in
such cases, the precise level of the target is of lesser
importance.
[0145] Such detection can be carried out by physical, chemical, and
biological reactions, such as hybridization, nucleic acid
amplification, immunoassays, and enzymatic reaction. In some
embodiments, this detection method can be used for qualitative
analysis of one or more target molecules. As describe above, in
some embodiments material may, after a reaction, processing, or
even a detection step, be transferred to another device, or even
transferred to another region of the same device. In some
embodiments, recovered material may be removed from the device. In
some embodiments, material after detection may be recovered from
device and further analyzed. Such recovery may be carried out, in
some embodiments, by accessing individual wells of a device, e.g.,
by a pipettor. In some embodiments, recovery may be achieved by
combining material from multiple wells, for example by actuating a
device to the loading position and using a carrier fluid to expel
the material from the device.
Exemplary Embodiments
[0146] The following illustrates an exemplary embodiment of the
disclosed devices. The embodiment comprises a
rotationally-configured device for quantifying RNA with a large
dynamic range by using multivolume digital RT-PCR (MV digital
RT-PCR).
[0147] Quantitative detection of RNA provides valuable information
for study of gene expression, and has the potential to improve
evaluation of diseases (including stroke, leukemia, and prostate
cancer), analysis of graft rejection in transplantation, and
vaccine development. Quantification of viral RNA has also become
useful for monitoring the progression of viral infection and
efficacy of applied treatment.
[0148] One such instance is in the treatment of HIV. More than 33
million people worldwide are living with HIV, and a large number of
them are in developing countries and resource-limited areas.
First-line antiretroviral treatment is becoming widely available,
and it greatly increases both the duration and quality of life of
HIV patients. However, this first-line treatment can fail as the
virus mutates and develops drug resistance. In order to stop the
global spread of drug resistance and provide proper treatment for
patients, it is critical to evaluate the HIV viral load at regular
intervals (every 3 to 4 months) after initial treatment is shown to
be effective. HIV viral load measurement is a particularly useful
tool for diagnosing and evaluating the status of HIV infection in
children under age 18 months.
[0149] The hepatitis C virus (HCV) infection is also a significant
global healthcare burden, as it has been identified as one of the
major causes of liver disease and is one of the most common
co-infections of HIV. HCV viral load may also need to be monitored
to determine the effectiveness of treatment.
[0150] The viral load for chronic HCV can range from about 50,000
to about 5 million international units per mL (IU/mL), while for
patients responding to antiviral treatment the load will be lower.
Successful treatment should result in essentially undetectable
levels of HCV viral RNA, and the assessment of such treatment may
require HCV viral load measurements capable of a wide dynamic
range.
[0151] As explained previously, real time quantitative RT-PCR is
one standard for monitoring viral load for HIV, HCV, and other
viral infections. However, this test is cost-prohibitive under
resource-limited settings and usually requires multiple
instruments, highly skilled technicians, and isolated rooms to
prevent contamination. Moreover, the efficiency of RT-PCR, the
quality of sample and selection of targets, and the methods for
interpretation of the data present concerns for the accuracy of
quantifying RNA using RT-PCR.
[0152] While a dipstick device has been developed that provides
semiquantitative measurements of HIV viral load after amplification
in resource limited settings, no quantitative test exists to
resolve a 3-fold (0.5 log.sup.10) change in HIV RNA viral load,
which is considered to be clinically significant. Digital PCR is
one method that performs quantitative analysis of nucleic acids by
detecting single molecule of DNA or RNA and can provide an absolute
count of the nucleic acid copy number with potentially higher
accuracy compared to real time PCR. Existing applications of
digital PCT, however, require significant skill and resource
commitments.
[0153] The exemplary, disclosed devices present a microfluidic
platform that can manipulate liquid samples from
picoliter-to-microliter scales by relative movement of different
plates without the need for complex control systems. These devices
may be used for multiplex PCR, digital PCR, and digital isothermal
amplification (RPA).
[0154] In place of using wells of uniform size, using wells of
multiple volumes to achieve the same dynamic range can reduce the
total number of wells and increase the spacing among wells to
simplify imaging and downstream analysis. A mathematical approach
for experimental design and statistical analysis for multivolume
digital PCR (MV digital PCR) has been characterized using DNA in
Kreutz et al., Anal. Chem. 2011, DOI 10.1021/ac201658s,
incorporated herein by reference for all purposes.
[0155] A disclosed devices was applied, as set forth below, to
quantitative analysis of RNA with large dynamic range by MV digital
RT-PCR. This device was characterized with a serial dilution of a
synthetic control RNA molecule of 906 nucleotides (906 nt). Also
described is a second design of the platform that maintains a large
dynamic range for five samples simultaneously, allowing for
multiplexed experiments. This system was validated by using HCV
control viral RNA and HIV viral RNA together with internal
controls. The system also displayed the use of multivolume designs
to quantify HIV viral load at a large dynamic range by quantifying
purified HIV viral RNA from clinical patients' samples.
[0156] Results
[0157] First characterized was a multivolume digital device (Design
1, Table 1, FIG. 11) with a large dynamic range suitable for viral
load testing. This device contained four different volumes (1 nL, 5
nL, 25 nL, 125 nL) with 160 wells each (FIG. 1A) for a theoretical
dynamic range (lower dynamic range, LDR, to upper limit of
quantification, ULQ) of 5.2.times.10.sup.2 to 4.0.times.10.sup.6
molecules/mL at 3-fold resolution and a lower detection limit (LDL)
of 1.2.times.10.sup.2 molecules/mL in the final RT-PCR mixture. The
LDR corresponds to the lowest concentration that can be resolved
from a 3- or 5-fold higher concentration; the ULQ is the
concentration that has a 95% chance of generating at least one
negative well and is equal to the concentration calculated from
three negative wells; the LDL is the concentration that has a 95%
chance of generating at least one positive well and is equal to the
concentration calculated from three positive wells.
[0158] Continuous fluidic paths are generated by partially
overlapping the wells in the top plate and the wells in the bottom
plate (e.g., FIG. 1B, FIG. 1E; FIG. 15). The design of this device
follows the general principles of dead-end filling for complete
filling of aqueous reagents (FIG. 1C,F). After complete loading,
the top plate is slipped (rotated) clockwise by .about.8.degree. to
break the fluidic path and overlay the wells filled with solution
with the wells in the facing plate used to control thermal
expansion (FIG. 1D, FIG. 1G). The device is then placed on a flat
in situ adaptor for thermal cycling.
[0159] The theory for design and analysis of this multivolume
device are described in detail and validated by using digital PCR
for DNA. Briefly, concentrations were calculated based on Most
Probable Number (MPN) theory by combining the results from each
volume (i=1, 2, 3, 4) in the first equation below and solving for
.lamda. (concentration, molecules/mL), where n.sub.i is the total
number of wells at each volume, b.sub.i is the number of negative
wells at that volume, and v.sub.i is the well volume (mL).
Combining results allows for more precise identification of the
"most probable" concentration and also improves the confidence
interval. To find the confidence interval, the standard deviation,
.sigma., for ln (.lamda.) is determined using the second equation
below, which was derived based on the Fisher information.
i = 1 m n i v i = i = 1 m ( n i - b i ) v i ( 1 - e - v i .lamda. )
##EQU00016## .sigma. = 1 .lamda. 2 v i 2 n i e v i .lamda. - 1
##EQU00016.2##
[0160] To validate the performance of the multivolume device with
RNA, digital RT-PCR was performed using a six order-of magnitude
serial dilution of synthetic control RNA template (906 nt). This
control RNA was synthesized from a control plasmid and purified by
using a commercial purification kit. The concentration of the stock
solution of control RNA was measured spectrophotometrically by a
NanoDrop.TM. device to be .about.1.8 ng/.mu.L, corresponding to
.about.4.1.times.10.sup.12 molecules/mL, which may contain some
background signal.
[0161] Using the device and through statistical analysis of all MV
digital RT-PCR results (FIG. 3), a nominal real concentration of
the control RNA in solution was obtained, 2.2.times.10.sup.12
molecules/mL, which value was used as the true concentration of all
MV digital RT-PCR results reported in FIG. 3. A RT-PCR master mix
containing EvaGreen Super-Mix, RT-transcriptase, bovine serum
albumin (BSA), and primers was mixed with the RNA template
solution. EvaGreen, an intercalating dye, was used for end-point
fluorescent imaging after thermal cycling (FIG. 2).
[0162] FIG. 1 illustrates a rotational multivolume device (well
volumes: 1 nL, 5 nL, 25 nL, 125 nL). (A) Bright field image of the
rotational device after slipping to form isolated compartments,
shown next to a U.S. quarter. (B-D) Schematics and (E-G) bright
field microphotograph show (B, E) the assembled rotational device.
(C, F) The device filled with food dye after dead-end filling. (D,
G) The device after rotational slipping: 640 aqueous droplets of
four different volumes (160 wells with volumes of 1 nL, 5 nL, 25
nL, 125 nL each) were formed simultaneously. In the schematics,
dotted lines indicate features in the top plate, and black solid
lines represent the features in the bottom plate.
[0163] FIG. 2 shows end-point fluorescence images of multivolume
digital RTPCR performed on a rotational device for synthetic RNA
template at five different concentrations. (A) Control, containing
no RNA template. (B-F) Serial dilution of 906 nt RNA template from
2.2.times.10.sup.2 to 2.2.times.10.sup.6 molecules/mL in the RT-PCR
mix.
[0164] No false positives were observed after amplification in four
negative control experiments, as there was no significant increase
of fluorescent intensity in wells (FIG. 2A). As the concentration
of RNA template increased (the dilution factor decreased), the
fraction of positive wells in each set of individual volumes was
counted and the concentration of template in the RT-PCR mix was
calculated as described above (FIG. 2B-F). The glass device was
reused after being thoroughly cleaned with piranha acid (3:1
sulfuric acid:hydrogen peroxide), plasma cleaned, and resilanized
with dichlorodimethylsilane. Four to five experiments were
performed for each concentration of template, and the calculated
concentration of template RNA showed good agreement within the
expected statistical distribution at each concentration and scaled
linearly with the expected concentration (FIG. 3A). The results at
the concentrations of 2.2.times.10.sup.6, 2.2.times.10.sup.5,
2.2.times.10.sup.4, 2.2.times.10.sup.3, 2.2.times.10.sup.2, and
7.3.times.10.sup.1 molecules/mL in the RT-PCR mix were used to
estimate an initial stock concentration of control RNA of
approximately 2.2.times.10.sup.12 molecules/mL. The experimental
results across the concentrations agree well with the theoretically
predicted distribution (FIG. 3A,B). Of the 26 experiments, 19 fall
within the 95% confidence interval and 22 fall within the 99%
confidence interval.
[0165] FIG. 3 presents the performance of digital RT-PCR with
synthetic RNA template on the multivolume device over a 4
log.sub.10 dynamic range, comparing the expected concentration of
RNA in RT-PCR mix to (A) the observed concentration, and (B) the
ratio of the observed/expected concentration. Individual
experimental results (crosses) and average results (crosses) for
concentration were plotted against the dilution level of the RNA
stock solution. Four to five experiments were performed at each
concentration, and some experimental results are overlapping. The
experimental results show a linear relationship with the dilution
level and fit within the expected distribution. The experimental
results were used to estimate an initial stock concentration, whose
distribution was then fit to the dilution level to provide the
expected value (black curve) and 95% confidence interval (gray
curves).
[0166] Over the dynamic range of the device, the contribution of
wells with different volumes to the calculated concentration
varies, approximated in FIG. 4A. As the concentration of control
RNA template increases (the dilution decreases), the major
contribution to the calculated final concentration shifts from
wells of large volume (125 nL) to wells of medium volume (25 nL and
5 nL) and then to wells of small volume (1 nL). The percent that
the result from each volume contributes to .sigma. serves as an
estimate of the relative contribution of that volume to the
concentration determined by all volumes on the entire chip. In FIG.
4A, the data bars for the 125 nL volume are for (left to right)
2.2.times.10.sup.2 molecules/mL, 2.2.times.10.sup.3 molecules/mL,
and 2.2.times.10.sup.4 molecules/mL. For 25 nL, data bars are (left
to right) 2.2.times.10.sup.2 molecules/mL, 2.2.times.10.sup.3
molecules/mL, 2.2.times.10.sup.4 molecules/mL, 2.2.times.10.sup.5
molecules/mL, and 2.2.times.10.sup.6 molecules/mL. For 5 nL
volumes, the data bars are (left to right) 2.2.times.10.sup.2
molecules/mL, 2.2.times.10.sup.3 molecules/mL, 2.2.times.10.sup.4
molecules/mL, 2.2.times.10.sup.5 molecules/mL, and
2.2.times.10.sup.6 molecules/mL. For 1 nL volumes, the data bars
are (left to right) 2.2.times.10.sup.4 molecules/mL,
2.2.times.10.sup.5 molecules/mL, and 2.2.times.10.sup.6
molecules/mL. The concentration calculated from analysis of
positive and negative wells of each of the volumes on the
individual device was selfconsistent and was consistent with the
calculated concentration determined by combining all wells with
different volumes (FIG. 4B). This result indicates that multivolume
digital approach is fully compatible with analysis of RNA by
RT-PCR.
[0167] FIG. 4A shows that for each dilution, the approximate
contributions of the results from each well volume toward
calculating the final concentration were calculated based on the
contributions of each volume to the standard deviation, .sigma..
FIG. 4(B), showing the concentration of RNA template calculated
from the overall chip (combining all well volumes, solid bars) and
individual volumes (patterned bars) is self-consistent on the MV
digital RT-PCR device. Four experiments were performed with
2.2.times.10.sup.4 molecules/mL of control RNA template (906 nt) in
the RT-PCR mix.
[0168] To illustrate incorporation of multiplexing into the device
while maintaining the high dynamic range, the design of the
multivolume device was modified by adding two additional volumes
(FIG. 11, Design 2A): 0.2 nL (160 wells) and 625 nL (80 wells).
When the rotational chip is split into five sections to quantify
five different analytes, the 0.2 nL wells extend the upper limit of
quantification with 3-fold resolution to 1.2.times.10.sup.7
molecules/mL in the RT-PCR mix, and the 625 nL wells maintain a
reasonable lower detection limit of 2.0.times.10.sup.2 molecules/mL
and lower dynamic range with 3-fold resolution at
1.8.times.10.sup.3 molecules/mL in the RT-PCR mix (FIG. 5A). The
higher upper limit of quantification is required to quantify HCV
viral RNA, and the lower dynamic range and lower detection limit
are required for the HIV viral load test. Five different solutions
can be introduced into the device simultaneously (FIG. 5A) for
multiplexed analysis.
[0169] As HCV is one of the most common co-infections for HIV
patients, validation was performed on the multiplexed device with a
five-plex panel: measurement of HIV viral RNA, measurement of HCV
control viral RNA, a negative control for HIV, a negative control
for HCV, and measurement of 906 nt control RNA in HCV sample for
quantification of sample recovery rate (FIG. 5B,C). The 906 nt
control RNA was the same one characterized by using digital RT-PCR
on the device (design 1; see FIGS. 1 and 11). HIV viral RNA was
purified from an archived sample of plasma containing HIV (viral
RNA estimated to be .about.1.5.times.10.sup.6 molecules/mL) from a
de-identified patient sample, and HCV control viral RNA was
purified from a commercial sample containing control HCV virus (25
million IU/mL, OptiQuant-S HCV Quantification Panel, Acrometrix)
using the iPrep purification instrument, as described elsewhere
herein. As the final elution volume of purified nucleic acid is
generally smaller than the starting volume of plasma, there is a
concentrating effect on viral RNA after sample purification. To
characterize this concentrating effect, the 906 nt control RNA with
known concentration was added to the lysed plasma and was
quantified again after sample preparation. The ratio of the
concentration of 906 nt control RNA after/before sample preparation
is defined as the concentrating factor. The concentrating factors
after sample purification were approximately 6.6 for HIV viral RNA
and approximately 4.5 for HCV control viral RNA. Primers for HIV
and HCV were selected. Only one pair of primers was added to each
sample, and the experiment was repeated six times. In those six
experiments, no false positives were observed in either HIV or HCV
negative control panels after thermal cycling, and no
crosscontamination was observed among different panels. From these
six experiments, the average calculated concentration of HIV viral
RNA after purification was 7.9.times.10.sup.6 molecules/mL with
standard deviation of 2.5.times.10.sup.6 molecules/mL,
corresponding to 1.2.times.10.sup.6 molecules/mL with standard
deviation of 3.7.times.10.sup.5 molecules/mL in the original plasma
sample. The average concentration of HCV control viral RNA after
purification was 1.0.times.10.sup.8 molecules/mL with standard
deviation of 4.4.times.10.sup.7 molecules/mL, corresponding to
2.3.times.10.sup.7 molecules/mL with standard deviation of
9.7.times.10.sup.6 molecules/mL in the original control plasma
sample. (Additional information may be found in "Multiplexed
Quantification of Nucleic Acids," Shen et al., JACS 2011.)
[0170] There is no universal conversion factor from international
units to copy number for HCV viral load; it is a value that depends
on the detection platform, including the protocols and equipment
used. Because the HCV concentration in the original commercial
sample was stated to be 2.5.times.10.sup.7 IU/mL, the conversion
factor from international units to copy number for HCV viral load
in the test is approximately 0.9. The same conversion number (0.9)
was published for the Roche Amplicor HCV Monitor v2.0 test when
using a manual purification procedure.
[0171] FIG. 5 illustrates a device for multiplexed, multivolume
digital RT-PCR with high dynamic range. (A) A photograph of a
multiplex device for up to five samples corresponding to designs 2A
and 2B in Table 1 (FIG. 11) with a total of 80 wells of 625 nL, 160
wells of 125 nL, 160 wells of 25 nL, 160 wells of 5 nL, 160 wells
of 1 nL, and 160 wells of 0.2 nL. (B) Fluorescent photograph of a
multiplexed digital RT-PCR detection panel: (I) measurement of
internal control of 906 nt RNA template in HCV sample; (II) HCV
control viral RNA measurement; (III) negative control for HIV (HIV
primers with no loaded HIV RNA template); (IV) HIV viral RNA
measurement; (V) negative control for HCV (HCV primers with no
loaded HCV RNA template). Inset shows an amplified area from HCV
viral load test.
[0172] FIG. 6 shows multivolume digital RT-PCR for quantification
of HIV viral load in two patients' samples. Input concentration was
calculated from a single clinical measurement for each patient
using the Roche CAP/CTM v2.0 system and was assumed to be the true
concentration. Each concentration was measured at least four times,
and each individual experiment is plotted as single point on the
graph. The black solid line is the predicted concentration based on
the assumption that the clinical measurement gave a true
concentration. The gray solid lines were calculated using MPN
theory and represent the 95% confidence interval for the predicted
concentration.
[0173] The tabular summary in FIG. 9 present the detection and
quantification data and dynamic range for the two designs
investigated here. Without being bound to any single theory, the
dynamic range of design 1 can be easily extended by adding a set of
wells smaller than 1 nL in volume and a set of wells larger than
125 nL in volume. Therefore, if a larger dynamic range is required,
the multiplexed design (Design 2A, FIG. 11) may be used for a
single sample (Design 2B, FIG. 11). When using the entire chip for
one sample, the 160 smallest wells (0.2 nL in volume) extend the
upper limit of quantification with 3-fold resolution to
2.0.times.10.sup.7 molecules/mL in the RT-PCR mix and the 80
largest wells (625 nL in volume) extend the lower detection limit
to 40 molecules/mL and lower dynamic range with 3-fold resolution
to 1.7.times.10.sup.2 molecules/mL in the RT-PCR mix (Table 1,
Design 2B, FIG. 11). This large dynamic range is useful for
quantification of viral load.
[0174] A RT-PCR mix containing an HIV viral RNA sample (prepared as
described above and then serially diluted) with an expected
concentration of 51 molecules/mL was used to test the lower
detection limit of design 2B (FIG. 11). Three negative control
experiments were performed (without HIV viral RNA) in parallel, and
no false positives were observed. Six experiments were performed to
quantify the viral RNA concentration (see FIG. 7), and the average
calculated HIV viral RNA concentration in the RT-PCR mix was 70
molecules/mL with standard deviation of 20 molecules/mL,
corresponding to 32 molecules/mL with standard deviation of 9
molecules/mL in the original plasma sample.
[0175] To further validate the feasibility of using a rotational
multivolume device to quantify HIV viral load, Design 1 (FIG. 11)
was used to measure HIV viral RNA purified from two archived
samples of HIV-infected blood plasma from two different anonymous
patients. The HIV viral RNA from each patient sample was extracted
and purified automatically using the iPrep purification instrument,
and concentrating factors of 7.1 and 6.6 were achieved for the two
different patient samples. Each patient sample of purified HIV
viral RNA was serially diluted and characterized by MV digital
RT-PCR on the device using previously published primers, and each
experiment was repeated at least four times (FIG. 6). The same
plasma samples were characterized in a single experiment using the
Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, v2.0 (CAP/CTM v2.0)
according to the manufacturer's recommendation, and these values
were treated as the standard for characterization. The data from
device were self-consistent for both patients (FIG. 6). Three
negative control experiments using the same primers but no HIV
template did not show false positive, as no increase of fluorescent
intensity was observed (see FIG. 10).
[0176] For patient 1, the results (FIG. 6, crosses) were on average
approximately 40% lower than that predicted by the single-point
measurement of the HIV viral load using Roche CAP/CTM v2.0 (see
FIG. 7). There were differences in the test designs: while the
present experiment targets a single LTR region of HIV RNA, the
Roche CAP/CTM v2.0 test includes two HIV sequences: one in gag and
another in LTR region. Further, the two tests use different
detection methods (EvaGreen in the present experiment vs TaqMan
probes in the Roche CAP/CTM v2.0 test) and different internal
controls. For patient 2, excellent agreement with the Roche
clinical measurement was observed over the entire range (FIG. 6,
plus marks; see also FIG. 10 (tabular summary). Without being bound
to any single theory, this difference in agreement between the two
methods for the two samples is not surprising, given that each
patient has a unique HIV viral genome, and the primers, internal
controls, and detection method used in one method may be better
suited to detect one patient's viral genome than another's.
Overall, taking into consideration the concentrating effect during
sample preparation, the lowest concentration of serially diluted
HIV viral RNA detected on the device corresponded to 37
molecules/mL in the patient plasma, and the highest concentration
corresponded to 1.7 million molecules/mL in the patient plasma.
[0177] Results Summary
[0178] Motivated by the problem of quantifying viral load under
point-of-care and resource-limited settings, here is shown
successful testing of the applicability of multivolume digital
assays to quantitative analysis of RNA over wide dynamic range via
digital RT-PCR on two rotational devices (Table 1). The first
device has a dynamic range (at 95% CI) of 5.2.times.10.sup.2 to
4.0.times.10.sup.6 molecules/mL with 3-fold resolution and lower
detection limit of 1.2.times.10.sup.2 molecules/mL. The device was
characterized using synthetic control RNA, demonstrating that MV
digital RT-PCR performs in agreement with theoretical predictions
over the entire dynamic range (FIG. 3). Results from wells of
different volumes were mutually consistent and enabled
quantification over a wide dynamic range using only 640 total wells
(FIG. 4). This chip was also validated with viral RNA from two HIV
patients (FIG. 6), demonstrating good agreement with single-point
measurements performed on a Roche CAP/CTM v2.0 clinical instrument.
Using this chip, positive wells were detected that corresponded to
a concentration of 81 molecules/mL HIV viral RNA purified from
patient plasma in the RT-PCR mix, which corresponds to around 37
molecules/mL in the original plasma samples. While below the
detection limit at 95% confidence interval, this concentration
should give at least one positive well 86% of the time, so it is
not surprising that all four of the experiments had at least one
positive well at this concentration.
[0179] A second chip was used to test the scalability and
flexibility of the multivolume approach by introducing both
multiplexed and higher-range quantification. Additional wells were
added with volumes of 0.2 nL and 625 nL and divided the device into
five individual regions. There was no evidence of
cross-contamination among samples on this rotational design, in
agreement with previous results on a translational device. This
multiplexed device was designed to test five samples, each at a
dynamic range (3-fold resolution) from 1.8.times.10.sup.3 to
1.2.times.10.sup.7 molecules/mL with a lower detection limit of
2.0.times.10.sup.2 molecules/mL. Multiplexing capability (FIG. 5)
enables a number of features on the same chip, including (i)
incorporating negative controls, (ii) measuring levels of control
RNA to quantify the quality of sample preparation, (iii) monitoring
co-infections, (iv) designing customized arrays for multiple
targets, i.e. for nucleic acid targets that require measurements
with different dynamic ranges and resolution, using wells of
different sizes with customized numbers of wells at each size for
each target, and (v) allowing for flexibility depending on
technical and economic constraints by using the same device to
perform either more analyses of lower quality, but at
proportionally lower cost, or a single analysis of high quality
including wider dynamic range and higher resolution. If this
multiplexed device is used for a single sample, the dynamic range
of the device with 3-fold resolution is designed to be
1.7.times.10.sup.2 to 2.0.times.10.sup.7 molecules/mL with a lower
detection limit of 40 molecules/mL. Even with only a modest
concentrating effect during sample preparation, this device would
enable detecting targets at 10-20 molecules/mL in the original
sample.
[0180] The high sensitivity of the this MV digital RT-PCR platform
is valuable for a number of applications beyond viral load,
including detecting rare cells and rare mutations, prenatal
diagnostics, and monitoring residual disease. Besides monitoring
the HIV viral load of patients on antiretroviral treatments, this
approach is a method to screen newborns whose mothers are carrying
HIV, where maternal HIV antibodies would potentially interfere with
the antibody test. In addition, similar molecular diagnostics
methods may be used to measure proviral DNA in infants. This
approach can also be applied to investigation of copy number
variation and gene expression, both for both for research and
diagnostic settings.
[0181] The rotational format of the device is useful for
resource-limited settings because the movement is easy to control
even manually; for a chip with a 2 in. (50 mm) diameter, a
8.degree. rotation moves the outer edge of the chip by .about.3.5
mm, a distance that is easily done by hand, especially with
internal stoppers and guides. At the same time, that rotation moves
the wells which are 2.8 mm from the center by 0.39 mm. This feature
is ideal for multivolume formats but also can be taken advantage of
in single-volume formats. The devices are also particularly
attractive for multivolume formats due to its lack of valves and
ease of operation. A number of additional developments will
increase the usefulness this chip. The considerations among
resolution, dynamic range, and the extent of multiplexing of the
multivolume device are described (Kreutz et al., Anal. Chem. 2011,
DOI 10.1021/ac201658s). The exemplary designs presented here were
fabricated in glass, and a functional device of a different design
made from plastic by hot embossing was previously demonstrated.
[0182] For applications to resource-limited settings, devices made
with inexpensive materials such as plastics are suitable. The
disclosed devices are compatible with other amplification
chemistries, including polymerization and depolymerization methods,
toe-hold initiated hybridization-based amplification, and other
amplifications including silver-based amplification. When combined
with isothermal amplification methods, such as recombinase
polymerase amplification, loop-mediated amplification,
strand-displacement amplification, helicase-dependent
amplification, rolling circle amplification, and visual readout
methods, the MV digital RT-PCR device makes quantitative molecular
diagnostics accessible in resource-limited settings.
[0183] Chemicals and Materials
[0184] All solvents and salts obtained from commercial sources were
used as received unless otherwise stated. SsoFast EvaGreen SuperMix
(2X) was purchased from Bio-Rad Laboratories (Hercules, Calif.).
One-Step SuperScript.RTM. III Reverse Transcriptase, iPrep.TM.
purification instrument, and iPrep.TM. PureLink.TM. virus kit were
purchased from Invitrogen Corporation (Carlsbad, Calif.). All
primers were purchased from Integrated DNA Technologies
(Coralville, Iowa). Bovine serum albumin (20 mg/mL) was ordered
from Roche Diagnostics (Indianapolis, Ind.). Mineral oil,
tetradecane, and DEPC-treated nuclease-free water were purchased
from Fisher Scientific (Hanover Park, Ill.). Dichlorodimethylsilane
was ordered from Sigma-Aldrich (St. Louis, Mo.). PCR Mastercycler
and in situ adapter were purchased from Eppendorf (Hamburg,
Germany). Spectrum food color was purchased from August Thomsen
Corp (Glen Cove, N.Y.). Soda-lime glass plates coated with layers
of chromium and photoresist were ordered from Telic Company
(Valencia, Calif.). Photomasks were designed using AutoCAD (San
Rafael, Calif.) and ordered from CAD/Art Services, Inc. (Bandon,
Oreg.). Microposit.TM. MF.TM.-CD-26 developer was purchased from
Rohm and Hass Electronic Materials LLC (Marlborough, Mass.).
Amorphous diamond coated drill bits were purchased from Harvey Tool
(0.030 inch cutter diameter, Rowley, Mass.). Adhesive PDMS film
(0.063 inch thick) was purchased from McMaster (Atlanta, Ga.). The
MinElute PCR purification kit and QIAamp Viral RNA mini kit were
purchased from Qiagen Inc. (Valencia, Calif.). The OptiQuant.RTM.-S
HCV RNA quantification panel was purchased from AcroMetrix
(Benicia, Calif.).
[0185] Fabrication of Devices for Multivolume Digital RT-PCR
[0186] The procedure for fabricating the devices from soda lime
glass was based on procedures described in previous work. To
fabricate devices for multivolume digital RT-PCR, wells of two
different depths were etched using a two-step exposing-etching
protocol. The soda lime glass plate pre-coated with chromium and
photoresist was first aligned with a photomask containing the
design for wells of 25 nL and 125 nL for Design 1 (Table 1, FIG.
11). For Design 2, this photomask also contained the designs of the
additional wells of 625 nL. The glass plate was then exposed to UV
light using standard exposure protocols. After exposure, the glass
plate was detached from the photomask and immersed in developer to
immediately remove the photoresist that was exposed to UV light.
The underlying chromium layer that was exposed was removed by
applying a chromium etchant (a solution of 0.6:0.365 mol/L
HClO.sub.4/(NH.sub.4).sub.2Ce(NO.sub.3).sub.6). The glass plate was
thoroughly rinsed with water and dried with nitrogen gas. The glass
plate was then aligned with a second photomask containing the
designs of wells of 1 nL and 5 nL for Design 1 (Table 1, FIG. 11)
by using a mask aligner. For Design 2 (FIG. 11), this second
photomask also contained the designs of the additional wells of 0.2
nL. The glass plate was then exposed to UV light a second time.
After the second exposure, the photomask was detached from the
glass plate, and the back side of the glass plate was protected
with PVC sealing tape. The taped glass plate was then immersed in a
glass etching solution (1:0.5:0.75 mol/L HF/NH.sub.4F/HNO.sub.3) to
etch the glass surface where chromium coating was removed in the
previous step (areas containing wells of 25 nL, 125 nL, and 625
nL), and the etching depth was measured by a profilometer. After
the larger features were etched to a depth of 70 .mu.m, the glass
plate was placed in the developer again to remove the previously
exposed photoresist in areas containing the patterns for the
smaller features (1 nL and 5 nL wells, and the additional wells of
0.2 nL for Design 2, FIG. 11). The underlying chromium layer was
removed by using the chromium etchant as describe above, and a
second glass etching step was performed to etch all features to a
further depth of 30 .mu.m. The final device contained wells of
depths of 100 .mu.m and 30 .mu.m was fabricated.
[0187] After the two-step etching, the glass plate was thoroughly
rinsed with Millipore water and ethanol and then dried with
nitrogen gas. The glass plate was oxidized using a plasma cleaner
and immediately placed in a desicator with dichlorodimethylsilane
for gas-phase silanization. For Design 2A (FIG. 11), circular inlet
reservoirs (4 mm inner diameter and 6 mm outer diameter) were made
by cutting adhesive PDMS film, then fixing the reservoirs around
the five inlets before plasma cleaning. After one hour, the
silanized glass plate was thoroughly rinsed with chloroform,
acetone, and ethanol, and then dried with nitrogen gas.
[0188] To re-use the glass devices, each device was thoroughly
cleaned with piranha acid (3:1 sulfuric acid:hydrogen peroxide),
then oxidized using a plasma cleaner and silanized as described
above.
[0189] Device Assembly
[0190] Devices were assembled under de-gassed oil (mineral
oil:tetradecane 1:4 v/v). The bottom plate was immersed into the
oil phase with the patterned wells facing up, and the top plate was
then immersed into the oil phase and placed on top of the bottom
plate with the patterned side facing down. The two plates were
aligned under a stereoscope (Leica, Germany) as shown in FIG. 1A
and stabilized using binder clips.
[0191] Device Loading
[0192] A through-hole was drilled in the center of the top plate to
serve as the solution inlet for Design 1 and Design 2B. The reagent
solution was loaded through the inlet by pipetting. For Design 2A,
five through-holes were drilled at the top left corner of the top
plate to serve as fluid inlets (FIG. 5A). For multiplex
experiments, five different reaction solutions were placed in the
inlet reservoirs, and a dead-end filling adapter was placed on top
of the devices to cover all the inlets. A pressure of 18 mmHg was
applied to load all the solutions simultaneously. The principle and
detailed method for dead-end filling are described in a previous
work..sup.3 Reservoirs were removed after the solution was
loaded.
[0193] Synthesis and Purification of Control RNA (906 nt)
[0194] The control RNA (906 nucleotide) was synthesized from the
LITMUS 28iMal Control Plasmid using a HiScribe.TM. T7 In Vitro
Transcription Kit with the manufacture's recommended procedures
(New England Biolabs, Ipswich, Mass.) and purified using MinElute
PCR purification kit with manufacture recommended protocols.
[0195] Automatic Viral RNA Purification from Plasma Sample
[0196] Plasma samples containing the HIV virus were obtained from
deidentified patients at the University of Chicago Hospital. Plasma
containing a modified HCV virus as a control (25 million IU/mL,
part of OptiQuant-S HCV Quantification Panel) was purchased from
AcroMetrix (Benicia, Calif.). A plasma sample of 400 .mu.L was
mixed with 400 .mu.L lysis buffer (Invitrogen Corporation,
Carlsbad, Calif.) to lyse the virus. Then 2 .mu.L of control RNA
(906 nt) was added to characterize the purification efficiency and
concentrating factor. The mixed sample was then transferred into
the iPrep.TM. PureLink.TM. virus cartridge. The cartridge was
placed in the iPrep.TM. purification instrument and the
purification protocol was performed according to the manufacturer's
instructions. The final elution volume was 50 .mu.L, therefore a
theoretical eight-fold concentrating factor was expected. The
initial concentration of control RNA and the concentration of
control RNA in the purified sample after preparation were
characterized on the device (Design 1). The final concentrating
factor was 4.5 for HCV and 6.6 for HIV in the multiplex RT-PCR
amplification (FIG. 5). The concentrating factors for the two HIV
samples were 7.1 and 6.6 for the experiments in FIG. 6.
[0197] Primer Sequences for RT-PCR Amplification
[0198] Primers for the control RNA (906 nt) were: GAA GAG TTG GCG
AAA GAT CCA CG and CGA GCT CGA ATT AGT CTG CGC. The control RNA
template was serially diluted in 1 mg/mL BSA solution. The RT-PCR
mix contained the following: 30 .mu.L of 2.times. EvaGreen
SuperMix, 1 .mu.L of each primer (10 .mu.mol/L), 3 .mu.L of BSA
solution (20 mg/mL), 1.5 .mu.L of SuperScript.RTM. III Reverse
Transcriptase, 17.5 .mu.L of nuclease-free water, and 6 .mu.L of
template solution.
[0199] Primer sequences for HIV viral RNA was selected from a
previous publication:.sup.4 GRA ACC CAC TGC TTA ASS CTC AA; GAG GGA
TCT CTA GNY ACC AGA GT. Primer sequences for control HCV viral RNA
were selected from a previous publication:.sup.5 GAG TAG TGT TGG
GTC GCG AA; GTG CAC GGT CTA CGA GAC CTC.
[0200] RT-PCR Amplification on the Devices
[0201] To amplify HIV viral RNA in FIG. 5, the RT-PCR mix contained
the following: 15 .mu.L of 2.times. EvaGreen SuperMix, 0.6 .mu.L of
each primer (10 .mu.mol/L), 1.5 .mu.L of BSA solution (20 mg/mL),
0.75 .mu.L of SuperScript.RTM. III Reverse Transcriptase, 10.05
.mu.L of nuclease-free water, and 1.5 .mu.L of template solution.
The template solution used here was diluted 250-fold from the
original HIV viral RNA stock solution purified from Patient sample
2 using 1 mg/mL BSA solution.
[0202] To amplify control HCV viral RNA in FIG. 5, the RT-PCR mix
contained the following: 15 .mu.L of 2.times. EvaGreen SuperMix,
0.25 .mu.L of each primer (10 .mu.mol/L), 1.5 .mu.L of BSA solution
(20 mg/mL), 0.75 .mu.L of SuperScript.RTM. III Reverse
Transcriptase, 10.25 .mu.L of nuclease-free water, and 2 .mu.L of
template solution. The template solution was diluted 5-fold from
the original control HCV viral RNA stock solution purified from
OptiQuant-S HCV Quantification Panel.
[0203] To amplify the control RNA (906 nt) in FIG. 5, the RT-PCR
mix contained the following: 15 .mu.L of 2.times. EvaGreen
SuperMix, 0.25 .mu.L of each primer (10 .mu.mol/L), 1.5 .mu.L of
BSA solution (20 mg/mL), 0.75 .mu.L of SuperScript.RTM. III Reverse
Transcriptase, 10.25 .mu.L of nuclease-free water, and 2 .mu.L of
template solution. The template solution was diluted 5-fold from
the original control HCV viral RNA stock solution purified from
OptiQuant-S HCV Quantification Panel.
[0204] The experiment in FIG. 5 was repeated six times, and the
resultant data were used to calculate the target concentration.
[0205] To amplify HIV viral RNA with expected final concentration
above 1000 molecules/mL in the RT-PCR mix in FIG. 6, the RT-PCR mix
contained the following: 20 .mu.L of 2.times. EvaGreen SuperMix, 1
.mu.L of each primer (10 .mu.mol/L), 2 .mu.L of BSA solution (20
mg/mL), 1 .mu.L of SuperScript.RTM. III Reverse Transcriptase, 13
.mu.L of nuclease-free water, and 2 .mu.L of template solution. The
template was serially diluted in 1 mg/mL BSA solution. For
experiments with HIV viral RNA concentration below 1000
molecules/mL in the final RT-PCR mix, the RT-PCR mix contained the
following: 30 .mu.L of 2.times. EvaGreen SuperMix, 1.5 .mu.L of
each primer (10 .mu.mol/L), 2 .mu.L of BSA solution (20 mg/mL), 1.5
.mu.L of SuperScript.RTM. III Reverse Transcriptase, 3.5 .mu.L of
nuclease-free water, and 20 .mu.L of template solution.
[0206] To amplify the control RNA (906 nt) in the HIV sample in
FIG. 5 and FIG. 6, the RT-PCR mix contained the following: 20 .mu.L
of 2.times. EvaGreen SuperMix, 1 .mu.L of each primer (10
.mu.mol/L), 2 .mu.L of BSA solution (20 mg/mL), 1 .mu.L of
SuperScript.RTM. III Reverse Transcriptase, 13 .mu.L of
nuclease-free water, and 2 .mu.L of HIV viral RNA stock solution
after sample preparation.
[0207] The concentration of control RNA (906 nt) before sample
preparation was characterized on device Design 1 (FIG. 11) with the
RT-PCR mix contained the following: 20 .mu.L of 2.times. EvaGreen
SuperMix, 1 .mu.L of each primer (10 .mu.mol/L), 2 .mu.L of BSA
solution (20 mg/mL), 1 .mu.L of SuperScript.RTM. III Reverse
Transcriptase, 13 .mu.L of nuclease-free water, and 2 .mu.L of
template solution. The template was prepared by diluting 2 .mu.L of
stock control RNA (906 nt) solution into 400 .mu.L of 1 mg/mL BSA
solution.
[0208] To amplify HIV viral RNA in FIG. 7, the RT-PCR mix for HIV
viral RNA contained the following: 90 .mu.L of 2.times. EvaGreen
SuperMix, 3.6 .mu.L of each primer (10 .mu.mol/L), 6 .mu.L of BSA
solution (20 mg/mL), 4.5 .mu.L of SuperScript.RTM. III Reverse
Transcriptase, 12.3 .mu.L of nuclease-free water, and 60 .mu.L of
template solution. The template solution used here was diluted
62500-fold from the original HIV viral RNA stock solution purified
from Patient sample 2 using 1 mg/mL BSA solution. This experiment
was repeated six times and all data was used to calculate HIV viral
RNA concentration. Three negative control experiments were
performed with the same primer pairs but no HIV viral RNA, and
showed no false positives.
[0209] The amplifications were performed using a PCR mastercycler
machine (Eppendorf). To amplify the RNA, an initial 30 min at
50.degree. C. was applied for reverse transcription, then 2 min at
95.degree. C. for enzyme activation, followed by 35 cycles of 1 min
at 95.degree. C., 30 sec at 55.degree. C. and 45 sec at 72.degree.
C. After the final cycle, a final elongation step was applied for 5
min at 72.degree. C. This thermal cycling program was applied to
all experiments except for those in FIG. 7, where 39 cycles were
adapted instead of 35 cycles.
[0210] Image Acquisition and Analysis
[0211] Bright-field images in FIG. 1 and FIG. 5 were acquired using
a Canon EOS Rebel XS digital SLR camera (Lake Success, N.Y.). Other
bright-field images were acquired using a Leica stereoscope. All
fluorescence images were acquired by Leica DMI 6000 B
epi-fluorescence microscope with a 5.times./0.15 NA objective and
L5 filter at room temperature. All fluorescence images were
corrected for background by using an image acquired with a standard
fluorescent control slide. All the images were then stitched
together using MetaMorph software (Molecular Devices, Sunnyvale,
Calif.).
[0212] FIG. 7 shows a representative experiment performing RT-PCR
of HIV viral RNA at an expected concentration of 51 molecules/mL in
RT-PCR mix on the Design 2B device to test the lower detection
limit of the device. This experiment was repeated six times to
quantify the viral RNA concentration.
[0213] FIG. 8 shows a representative negative control for HIV viral
load (HIV primers with no loaded HIV RNA template) on device Design
1, corresponding to experiments shown in FIG. 6.
[0214] FIG. 9 (table) presents performance of quantification of HIV
viral RNA concentration from patient 1 on device comparing to Roche
COBAS.RTM. AmpliPrep/COBAS.RTM. TaqMan.RTM. HIV-1 Test, v2.0 system
(CAP/CTM v2.0). Each experiment was repeated at least four times on
device. Only 2 significant digits are shown. The expected HIV
concentration of patient plasma was calculated based on dilution
factors and a single result from Roche CAP/CTM v2.0. The results
from device are obtained with serial diluted purified patient HIV
viral RNA and are converted to the original concentration in
patient plasma (with or without dilutions) using the purification
concentrating factor.
[0215] FIG. 10 (table) shows performance of quantification of HIV
viral RNA concentration from patient 2 on device comparing to Roche
COBAS.RTM. AmpliPrep/COBAS.RTM. TaqMan.RTM. HIV-1 Test, v2.0 system
(CAP/CTM v2.0). Each experiment was repeated at least four times on
device. Only 2 significant digits are shown. The expected HIV
concentration of patient plasma was calculated based on dilution
factors and a single result from Roche CAP/CTM v2.0. The results
from device are obtained with serial diluted purified patient HIV
viral RNA and are converted to the original concentration in
patient plasma (with or without dilutions) using the purification
concentrating factor.
[0216] LAMP Amplification
[0217] Digital reverse transcription loop mediated isothermal
amplification (RT-LAMP) can be performed on a device according to
the present disclosure. In some embodiments, digital RT-LAMP is
performed on a multivolume device. In one embodiment, one-step
digital RT-LAMP is carried out by mixing template, primers,
detection reagent, reaction mix and enzyme, then loading the
solution onto a device and heating up the device to a proper
temperature for a period of time.
[0218] For example, the following mixture of reagents has been
used: 20 .mu.L reaction mix, 2 .mu.L enzyme mix (Loopamp RNA
Amplification Kit from Eiken Chemical Co., LTD.), 2 .mu.L detection
reagent (Eiken Chemical Co., LTD.), 2 .mu.L 20 mg/mL BSA, 8 .mu.L
RNase free water, 4 uL primer mix and 2 .mu.L HIV RNA purified from
AcroMetrix.RTM. HIV-1 Panel 1E6. The final concentration of primers
was 2 .mu.M for BIP/FIP, 1 .mu.M for LOOP primers, 0.25 .mu.M for
B3/F3. All solutions were operated on ice.
[0219] The solution was loaded onto a multivolume device (design
published in Shen et al., JACS 2011 133: 17705) and the relative
position of the plates of the device were fixed by wax. The whole
device was heated on a thermal cycler block (Eppendorf) for about 1
hour then terminated at 95.degree. C. for 2 minutes. The
fluorescence image was acquired by Leica DMI 6000 B
epi-fluorescence microscope with a 5.times./0.15 NA objective and
L5 filter at room temperature. The measured concentration of
digital RT-LAMP was 10% of that from digital RT-PCR using B3/F3 as
primers.
[0220] In another embodiment, two-step digital RT-LAMP is carried
out in two separate steps. Reverse Transcription is done by mixing
template, BIP/FIP primers, reverse transcriptase, and reaction mix
in a tube, and heating to a proper temperature. Digital LAMP is
performed by mixing cDNA solution with all other components,
loading the solution onto a device, and heating the device at a
proper temperature for a period of time.
[0221] In another embodiment, digital RT-LAMP is performed by
running the reverse transcription step on the device in a digital
format, mixing the product with other components of LAMP on-chip
and heating the device. The result of this protocol has been
experimentally observed to be the same as when performing the RT
step in a test tube.
[0222] In one set of experiments performed with two-step digital
RT-LAMP, 10 .mu.L reaction mix, 1 .mu.L 20 mg/mL BSA, 0.5 .mu.L
Superscript III reverse transcriptase (Invitrogen), 6 .mu.L RNase
free water, 0.5 uL BIP/FIP primer mix (10 .mu.M) and 2 .mu.L HIV
RNA purified from AcroMetrix.RTM. HIV-1 Panel 1E6 were mixed
together in a test tube. All solutions were operated on ice. The
solution was heated to 50.degree. C. for 15 min for reverse
transcription.
[0223] All other components of LAMP mixture (2 .mu.L enzyme mix, 2
.mu.L detection reagent, 10 .mu.L reaction mix, 1 .mu.L 20 mg/mL
BSA, all other primers and RNase free water to make up the volume
to 20 .mu.L.) were mixed together with the solution obtained from
reverse transcription and loaded on a device immediately. The whole
device was heated on a thermal cycler block (Eppendorf) for about 1
hour then terminated at 95.degree. C. for 2 minutes. Imaging
settings were the same as described for the one-step RT-LAMP
experimental protocol above. The measured concentration obtained
after performing digital RT-LAMP was found to be 30% of that from
digital RT-PCR using B3/F3 as primers.
[0224] In another set of experiments, the efficiency of two-step
digital RT-LAMP was found to be improved by adding only BIP/FIP
primer in the RT step, adding RNase H after the RT step and
removing B3 from the primer mixture.
[0225] For example, 10 .mu.L reaction mix, 1 .mu.L 20 mg/mL BSA,
0.5 .mu.L Superscript III reverse transcriptase (Invitrogen), 6
.mu.L RNase free water, 0.5 uL BIP/FIP primer mix (10 .mu.M) and 2
.mu.L HIV RNA purified from AcroMetrix.RTM. HIV-1 Panel 1E6 were
mixed together. All solutions were operated on ice. The solution
was heated to 50.degree. C. for 15 min for reverse transcription
then followed by the addition of 0.5 .mu.L RNase H (NEB) and
incubation at 37.degree. C. for 10 minutes.
[0226] All other components of LAMP mixture (2 .mu.L enzyme mix, 2
.mu.L detection reagent, 10 .mu.L reaction mix, 1 .mu.L 20 mg/mL
BSA, all other primers except for B3 and RNase free water to make
up the volume to 20 .mu.L) were mixed together with the solution
obtained from reverse transcription and loaded on a device
immediately. Heating and imaging settings were the same as
described for the two-step RT-LAMP experimental protocol above. The
measured concentration after performing digital RT-LAMP was found
to be 60% of that obtained via digital RT-PCR using B3/F3 as
primers.
[0227] In another set of experiments, the efficiency of two-step
digital RT-LAMP was found to be improved by adding only BIP/FIP
primer in the RT step, adding thermostable RNase H into the LAMP
mixture and removing B3 from the primer mixture.
[0228] For example, 10 .mu.L reaction mix, 1 .mu.L 20 mg/mL BSA,
0.5 .mu.L Superscript III reverse transcriptase (Invitrogen), 6
.mu.L RNase free water, 0.5 uL BIP/FIP primer mix (10 .mu.M) and 2
.mu.L HIV RNA purified from AcroMetrix.RTM. HIV-1 Panel 1E6 were
mixed together. All solutions were operated on ice. The solution
was heated to 50.degree. C. for 15 min for reverse
transcription.
[0229] All other components of LAMP mixture (2 .mu.L enzyme mix, 2
.mu.L detection reagent, 10 .mu.L reaction mix, 1 .mu.L 20 mg/mL
BSA, all other primers except for B3 and RNase free water to make
up the volume to 20 .mu.L) and 0.5 uL Hybridase.TM. Thermostable
RNase H (Epicenter) were mixed together with the solution obtained
from reverse transcription and loaded on a device immediately. The
heating and imaging settings were the same as described for the
two-step RT-LAMP experimental protocols above. The measured
concentration after performing digital RT-LAMP was found to be 60%
of that obtained from digital RT-PCR using B3/F3 as primers.
[0230] Imaging with Mobile Device Camera
[0231] In one embodiment, an imaging device with wireless
communication capability may be used to capture the results of both
isothermal and non-isothermal methods such as digital LAMP and
digital NASBA performed on a microfluidic device as disclosed
herein.
[0232] As one example, an iPhone 4S.TM. is used to capture results
on a disclosed device. The fluorescence readout is achieved by a
standard iPhone 4S.TM. 8MP camera equipped with a yellow dichroic
long-pass filter 10CGA-530 (Newport, Franklin, Mass.). Fluorescence
excitation was achieved by shining blue light on a device at an
oblique angle of approximately 30.degree.. The light source was a
blue LED (LIU003) equipped with a blue short-pass dichroic filter
FD1B (Thorlabs, Newton, N.J.). Excitation light reached the sample
in two ways: by direct illumination and by multiple reflections
between the device plates.
[0233] A device of a design described in a previous publication
(Shen et al., JACS 2011 133: 17705) was imaged in the experiments.
Soda-lime glass plates with chromium and photoresist coating (Telic
Company, Valencia, Calif.) were used to fabricate devices. The
method for making a glass device described in a previous
publication (Du, Lab Chip 2009, 2286-2292), was used. Briefly, the
photoresist-coated glass plate was exposed to ultraviolet light
covered by a photomask with designs of the wells and ducts.
Following removal of the photoresist using 0.1 M NaOH solution, the
exposed chromium coating was removed by a chromium-etching
solution. The patterns were then etched in glass etching solution
in a 40.degree. C. shaker. After glass etching, the remaining
photoresist and chromium coatings were removed by ethanol and
chromium-etching solution, respectively. The surfaces of the etched
glass plates were cleaned and subjected to an oxygen plasma
treatment, and then the surfaces were rendered hydrophobic by
silanization in a vacuum desiccator as previously described (Roach,
Analytical Chemistry 2005, 785-796). Inlet holes were drilled with
a diamond drill bit 0.035 inch in diameter.
[0234] A fluorescent reaction mix for digital LAMP was prepared,
loaded in the device, and allowed to react, as described elsewhere
in this application.
[0235] An image was produced using an iPhone application,
Camera+.TM. (obtained via taptaptap.com) in automatic mode; no
tripod was used. The excitation light was shined from one side of
the device under an oblique angle of approximately 30.degree.. The
resulting illumination was relatively uniform, suggesting that
light spreads by multiple reflections inside the analysis
device.
[0236] FIG. 12 shows an image of a multivolume device filled with
LAMP reaction mix obtained with a iPhone 4S.TM. camera. Image size
is 8 MP. The total number of wells of each kind is 160. In total
there are 122 positive largest wells, 42 of the second largest
positive wells, 5 of the second smallest positive wells and 2 of
the smallest positive wells. Well count was done automatically
using Metamorph software. The signal/noise ratio is over 20 even
for the smallest wells.
[0237] FIG. 13 shows a magnified portion of the image in FIG. 12.
In this image, the smallest wells in the image are approximately
15-20 pixels wide and the signal/noise ratio is over 20.
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Sequence CWU 1
1
6123DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1gaagagttgg cgaaagatcc acg 23221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2cgagctcgaa ttagtctgcg c 21323DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 3graacccact gcttaassct caa
23423DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 4gagggatctc tagnyaccag agt 23520DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
5gagtagtgtt gggtcgcgaa 20621DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 6gtgcacggtc tacgagacct c
21
* * * * *
References